TWI543760B - Application of Antelope Antioxidant in the Preparation of Drugs for the Treatment of Kidney Diseases (1) - Google Patents

Description

Use of Antrodia camphorata compound in preparing medicine for treating kidney disease (1)

The present invention relates to the medical use of a compound isolated from Antrodia camphorata, and more particularly to the use of an Antrodia camphorata compound for the preparation of a medicament for the treatment of kidney disease.

Antrodia camphorata , also known as Antrodia camphorata , Burdock mushroom or Rhododendron chinense , is a perennial fungus of the genus Aphyllophorales and Polyporaceae . It is a unique species of fungi in Taiwan and only grows in Taiwan. On the inner wall of the hollow decayed heartwood of the genus Cinnamoum kanehirai Hay. Due to the extremely rare distribution of burdock trees and the artificial piracy, the number of wild burdocks that can grow in the middle of the burdock is even rarer, and because the growth of its fruiting bodies is rather slow, the growth period is only from June to October. Between, so the price is very expensive. In Taiwan folk medicine, Niu Zhizhi has the functions of detoxification, alleviating diarrhea, anti-inflammatory, treating liver-related diseases and anti-cancer. Antrodia camphorata has many complex components, such as triterpenoids, polysaccharides (such as β-D-glucan), and many other physiologically active ingredients. Adenosine, vitamins (such as vitamin B, nicotinic acid), protein (including immunoglobulin), superoxide dismutase (SOD), trace elements (such as: calcium, phosphorus, strontium), nucleic acids, Sterols and blood pressure stabilizing substances (such as antodia acid), etc., these physiologically active ingredients are considered to have anti-tumor, immune-enhancing, anti-allergic, anti-pathogenic, anti-hypertensive, hypoglycemic and cholesterol-lowering effects. At the same time, in recent years, it has been discovered that certain ingredients may have anti-inflammatory effects, especially for treating kidney diseases.

Commonly common kidney diseases often affect kidney function by attacking the kidney skein. Kidney spheroid disease includes many diseases caused by a variety of genes and environmental factors, but it is mainly divided into two major categories of glomerulosclerosis and glomerulonephritis. Kidney spheroid hard Symptoms refer to glomerular sclerosis in the kidney. Generally refers to the microvascular of the kidney, the glomerulus of the kidney, and the damage to the renal functional unit used to filter urine from the blood. Urine protein (the most abundant protein in urine) is one of the signals of glomerulosclerosis. Kidney damage can affect kidney filtration and cause protein to leak from the blood into the urine. However, glomerulosclerosis is only one of the many causes of urinary protein. Kidney sectioning may be a necessary basis for judging whether a patient has kidney sclerosing or other kidney disease. Renal glomerulosclerosis may specifically mean localized glomerulosclerosis (FSGS) and nodular glomerulosclerosis.

Localized glomerulosclerosis (FSGS) is judged by local lesions of glomerular sclerosis and foot process effacemen. Nearly 20 years of research have shown that the incidence of end-stage renal disease in patients with FSGS is between 13 and 78%. Although the etiology and pathogenesis of FSGS are not clear, it should be caused by the damage of the glomerular epithelial cells themselves, which leads to a complex reaction in the glomerulus, which leads to hardening of the glomerulus.

Nodular glomerulosclerosis or intercapillary glomerulonephritis, also known as diabetic nephropathy (diabetic nephropathy) or Kimmelstiel-Wilson syndrome, is a progressive renal syndrome. It is caused by capillary lesions of the glomerulus of the kidney. The disease is characterized by nephropathy and diffuse glomerular sclerosis; it is caused by diabetes for a long time, and the main treatment in each country is dialysis.

So far, although steroids and immunological preparations are mainly used for the treatment of primary FSGS, the therapeutic effects of these therapies on the development of renal injury are still wired, and because they produce various side effects, the treatment is only Based on rule of thumb, and less based on pathological evidence.

Renal glomerulonephritis refers to the inflammatory response of the renal membrane tissue, in which the membrane tissue system has a filtering function, and the waste and more fluid can be separated from the blood.

The most common renal glomerulonephritis is immunoglobulin A type glomerulonephritis (IgAN), the cause of this accelerated development of nephritis is still unpredictable, and clinically still unable to prevent and treat, it is considered to be one of the main factors that may cause chronic renal failure of kidney disease. Therefore, in the kidney of IgAN patients, even if other immune, clinical and pathological factors cause renal failure, systemic T cell activation and lymphocyte/macrophage/neutrophil infiltration are abnormal. Increased is considered to be one of the main processes leading to the conversion of IgAN into chronic renal failure. Furthermore, oxidative stress is also a major cause of the increase and development of IgAN in patients and animal models; it has been reported that reactive oxygen species (ROS) are directly induced in most human and experimental kidney diseases (including IgAN). disease.

Although glucocorticoid steroids have been widely used in the treatment of IgAN, their effects on IgAN's ability to maintain kidney function and slow down urinary protein remain unclear and potentially uncontrollable inhibition due to prolonged use. The side effects of the immune response still cause problems in medication.

At present, many experiments have learned that the extract of Antrodia camphorata has many medical effects, and its components have been analyzed one after another. However, whether there are other compounds in the extract of Antrodia camphorata that can effectively treat kidney diseases is still an urgent problem to be solved.

Accordingly, the present invention is directed to two compounds extracted from Antrodia camphorata, and whether it has an effect of treating kidney diseases and anti-kidney inflammation.

It is an object of the present invention to provide a use of an antrodia camphorata compound for the preparation of a medicament for the treatment of a kidney disease, the compound being represented by the formula I: The kidney disease is renal glomerulosclerosis or renal glomerulonephritis.

Preferably, the glomerulosclerosis is local glomerulosclerosis (FSGS) or nodular glomerulosclerosis.

Preferably, wherein the renal glomerulonephritis is immunoglobulin type A glomerulonephritis (IgAN).

Among the above compounds, LT1 has an antioxidant effect on nephritis cells, and it can be understood that the above-mentioned Astragalus lucidum compound can be used in the preparation of a drug for treating kidney diseases, and it is expected that the currently known kidney diseases, including glomerular sclerosis Syndrome and renal glomerulonephritis, especially local glomerulosclerosis (FSGS), nodular glomerulosclerosis, immunoglobulin A glomerulonephritis (IgAN) have anti-inflammatory effects.

The first (A) to the first (B) graphs show the results of the MTT cell viability assay of the two kinds of Antrodia camphorata compounds, respectively.

The second (A) to the second (B) graphs show the results of the determination of antioxidants by two species of Antrodia camphorata compounds, respectively.

The third (A) to three (B) graphs show the results of analysis of anti-inflammatory response (MCP-1) of two kinds of Antrodia camphorata compounds, respectively.

The present invention is directed to two compounds extracted from Antrodia camphorata, and whether it has an effect of treating kidney diseases and anti-kidney inflammation. Antrocamol LT1 and Antrocamol LT2 are new compounds discovered by the inventors. The above extraction and structural identification data will be provided below.

Extraction of burdock

Antrodia camphorata mycelium, fruiting body or a mixture of the two (1.0kg) is extracted twice with 10 times the amount of alcohol, and then concentrated to obtain about 230g (LT-E) of crude extract, crude extract The extraction method was carried out three times with dichloromethane/water (1:1), and divided into a dichloromethane layer of about 102.6 g (LT-ED) and an aqueous layer of about 127.4 g (LT-EW). g is separated into the ANCA-ED-1, ANCA-ED- by gel column chromatography through a solvent of n-hexane/dichloromethane (1:4), dichloromethane, methanol/dichloromethane (5:95). 2. Four layers of ANCA-ED-3 and ANCA-ED-4.

According to the above results, ANCA-ED-2 and ANCA-ED-3 were further purified, and the ANCA-ED-3 layer was subjected to preparative reverse phase column chromatography (C-18 reverse phase chromatography column) at 80%. MeOH/H20 obtained a new compound Antrocamol LT1 about 150 mg in the vicinity of 18.75 minutes, while the LT-ED-2 layer was prepared by reverse phase column chromatography (C-18 reverse phase chromatography column) with 80% MeOH/H20. Another new compound, Antrocamol LT2, was obtained at about 170 mg in the vicinity of 25.10 minutes.

Antrocamol LT1 is a colorless liquid product. The compound has a molecular formula of C ₂₄ H ₃₈ O ₅ and a molecular weight of 406. The complete Chinese name is 4-hydroxy-2,3-dimethoxy-6-methyl-5. -(9-hydroxy-3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, 4-hydroxy-5-[9-hydroxy-3,7 , 11-trimethyldodeca-2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone.

LT1 structural identification data: ¹ H-NMR (400 MHz, CDCl ₃ ): 1.12 (3H, d, J = 7.2 Hz,), 1.61 (3H, s), 1.64 (3H, s), 1.66 (3H, s), 1.68 (3H, s), 1.72 (1H, m), 1.98-2.30 (8H), 2.51 (1H, dq, J = 11.6, 7.2 Hz), 3.62 (3H, s), 4.02 (3H, s), 4.33 (1H, d, J = 2.8 Hz), 4.35 (1H, dt, J = 9.2, 4.0 Hz), 5.09 (1H, d, J = 8.4 Hz), 5.14 (1H, t, J = 7.2 Hz), 5.15 (1H, t, J = 7.2 Hz); ¹³ C-NMR (100 MHz, CDCl ₃ ): .12.17 (q), 15.95 (q), 16.19 (q), 18.13 (q), 25.72 (q), 25.93 ( t), 26.78(t), 39.41(t), 39.98(d), 43.29(d), 47.94(t), 58.81(q), 60.48(q), 65.35(d), 67.24(d), 121.64( d), 127.64(d), 128.42(d), 132.03(s), 134.99(s), 135.97(s), 137.42(s), 160.82(s), 197.15(s).

Antrocamol LT2: a colorless liquid product. The compound has a molecular formula of C ₂₆ H ₄₀ O ₆ ; the molecular weight is 448. The complete Chinese name is 4-acetyl carboxy-2,3-dimethoxy-6- 5-(9-hydroxy-3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, 4-acetoxy-5-[9-hydroxy- 3,7,11-trimethyldodeca-2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone.

LT2 structure identification data: ¹ H-NMR (400 MHz, CDCl ₃ ): 1.18 (3H, d, J = 7.2 Hz,), 1.54 (3H, s), 1.64 (3H, s), 1.67 (3H, s), 1.69 (3H, s), 1.72 (1H, m), 1.80-2.40 (8H), 2.50 (1H, dq, J = 11.6, 7.2 Hz), 3.65 (3H, s), 3.98 (3H, s), 4.36 (1H, m), 5.10 (1H, t, J = 6.8 Hz), 5.12 (1H, d, J = 8.0 Hz), 5.20 (1H, t, J = 6.4 Hz), 5.72 (1H, t, J = 3.2 Hz); ¹³ C-NMR (100 MHz, CDCl ₃ ): .12.80 (q), 15.96 (q), 16.09 (q), 18.14 (q), 20.93 (q), 25.72 (q), 26.19 (t) , 26.76(t), 39.47(t), 41.25(d), 42.98(d), 48.12(t), 59.65(q), 60.67(q), 65.53(d), 68.98(d), 120.74(d) , 127.42(d), 128.25(d), 131.74(s), 134.70(s), 137.31(s), 137.56(s), 158.21(s), 169.73(s), 196.84(s).

Cell culture method

In this case, subsequent experiments using mouse kidney sputum cells (Mouse MC line CRL-1927) were used. The cell line was obtained from American Type Culture Collection (Rockville, MD, USA) in a 3:1 ratio of Dulbecco's modified Eagle's medium and Ham's F-12 medium, and 5% fetal bovine serum and 14 mM HEPES were added for regular culture. .

Cell viability analysis

The above cell lines were separately cultured in an appropriate culture solution for 24 hours. The proliferated cells were washed once with PBS, and the cells were treated with 1× trypsin-EDTA, followed by centrifugation at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then, 10 ml of the new culture solution was added, the cells were resuspended by shaking slightly, and the cells were placed in a 96-well microplate. During the test, 0.01 to 200 μg/ml of Antrodia camphorata compound was added to each well, and cultured at 37 ° C, 5% CO ₂ for 48 hours. Thereafter, 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis) was added to each well in the dark. , MO) reaction for 2 hours. The absorbance was measured by an enzyme immunoassay analyzer at an absorption wavelength of 570 nm to calculate the cell survival rate, and the concentration required for the growth half inhibition rate (ie, IC50 value) was calculated. All experimental data were expressed as mean ± standard error. . The experimental data were statistically analyzed by a paired-t test. A statistical difference was considered with a p value of less than 0.05.

For the cell viability assay of two kinds of Antrodia camphorata compounds, the results are shown in Figure 1 (A) ~ one (B). The results showed that Antrocamol LT1 and Antrocamol LT2 did not affect the growth of renal sputum cells at the current concentration (≦0.048 μg/ml).

Anti-oxidation/anti-inflammatory effect of Antrodia camphorata compound

As it is currently known, glomerular sclerosis or glomerulonephritis is caused by a severe inflammatory reaction, and white blood cells accumulated by the immune response release a large amount of ROS and cause oxidative stress in the kidney cells, which in turn causes damage to the kidneys. Injury, so reducing or even eliminating oxidative stress in kidney cells is one of the methods for treating pyelonephritis, so reducing intracellular ROS is also one of the important indicators for the treatment of kidney diseases mentioned above.

Determination of intracellular reactive oxygen species (ROS)

The measurement of intracellular ROS was determined by measuring the fluorescence intensity of 2',7'-dichlorofluorescein diacetate oxide (2',7'-dichlorofluorescein). After the renal sputum cells were treated with the test compound, 2', 7'-dichlorofluorescein diacetate (2 μM) was added for 30 minutes, and LPS was added at the specified time. Next, an absorption spectrum reader (Bio-Rad Laboratories, Inc.) having an excitation wavelength of 485 nm and a radiation wavelength of 530 nm was used for reading, and the stronger the fluorescence, the higher the ROS content.

Since reactive oxygen species (ROS) is inextricably linked to nephritic diseases, the above-described measurement of intracellular ROS is carried out, and the results are shown in the second (A) to (B) graphs.

Referring to the second (A) diagram, inhibition of oxidative stress (ROS) was performed at 0.012 μg/ml and 0.048 μg/ml of Antrocamol LT1, respectively. The results showed that 0.012 μg/ml inhibited the oxidation pressure by 3% at 5 minutes. , 0.048 μg / ml can inhibit the oxidation pressure by 14%.

See Figure 2 (B) for 0.012μg/ml and 0.048μg/ml respectively. The Antrocamol LT2 was subjected to inhibition of oxidative stress (ROS) analysis and the results showed no inhibition at the current concentration at 5 minutes.

The results are organized as shown in Table 1 below:

Anti-inflammatory effect of Antrodia camphorata compound on nephritis cells

It is known that MCP-1 plays an important role in promoting renal tubular interstitial inflammation, tubular atrophy, and renal fibrosis. Therefore, this indicator protein is tested to determine the anti-inflammatory effect of Antrodia camphorata compounds on kidney cells. .

The ELISA assay kit (Biosciences, Los Angeles, CA, USA) was used to detect the MCP-1 protein of the cell supernatant according to the instructions, and the ELISA reader (Bio-Tek) was used for reading (absorption wavelength was 450 nm). . *** indicates p < 0.005, NS indicates.

Referring to the third (A) diagram, anti-inflammatory reaction (MCP-1) analysis was performed at LT1 of 0.048 μg/ml and 0.012 μg/ml, and the results showed that the current concentration had no inhibitory effect.

Referring to the third (B) diagram, anti-inflammatory reaction (MCP-1) analysis was performed at LT2 of 0.048 μg/ml and 0.012 μg/ml, and the results showed that the current concentration had no inhibitory effect.

The results are organized as shown in Table 2 below: Table 2

Based on the above experimental results, the results of the intracellular ROS assay showed that LT1 has an antioxidant effect on nephritis cells, and indirectly confirms its potential for the preparation of a medicament for treating kidney diseases, which is expected to be known for the time being. Kidney disease, including glomerulosclerosis and glomerulonephritis, especially local glomerulosclerosis (FSGS), nodular glomerulosclerosis, immunoglobulin A glomerulonephritis (IgAN), etc. All have anti-inflammatory effects.

In addition, LT2 has not found any anti-oxidation or anti-inflammatory effect on nephritis cells, and is not included in the scope of protection of the present invention. However, it can be confirmed that the LT1 compound also extracted from Antrodia camphorata has anti-inflammatory effect. Preciousness.

The use of the Antrodia camphorata compound provided by the present invention in the preparation of a medicament for treating kidney diseases is of industrial value, but the above description is only for the preferred embodiment of the present invention, and those skilled in the art can Other modifications are possible in the description, but such modifications are still within the spirit of the invention and the scope of the invention as defined below.

Claims (3)

A use of an antrodia camphorata compound for the preparation of a medicament for the treatment of a kidney disease, the compound being represented by the formula I: The kidney disease is renal glomerulosclerosis or renal glomerulonephritis. The use according to claim 1, wherein the glomerulosclerosis is local glomerulosclerosis (FSGS) or nodular glomerulosclerosis. The use according to claim 1, wherein the glomerulonephritis is immunoglobulin type A glomerulonephritis (IgAN).