KR102615399B1 - A composition for the prevention or treatment of hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract - Google Patents

Abstract

The present invention relates to a composition for preventing or treating hepatitis C virus infection disease, comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract, which is isolated from Agrimonia pilosa extract and Galla rhois extract according to the present invention. One compound may be useful as a pharmaceutical composition and food composition that can effectively prevent or treat hepatitis C virus infection disease and liver disease by inhibiting the expression of HCV Core 1b and HCV NS5A.

Description

A composition for the prevention or treatment of hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract {A composition for the prevention or treatment of hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract}

The present invention relates to a preventive or pharmaceutical composition for hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract.

Hepatitis C virus (HCV: hereinafter referred to as “HCV”) is mainly caused by blood transfusion and community-acquired infection. When infected with HCV and symptoms appear, approximately 20% of cases progress to acute hepatitis, approximately 80% progress to chronic hepatitis, and approximately 30% of these progress to cirrhosis and liver cancer. According to recent reports, more than 200 million people worldwide are infected with HCV, with an estimated number of over 4.5 million in the United States (estimated to be up to 15 million) and more than 5 million in Europe. It is known that he is a hepatitis patient.

To date, there is no excellent vaccine or effective treatment for hepatitis C, so many pharmaceutical companies and research institutes around the world are developing hepatitis C treatments. Hepatitis C shows an even distribution worldwide compared to hepatitis B, and the rate of metastasis to cirrhosis and liver cancer is much higher than that of hepatitis B. And the rate of progression to chronic hepatitis is much higher, and research on the mechanism of this progression is still in progress. In addition, hepatitis C can be transmitted not only through blood transfusions, but also through intravenous drug use or tattooing, but is mainly transmitted through direct blood contact. When infected with the hepatitis C virus, most patients progress to chronic disease, which in turn progresses to cirrhosis and liver cancer. Therefore, the development of effective vaccines and treatments is urgently needed. The genotype of HCV varies between virus strains and mutations often occur. In the case of chronic hepatitis caused by HCV, reinfection and simultaneous hepatitis can occur due to genetic variants. Coinfection may also occur. For this reason, it is very difficult to successfully develop an effective vaccine for hepatitis C.

Currently, a combination of interferon-α and ribavirin is being used to treat hepatitis C, but the cure rate is low and the side effects are very severe. In the case of interferon therapy, about 25% of cases show no response at all, and about 25% of cases respond temporarily and then relapse. In the remaining 50% of patients, the ALT level remains normal and HCV RNA becomes negative until after the end of treatment. Among them, about 50% relapse within 3-6 months after the end of treatment, so only about 25% of the patients relapse after 6 months. This shows a sustained viral response that maintains the therapeutic effect. In addition, there are six genotypes of the hepatitis C virus, and type 1b, which is the most common in Korea, does not respond well to interferon treatment compared to types 2 and 3. For this reason, combination therapy with ribavirin is used, and in this case, the treatment effect has been shown to increase by about two-fold. However, when ribavirin is used alone, it has little effect and causes side effects such as anemia due to destruction of red blood cells, so interferon treatment is mainly used. It is known to be prescribed only in cases of no response or recurrence. To date, an antiviral agent that effectively treats hepatitis C by acting specifically on HCV and inhibiting its proliferation has not been developed. Interferon treatment is effective because it increases sensitivity to HCV Core 1b when combined with ribavirin, a treatment for HCV NS5A, whereas ribavirin alone has no inhibitory effect on HCV core 1b, so HCV CORE 1b itself We sought to develop a highly effective treatment for hepatitis C virus disease that simultaneously inhibits HCV NS5A.

Accordingly, the present inventors confirmed that compounds isolated from Agrimonia pilosa extract and Galla rhois extract can effectively prevent or treat hepatitis C virus infection disease by inhibiting the expression of HCV Core 1b and HCV NS5A. The present invention has been completed.

Korean Patent No. 10-0901761

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating hepatitis C virus infection disease.

Another object of the present invention is to provide a health functional food composition for preventing or improving hepatitis C virus infection disease.

Another object of the present invention is to provide a health food composition for preventing or improving hepatitis C virus infection disease.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease.

Another object of the present invention is to provide a health functional food composition for preventing or improving liver disease.

Another object of the present invention is to provide a health food composition for preventing or improving liver disease.

The present invention provides a pharmaceutical composition for preventing or treating hepatitis C virus infection disease, comprising nicotiflorin as an active ingredient.

In the present invention, the compound preferably inhibits the expression of HCV Core 1b, NS5A or HCV 214.

In addition, the present invention provides a pharmaceutical composition for enhancing the treatment effect of hepatitis C virus infection disease through co-administration with ribavirin, which contains nicotiflorin as an active ingredient.

In the present invention, the compound preferably inhibits the expression of HCV Core 1b, NS5A or HCV 214.

In addition, the present invention provides a health functional food composition for preventing or improving hepatitis C virus infection disease, comprising nicotiflorin as an active ingredient.

In addition, the present invention provides a health functional food composition containing nicotiflorin as an active ingredient for enhancing the treatment effect of hepatitis C virus infection disease through co-administration with ribavirin.

The compounds isolated from Seonhakcho extract and Chinese gallbladder extract according to the present invention are useful as pharmaceutical compositions and food compositions that can effectively prevent or treat hepatitis C virus infection disease and liver disease by suppressing the expression of HCV Core 1b and HCV NS5A. can do.

Figure 1 shows the results confirming the inhibition of HCV protein expression by Seonhakcho extract (AP), Chinese gallbladder extract (RG), and a mixture of Seonhakcho extract and gallbladder extract (APRG64).
Figure 2 shows the total ion chromatograms (TIC) of a mixture of Seonhakcho extract and gallbladder extract (APRG64).
Figure 3 shows the results of confirming the cytotoxicity of 14 compounds isolated from a mixture (APRG64) of Seonhakcho extract and Brassica chinensis extract of the present invention.
Figure 4 shows the results of confirming the expression of HCV Core 1b of 14 compounds isolated from the mixture (APRG64) of Seonhakcho extract and gallbladder extract of the present invention.
Figure 5 shows the results of confirming the expression of NS5A of 14 compounds isolated from the mixture (APRG64) of Seonhakcho extract and gallbladder extract of the present invention.
Figure 6 shows the results of confirming the HCV inhibitory activity of 14 compounds isolated from the mixture of Seonhakcho extract and gallbladder extract (APRG64) of the present invention when treated in combination with interferon gamma treatment.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating hepatitis C virus infection disease

The present invention provides a pharmaceutical composition for preventing or treating hepatitis C virus infection disease, comprising a compound represented by Formula 1 or Formula 2 isolated from Agrimonia pilosa extract and Galla rhois extract.

[Formula 1]

(In Formula 1 above,

R ¹ to R ⁶ and R ⁸ to R ¹¹ are independently -H, -OH, C _1-5 straight or branched alkoxy, . , , , or ego;

R ⁷ is independently hydrogen or an oxo group (=O)).

[Formula 2]

.

In the pharmaceutical composition of the present invention, R ¹ to R ⁶ and R ⁸ to R ¹¹ are independently -H, -OH, C _1-3 linear or branched alkoxy, . , , , or ego;

R ⁷ may independently be hydrogen or an oxo group (=O).

In the pharmaceutical composition of the present invention, preferred examples of the compound represented by Formula 1 include the following compounds.

(1) procyanidin B2;

(2) taxifolin;

(4) routine;

(5) hyperin;

(6) luteolin;

(7) astragalin;

(8) quercetin;

(9) Apigenin 7-glucuronide;

(10) baicalin;

(11) afzelin;

(12) nicotiflorin;

(13) Tiliroside; and

(14) Apigenin.

More preferably

(2) Taxifolin;

(9) Apigenin 7-glucuronide;

(10) baicalin;

(11) afzelin;

(12) nicotiflorin; and

(13) It may be tiliroside.

In the pharmaceutical composition of the present invention, the compound represented by Formula 2 may be (3) coumarin.

In the pharmaceutical composition of the present invention, the Agrimonia pilosa extract and the Galla rhois extract can be extracted with an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof to prepare an extract, preferably 50 It can be extracted using % ethanol aqueous solution.

The term "extract" used in the present invention refers to a preparation obtained by squeezing the extraction target into an appropriate leachate and evaporating the leachate to concentrate, but is not limited thereto, the extract, dilution or concentrate of the extract obtained through extraction treatment. , it may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The Seonhakcho extract and the Chrysanthemum chinensis extract can be prepared using general extraction, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably includes boiling water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

In the pharmaceutical composition of the present invention, the Agrimonia pilosa extract and Galla rhois extract can be mixed and used at a weight ratio of 5:5 to 8:2, preferably Agrimonia pilosa extract and Galla rhois extract can be mixed and used at a weight ratio of 6:4.

The compounds represented by Formula 1 or Formula 2 below, isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention, can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, It is manufactured using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc. These solid preparations are one or more of Agrimonia pilosa extract and Galla rhois extract of the present invention. It is prepared by mixing the compound represented by Formula 1 or Formula 2 below with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

The effective dosage for the human body of the compound represented by Formula 1 or Formula 2 isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention depends on the patient's age, weight, gender, dosage form, and health status. It may vary depending on the degree of disease, and is generally about 0.01-1000 mg/kg/day, preferably 0.1-500 mg/kg/day, at regular intervals according to the judgment of a doctor or pharmacist. It may also be administered in divided doses.

Health functional food or health food composition for preventing or improving hepatitis C virus infection disease

The present invention provides a health functional food or health food composition for preventing or improving hepatitis C virus infection disease, comprising a compound represented by the following formula 1 or formula 2 isolated from Agrimonia pilosa extract and Galla rhois extract. provides.

[Formula 1]

(In Formula 1 above,

R ¹ to R ¹¹ are as defined in paragraph 1).

[Formula 2]

.

In the composition of the present invention, preferred examples of the compound represented by Formula 1 include the following compounds.

(1) procyanidin B2;

(2) taxifolin;

(4) routine;

(5) hyperin;

(6) luteolin;

(7) astragalin;

(8) quercetin;

(9) Apigenin 7-glucuronide;

(10) baicalin;

(11) afzelin;

(12) nicotiflorin;

(13) Tiliroside; and

(14) Apigenin.

More preferably

(2) Taxifolin;

(9) Apigenin 7-glucuronide;

(10) baicalin;

(11) afzelin;

(12) nicotiflorin; and

(13) It may be tiliroside.

In the composition of the present invention, the compound represented by Formula 2 may be (3) coumarin.

In the composition of the present invention, the Agrimonia pilosa extract and the Galla rhois extract can be extracted with an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and preferably 50% ethanol. It can be extracted using an aqueous solution.

The term "extract" used in the present invention refers to a preparation obtained by squeezing the extraction target into an appropriate leachate and evaporating the leachate to concentrate, but is not limited thereto, the extract, dilution or concentrate of the extract obtained through extraction treatment. , it may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The Seonhakcho extract and the Chrysanthemum chinensis extract can be prepared using general extraction, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably includes boiling water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

In the composition of the present invention, the Agrimonia pilosa extract and the Galla rhois extract can be mixed and used at a weight ratio of 5:5 to 8:2, preferably the Agrimonia pilosa extract and the Galla rhois extract ( Galla rhois ) extract can be mixed and used at a weight ratio of 6:4.

When the compounds isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention are used as health functional foods and health food compositions, there is no particular limitation on the type of food. Examples of health functional foods to which compounds isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention can be added may be in the form of tablets, capsules, pills, or liquid. Examples of health foods include various Drinks, meat, sausages, bread, candies, snacks, noodles, ice cream, dairy products, soups, isotonic drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes, etc., and health functional foods and health food compositions in the conventional sense. Includes all.

Health functional foods and health food compositions containing compounds isolated from Seonhakcho ( Agrimonia pilosa ) extract and Galla rhois extract according to the present invention can be added as is to food or used together with other foods or food ingredients, and can be used as usual It can be used appropriately depending on the method. The mixing amount of compounds isolated from Agrimonia pilosa extract and Galla rhois extract can be appropriately determined depending on the purpose of use (prevention or improvement). In general, the amount of the composition in health functional foods and health food compositions can be 0.1 to 90 parts by weight of the total weight of the food. However, in the case of long-term intake for the purpose of maintaining health or regulating health, the amount may be below the above range, and since there is no problem in terms of safety, it can be used from Agrimonia pilosa extract and Galla rhois extract. The isolated compound may be used in amounts above the above range.

The health functional food and health food composition of the present invention have no special restrictions on other ingredients other than containing the compounds isolated from the Agrimonia pilosa extract and Galla rhois extract of the present invention as essential ingredients in the indicated ratio. Like beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 of the health functional food and health food composition of the present invention.

In addition to the above, the health functional food and health food composition containing compounds isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention include various nutrients, vitamins, minerals (electrolytes), synthetic flavors, and natural flavors. Flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. It may contain carbonating agents, etc. to be used. In addition, the health functional food and health food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

These ingredients can be used independently or in combination. The ratio of these additives is not that critical, but ranges from 0.1 to about 20 parts by weight per 100 parts by weight of the health functional food and health food composition containing the compounds isolated from Agrimonia pilosa extract and Galla rhois extract of the present invention. It is common to select from .

Pharmaceutical composition for preventing or treating liver disease

The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising a compound represented by the following formula (1) or formula (2) isolated from Agrimonia pilosa extract and Galla rhois extract.

[Formula 1]

(In Formula 1 above,

R ¹ to R ¹¹ are as defined in paragraph 1).

[Formula 2]

.

In the pharmaceutical composition of the present invention, examples of the liver disease may be fatty liver, liver fibrosis, cirrhosis, or liver cancer.

Health functional food or health food composition for preventing or improving liver disease

The present invention provides a health functional food or health food composition for preventing or improving liver disease, comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 isolated from Agrimonia pilosa extract and Galla rhois extract.

[Formula 1]

(In Formula 1 above,

R ¹ to R ¹¹ are as defined in paragraph 1).

[Formula 2]

.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

<Example 1> Preparation of extract and isolation of compounds

<1-1> Preparation of extract

Leaves of Agrimonia Pilosa and Galla rhois were purchased from BioKorea Co., LTd (Seoul), and voucher specimens (BMRI-AP-1601, BMRI-RG-1602) were purchased from Kyung Hee University Biomedical Sciences, Yongin, Korea. Deposited to the research center. The dried sample (20 kg) was extracted with 50% ethanol for 6 hours at 80 ± 2°C and then filtered. Afterwards, it was concentrated using a rotary evaporator and lyophilized. The final product yield was 1.57kg of Seonhakcho and 11.59kg of Five-spotted Chrysanthemum. A mixture of Seonhakcho and Chinese gallbladder was used in a ratio of 6:4, respectively, and was abbreviated as APRG64. All samples were stored at 4 °C until use.

<1-2> Compound separation

To separate compounds from the mixture (APRG64) of the extracts of Seonhakcho and Brassica chinensis, tiMate™3000 Rapid Separation Binary System (Thermo fisher scientific Inc., Waltham, MA, USA) and Kinetex F5 C18 column (2.6 μm, 2.1 × 100 mm) HPLC was performed using . The column oven was maintained at 25°C, and the mobile phase contained solvent A (0.1% aqueous formic acid solution (v/v)) and solvent B (0.1% formic acid dissolved in acetonitrile (v/v)). The elution gradient was as follows. 0-2 min, B 5%; 2-8 min, B 5-20%; 8-16 min, B 20%; 16-28 min, B 20-35%; 28-32 min, B 35-100% ; and 32-35 min, B 100%.

The flow rate was 250 μl/min and the injection volume was 5 μl for each run. MS analysis was performed using a SCIEX Triple TOF 5600 (SCIEX, Framingham, MA, USA) operating in positive ion mode. The mass spectrometer performed alternating high- and low-energy scans, known as MSE acquisition mode. Operating parameters were set as follows: cone voltage 40 V; capillary 3.0 kV; Source temperature 500°C, cone gas flow 30 L/h; and desolvation gas flow at 800 L/h. Data were collected from 100 to 1000 m/z.

Example 1 Total ion chromatograms (TIC) of APRG64 are shown in Figure 1. Table 1 below shows information on the name, molecular formula, retention time, mass value, and mass accuracy of a total of 14 compounds. Peaks were selected based on shared characteristics with values higher than 0.95 in the Analyst library and mass accuracy for each peak less than 40 ppm.

RT (min) name Molecular
Formula Observed Mass (Da) Exposed Mass (Da) Mass Accuracy (ppm) One 1.14 procyanidin B2 C ₃₀ H ₂₆ O ₁₂ + H 579.1488 579.1502 2.5 2 7.12 dihydroquercetin C ₁₅ H ₁₂ O ₇ +H 305.0659 305.0661 0.8 3 8.48 coumarin C ₉ H ₆ O ₂ +H 147.0437 147.0446 6.3 4 9.21 rutin C ₂₇ H ₃₀ O ₁₆ + H 611.1605 611.1612 1.2 5 9.44 hyperin C ₂₁ H ₂₀ O ₁₂ +H 465.1030 465.1033 0.7 6 10.25 luteolin C ₁₅ H ₁₀ O ₆ + H 287.0552 287.0555 1.1 7 10.30 astragalin C ₂₁ H ₂₀ O ₁₁ +H 449.1073 449.1084 2.5 8 10.33 quercetin C ₁₅ H ₁₀ O ₇ +H 303.0498 303.0505 2.4 9 10.70 apigenin 7-O-β-D-glucuronide C ₂₁ H ₁₈ O ₁₁ + H 447.0923 447.0927 1.0 10 10.76 baicalin C ₂₂ H ₂₀ O ₁₀ + H 447.0936 447.0927 -2.0 11 11.46 afzelin C ₂₁ H ₂₀ O ₁₀ +H 433.1126 443.1135 2.1 12 12.96 nicotiflorin C ₂₇ H ₃₀ O ₁₅ +H 595.1446 595.1663 36.5 13 16.28 tiliroside C ₃₀ H ₂₆ O ₁₃ +H 595.1445 595.1451 1.1 14 19.83 apigenin C ₁₅ H ₁₀ O ₅ + H 271.0602 271.0606 1.6

Based on the results of FIG. 2 and Table 1, the structural formulas of the compounds isolated from Seonhakcho and Brassica chinensis extracts are shown in Table 2 below.

name structure IUPAC Name One procyanidin B2 (2R,3R)-2-(3,4-dihydroxyphenyl)-8-[(2R,3R,4R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro -2H-chromen-4-yl]-3,4-dihydro-2H-chromene-3,5,7-triol 2 Taxifoline
(taxifolin) 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydrochromen-4-one 3 Coumarin
(coumarin) chromen-2-one 4 Routine
(rutin) 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R, 5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one 5 hyperine
(hyperin) 4-[5,7-dihydroxy-4-oxo-3-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-2 -yl]-2-hydroxyphenolate 6 luteolin
(luteolin) 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one 7 Astragalin
(astragalin) 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen- 4-one 8 Quercetin
(quercetin) 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one 9 Apigenin-7-glucuronide
(Apigenin 7-glucuronide) (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-[5-hydroxy-2-(4-hydroxyphenyl)-4-oxochromen-7-yl]oxyoxane-2-carboxylic acid 10 baicalin
(baicalin) (2S,3S,4S,5R,6S)-6-(5,6-dihydroxy-4-oxo-2-phenylchromen-7-yl)oxy-3,4,5-trihydroxyoxane-2-carboxylic acid 11 Afzeline
(afzelin) 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one 12 Nicotiflorine
(nicotiflorin) 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R, 6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one 13 Tiliroside
(tiliroside) [(2R,3S,4S,5R,6S)-6-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxan-2- yl]methyl (E)-3-(4-hydroxyphenyl)prop-2-enoate 14 apigenin
(apigenin) 5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one

<Preparation Example 1> Cell culture

Huh 7.5 cells were grown in DMEM (Dulbecco's Modified Eagle's Medium, WELGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences) and 1% penicillin-streptomycin (PS, WELGENE, Daegu, Korea). Cultured. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO ₂ .

<Preparation Example 2> HCV (Hepatitis C virus) RNA preparation

<2-1> Transformation

Cells were transformed according to the manufacturer's instructions (ECOS 101, Yeastern Biotech Co., Ltd, Taipei, Taiwan). Briefly, 1 μl of plasmid DNA was mixed with 50 μl of cells and then incubated on ice for 5 min. The cultured cells were applied to an agar plate containing ampicillin and cultured in an incubator at 37°C until bacterial colonies appeared.

<2-2> Midiprep

Midiprep was performed using PureLink™HiPure Plasmid DNA Purification Kits (Invitrogen, CA, USA). Plasmid DNA was dissolved in an appropriate volume of TE buffer using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher, Waltham MA, USA) to measure concentration and purity. 1 μg of the obtained DNA was applied to an agarose gel containing GelRed®Nucleic Acid Gel Stain (Biotium, CA, USA) in a Mupid-2 Plus electrophoresis system (ADVANCE).

<2-3> DNA linearization

JFH-1 plasmid was provided by Dr. Takaji Wakita (National Institute of Infectious Diseases, Japan). The plasmid was digested with Linearized plasmid DNA was extracted twice with phenol:chloroform:isoamyl alcohol and precipitated with pH 5.2 sodium acetate and 100% ethanol. After washing once with 70% ethanol, concentration and purity were measured using a spectrophotometer.

<Preparation Example 3> In vitro transcription

In vitro transcription was performed using the MEGAscript®kit (Life Technology, CA, USA) according to the manufacturer's instructions. After transcription, the extracted RNA was precipitated with an equal volume of isopropanol. After sufficiently dissolving the precipitated RNA pellet in RNase-free water, the concentration and purity were confirmed using a spectrophotometer.

<Preparation Example 4> Transfection

Electroporation was performed using the Neon®transfection system (Invitrogen, CA, USA). Briefly, Huh 7.5 cells were resuspended in 100 μl R buffer and extracted RNA was added. After adding the cell mixture to a 100㎕ Neon® tip, a shock (1400 V·20 ms ^-1 pulse) was applied. Shocked cells were resuspended in antibiotic-free medium and cultured at 37°C in a humidified atmosphere containing 5% CO ₂ .

<Experimental Example 1> Anti-HCV activity of extract

To confirm whether the extract of Seonhakcho and Shipwreck (APRG64) of Example 1-1 exhibits anti-HCV activity, Huh 7.5 cells were transfected with full-length HCV RNAs transcribed from the pJFH-1 plasmid. Two days later, Huh 7.5 cells were treated with each extract for 2 days. Since the expression levels of HCV Core 1b and NS5A indicate replication of HCV, the expression levels of these proteins were analyzed by Western blotting.

As a result, as shown in Figure 1, Seonhakcho extract (AP) and gallbladder extract (RG) inhibited the expression of HCV Core 1b and NS5A, and the mixture of Seonhakcho extract and gallbladder extract (APRG64) also strongly inhibited the expression of HCV protein. suppressed.

<Experimental Example 2> Cytotoxicity

First, Huh 7.5 cells were treated with each of the 14 compounds isolated in Example 1-2 at various concentrations for 24 hours, and cytotoxicity was analyzed using a dye exclusion method taking advantage of the fact that trypan blue does not stain living cells.

As a result, as shown in Figure 3, the optimal treatment concentrations of the 14 compounds isolated in Example 1-2 were Procyanidin B2 (1, 5 μg/mL), Taxifolin (2), respectively. , 5 μg/mL), Coumarin (3, 50 μg/mL), Rutin (4, 5 μg/mL), hyperin (5, 5 μg/mL), luteolin ( luteolin (6, 2.5 μg/mL), astragalin (7, 50 μg/mL), Quercetin (8, 2.5 μg/mL), Apigenin 7-glucuronide (Apigenin 7) -glucuronide) (9, 50 μg/mL), Baicalin (10, 50 μg/mL), afzelin (11, 50 μg/mL), Nicotiflorin (12, 50 μg/mL), Tiliroside (13, 50 μg/mL), and apigenin (14, 5 μg/mL), and the concentrations confirmed above were used in subsequent experiments.

<Experimental Example 3> HCV inhibitory activity

To confirm the anti-HCV activity of the 14 compounds isolated in Example 1-2, compound 14 of Example 1-2 was added to untransfected (Mock) Huh 7.5 cells or Huh 7.5 cells transfected with HCV RNA. Each species was treated for 48 hours, cell lysates were prepared, and the expression of HCV Core 1b and NS5A was analyzed by Western blot. GAPDH was used as a control. The band intensities of proteins were quantified by Image J software and relative expression to GAPDH was shown (*, p ≤ 0.05).

As a result, as shown in Figure 4, all compounds inhibited the expression of HCV Core 1b protein compared to the untreated control group. Hyperin, luteolin, astragalin, afzelin, and apigenin showed relatively strong inhibitory activity compared to other compounds.

Additionally, as shown in Figure 5, the expression of NS5A was also significantly suppressed. In particular, among the 14 compounds, luteolin and tiliroside showed relatively strong inhibitory activity against HCV NS5A expression.

<Experimental Example 4> HCV inhibition when combined with interferon gamma treatment

In order to confirm the anti-HCV activity of the 14 compounds isolated in Example 1-2 when combined with interferon treatment, each of the 14 compounds of Example 1-2 was added to Huh 7.5 cells transfected with HCV RNA. 14 compounds of 1-2 and interferon gamma treatment (ribavirin) were treated in combination (CO-Treat) for 48 hours, RNA was extracted from cells and cDNA was synthesized, and the expression of HCV 214 was analyzed by qRT-PCR. did. Beta-actin was used as a control.

As a result, as shown in Figure 6, when comparing the group treated with the compound alone and the group treated with the compound and interferon gamma treatment (ribavirin) in combination, luteolin and astragalin (astragalin), apigenin-7-glucuronide (Apigenin 7-glucuronide) and apigenin (apigenin) compounds inhibit Core1B, so it was confirmed that there was no synergistic effect when combined with interferon gamma treatment (ribavirin). It was confirmed that the expression of HCV 214 was significantly suppressed when treated alone.

In addition, coumarin, afzelin, and nicotiflorin compounds inhibit both NS5A and Core1B, so when treated alone, the expression of HCV 214 was slightly inhibited, but interferon gamma treatment (ribavirin) It was confirmed that the expression of HCV 214 was excellently suppressed when treated in combination.

Therefore, based on the above experimental results, it was confirmed that the compound of Example 1-2 isolated from the extract of Seonhakcho and Chinese gallbladder extract can be treated in combination with an interferon gamma treatment (ribavirin), and that it exhibits HCV inhibitory activity even when treated alone.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Preparation example of medicine

The compound represented by Formula 1 or Formula 2 according to the present invention can be formulated in various forms depending on the purpose. The following is an example of several formulation methods containing the compound represented by Formula 1 or Formula 2 as an active ingredient according to the present invention, but the present invention is not limited thereto.

<Drug Preparation Example 1> Preparation of powder

2 g of compound of formula 1 or formula 2

1 g lactose

After mixing the above ingredients, they were filled into an airtight bubble to prepare a powder.

<Drug Preparation Example 2> Preparation of tablets

100 mg of compound of formula 1 or formula 2

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

<Drug Preparation Example 3> Preparation of capsules

100 mg of compound of formula 1 or formula 2

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

<Pharmaceutical Preparation Example 4> Preparation of injections

10 μg/ml of compound of Formula 1 or Formula 2

Dilute hydrochloric acid BP until pH 3.5

Sodium Chloride BP for Injection up to 1 ml

Dissolve the compound of formula 1 or formula 2 according to the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP, and adjust the volume using sodium chloride BP for injection. Adjusted and mixed thoroughly. The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120°C for 15 minutes or more to prepare an injection solution.

<Drug Preparation Example 5> Preparation of nasal absorbent (Nasal spray)

1.0 g of compound of formula 1 or formula 2

Sodium acetate 0.3 g

Methylparaben 0.1 g

Propylparaben 0.02 g

Sodium chloride dosage

HCl or NaOH pH adjustment appropriate amount

Proper amount of purified water

According to the manufacturing method of a conventional nasal absorbent, 3 mg of the compound of Formula 1 or Formula 2 is prepared per 1 mL of saline water (0.9% NaCl, w/v, solvent is purified water), and filled into an opaque spray container. A nasal absorbent was prepared by sterilization.

<Drug Preparation Example 6> Preparation of liquid preparation

100 mg of compound of formula 1 or formula 2

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add lemon zest, mix the above ingredients, add purified water to adjust the total to 100 mL, fill in a brown bottle, and sterilize to prepare the liquid. did.

Manufacturing example of health food

The compound of Formula 1 or Formula 2 according to the present invention can be manufactured into various types of health foods depending on the purpose. The following illustrates a method for producing several health foods containing the compound of Formula 1 or Formula 2 as an active ingredient according to the present invention, but the present invention is not limited thereto.

<Health food manufacturing example 1> Manufacturing of dairy products

0.01-1 part by weight of the compound of Formula 1 or Formula 2 of the present invention was added to milk, and various dairy products such as butter and ice cream were manufactured using the milk.

<Health food production example 2> Production of sun food

Brown rice, barley, glutinous rice, and coix seed were gelatinized and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder. Black beans, black sesame seeds, and perilla seeds were steamed and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder. The compound of Formula 1 or Formula 2 of the present invention was concentrated under reduced pressure in a vacuum concentrator to obtain a dry powder. The dry powder of the grains, seeds, and compounds of Formula 1 or Formula 2 prepared above was mixed in the following ratio.

Grains (34 parts by weight of brown rice, 19 parts by weight of coix radish, 20 parts by weight of barley),

Seeds (7 parts by weight perilla seeds, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Compound of formula 1 or formula 2 (2 parts by weight),

Reishi (1.5 parts by weight), and

Rehmannia glutinosa (1.5 parts by weight).

Manufacturing example of health functional food

The compound of Formula 1 or Formula 2 according to the present invention can be manufactured into various types of health functional foods depending on the purpose. The following is an example of a method for manufacturing several health functional foods containing the compound of Formula 1 or Formula 2 as an active ingredient according to the present invention, but the present invention is not limited thereto.

<Example 1 of manufacturing health functional food> Manufacturing of health functional food

100 mg of compound of formula 1 or formula 2

Vitamin mixture dosage

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients relatively suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a typical health functional food manufacturing method. Then, granules can be prepared and used to manufacture health functional food compositions according to conventional methods.

<Example 2 of manufacturing health functional food> Manufacturing of health functional beverage

100 mg of compound of formula 1 or formula 2

100 mg citric acid

100 mg of oligosaccharides

Plum concentrate 2 mg

Taurine 100 mg

Add purified water to make a total of 500 mL.

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution was filtered, obtained in a sterilized container, sealed, sterilized, and stored in the refrigerator. Then, the present invention It is used in the production of health drink compositions. The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for preferred drinks, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demand country, and use purpose.

Claims (6)

delete delete In a pharmaceutical composition for enhancing the treatment effect of hepatitis C virus infection disease mediated by the expression of HCV Core 1b, NS5A and HCV 214 through co-administration with ribavirin, comprising nicotiflorin as an active ingredient,
The nicotiflorine is
A composition that simultaneously inhibits the expression of HCV Core 1b, NS5A, and HCV 214. delete delete In a health functional food composition for enhancing the treatment effect of hepatitis C virus infection disease mediated by the expression of HCV Core 1b, NS5A and HCV 214 through co-administration with ribavirin, containing nicotiflorin as an active ingredient,
The nicotiflorine is
A composition that simultaneously inhibits the expression of HCV Core 1b, NS5A, and HCV 214.