CN105497005A - Application of flavonoid compound in treatment of inflammatory diseases - Google Patents

Abstract

The invention discloses an application of a flavonoid compound 7-hydroxy-5,3',4'-trimethoxyflavone as an active component in treatment of inflammatory diseases. 7-hydroxy-5,3',4'-trimethoxyflavone as the anti-inflammation active component has the characteristics of small toxicity and good curative effect and has a remarkable inhibition effect on inflammatory factors, especially macrophage factors, so that the compound can be used as an anti-inflammatory preparation for treating the inflammatory diseases such as sepsis, arthritis, gastritis, immune type enteritis, diabetes and the like related with multiple other macrophages.

Description

Application of a flavonoid compound in the treatment of inflammatory diseases

technical field

The invention belongs to the technical field of biopharmaceuticals, and specifically relates to the application of a flavonoid compound 7-hydroxy-5,3',4'-trimethoxyflavone as an active ingredient in the treatment of inflammatory diseases, and is mainly used in various macrophages Inflammatory diseases involved, such as arthritis, gastritis, immune enteritis, diabetes, etc.

Background technique

Inflammation is a common disease that affects people's health in all aspects. In a series of medical breakthroughs, scientists have discovered that inflammation is linked to osteoarthritis, sepsis, arteriosclerosis, heart disease, stroke, cancer, diabetes, asthma, migraines, Alzheimer's disease, periodontitis, irritable bowel syndrome Syndrome, chronic fatigue syndrome, etc. are closely related. As you can see, inflammation affects the entire body. Some inflammatory diseases such as sepsis remain a huge medical problem worldwide, with severe systemic inflammatory syndromes leading to multi-organ failure and high mortality rates. In North American countries, it is estimated that the incidence of sepsis exceeds 600,000 cases per year, and the mortality rate is as high as 30%-50%. Currently, there is no effective cure for sepsis clinically. In addition, many chronic inflammatory diseases are very harmful: one can cause high cholesterol, high blood pressure, and heart disease. Studies have found that inflammation and heart disease are one of the diseases with the strongest correlation, especially arteriosclerosis. In the past, we always thought that the cause of arteriosclerosis was excess cholesterol, so scientists focused on how to control the lower cholesterol content. It is now known that cholesterol only plays an indirect role and that inflammation is the causative factor. Inflammation causes white blood cells, low-density lipoprotein cholesterol, and platelets to accumulate on the blood vessel wall through chemical attraction, resulting in atherosclerosis. We call these generated substances plaques. With the accumulation of plaque, the damage of blood vessels is increased, resulting in more damage, leading to more inflammatory plaques, and endangering the cardiovascular system; second, it can cause diabetes. Scientists have long known that type 1 diabetes is caused by A disease caused by an autoimmune response that attacks and destroys insulin-producing cells in the pancreas, recent studies have shown that type 2 diabetes is also associated with inflammation. A survey in Scotland found that a high amount of an inflammatory product called C-reactive protein may develop type 2 diabetes within 5 years. It is absolutely necessary to control the occurrence of type 2 diabetes by controlling inflammation; three can cause autoimmune diseases such as rheumatoid arthritis, lupus erythematosus or multiple sclerosis syndrome, all of which are caused by uncontrollable inflammatory reactions; four can Leading to gingivitis or periodontal disease, many physiological signals of inflammation are hidden, but gingivitis can cause lesions in other parts of the body, such as arteriosclerosis, coronary heart disease, etc., which should be paid close attention to; five can cause cancer lesions , any attack of chronic inflammation can trigger cancer. Cells and chemicals involved in the inflammatory process can cause changes in cells: making cells more prone to cancer, turning precancerous cells into activated cancer cells, leading to the growth of cancer cells. And more and more inflammatory diseases are difficult to cure, and more new anti-inflammatory drugs need to be developed to meet people's needs.

Inflammation is a defense-based response of the body to damage caused by various inflammatory stimuli, and is an extremely common and important pathological process. Macrophages are an essential component of innate immunity and play an important role in resisting inflammation and host defense responses, and macrophages also have the function of maintaining body homeostasis, including organ remodeling and metabolic functions during individual development adjustment. Macrophages have a high degree of diversity and specificity, are widely distributed in various tissues and organs, sense and respond to different microenvironmental stimuli, and thus differentiate into different phenotypes. M1-type macrophages, that is, classically activated macrophages, express high levels of pro-inflammatory factors, reactive nitrogen, and reactive oxygen species, have high microbial and tumor killing effects, and promote TH1 responses. M2-type macrophages, alternatively activated macrophages, have enhanced antiprotozoal ability, promoting tissue regeneration and tumor progression. The process of macrophage differentiation to M1 and M2 is called polarization. M1 and M2 are the two extremes of macrophage polarization. In practice, macrophages may be in any stage of polarization. M1-M2 macrophages can transform into each other to a certain extent. The pathological state of the disease is also closely related to the dynamic changes in the activation state of macrophages. Classically activated M1 macrophages are involved in the initiation and persistence of inflammation, and M2 or M2-like macrophages are involved in the resolution of chronic inflammation. Different functions of M1 and M2 Monocytes and macrophages are the first line of defense of the body. Therefore, targeting macrophage-mediated inflammation can be used as a screening index for anti-inflammatory drugs.

The body's inflammatory response is mainly generated by endotoxin entering and acting on macrophages. Endotoxin is present in the outer membrane of Gram-negative bacteria, and its main component is lipopolysaccharide (LPS). It is a pathogenic source that widely exists in nature. LPS acts on cells, especially monocytes, macrophages and neutrophils, and induces macrophages and neutrophils to produce bioactive molecules through signal transduction pathways, which plays a role in the pathogenic mechanism of Gram-negative bacteria. important role. When endotoxin enters the human body, it can cause fever, diarrhea, dysentery, disseminated intravascular hemolysis, septic shock, and even death. The main cause of endotoxin shock is that a large amount of endotoxin acts on the body's macrophages and other systems to produce biologically active substances such as IL-1beta, IL-6, and TNF-alpha. These substances act on small blood vessels to cause dysfunction, which leads to microcirculatory disturbance. The clinical manifestations are microcirculatory failure, hypotension, hypoxia and acidosis, etc., which eventually lead to shock or even death of the patient. It can be seen that macrophages, as important cells in endotoxic shock, participate in the occurrence and progression of diseases. Therefore, a comprehensive and in-depth understanding of the mechanism of endotoxin and the study of rapid and effective endotoxin antagonists are of great significance for the prevention and treatment of inflammatory diseases.

Flavonoids have been widely studied as active ingredients of various traditional Chinese medicine preparations. 7-Hydroxy-5,3',4'-trimethoxyflavone is soluble in organic solvents such as methanol, ethanol, acetone and chloroform, but not See the research that this compound improves inflammatory diseases by regulating the function of macrophages, and this compound has not been used to treat any inflammatory diseases.

7-hydroxy-5,3′,4′-trimethoxyflavone, chemical name: 7-hydroxy-5,3′,4′-trimethoxyflavone, C ₁₈ H ₁₆ O ₆ , molecular structure:

Contents of the invention

The purpose of the present invention is to explore the application of 7-hydroxy-5,3',4'-trimethoxyflavone as an anti-inflammatory drug in the treatment of various inflammatory diseases involving macrophages.

The present invention proves through research that the main anti-inflammatory mechanism of 7-hydroxy-5,3',4'-trimethoxyflavone is to adjust the polarization of macrophages, and can be used for treating various inflammatory diseases in which macrophages participate.

7-Hydroxy-5,3',4'-trimethoxyflavone (HTMF) is isolated and purified from natural plants or synthesized. This compound is not toxic to normal macrophages, which indicates that the application of HTMF treatment can avoid the side effects of some common immunomodulatory compounds. At the same time, HTMF can inhibit the characteristic inflammatory factors IL-1beta, IL-6, and NO released by M1 macrophages without affecting the survival rate of macrophages, indicating that HTMF has a good anti-inflammatory effect.

Compared with the prior art, the beneficial effect of the present invention: 7-hydroxy-5,3',4'-trimethoxyflavone (HTMF) is derived from the extract of natural plants, and as an anti-inflammatory active ingredient, it has low toxicity and good curative effect It has good characteristics and has a significant inhibitory effect on inflammatory factors, especially macrophage cytokines, so it can be used as an anti-inflammatory agent for other inflammatory diseases in which macrophages participate, such as sepsis, arthritis, immune enteritis, Gastritis, diabetes, etc.

detailed description

The effects of the present invention are further described below through specific embodiments:

Embodiment 1.HTMF toxicity detection

After the normally cultured mouse macrophage cell line Raw264.7 was co-incubated with different concentrations of HTMF for 24 hours, the survival rate was detected by MTT method. Phage strains are not toxic. Raw264.7 cells were planted in a 96-well plate at 5*10 ⁴ cells/well, 100ng/ml LPS was added, and HTMF was added to incubate at 37°C for 24 hours. The effect of the compound on the survival of the cell line was detected by MTT method. Experiments showed that HTMF also had no inhibitory effect on the survival rate of LPS-activated macrophages (Figure 1, compound HTMF is 18).

Example 2. Inhibitory effect of HTMF on NO secretion from macrophages activated by lipopolysaccharide (LPS)

Raw264.7 cells were planted in a 96-well plate at 5*10 ⁴ cells/well, added HTMF, and incubated in a 37°C incubator for 24 hours, and the effect of the compound on the secretion of NO by the cells was detected by the Gress reagent method. The experimental results show that HTMF has a better inhibitory effect on NO, and the inhibition rate is close to 40% at 30uM. (Figure 2, compound HTMF is 18)

Example 3. Inhibition of IL-1β and IL-6mRNA levels in lipopolysaccharide (LPS)-activated macrophages by HTMF

Raw264.7 cells were planted in a 6-well plate at 1*10 ⁶ cells/well, and HTMF and 100ng/ml lipopolysaccharide (LPS) were added at the same time and incubated at 37°C for 6 hours. Compared with no HTMF, IL was detected by QPCR -1β, the change of IL-6mRNA level. The experimental results showed that after LPS stimulation, macrophages were activated, and the mRNA levels of cytokines IL-1β and IL-6 increased (Figure 3, compound HTMF is 18). Under the action of HTMF, the mRNA levels of IL-1β and IL-6 were significantly reduced (Figure 3, compound HTMF is 18).

Combining the results of Figure 1, Figure 2, and Figure 3, it shows that HTMF inhibits macrophage activation without affecting the survival of macrophages, revealing that HTMF has a significant inhibitory effect on inflammatory factors, especially macrophage cytokines, so it can be used as a Anti-inflammatory preparations are used for other inflammatory diseases involving macrophages, such as sepsis, arthritis, immune enteritis, diabetes, gastritis, etc.

Description of drawings

Figure 1 shows the toxicity test results of HTMF (compound HTMF is No. 18).

(A) After the normal cultured mouse macrophage cell line Raw264.7 was co-incubated with different concentrations of HTMF for 24 hours, the survival rate was detected by MTT method. The dose reached 30 μM, and the cell survival rate did not decrease significantly compared with the normal group, indicating that HTMF (Compound HTMF No. 18) has no toxicity to macrophage cell lines.

(B) Raw264.7 cells were seeded in a 96-well plate at 5*10 ⁴ cells/well, 100ng/ml LPS was added, and HTMF was added to incubate at 37°C for 24 hours. The effect of the compound on cell line survival was detected by MTT method. Experiments showed that HTMF (compound HTMF No. 18) had no inhibitory effect on the survival rate of LPS-activated macrophages.

Figure 2 shows the inhibitory effect of HTMF on the secretion of NO from macrophages activated by lipopolysaccharide (LPS) (No. 18 in the figure is the compound HTMF)

Raw264.7 cells were planted in a 96-well plate at 5*10 ⁴ cells/well, added HTMF, and incubated in a 37°C incubator for 24 hours, and the effect of the compound on the secretion of NO by the cells was detected by the Gress reagent method. The experimental results show that HTMF has a better inhibitory effect on NO, and the inhibition rate is close to 40% at 30uM. (Figure 2, compound HTMF is 18)

Figure 3 is the inhibitory effect of HTMF on IL-1β and IL-6 mRNA levels in lipopolysaccharide (LPS)-activated macrophages (No. 18 in the figure is compound HTMF)

Raw264.7 cells were planted in a 6-well plate at 1*10 ⁶ cells/well, and HTMF and 100ng/ml lipopolysaccharide (LPS) were added at the same time and incubated at 37°C for 6 hours. Compared with no HTMF, IL was detected by QPCR -1β, the change of IL-6mRNA level. The experimental results showed that after LPS stimulation, macrophages were activated, and the mRNA levels of cytokines IL-1β and IL-6 increased (Figure 3, compound HTMF is 18). Under the action of HTMF, the mRNA levels of IL-1β and IL-6 were significantly reduced (Figure 3, compound HTMF is 18).

Claims (2)

1. 7-Hydroxy-5,3',4'-trimethoxyflavone is used as an active ingredient in the treatment of inflammatory diseases. 2. The use according to claim 1, characterized in that 7-hydroxy-5,3',4'-trimethoxyflavone as an active ingredient can treat inflammatory diseases involving macrophages.