WO2020209690A1 - Pharmaceutical composition containing wild-walnut extract, fraction thereof, or physiologically active substance derived therefrom as active ingredient, and having effects of inhibiting production of advanced glycation end products and decomposing advanced glycation end products - Google Patents

Abstract

The present invention pertains to a pharmaceutical composition containing a wild-walnut extract, a fraction thereof, or a compound isolated from the fraction. A wild-walnut extract, a fraction thereof, and a compound isolated from the fraction according to the present invention have the effects of inhibiting the production of advanced glycation end products (AGEs) and decomposing advanced glycation end products that are produced, and can thus be advantageously used in a pharmaceutical composition for preventing or treating diabetic complications, nonalcoholic steatohepatitis, or degenerative brain disease caused by advanced glycation end products (AGEs).

Description

Pharmaceutical composition having the effect of inhibiting and decomposing the production of final saccharified products containing sputum extract, fractions or physiologically active substances derived therefrom as active ingredients

The present invention relates to a pharmaceutical composition having an effect of inhibiting and decomposing a final saccharified product containing a sputum extract, a fraction thereof, or a physiologically active substance derived therefrom as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including sputum extract, fractions thereof, or physiologically active substances derived therefrom. will be.

Diabetes is a chronic disease in which the amount of insulin secretion is insufficient due to genetic or environmental factors, or the concentration of glucose in the blood is high because the normal function is not performed, and glucose is discharged into the urine. As the state of elevated blood sugar in the body continues for a long time due to diabetes, the glycated product invades large and small blood vessels in the retina, kidneys, nerves, or whole body, leading to chronic complications. Because diabetes is more risky for diabetic complications than itself, the primary goal of diabetes treatment today is to suppress the initiation or progression of diabetic complications. Typical diabetes complications include retinopathy, nephropathy, and neurosis. Diabetes, including the complications of diabetes, is a serious chronic disease that generally occurs in the modern era as it is known to be the fourth highest cause of death in Korea.

The mechanisms that induce these diabetes complications are largely osmotic stress due to non-enzymatic glycation of protein and changes in the mechanism of the polyol pathway, and oxidative stress due to free radicals. ), etc.

In the glycosylation of proteins, proteins and sugars react to form a schiff base first, and as the reaction proceeds further, amadori product is generated.Continuing through condensation and dehydration, the final glycosylation product (advanced glycation) is performed. end product, AGEs). Since this reaction occurs almost spontaneously without supplying energy at the beginning of the reaction, it occurs in food or in our body, and has an irreversible characteristic after a certain stage. Therefore, once the final glycated product is produced, it is not degraded even if blood sugar is restored to normal, but accumulates in the tissue during the protein survival period, causing abnormal changes in the structure and function of the tissue. In this way, by the non-enzymatic protein saccharification reaction, saccharification occurs in proteins such as basement membrane, plasma albumin, lens protein, fibrin, and collagen, and the resulting final glycated products (AGEs) abnormally change the structure and function of tissues, resulting in complications of chronic diabetes. Causes. In this way, the polyol pathway, the non-enzymatic saccharification reaction and the oxidative stress mechanisms are related to each other to cause diabetic complications, so in order to delay the onset of, prevent or treat the onset of diabetic complications, It is important.

The final glycated product was recently found to be associated with non-alcoholic steatohepatitis (NASH) disease in addition to complications of diabetes. Although the pathogenesis of nonalcoholic steatohepatitis has not been fully elucidated, it is widely accepted that at least it is closely related to insulin resistance. When insulin resistance increases due to a combination of genetic factors and environmental factors related to lifestyle, such as diet and exercise, excess free fatty acid (FAA) is produced in the liver. Free fatty acids are converted into non-toxic triglycerides (TG) in hepatocytes, and are primarily in a state of simple steatosis (Non-Patent Documents 1 and 2). As various oxidative stresses are added, it is known that fat peroxidation and overproduction of inflammatory cytokines lead to liver cell damage and inflammatory reactions, leading to non-alcoholic steatohepatitis. Interestingly, non-alcoholic steatohepatitis often accompanies type 2 diabetes, another insulin resistance-related disease, and this suggests a link between the final glycated product, which is known to be a major causative agent of diabetic complications, and non-alcoholic steatohepatitis. In particular, glyceraldehyde (GA)-derived final glycosylation products (GA-AGEs) are the most toxic among final glycosylation products produced in the body, so they are called TAGEs (toxic AGEs). Recently, TAGEs are used in patients with non-alcoholic steatohepatitis. It has been found to be present in high concentrations in serum and liver tissue. In addition to glyceraaldehyde, final glycated products derived from dicarbonyl compounds such as glyoxal (GO) and methylglyoxal (MGO) will also promote non-alcoholic steatohepatitis.

The final glycated product is also reported to be increased in the brain of the elderly or aged animals, and it is known that it can affect degenerative brain diseases such as Alzheimer's disease by promoting the death of nerve cells (Non-Patent Documents 3 and 4 ).

Final glycated product inhibitors related to diabetes complications and non-alcoholic steatohepatitis have been synthesized, but show toxicity when administered for a long time. For example, aminoguanidine, a synthetic drug, is a nucleophilic hydrazine that binds to the product of the condensation reaction and inhibits the production of the final glycated product, thereby preventing the development of diabetes complications. This is the most promising drug for the prevention and treatment of diabetic complications, and has been conducted up to phase 3 clinical trials. However, there is a problem that toxicity is induced when administered for a long period of time, so the development of a safer drug is required. Furthermore, since there is no clear treatment method for non-alcoholic steatohepatitis other than liver transplantation, there is a need to develop a safer new treatment that can solve this problem.

Juglans mandshurica Maxim. is classified as Juglandaceae or walnut family, and exists in the form of a tree or shrub, and is native to the Americas, Eurasia or South Asia. It is similar to a walnut tree native to China. It is mainly inhabited in the northeastern part of Korea or northeastern China. It is used as a medicinal plant and herbal medicinal material. The one called Chupi refers to collecting and drying the bark of a sputum tree between spring and autumn in oriental medicine. , Fever and clearing eyes. It is prescribed for enteritis, diarrhea, alleles, redness of the eyes, and swelling pain. It is also used in the treatment of skin diseases, cancer, gastritis, etc., and the fruit of the sputum tree (sputum; chuja) is 9 What is ripe in October is used for medicinal or edible use, and the seeds of the fruit are eaten or used as medicinal materials.

In China and North Korea, sputum bark and fruit are used as cancer treatment and are used for chronic enteritis, dysentery, hepatitis, cirrhosis, low back pain, neuralgia and various skin diseases. Home remedies include gastritis (duodenal ulcer; crushed fruit and eaten with juice), athlete's foot (decoction of sputum branches with water and soaking feet), acute enteritis (severe leaves and branches are cut off with water and decoated with water. Taking), etc.

Among the documents reported as the efficacy of sputum, Republic of Korea Patent No. 10-1292837 (Patent Document 1) discloses the use of separating the active compound from the extract of sputum leaves and improving skin wrinkles thereof. In addition, it has been known about the inhibitory effect of HIV-1 reverse transcriptase and Rnase activity of the active compound isolated from the extract of Sputum bark (Non-Patent Document 5). Regarding the anti-obesity effect of the sputum tree extract, it is known that the water extract of sputum tree can exhibit an anti-obesity effect through the regulation of the activity of pancreatic lipolytic enzymes (Non-Patent Document 6). However, the effect of inhibiting or decomposing the production of final glycated products of sputum sputum, in particular, sputum extract as well as novel compounds isolated therefrom have various physiological activities, especially anti-diabetic complications, anti-alcoholic steatohepatitis, degenerative brain diseases It has not been reported that it could be active.

[Prior technical literature]

[Patent Literature]

1. Korean Patent Registration No. 10-1292837 (2013.07.29)

[Non-patent literature]

1. Yamaguchi K, et al. "Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis." Hepatology. 2007 Jun;45(6):1366-74.

2. Feldstein AE, et al. "Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway." Hepatology. 2004 Jul;40(1):185-94.

3. Final glycation product and diet management for diabetics (National Food Plus Support Center, 2013),

4.Alzheimer's Disease and Type 2 Diabetes: A Critical Assessment of the Shared Pathological Traits (Front Neurosci. 2018 Jun 8;12:383.doi: 10.3389/fnins)

5. Min, Byung-Sun, et al. "Inhibition of human immunodeficiency virus type 1 reverse transcriptase and ribonuclease H activities by constituents of Juglans mandshurica." Chemical and pharmaceutical bulletin 48.2(2000): 194-200.

6. Han, Likun, et al. "Inhibitory effects of compounds isolated from fruit of Juglans mandshurica on pancreatic lipase." Journal of Natural Medicines 61.2 (2007): 184-186.

An object of the present invention is a pharmaceutical composition for preventing or treating diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases by inhibiting the production of final glycated products or decomposing the resulting final glycated products Or to provide a health functional food composition for improving or preventing the above diseases.

In order to achieve the above object, the present invention is for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including a sputum extract, a fraction thereof, or a compound isolated from the fraction Provides a pharmaceutical composition.

In addition, the present invention provides a health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract, a fraction thereof, or a compound isolated from the fraction. to provide.

In one embodiment, the compound may be represented by any one of the following Formulas 1 to 10.

Phalaenopsis extract according to the present invention, a fraction thereof, or a compound isolated from the fraction inhibits the production of final saccharified products (AGEs) and has the effect of decomposing the final saccharified products, thus final saccharified products (AGEs) Diabetic complications caused by, non-alcoholic steatohepatitis, inflammatory disease, fibrosis or degenerative brain disease can be usefully used in improving, preventing or treating.

Figures 1 and 2 show the effect of inhibiting the production of final saccharified products (AGEs) of the sputum extract and its fractions.

3 and 4 show the effect of decomposing sputum extract, its fractions, and the final saccharified products (AGEs) of the compounds isolated from the fractions.

Figure 5 is a graph showing the result of measuring the NO concentration of the sputum extract, a fraction thereof in macrophages.

6A and 6B are graphs showing the results of measuring cell viability of sputum extract and fractions thereof in macrophages.

7A and 7B are graphs showing the amount of IL-6 and TNF-α production.

Figure 8 is a graph showing the result of measuring the NO concentration of the sputum extract, a fraction thereof in microglia.

9A and 9B are graphs showing the vascular endothelial cell protective efficacy of a sputum extract and a fraction thereof.

Figure 10 is a picture and graph showing the expression degree of α-SMA of a sputum extract, a fraction thereof.

11 is a diagram and a graph showing the expression level of BDNF of a sputum extract, a fraction thereof.

12a and 12b are graphs showing the neuroblastic protective efficacy of a sputum extract, a fraction thereof.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcholic steatohepatitis (NASH), inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract or a fraction thereof Provides.

In the present application, the bark, root, leaf, flower, or fruit can be used as the sputum tree, and it is more preferable that it is a fruit. In addition, the sputum tree can be used without limitation, such as cultivated or commercially available ones.

The extract is preferably extracted using water, a C1 to C4 lower alcohol or a mixture thereof as a solvent, and the lower alcohol may preferably be ethanol, methanol or butanol, but is not limited thereto.

The extract may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) extracting by adding an extraction solvent to the sputum tree;

2) filtering the extract of step 1); And

3) A step of drying after concentrating the filtered extract of step 2) under reduced pressure.

In the above method, conventional methods in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used as the extraction method of the sputum extract.

In the above method, the vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

The fraction may be prepared by adding a solvent to the sputum extract. The solvent may be water, hexane, ethyl acetate, or chloroform, but is not limited thereto.

In another embodiment, the present invention relates to diabetes complications, nonalcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases, including any one of the following formulas 1 to 10, or a pharmaceutically acceptable salt thereof. It provides a pharmaceutical composition for prevention or treatment.

The compound may be isolated from a sputum extract or a fraction thereof.

The diabetic complication of the present invention may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic osteoporosis or diabetic atherosclerosis.

Inflammatory diseases of the present invention include uveitis, conjunctivitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, infectious arthritis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, glomerulonephritis, meningitis, peritonitis, osteomyelitis, cellulitis, pancreatitis, atopic dermatitis, asthma, Traumatic shock, allergic rhinitis, mumps, acute bronchitis, chronic bronchitis, tendonitis, hemoglobinosis and inflammatory bowel disease.

Fibrosis of the present invention is idiopathic pulmonary fibrosis, cystic fibrosis, drug-induced fibrosis, environment-induced fibrosis, lymphangioleiomyomatosis, radiation-induced fibrosis, renal fibrosis, liver fibrosis, skin fibrosis, skin sclerosis, systemic sclerosis, or myelofibrosis. Can be

The degenerative brain disease of the present invention may be Alzheimer's disease or stroke.

Diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases of the present invention may be caused by advanced glycation end products (AGEs).

As confirmed through the following examples, the sputum tree extract of the present invention, its fraction, or the compounds of Formulas 1 to 10 inhibit the production of final saccharified products or decompose the resulting final saccharified products, so that the sputum of the present invention The tree extract, a fraction thereof, or the compounds of Formulas 1 to 10 are effective in treating or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases.

The present invention also provides a health functional food composition for the improvement or prevention of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis, or degenerative brain diseases comprising a sputum extract, a fraction thereof, or the compound of Formulas 1 to 10. to provide.

Hereinafter, examples and the like will be described in detail to aid in understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples.

[Example 1] Separation of Phalaenopsis extract, its fraction, and active compound

As for the sputum tree, the fruit of the sputum tree was purchased from the Gyeong-dong medicinal market in Seoul, Korea in 2014. After sonicating the sputum fruit (10 kg) for 4 hours, it was extracted with methanol (5L, 3 times) to obtain an extract (200g) and then suspended to obtain a suspension (JM1). The suspension (JM1) was added to water and continuously distributed with chloroform and ethyl acetate, and then the solvent was removed, and the chloroform fraction (JM1, 17.5 g), the ethyl acetate fraction (JM2, 45.7 g) and the water fraction (JM3, 78.6 g) were added. g) was obtained. The chloroform fraction was loaded on a silica gel column and eluted with a gradient of hexane-acetone (20:1 → 1:1, /v) to obtain a total of 5 fractions, which were JM1A (3.4 g) and JM1B ( 4.1 g), JM1C (2.2 g), JM1D (1.2 g) and JM1E (2.5 g).

The JM1C fraction was again loaded on a silica gel column, and five small fractions were eluted using a mobile phase solvent mixed with chloroform:methanol at 5:1 (v/v), and these were JM1C1 (0.4 g) and JM1C2 ( 0.3 g), JM1C3 (0.8 g), JM1C4 (0.8 g) and JM1C5 (0.3 g). Then, the JM1C2 small fraction was again loaded on an HPLC equipped with a J'sphere ODS H-80 column (250 ㎜ X 20 ㎜), and a 22% MeCN aqueous solution was flowed at a flow rate of 3 mL/min. 8B3 (4.3 mg) was obtained. In addition, the JM1C3 small fraction was loaded on HPLC under the same conditions as above to obtain compounds 8C10 (6.3 mg), 8C11 (3.7 mg), 8D5 (3.5 mg), and 8D4 (5.2 mg). Compound 8C13 (2.7 mg) and 8C14 (7.3 mg) were obtained from the JM1C4 fraction under the same conditions. In addition, the JM1C5 small fraction was loaded on an HPLC equipped with a J'sphere ODS H-80 column (250 mm x 20 mm), and a 15% MeCN aqueous solution was flowed at a flow rate of 3 ml/min to finally compound 8E4 ( 5.7 mg) was obtained.

The JM2 fraction was loaded on a silica gel column, eluted with a gradient of chloroform:methanol (20:1 → 1:1, /v), JM2A (7.0 g), JM2B (6.3 g), JM2C (5.8 g) ), JM2D (4.3 g), JM2E (8.2 g) were obtained in five small fractions. The JM2C fraction was loaded on a YMC RP-18 column and eluted with acetone:water (1:1, v/v) to obtain two small fractions of JM2C1 (1.6 g) and JM2C2 (2.3 g). The JM2C1 fraction was eluted with a 15% MeCN aqueous solution on a J'sphere ODS H-80 column (250 ㎜ x 20 ㎜) to obtain compounds 9C2 (2.3 mg) and 9B (3.3 g). The purity of the isolated compounds was more than 95%.

[Example 2] Confirmation of the effect of inhibiting the production of final saccharified products (AGEs)

In this example, the effect of inhibiting the formation of final saccharified products (AGEs) of the sputum extract, its fraction, and the compound isolated from the fraction was examined.

The final glycosylated product (AGEs) reagent is 5 mg/ml bovine serum albumin (BSA), 0.025% sodium azide to prevent bacterial growth, and methylglyoxal (MGO) or glyoxal (GO). Was prepared. The final glycated products derived from methylglyoxal were referred to as MGO-AGEs, and the final glycated products derived from glyoxal were referred to as GO-AGEs.

Each of 1, 5, and 10 mg/ml of sputum extract and its fractions were incubated with a final saccharified product (AGEs) reaction reagent for 7 days at 37°C. After incubation, the fluorescence intensity of the reaction product was measured at 355 nm and 460 nm, respectively, using a VICTOR ^TM X3 multilabel plate reader.

Aminoguanidine (AG), an inhibitor of the final glycated product, was used as a positive control.

sample	Treatment concentration	Final saccharified products (AGEs) production%
Control (MGO-AGEs)	-	100.00 ± 0.39
Positive control group (AG)	1 mM	38.52 ± 0.77
Phalaenopsis methanol extract	1 mg/ml	104.62 ± 0.46
	5 mg/ml	55.01 ± 0.31
	10 mg/ml	42.00 ± 0.25
Phalaenopsis fraction (chloroform layer)	1 mg/ml	74.54 ± 3.11
	5 mg/ml	42.38 ± 2.67
	10 mg/ml	31.61 ± 0.87

sample	Treatment concentration	Final saccharified products (AGEs) production%
Control (GO-AGEs)	-	100.00 ± 0.29
Positive control group (AG)	1 mM	61.32 ± 0.56
Phalaenopsis methanol extract	1 mg/ml	90.08 ± 1.71
	5 mg/ml	77.03 ± 1.52
	10 mg/ml	67.43 ± 2.68
Phalaenopsis fraction (chloroform layer)	1 mg/ml	92.48 ± 2.54
	5 mg/ml	83.83 ± 1.64
	10 mg/ml	58.09 ± 1.60

As a result, it was confirmed that the production of the final glycation product was suppressed compared with the final glycation product produced by reaction with albumin and methylglyoxal or albumin and glyoxal when treating the sputum tree extract and its fractions according to the present invention. (Table 1 and Table 2).

As shown in Figs. 1 and 2, when the methanol extract (Total) and the chloroform fraction (CHCl ₃ ) were treated, the effect of inhibiting the production of final saccharified products (AGEs) was confirmed.

[Example 3] Confirmation of the decomposition effect of final saccharified products (AGEs)

In this example, the effect of decomposing sputum extract, its fraction, or the compound isolated from the fraction was confirmed.

Methylglyoxal (MGO) or glyoxal (GO) was mixed with bovine serum albumin (BSA) to produce final glycosylated products (AGEs), and then sputum extract and its fractions were each 0.1 mg/ml in the final glycation product. , At concentrations of 0.5 and 1.0 mg/ml, the compounds isolated from the fractions were treated at concentrations of 0.1 and 0.4 mM, respectively, for 24 hours. After the reaction, a reagent including 2,4,6-trinitrobenzene sulfonic acid (TNBSA), 4% sodium bicarbonate, 10% sodium dedecyl sulfate and 1N hydrochloric acid solution was added. Using a microplate reader, the amount of free amine, which is a decomposition product of the final saccharified product, was measured at 335 nm, and the degree of decomposition of the final saccharified product was confirmed.

Aminoguanidine (AG), an inhibitor of the final glycated product, was used as a positive control.

sample	Treatment concentration	Amount of free amine%
Control (BSA)	-	100.00 ± 1.86
Negative controls (MGO-AGEs)	-	45.45 ± 0.90
Positive control (AG)	1.0 mM	61.64 ± 0.77
Phalaenopsis methanol extract	0.1 mg/ml	53.15 ± 0.55
	0.5 mg/ml	57.70 ± 3.94
	1.0 mg/ml	64.24 ± 0.91
Phalaenopsis fraction (chloroform layer)	0.1 mg/ml	59.33 ± 0.66
	0.5 mg/ml	79.27 ± 1.00
	1.0 mg/ml	94.85 ± 1.84
JM-8C13	0.1 mM	59.21 ± 1.41
	0.4 mM	78.91 ± 3.28

sample	Treatment concentration	Amount of free amine%
Control (BSA)	-	100.00 ± 3.15
GO-AGEs	-	62.19 ± 3.67
Positive control group (AG)	1.0 mM	76.44 ± 0.99
Phalaenopsis methanol extract	1.0 mg/ml	76.80 ± 3.90
Phalaenopsis fraction (chloroform layer)	0.5 mg/ml	76.38 ± 1.28
	1.0 mg/ml	91.96 ± 5.03
JM-8C13	0.4 mM	71.56 ± 2.98

As shown in Figure 3, in the methanol extract (Total), chloroform fraction (CHCl ₃ ), and the compound isolated from the fraction (JM-8C13), as compared to the negative control (MGO-AGEs), more free amines It was confirmed that the amount was detected.

In addition, as shown in Figure 4, when treated with the methanol extract (Total) 1.0 mg/ml, the chloroform fraction (CHCl ₃ ) 0.5 mg/ml and 1.0 mg/ml, and the isolate (JM- 8C13) In the case of treatment with 0.4 mM, it was confirmed that a higher amount of free amine was detected as compared to the negative control (GO-AGEs).

Therefore, it can be seen that the extract, the fraction and the compound isolated from the fraction have excellent decomposition effects of the final saccharified product (Tables 3 and 4).

[Example 4] Confirmation of NO production inhibitory effect in macrophages

In this example, in order to determine whether the sputum tree extract of the present invention, its fraction, and the compound isolated from the fraction have an inhibitory effect on NO generation, cells are treated with a sputum tree extract, a fraction thereof, or a compound isolated from the fraction. After that, the amount of NO generation was confirmed. Since NO is highly reactive and very unstable, the concentration of nitrite was measured indirectly instead of directly measuring the NO concentration.

Raw 264.7 cells, which are macrophages, were seeded in a 96-well plate at 5×10 ⁴ each and incubated for 24 hours in a 37° C., 5% O ₂ incubator, and then sputum extract, a fraction thereof (10, 50 μg/ml), from the fraction The isolated compounds (10, 20 μg/ml) and positive control L-NMMA (20 μM) were pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, 50 μl of the supernatant was taken and transferred to a 96-well plate, and then Griess reagent (50 μl,% sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water) was added 1:1. After mixing with, it was treated on a plate containing 50 μl supernatant. After the reaction, the wavelength range of the microplate reader was measured to be 540 nm, and a conditioned medium was used for NO measurement. At this time, L-NMMA used as a positive control is an inhibitor of NO production.

As a result, as shown in Figure 5, it was confirmed that NO generation was suppressed when the methanol extract (Total), water fraction (H ₂ O), EtOAc fraction, and CHCl ₃ fraction were treated with 50 μg/ml, respectively. .

[Example 5] Confirmation of expression of cytotoxicity in macrophages

In this example, in order to determine whether the sputum sputum extract and its fractions of the present invention express cytotoxicity, cells were treated with the sputum sputum extract and its fractions of the present invention, and then cell viability was confirmed.

Raw 264.7 cells, which are macrophages, were seeded in a 96-well plate at 5×10 ⁴ each and incubated for 24 hours in a 37° C., 5% O ₂ incubator, and then sputum extract, its fraction (10, 50 μg/ml) and positive control L- NMMA (20 μM) was pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After incubation for 24 hours, 0.5 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5- bromide) was treated for 1 hour. Thereafter, cells were lysed using 100 μl of DMSO, and then the 570 nm wavelength value was measured with a microplate reader to evaluate the cytotoxicity. At this time, L-NMMA used as a positive control is an inhibitor of NO production.

As a result, as shown in Figs. 6a and 6b, when the methanol extract (Total), H2O fraction, Hexane fraction, EtOAc fraction, and CHCl ₃ fraction are treated with 50 μg/ml, respectively, it can be safely used without cytotoxicity. Confirmed.

[Example 6] Confirmation of the effect of inhibiting the production of inflammation-related factors in macrophages

In this example, in order to confirm whether the sputum extract of the present invention and its fractions inhibit the production of IL-6 and TNF-α, which are inflammation-related factors, cells were treated with the sputum sputum extract of the present invention and a fraction thereof, 6 and TNF-α production amount was confirmed.

Raw 264.7 cells, which are macrophages, were seeded in a 24-well plate at 3×10 ⁵ each and incubated for 24 hours in a 37° C., 5% O ₂ incubator, and then sputum extract, its fraction (50 μg/ml) and positive control L-NMMA (20 μM) was pretreated for 30 minutes and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, the production amount of IL-6 and TNF-α was tested according to the manufacturer's protocol using an ELISA kit (R&D system, USA) using the culture medium of the cultured cells. At this time, L-NMMA used as a positive control is an inhibitor of NO production.

As a result, as shown in Figs. 7a and 7b, it was confirmed that the sputum extract and the fraction inhibited IL-6 production, and the EtOAc fraction and the CHCl ₃ fraction inhibited TNF-α production.

[Example 7] Confirmation of NO production inhibitory effect in microglia

In this example, in order to check whether the sputum tree extract and its fractions of the present invention have the effect of inhibiting NO generation, cells were treated with the sputum tree extract and its fractions of the present invention, and then the amount of NO production was confirmed. Since NO is highly reactive and very unstable, the concentration of nitrite was measured indirectly instead of directly measuring the NO concentration.

BV-2 cells, which are microglial cells, were seeded in a 96-well plate at ⁴ ×10 ⁴ each and incubated in an incubator at 37° C., 5% O ₂ for 24 hours, and then sputum extract, fractions thereof (10, 50, 100 μg/ml) and Positive control L-NMMA (20 μM) was pretreated for 1 hour and then stimulated with 100 ng/ml LPS for 24 hours. After 24 hours, 50 μl of the supernatant was taken and transferred to a 96-well plate, and then Griess reagent (50 μl,% sulfanilamide in 5% phosphoric acid and 50 μl 0.1% N-1-napthylethylenediamine dihydrochloride in water) was added to each of 1:1. After mixing, the plate was treated with 50 μl supernatant. After the reaction, the wavelength range of the microplate reader was measured to be 540 nm, and a conditioned medium was used for NO measurement. At this time, L-NMMA used as a positive control is an inhibitor of NO production.

As a result, as shown in Figure 8, it was confirmed that the fraction of the sputum extract inhibits NO generation. In addition, it was confirmed that more excellent effects were exhibited as the concentration increased.

[Example 8] Confirmation of vascular endothelial cell protection efficacy

In this example, it was confirmed whether the effect of protecting vascular endothelial cells against methylglyoxal (MGO)-induced toxicity of the sputum tree extract and its fractions of the present invention. To this end, cells were treated with the sputum extract of the present invention and a fraction thereof, and then cell viability was confirmed. Methylglyoxal (MGO) is the final glycation product.

After seeding vascular endothelial cell HUVECs in a 96-well plate (12X10 ³ cells/well) and incubating for 24 hours in a 37°C, 5% O ₂ incubator, a sample was prepared with EBM-2 culture solution and pretreated for 1 hour. Glyoxal (MGO) was treated with 500 μM. After incubation for 24 hours, 0.1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-bromide) was treated for 2 hours and 30 minutes. Thereafter, cells were lysed using 100 μl of DMSO and then measured with an ELISA plate reader having a wavelength of 570 nm to evaluate whether cytotoxicity was present.

As a result, as shown in Figs. 9a and 9b, it was confirmed that the sputum extract and the EtOAc fraction of the fractions thereof showed cell protective efficacy against methylglyoxal (MGO) and can be safely used without cytotoxicity.

[Example 9] Antifibrotic activity confirmation

In this example, antifibrotic activity was confirmed by measuring the effect of the sputum extract of the present invention and its fractions on the expression level of α-SMA (α-Smooth muscle actin), which is an index of fibrosis. The increase in α-SMA means fibrosis.

LX2 cells (Human hepatic stellate cells) were seeded with 1X10 ⁶ each in a 60 mm dish and incubated for 24 hours in a 37°C, 5% O ₂ incubator, and then the sputum extract and its fraction were pretreated at a concentration of 50 μg/ml. After incubation for 1 hour, TGF-β was treated at a concentration of 5 ng/ml and cultured for 24 hours. After collecting the cultured cells, spin down the cell pellet with a centrifuge (5,000 rpm, 5 min, 4℃), add lysis buffer, dissolve for 1 day, and then centrifuge (12,000 rpm, 25 min, 4℃) Thus, cell membrane components and the like were removed. After denatured separation by SDS-PAGE, it was transferred to a PVDF membrane. Then, after reacting with the primary antibody overnight, the secondary antibody was bound and the expression of α-SMA was measured using a ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA).

As a result, as shown in Figure 10, it can be seen that both the extract and the fraction have excellent anti-fibrosis effect.

In addition, it was confirmed that the amount of α-SMA significantly decreased in the H ₂ O fraction and the CHCl ₃ fraction.

[Example 10] BDNF expression level confirmation

In this example, the effect of the sputum extract of the present invention and a fraction thereof on the expression level of brain-derived neurotrophic factor (BDNF) was measured to confirm synaptic plasticity and neuronal activity.

BDNF is a factor that regulates nerve function and is essential for the survival, differentiation and molecular mechanisms of neurons in the central nervous system, and is involved in various intercellular signaling pathways.

The neuroblastoma cell line SH-SY5Y cells were seeded with 1×10 ⁶ each in a 60 mm dish and incubated for 24 hours in a 37° C., 5% O ₂ incubator, and then a sputum extract, a fraction thereof, at a concentration of 50 μg/ml for 24 hours. Cultured. After collecting the cultured cells, spin down the cell pellet with a centrifuge (5,000 rpm, 5 min, 4℃), add lysis buffer, dissolve for 1 day, and then centrifuge (12,000 rpm, 25 min, 4℃) Thus, cell membrane components and the like were removed. After denatured separation by SDS-PAGE, it was transferred to a PVDF membrane. Then, after reacting with the primary antibody overnight, the secondary antibody was bound and the expression of BDNF was measured using a ChmiDoc XRS+ imaging system (Bio-Rad, CA, USA).

As a result, as shown in FIG. 11, it was confirmed that the amount of BDNF increased in the H ₂ O fraction, the EtOAC fraction, and the CHCl ₃ fraction.

[Example 11] Whether or not cytotoxicity was expressed in neuroblastic cells

In this example, it was confirmed whether the effect of protecting a neuroblastic cell line against methylglyoxal (MGO)-induced toxicity of the sputum sputum extract and its fractions of the present invention. To this end, the cells were treated with the sputum extract of the present invention, a fraction thereof, and then the cell viability was confirmed.

The neuroblastic cell line SH-SY5Y was seeded in a 96-well plate (4X10 ⁴ cells/well) and incubated for 24 hours in a 37°C, 5% O ₂ incubator, and then a sample was prepared with DMEM culture (serum-free) and pretreated for 1 hour. After performing, methylglyoxal (MGO) was treated with 500 μM. After incubation for 24 hours, 0.1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-bromide) was treated for 2 hours and 30 minutes. Thereafter, cells were lysed using 100 ul of DMSO and then measured with an ELISA plate reader having a wavelength of 570 nm to evaluate whether or not cytotoxicity.

As a result, as shown in Figs. 12a and 12b, it was confirmed that the cell protective effect against methylglyoxal (MGO) was shown in the sputum extract, and can be safely used without cytotoxicity.

Claims (15)

1. A pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including sputum extract or fractions.
2. A pharmaceutical composition for the prevention or treatment of diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or brain diseases, including a compound of any one of the following Formulas 1 to 10, or a pharmaceutically acceptable salt thereof.

   
3. The pharmaceutical composition of claim 2, wherein the compound of any one of Formulas 1 to 10 is isolated from a sputum extract or fraction.
4. The pharmaceutical composition according to claim 1 or 3, wherein the extract is extracted using water, C1 to C4 lower alcohol, or a mixture thereof as a solvent.
5. The pharmaceutical composition according to claim 1 or 3, wherein the fraction is fractionated by adding a solvent to the sputum extract.
6. The pharmaceutical composition of claim 5, wherein the solvent is selected from the group consisting of water, hexane, ethyl acetate, and chloroform.
7. The pharmaceutical according to claim 1 or 2, wherein the diabetic complication is diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic heart disease, diabetic osteoporosis, or diabetic atherosclerosis. Composition.
8. The pharmaceutical composition according to claim 1 or 2, wherein the degenerative brain disease is Alzheimer's disease or stroke.
9. The method of claim 1 or 2, wherein the inflammatory disease is uveitis, conjunctivitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, infectious arthritis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, glomerulonephritis, meningitis, peritonitis, osteomyelitis, cellulitis , Pancreatitis, atopic dermatitis, asthma, allergic rhinitis, mumps, acute bronchitis, chronic bronchitis, systemic lupus erythematosus, tendonitis, hemoglobinosis and inflammatory bowel disease.
10. 3. A pharmaceutical composition, characterized in that it is sclerosis, systemic sclerosis, or myelofibrosis.
11. The pharmaceutical composition according to claim 1 or 2, wherein the diabetic complication, non-alcoholic steatohepatitis, inflammatory disease, fibrosis or degenerative brain disease is caused by the final glycated product.
12. The pharmaceutical composition according to claim 1 or 2, wherein the production of the final glycation product is inhibited or the resulting final glycation product is decomposed.
13. The pharmaceutical composition according to claim 1 or 2, which does not exhibit cytotoxicity.
14. A health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including sputum extract or fractions thereof.
15. A health functional food composition for improving or preventing diabetic complications, non-alcoholic steatohepatitis, inflammatory diseases, fibrosis or degenerative brain diseases, including a compound of any one of the following Formulas 1 to 10, or a pharmaceutically acceptable salt thereof.

    

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