Application of campsis grandiflora flavonoids in preparation of anti-hepatic fibrosis drugs

Abstract

The application discloses application of campion flower flavones in preparation of anti-hepatic fibrosis drugs, and an extraction method of the campion flower flavones, which comprises the following steps: reflux extraction of campion flowers by using ethanol to obtain an extract; recovery of ethanol from the extract under reduced pressure until there is no alcohol smell to obtain campion flower extract; and elution of the campion flower extract through a resin column, and then elution of the resin column by using distilled water and ethanol solution in sequence, recovery of ethanol, and evaporation to dryness to obtain the campion flower flavones. The campion flower flavones have the effect of improving hepatic fibrosis, and with the increase of the drug concentration, the indexes related to liver injury of mice are significantly reduced, and the high-dose campion flower flavones can achieve a similar effect to that of the positive drug colchicine. In addition, the in-vitro experiment shows that the campion flower flavones can induce mitochondrial oxidative stress of hepatic stellate cells, inhibit the activation of the hepatic stellate cells, and reduce collagen deposition in the liver and indexes of serum liver injury, thereby providing a new drug selection for the treatment of hepatic fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to application of a campsis grandiflora flavone in preparation of an anti-hepatic fibrosis drug. BACKGROUND

[0002] Hepatic fibrosis is a pathological and physiological process of abnormal proliferation of connective tissue of the liver caused by various pathogenic factors such as viral or parasitic infection, bile stasis, metabolic disease and long-term intake of excessive alcohol. When the liver is damaged, the dynamic balance between the synthesis, deposition, degradation and absorption of collagen fibers is destroyed, leading to excessive secretion and deposition of extracellular matrix, and further damaging the normal function and structure of the liver. Hepatic fibrosis is a necessary pathological process for various chronic liver damage to liver cirrhosis and eventually to liver failure, and its complex regulation mechanism forms a microenvironment prone to tumorigenic nodules, increasing the risk of cancer.

[0003] Hepatic fibrosis is characterized by activation of hepatic stellate cells (HSC) into myofibroblast-like phenotype, and HSC activation leads to increased expression of alpha-SMA and extracellular matrix proteins such as collagen I, resulting in the occurrence of hepatic fibrosis. Current research suggests that inhibiting HSC activation or eliminating activated HSC is an effective means of treating hepatic fibrosis. Recent studies have found that activated HSCs contain a large number of mitochondria, which may provide more energy for HSC activation, and when mitochondrial function is impaired, activated HSCs may be more susceptible to mitochondrial reactive oxygen species (ROS) attack. The pathological mechanism of hepatic fibrosis is complex, and there is currently no effective western medicine for the treatment of hepatic fibrosis.

[0004] Traditional Chinese medicine theory believes that hepatic fibrosis is caused by deficiency of vital qi, invasion of multiple pathogenic factors such as qi, blood, dampness, heat, stasis and toxins in the body, resulting in obstruction of blood vessels, stagnation of qi and blood stasis, and the disease is located in the liver and spleen, which leads to liver function damage over time. Therefore, strengthening the body and promoting blood circulation are the main principles of treating hepatic fibrosis in traditional Chinese medicine. The mild nature of traditional Chinese medicine has a unique advantage in the treatment of hepatic fibrosis, with less side effects compared to western medicine, and lower treatment cost. However, there is currently no effective treatment strategy for hepatic fibrosis, and finding effective anti-hepatic fibrosis drugs is the top priority in the treatment of fibrosis.

[0005] Campsis grandiflora is the dried flower of Campsis grandiflora (Thunb.) K. Schum. of Bignoniaceae, with sweet and sour taste and cold nature. It belongs to the liver and pericardium channels. The Divine Husbandman's Materia Medica records that campsis grandiflora has the effects of promoting blood circulation and cooling blood to dispel wind. There is currently no research on campsis grandiflora in the treatment of hepatic fibrosis. SUMMARY

[0006] The application aims at overcoming the defects in the prior art, and provides application of campion flower flavones in preparation of an anti-hepatic fibrosis drug.

[0007] The application provides the following technical scheme:

[0008] Application of campion flower flavones in preparation of an anti-hepatic fibrosis drug.

[0009] Further, the extraction method of the campion flower flavones comprises:

[0010] The campion flowers are extracted by refluxing with ethanol to obtain an extraction liquid;

[0011] The extraction liquid is subjected to vacuum recovery of ethanol until no alcohol taste is left, to obtain a campion flower extraction liquid;

[0012] The campion flower extraction liquid is passed through a resin column, and then eluted with distilled water and an ethanol solution in sequence, and the ethanol is recovered, and evaporated to dryness, to obtain the campion flower flavones.

[0013] Further, the method for extracting the campion flowers by refluxing with ethanol comprises:

[0014] The campion flowers are taken, 8 times the mass of 80% ethanol is added, and the extraction is performed twice for 1.5 h each time, and the extraction liquids of the two times are combined after the refluxing is completed.

[0015] Further, before the campion flower extraction liquid is passed through the resin column, the concentration is adjusted to 0.5 g·mL -1 .

[0016] Further, the method for passing the campion flower extraction liquid through the resin column comprises:

[0017] The campion flower extraction liquid is passed through a D101 macroporous adsorption resin column with a ratio of medicinal material amount to resin amount of 1:8 and a ratio of diameter to height of 1:12.

[0018] Further, the method for eluting with distilled water and an ethanol solution in sequence comprises:

[0019] First, 10 times the column volume of distilled water is used for elution; then, 20% ethanol is used for elution; and then, 80% ethanol is used for elution.

[0020] Further, the mass fraction of the campion flower flavones is 10%-15%.

[0021] Compared with the prior art, the application has the beneficial effects:

[0022] The clematis flower flavone provided in the present application has the effect of improving liver fibrosis, and with the increase of the drug concentration, the liver damage related indexes of mice are significantly reduced, and the high-dose clematis flower flavone can achieve similar effects to the positive drug colchicine; and in vitro experiments show that the clematis flower flavone can induce mitochondrial oxidative stress of hepatic stellate cells, inhibit the activation of hepatic stellate cells, and reduce liver collagen deposition, and reduce serum liver damage indexes, thereby providing a new drug selection for the treatment of liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a result graph of the influence of the clematis flower flavone on the liver fibrosis of mice in the embodiment of the present application;

[0024] Figure 2 is a result graph of the influence of the clematis flower flavone on the liver collagen fibers of mice in the embodiment of the present application;

[0025] Figure 3 is a result graph of the influence of the clematis flower flavone on the serum liver damage indexes of mice in the embodiment of the present application;

[0026] Figure 4 is a result graph of the cell viability and cytotoxicity detection experiment in the embodiment of the present application;

[0027] Figure 5 is a result graph of the expression change of the hepatic stellate cell activation related indexes ASCT2, COL1A1 and FN1 detected by the real-time fluorescence quantitative PCR instrument in the embodiment of the present application;

[0028] Figure 6 is a result graph of the protein expression of the hepatic stellate cell activation related indexes alpha-SMA, Collagen I and Fibronectin detected by the Western blot method in the embodiment of the present application;

[0029] Figure 7 is a result graph of the regulation effect of the clematis flower flavone on the mitochondrial reactive oxygen species of hepatic stellate cells in the embodiment of the present application. DETAILED DESCRIPTION

[0030] The application of the clematis flower flavone in preparing an anti-liver fibrosis drug is further described below in combination with the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0031] Example 1: Extraction of clematis flower flavone

[0032] 1.1 Experimental materials

[0033] Clematis (Bozhou Guangyuantang Traditional Chinese Medicine Herbal Pieces Co., Ltd.), anhydrous ethanol, D101 macroporous resin, rutin reference substance.

[0034] 1.2 Main instruments

[0035] Freeze dryer, UV spectrophotometer.

[0036] 1.3 Extraction of campsis grandiflora flavonoids

[0037] Take a certain amount of campsis grandiflora, add 8 times the mass of 80% ethanol for reflux extraction 2 times, 1.5h each time, combine the two extraction liquids, recover ethanol under reduced pressure until there is no alcohol smell, obtain campsis grandiflora extract, and adjust the mass concentration of campsis grandiflora extract to 0.5g·mL -1 , pass through D101 macroporous adsorption resin column with diameter-height ratio of 1:12 according to medicinal material amount:resin amount=1:8, first wash with 10 times the column volume of distilled water, 20% ethanol, discard, then wash with 80% ethanol to obtain total flavonoid fraction. Recover ethanol and evaporate to dryness to obtain campsis grandiflora flavonoids.

[0038] 1.4 Results

[0039] Using UV spectrophotometry, with rutin as the control, the mass fraction of campsis grandiflora flavonoids was measured to be 11.85%, and it was stored at -20℃ for use.

[0040] Example 2: In vivo experiment

[0041] 2.1 Animals

[0042] 48 SPF male ICR mice, 6-8 weeks old, weighing 18-22g, provided by the Comparative Medicine Center of Yangzhou University, production license number: SCXK(Su)2022-0009.

[0043] 2.2 Drugs and reagents

[0044] Campsis grandiflora flavonoids, colchicine, carbon tetrachloride (CCl4).

[0045] 2.3 Main instruments

[0046] Microplate reader, PCR instrument, automatic serum biochemical analyzer.

[0047] 2.4 Animal grouping

[0048] 48 male ICR mice were randomly divided into normal control group (n=8), liver fibrosis model group (n=8), campsis grandiflora flavonoids low-dose group (50mg / kg / d; n=8), campsis grandiflora flavonoids medium-dose group (100mg / kg / d; n=8), campsis grandiflora flavonoids high-dose group (150mg / kg / d; n=8), colchicine positive drug group (1.5mg / kg / d; n=8).

[0049] 2.5 Animal model establishment and drug treatment

[0050] Model establishment: A liver fibrosis model was established using the internationally recognized intraperitoneal injection method of carbon tetrachloride (CCl4). All mice underwent a one-week acclimatization period before subsequent experiments. A 10% CCl4 solution was prepared by mixing CCl4 and olive oil at a volume ratio of 1:9 and used to establish the liver fibrosis model. The solution was injected intraperitoneally three times a week at a dose of 0.1 ml / 20 g for four weeks.

[0051] Treatment methods: The patient was treated once daily by gavage with 50, 100, or 150 mg / kg of Campsis grandiflora flavonoids and intraperitoneal injection of colchicine (1.5 mg / kg) as a positive control. The normal control group and the model group were given the same volume of solvent. The treatment lasted for 4 weeks.

[0052] 2.6 Indicator Testing

[0053] Mouse livers were harvested, and their complete morphology was recorded using a camera. Mouse blood samples were collected and allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 15 minutes. The supernatant was used for subsequent serological tests of TBIL, ALT, AST, and ALP. A small portion of each mouse liver was fixed in 4% paraformaldehyde solution for subsequent histopathological examination: HE staining was used to observe changes in liver tissue cell structure, Masson staining was used to observe collagen deposition, Sirius red staining was used to detect type I and IV collagen deposition, and immunohistochemistry was used to observe the expression of α-SMA in the hepatic sinusoidal region.

[0054] 2.7 Statistical Methods

[0055] In all analyses, one-way ANOVA was used to analyze differences between groups (two or more groups), and P < 0.05 was considered statistically significant. Data were plotted using GraphPad Prism 8 (GraphPad software version 8.0).

[0056] 2.8 Results and Conclusions

[0057] like Figure 1 The images show the complete morphology of the mouse liver as recorded by a camera after the experiment, as well as the structural changes in liver tissue cells stained with hematoxylin and eosin (HE). Figure 1 It can be seen that CCl4-induced liver fibrosis in mice resulted in significant fibrotic pathological changes in the liver, characterized by a rough liver surface and a lighter color. Treatment with flavonoids from trumpet creeper alleviated the morphological changes in the liver induced by CCl4, resulting in the liver regaining its dark brown appearance and a glossy surface.

[0058] like Figure 2 Masson staining was used to observe collagen deposition; Sirius red staining was used to examine type I and IV collagen deposition; and immunohistochemistry was used to observe the expression of α-SMA in liver tissue. Figure 2It was found that flavonoids from trumpet creeper flower can reduce hepatic collagen deposition in a dose-dependent manner, as evidenced by a decrease in the area of ​​collagen staining with Masson's red and Sirius red, and can inhibit the expression of α-SMA, a key marker of fibrosis. These experiments demonstrate that flavonoids from trumpet creeper flower can significantly inhibit hepatic collagen deposition.

[0059] like Figure 3 These are the results of serum TBIL, ALT, AST, and ALP tests. Figure 3 It was found that the dose-dependent reduction of serum liver injury-related markers, including TBIL, ALT, AST, and ALP, by trumpet creeper flavonoids indicated that they could alleviate serum liver injury-related markers in mice.

[0060] The above results indicate that flavonoids from trumpet creeper can dose-dependently improve liver fibrosis-related indicators in mice, specifically by improving liver tissue structure, reducing collagen deposition, and decreasing the expression of liver injury-related indicators, and are slightly better than the positive control drug treatment effect.

[0061] Conclusion: Flavonoids from trumpet creeper flower can improve the pathological changes of liver fibrosis in mice.

[0062] Example 3: In vitro experiment

[0063] 3.1 Main cells used in the experiment

[0064] Human hepatocytes (LO2), mouse hepatocytes (AML12), human hepatic stellate cell line (HSC-LX2), and mouse hepatic stellate cell line (mHSC) were purchased from BeNa Culture Collections (Beijing, China).

[0065] 3.2 Experimental drugs and reagents

[0066] Trumpet creeper flavonoids, DMEM (Biotech), CCK8 (Abbkine, BMU106-CN), LDH detection kit (Beyotine, C0016), RIPA lysis buffer, reverse transcription kit II 1st Strand cDNASynthesisSuper Mix (11123ES60, YEASEN), fluorescent dye QuantiTect SYBR Green PCR Kit Hieff qPCRSYBR Green Master Mix (11202ES08, YEASEN).

[0067] 3.3 Main Instruments

[0068] Q3 Real-time PCR instrument, microplate reader (BIOTECH, USA).

[0069] 3.4 Experimental Methods

[0070] 3.4.1 Cell viability assay

[0071] HSC-LX2 and mHSC cells in logarithmic growth phase were seeded in 96 empty plates at a density of 1*102. 4 Cells were cultured at 37℃ in a 5% CO2 incubator. When the cell density reached 60%-70%, they were treated with 0, 10, 20, 40, 80, 100, 150, 200, and 300 μg / ml of Campsis grandiflora flavonoids for 24 hours. 10 μl of CCK8 was added to each well, and the cells were incubated for another 4 hours. The absorbance was measured at 450 nm using a microplate reader. The inhibition rate of cell viability was calculated based on the ratio of absorbance between the drug group and the control group, thus exploring the effect of Campsis grandiflora flavonoids on cell viability.

[0072] 3.4.2 Cytotoxicity Detection

[0073] LO2 and AML12 cells in logarithmic growth phase were seeded into 96 empty plates at a density of 1*102. 4 Each well was treated with 0, 10, 20, 40, 80, 100, 150, 200, and 300 μg / ml of trumpet creeper flavonoids for 24 hours. 150 μl of the LDH release reagent provided in the kit, diluted 10 times with PBS, was added, and the mixture was incubated for another hour. Subsequently, 60 μl of LDH detection working solution was added to each well, and the absorbance was measured at 450 nm using a microplate reader.

[0074] 3.4.3 Real-time PCR detection of gene expression related to hepatic stellate cell activation

[0075] Take the HSC-LX2 during the logarithmic growth phase, 1*10 6 Cells were seeded in 6 mm petri dishes and treated with 40, 80, and 160 μg / ml of trumpet creeper flavonoids for 24 hours. mRNA was extracted using the Trizol method, and the expression changes of hepatic stellate cell activation-related indicators ASCT2, COL1A1, and FN1 were detected by real-time quantitative PCR.

[0076] 3.4.4 Western blot detection of changes in hepatic stellate cell activation-related indicators

[0077] Take the HSC-LX2 during the logarithmic growth phase, 1*10 6 Cells were seeded in 6 mm petri dishes and treated with 40, 80, and 160 μg / ml of trumpet creeper flavonoids for 24 hours. Cell proteins were collected, and the expression of hepatic stellate cell activation-related markers α-SMA, Collagen I, and Fibronectin was detected by Western blot.

[0078] 3.4.5 Detection of key indicators of oxidative damage in hepatic stellate cells mitochondria

[0079] HSC-LX2 cells in the logarithmic growth phase were used to set up a control group, a group treated with trumpet flavonoids (160 μg / ml), a group treated with TEMPO (mitochondrial ROS scavenger), and a group treated with a combination of TEMPO and trumpet flavonoids. Mito-SOX staining was used to detect mitochondrial ROS levels.

[0080] 3.5 Statistical Methods

[0081] In all analyses, one-way ANOVA was used to analyze differences between groups (two or more groups), and P < 0.05 was considered statistically significant. Data were plotted using GraphPad Prism 8 (GraphPad software version 8.0).

[0082] 3.6 Results and Conclusions

[0083] like Figure 4 To screen the optimal concentration of flavonoids from *Campsis grandiflora* for in vitro cell viability and cytotoxicity assays, the results showed that human hepatic stellate cells (LX2) and mouse hepatic stellate cells (mHSC) began to inhibit hepatic stellate cell viability at concentrations of 80 μg / ml and 20 μg / ml, respectively. Therefore, we used low, medium, and high doses of 40 μg / ml, 80 μg / ml, and 160 μg / ml in vitro. Cytotoxicity assays showed no cytotoxicity to human and mouse hepatocytes at these concentrations.

[0084] like Figure 5 To detect the expression changes of hepatic stellate cell activation-related markers ASCT2, COL1A1, and FN1 using real-time quantitative PCR; Figure 6 The protein expression of hepatic stellate cell activation-related markers α-SMA, Collagen I, and Fibronectin was detected by Western blot. Figure 5 and Figure 6 It was found that flavonoids from trumpet creeper flower could inhibit the expression of hepatic stellate cell activation-related markers α-SMA, Collagen I, and Fibronectin in a dose-dependent manner. These experiments demonstrate that flavonoids from trumpet creeper flower can significantly inhibit hepatic stellate cell activation.

[0085] like Figure 7 To label mitochondria using Mito-tracker, Mito-SOX was used for mitochondrial ROS staining to detect mitochondrial ROS levels. Figure 7It was found that trumpet creeper flavonoids could upregulate the level of mitochondrial ROS in hepatic stellate cells, suggesting that trumpet creeper flavonoids induced mitochondrial damage. Furthermore, the mitochondrial ROS inhibitor TEMPO could reverse the mitochondrial ROS accumulation induced by trumpet creeper flavonoids. These experiments demonstrate that trumpet creeper flavonoids can significantly upregulate the mitochondrial ROS level in activated hepatic stellate cells, leading to mitochondrial oxidative damage.

[0086] The above results indicate that: Trumpet creeper flavonoids can inhibit hepatic stellate cell activity in a dose-dependent manner without significant toxicity to hepatocytes; Trumpet creeper flavonoids can inhibit the expression of hepatic stellate cell activation-related markers α-SMA, CollagenI, and Fibronectin in a dose-dependent manner; and Trumpet creeper flavonoids can induce upregulation of mitochondrial oxidative stress levels in hepatic stellate cells.

[0087] Conclusion: Flavonoids from trumpet creeper flower have an anti-hepatic fibrosis effect by inducing mitochondrial oxidative stress and inhibiting hepatic stellate cell activation.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of campnosperma grandiflora flavonoids in the preparation of a medicament for the treatment of liver fibrosis, characterized in that, The campsis grandiflora flavone has the effect of inhibiting the activation of hepatic stellate cells by inducing mitochondrial oxidative stress, thereby playing the role of resisting liver fibrosis. The extraction method of the campsis grandiflora flavone comprises the following steps: The campsis grandiflora is taken, 8 times of 80% ethanol of the mass of the campsis grandiflora is added, and the campsis grandiflora is extracted by reflux for 2 times, 1.5 h each time; after the reflux is finished, the two times of extraction liquid are combined, and the extraction liquid is subjected to alcohol recovery under reduced pressure until there is no alcohol taste, so that the campsis grandiflora extraction liquid is obtained. The extraction solution of campion flower was adjusted to a mass concentration of 0.5 g·mL -1 Then, the medicinal material was passed through a D101 macroporous adsorption resin column with a height-diameter ratio of 1:12 at a ratio of medicinal material to resin of 1:

8. The column was first washed with 10 times the column volume of distilled water, then with 20% ethanol, and then with 80% ethanol. The total flavone fraction was obtained by discarding the eluate. The ethanol was recovered, and the campion flower flavone was obtained by drying. 2.The application of campsis radicans flavones in the preparation of anti-liver fibrosis drugs according to claim 1, characterized in that, The mass fraction of the campsis grandiflora flavone is 10%-15%.

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