Application of gynostemma pentaphylla extract monomer GI in preparation of medicine for treating and / or preventing colitis

Abstract

The invention discloses application of a fiveleaf gynostemma herb extract monomer GI in preparation of a medicine for treating and / or preventing colitis, the fiveleaf gynostemma herb extract can obviously protect damage caused by DSS, the colon length is obviously improved, oxidative stress caused by DSS can be reduced by regulating and controlling an Nrf-2 signal channel, and the colitis treatment and / or prevention can be realized by the fiveleaf gynostemma herb extract monomer GI in preparation of a medicine for treating and / or preventing colitis. The gynostemma pentaphyllum extract can significantly improve an LPS-induced RAW264.7 cell inflammation model, and treat and / or prevent colitis. The fiveleaf gynostemma herb extract disclosed by the invention has a very good effect of preventing and treating the DSS-induced colitis.

Description

Technical Field

[0001] This invention relates to the use of the dammarane-type triterpenoid monomer GI from Gynostemma pentaphyllum extract in pharmaceuticals or health foods for the treatment of DSS-induced colitis and LPS-induced RAW264.7 cells. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract, encompassing two main forms: ulcerative colitis (UC) and Crohn's disease (CD). In the disease state, macrophages are activated and release inflammatory factors, which are significantly induced in patients with colitis and significantly influence the inflammatory process, such as IL-6 and TNF-α. Cellular oxidative stress is present in the initiation and progression of inflammation, greatly affecting the inflammatory environment. Nrf2 is a key target for regulating oxidative stress in response to increased susceptibility to colitis and colorectal cancer, playing a crucial role not only in cellular defense against oxidative stress but also in the negative regulation of inflammatory responses.

[0003] Gynostemma pentaphyllum (Thunb.) Makino is the whole herb of the genus Gynostemma in the Cucurbitaceae family. Due to its dammarane-type saponin structure, similar to ginsenosides, it is known as "Southern Ginseng." Previous research by our group has discovered a large number of novel secondary saponins with significant pharmacological activity, exhibiting various effects such as anti-inflammatory, antioxidant, anti-tumor, diabetes treatment, and intestinal regulation. Among these, GI is the most abundant dammarane-type triterpenoid structure discovered for the first time. Therefore, exploring whether GI can effectively and gently treat colitis is of great significance. Summary of the Invention

[0004] The purpose of this invention is to propose the use of GI in the preparation of medicines or health foods for treating colitis.

[0005] This invention establishes a mouse model of DSS-induced colitis and explores for the first time the protective mechanism of GI treatment in this model. The study found that the mechanism of GI in treating colitis may be through the regulation of inflammatory response and oxidative stress.

[0006] This invention establishes an LPS-induced RAW264.7 macrophage inflammation model and explores for the first time the protective mechanism of GI treatment in an LPS-induced inflammation model. The study found that GI may work by regulating the inflammatory response and oxidative stress.

[0007] The objective of this invention is achieved by at least one of the following technical solutions:

[0008] The disease described is colitis.

[0009] The GI is used for preparing a drug for treating and / or preventing colitis.

[0010] The colitis is inflammatory bowel disease.

[0011] The drug is a drug capable of improving the damage of the colon tissue.

[0012] The drug is a drug capable of improving the damage of the colon tissue.

[0013] The drug is a drug capable of regulating the expression of the gene related to the inflammatory signal path.

[0014] Preferably, the drug is capable of treating colitis by regulating TNF-alpha, IL-1beta and NO inflammatory factors.

[0015] The drug is a drug capable of regulating the expression of the gene related to the oxidative stress signal path.

[0016] Preferably, the oxidative stress signal path is Nrf-2.

[0017] Preferably, the drug is capable of treating colitis by regulating the gene related to the oxidative stress signal path.

[0018] The drug is a drug capable of regulating serum CAT and MDA and ROS in cells.

[0019] The drug is a drug capable of regulating the intestinal barrier.

[0020] The drug is a drug capable of regulating the Occludin level in the tissue.

[0021] The drug is a drug capable of regulating the intestinal active bacteria abundance influence map.

[0022] The structural formula of the GI is as follows:

[0023]

[0024] The chemical structural formula and structural identification data of the compound GI are recorded in the literature: "Novel dammarane-type triterpenes isolated from hydrolyzate of total Gynostemma pentaphyllum saponins", X.-S. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3095-3099.

[0025] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0026] (1) GI is a stable and safe monomer extracted from functional food Gynostemma pentaphyllum. The establishment of mature LPS-induced RAW264.7 macrophage inflammation model and DSS-induced animal IBD model greatly guarantees the reliability of the experiment.

[0027] (2) Through further experimental research, it is found that after GI treatment, the CAT and MDA in the serum of mice can be significantly improved, and the levels of TNF-α and IL-1β can be significantly reduced, which has a good effect of preventing and treating colitis, and GI can be applied in the preparation of drugs or health care food for protecting colitis.

[0028] (3) Since Gynostemma pentaphyllum has good anti-inflammatory effect, and inflammation and oxidative stress are also important in the pathogenesis of colitis, GI can significantly inhibit the expression of inflammation and regulate Nrf-2 signal pathway related genes, so GI has a wide application prospect in the treatment of DSS-induced colitis.

[0029] The present application first discusses the mechanism of GI in treating colitis and can be applied in the treatment of colitis drugs or health care food. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The figure is the weight result of GI in DSS model mice.

[0031] Figure 2 The figure is the change graph of colon length of GI in DSS model mice.

[0032] Figure 3 The figure is the effect graph of GI on tissue damage of DSS model mice.

[0033] Figure 4 The figure is the effect result graph of GI on the expression of inflammatory factors in DSS model mice, wherein (a) is the TNF-α gene expression result graph; (b) is the IL-1β gene expression result graph.

[0034] Figure 5 The figure is the CAT, MDA level content detection result graph and Nrf2 protein expression level result graph, wherein (a) is the CAT result graph; (b) is the MDA result graph; (C) is the Nrf2 protein expression level result graph.

[0035] Figure 6 The figure is the expression result graph of GI on the immunohistochemical chemical staining Occludin of DSS model mice.

[0036] Figure 7 The figure is the influence graph of the intestinal live bacteria abundance of mice.

[0037] Figure 8Figure for the GI on the normal RAW264.7 cell survival rate results chart.

[0038] Figure 9 Figure for the LPS-induced inflammation model NO content results chart.

[0039] Figure 10 Figure for the GI in the LPS model immunofluorescence staining ROS expression results chart. DETAILED DESCRIPTION

[0040] The following specific embodiments of the present application are further described in conjunction with examples, but the implementation and protection of the present application are not limited thereto. It should be noted that if the following processes are not specifically described in detail, they can be implemented or understood by those skilled in the art with reference to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be purchased on the market.

[0041] The present application will be further described in detail below in conjunction with specific examples.

[0042] Example 1

[0043] The GI: The GI extracted by the laboratory earlier was determined by DAD method for purity determination, and the purity was ≥98%.

[0044] Example 2

[0045] Study on the therapeutic effect of GI on colitis in DSS model of mice.

[0046] Example 3

[0047] Study on the anti-inflammatory and antioxidant effects of GI in LPS-induced RAW264.7 macrophage inflammation model.

[0048] Experimental materials

[0049] Experimental material 1

[0050] The GI in Example 1 was used as the main research sample to further explore the treatment of colitis and the mechanism of action by adjusting Nrf2.

[0051] Experimental material 2

[0052] The GI in Example 1 was used as the main research sample to further explore the anti-inflammatory and antioxidant mechanism in the LPS-induced RAW264.7 macrophage inflammation model.

[0053] Experimental method

[0054] Experimental method 1

[0055] Firstly, the mouse DSS model was established, 6-7 weeks old male C57BL / 6 mice weighing 22-24 g (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were fed with standard pellet feed with free access to water, temperature 21-25℃, humidity 50-70%, light and dark alternation 12 hours. Randomly divided into blank control group, DSS model group, GI low dose group (GI 2.5 mg / kg), GI medium dose group (GI 5 mg / kg), GI high dose group (GI 10 mg / kg) and SCC positive drug group (SCC).

[0056] From 1-7 days, all mice except the CON group were fed with 2.8% DSS (dextran sulfate sodium). After the model was successfully established, the CON and DSS groups were given the same amount of water, and the mice in the GI 2.5 group, the GI 5 group, the GI 10 group and the SCC group were given the corresponding drugs by gavage from 8-14 days. The mice were weighed every day, and the clinical symptoms of colitis were monitored. On day 15, blood was collected from anesthetized mice, serum was obtained by centrifugation, and colon tissue was processed and measured for length. Paraffin-embedded colon tissue or frozen at -80℃ was used for subsequent analysis.

[0057] The contents of various indicators in serum were detected using a commercial kit to determine the changes of GI in DSS model of colitis.

[0058] Experimental method 2

[0059] Firstly, the LPS-induced RAW264.7 macrophage inflammation model was established. The RAW264.7 macrophage cell line was cultured in a sterile cell incubator at 37℃, 5% CO2, with DMEM high glucose medium containing 10% FBS and 1% double-antibiotic (penicillin and streptomycin).

[0060] The effect of GI on the survival rate of RAW264.7 cells was detected by CCK8 method. When the cells grew to the logarithmic growth phase, the RAW264.7 cell suspension was inoculated in a 96-well plate and cultured for 24 hours before the experiment. Each group was set up with 6 replicate wells. Cells were treated with different GI concentrations (2.5, 5, 10, 12.5, 15, 17.5, 20 μM) for 24 hours, and cell viability was measured by CCK8. Then the absorbance of each well at 450 nm wavelength was detected by a microplate reader. The cell survival rate was calculated according to the formula: cell survival rate = OD drug group / OD blank group x 100%.

[0061] RAW264.7 cells were inoculated in 6-well plates and cultured for 24 hours. When the cells were in good condition and in the logarithmic growth phase, the cells were treated with LPS (1 μg / mL) inducer for 24 hours, except for the CON group. The blank group was the control group, the LPS model group, the GI low-dose group (GI 2.5 μM), the GI medium-dose group (GI 5 μM), the GI high-dose group (GI 10 μM), and the SCC positive drug group (SCC).

[0062] The indicators of the cell supernatant and the cells were detected using a commercial kit to determine the changes of GI to the LPS-induced RAW264.7 cell model.

[0063] Experimental results

[0064] (1) In the mouse DSS model, the body weight of the mice in the different dose GI treatment groups and the SCC group was significantly higher than that in the DSS model group, and showed a dose-dependent effect. Figure 1 ).

[0065] (2) In the mouse DSS model, compared with the DSS model group, the different dose GI treatment groups and the SCC group significantly improved the change in colon length, and showed a dose-dependent effect. The experimental results showed that GI treatment of mice could effectively inhibit the shortening of colon length Figure 2 ).

[0066] (3) The pathological changes of DSS-induced intestinal tissues of mice were evaluated by HE staining. The experimental results observed that the colon mucosa structure of the mice in the DSS model group was severely damaged, the crypts and goblet cells were widely absent, and a large number of inflammatory cells infiltrated. Compared with the DSS model group, the colon structure of the mice treated with different doses of GI and SCC was clear, and the inflammatory cells were significantly reduced Figure 3 ). The results showed that GI could significantly reduce the colon tissue damage caused by DSS.

[0067] (4) The changes of inflammatory factors IL-1β and TNF-α in the serum of DSS-induced mice with colitis were detected at the protein level by ELISA. In the ELISA experimental results, compared with the DSS model group, the expression of inflammatory factors showed a downward trend after treatment with different doses of GI and SCC, and showed a dose-dependent effect Figure 4 a-b). The results showed that GI could inhibit the expression of IL-1β and TNF-α and reduce intestinal inflammation.

[0068] (5) Colitis is closely related to oxidative stress. In the mouse DSS model, the CAT content in the serum of mice in the different dose GI and SCC treatment groups was significantly higher than that in the DSS model group, and the MAD was lower than that in the DSS model group, and showed a dose-dependent effect Figure 5a-b). In addition, Nrf2 is an antioxidant factor that maintains the intracellular redox balance by regulating some antioxidant responses. The expression of Nrf2 in the DSS model group was significantly lower than that in the blank control group, and after treatment with different doses of GI and SCC, the expression was restored in a dose-dependent manner Figure 5 c).

[0069] (6) The intestinal barrier maintains intestinal homeostasis, and the mucus barrier is composed of a mucus protein secreted by the widely existing goblet cells in intestinal tissue. The expression of Occludin was detected by immunohistochemical staining. The immunohistochemical staining results showed that the expression of Occludin in the DSS model group was significantly lower than that in the blank control group, and compared with the DSS model group, the expression level of Occludin gradually recovered to the blank control group after treatment with different doses of GI and SCC, and showed a dose-dependent manner Figure 6 ).

[0070] (7) In order to study the response of intestinal microbiota of DSS-induced colitis mice, we analyzed the species by 16S rRNA high-throughput sequencing. Compared with the blank control group, the composition of intestinal microbiota in the DSS group changed significantly at the level of door ( Figure 7 a) and genus ( Figure 7 b). The improvement of the structural composition and diversity of the intestinal flora of the DSS model group after treatment with GI10 had a certain effect on the blank control group. This indicates that DSS causes considerable changes in the structure of intestinal microbiota, and GI intervention can reverse it.

[0071] (8) The normal RAW264.7 cells were treated with GI at a concentration of 0-15 μM for 24 hours, and there was no significant difference in cell survival rate after 24 hours of treatment with 2.5-12.5 μM GI compared with the blank control group. The experiment showed that within this range, GI had no effect on the survival rate of normal RAW264.7 cells Figure 8 ).

[0072] (9) The establishment of an inflammatory model by inducing RAW264.7 cells with 1 μg / mL LPS led to an increase in NO content. In the experiment, the NO content was detected by Griess method, and compared with the LPS model group, the NO content was reduced after treatment with different doses of GI and SCC, and showed a dose-dependent manner Figure 9 ). The results showed that GI could inhibit the expression of NO inflammatory factors and improve the damage of LPS-induced RAW264.7 cells.

[0073] (10) The release of intracellular reactive oxygen species was detected by DCFH-DA fluorescent probe. The results of immunofluorescence staining showed that the green fluorescence of the LPS model group cells was significantly enhanced. Compared with the LPS model group, after being treated with different doses of GI and SCC, the enhancement of green fluorescence induced by LPS was significantly reduced. The statistical results showed that compared with the LPS model group, the intracellular ROS content was significantly reduced after being treated with different doses of GI and SCC Figure 10 ).

Claims

1. The application of Gynostemma pentaphyllum extract monomer GI having the following structure in the preparation of drugs for the treatment and / or prevention of colitis.

2. The application according to claim 1, characterized in that, The colitis mentioned is inflammatory bowel disease.

3. The application according to claim 1, characterized in that, The drugs mentioned for treating and / or preventing colitis are those that can regulate TNF-α and IL-1β-related inflammatory factors.

4. The application according to claim 1, characterized in that, The drugs for treating and / or preventing colitis are those capable of modulating the Nrf-2 pathway of oxidative stress.

5. The application according to claim 1, characterized in that, The medications used to treat and / or prevent colitis are those capable of regulating serum CAT and MDA levels.

6. The application according to claim 1, characterized in that, The medications used to treat and / or prevent colitis are those that can regulate the composition of the gut microbiota.

7. A pharmaceutical composition for treating and / or preventing colitis, characterized in that, It contains the monomer GI from Gynostemma pentaphyllum extract.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further contains one or more pharmaceutically acceptable excipients or carriers.

9. Application of Gynostemma pentaphyllum extract monomer GI in the preparation of health products.

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