KR20130038000A - The pharmaseutical compositions for prevention or treatment of hepatitis b containing cinnamomum extracts, its fractions, or trans-cinnamaldehyde or 2-methoxycinnamaldehyde isolated from cinnamomum as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a cinnamon extract, a fraction thereof, or trans-cinnamaldehyde or 2-methoxycinnamaldehyde isolated from cinnamon for preventing and treating hepatitis type B is provided to suppress the generation of hepatitis B virus e antigen and s antigen. CONSTITUTION: A pharmaceutical composition for preventing and treating hepatitis type B contains trans-cinnamaldehyde or 2-methoxycinnamaldehyde, which is isolated from cinnamon, as an active ingredient. A cinnamon extract is prepared using water, alcohol, or a mixture thereof. [Reference numerals] (AA) Suppression rate(%); (BB) Treatment concentration(uM);

Description

Cinnamon extract, fractions thereof, or trans-cinnaaldehyde or 2-methoxycinnaaldehyde isolated from cinnamon as an active ingredient The Pharmaseutical compositions for prevention or treatment of hepatitis B containing Cinnamomum extracts ， its fractions, or trans--cinnamaldehyde or 2-methoxycinnamaldehyde isolated from Cinnamomum as an active ingredient}

The present invention provides a method for preventing and treating B-type infections containing Cinnamomum extract, fractions thereof, or trans-cinnamaldehyde or 2-methoxycinnamaldehyde isolated from cinnamon as an active ingredient. It relates to a pharmaceutical composition, and a composition for health food.

Human hepatitis B virus (HBV) causes acute and chronic hepatitis, and can progress to diseases such as self-controlling acute hepatitis, blunt hepatitis or chronic active hepatitis. Chronic active hepatitis develops, causes cirrhosis and liver cancer, and can be acutely acute with liver failure (Brechot, C., J. Hepatol., 4, 269-279, 1987). When infected with chronic hepatitis, most patients show persistent or active hepatitis, while some do not. The pathogenesis or causative factors of the viral hepatitis are not clear yet. Since some evidence suggests that liver damage caused by the hepatitis B virus is mediated by the host's immune cells (Milich, DR et al., J. Immunol., 143, 3141-3147, 1989). Intensive studies have been conducted on the immune response of specific B and T cells and the direct or pathogenic mechanisms of viral proteins (R. Zschke, O. et al., Nature, 348, 252-254 (1990). ). However, the causes of the clinical pathways that still vary from patient to patient are not clear.

Hepatitis B has been studied with great interest in its treatment because of its severity. Currently, several drugs and vaccines have been developed and have received good reviews in preclinical and clinical trials, but few drugs have been applied as actual therapeutics. To date, the only treatment for hepatitis B virus (HBV) hepatitis patients is human immunity. It depends on the system. Human immune action against HBV removes HBV particles, but on the other hand, destroys hepatocytes, so side effects of these immune actions can lead to fatal diseases. In addition, since these problems make it difficult to predict disease progression, it is desirable to use a combination of antiviral therapy and immunotherapy for fast and efficient hepatitis treatment. In other words, during the treatment of viral hepatitis, infected hepatocytes are destroyed by cytotoxic T lymphocytes (CTLs), and virus particles released from the destroyed hepatocytes remove the neutralizing antibodies against the virus to prevent reinfection into the hepatocytes. Proliferation of should also be suppressed. Several methods have been tried to treat the hepatitis B virus, including interferon, nucleic acid derivatives or immunomodulators, but only interferon-α has been shown to be the only therapeutic effect. have.

Interferon-α was first tested by Sculled et al in the mid-1970s as a treatment for chronic hepatitis B. It has been shown to be effective in inhibiting HBV replication. However, when interferon-α is administered three times a week for at least three months, on average, 20% of patients show a therapeutic effect that inhibits virus growth but do not show sustained inhibitory effects. Patients or children are resistant to interferon-α and therefore have a low therapeutic effect. In addition, it has recently been reported that only 50% or less of acute and chronic patients infected with hepatitis B virus are suitable for the treatment of interferon-α. Among the patients, various enzymes (AST, Only if the amount of SGOT, ALT, SGPT, etc. is increased, the interferon-α treatment can be expected to be effective. Therefore, there is an urgent need to develop a hepatitis B virus therapeutic agent that can treat patients with resistance to interferon-α. In particular, as a method of inhibiting the proliferation of hepatitis B virus, research has been conducted to find a substance that inhibits the attachment of a virus to a receptor or specifically inhibits an active protein or a polymerase of a virus. Hepatitis B virus replication produces three major antigens (c, s and e antigens), the c (core) antigen is a structural antigen crystal and the s (surface) antigen is an antigenic crystal that is represented by viral surface proteins. The e antigen is a marker in the blood to detect hepatitis B virus infection.

The e antigen is not a prerequisite for the propagation of hepatitis B and its role in the life history of the hepatitis B virus is not yet clear. However, it is speculated that the antigen plays an important role in view of the conserved gene of the e antigen. Thus, the e antigen is an immunological target for virus removal, in particular the host plays an important role in virus removal (Pignatelli, M. et al., J. Hepal. 4, 15-16, 1987).

The only treatment given to the patient is to rely on the human immune system. Human immune action against HBV removes HBV particles, but on the other hand destroys hepatocytes, a side effect of this immune action can lead to fatal diseases. In addition, since these problems make it difficult to predict disease progression, it is desirable to use a combination of antiviral therapy and immunotherapy for fast and efficient hepatitis treatment. In other words, during the treatment of viral hepatitis, infected hepatocytes are destroyed by cytotoxic T lymphocytes (CTLs), and virus particles released from the destroyed hepatocytes remove the neutralizing antibodies against the virus to prevent re-infection into the hepatocytes. Proliferation of should also be suppressed.

Cinnamon is a native of China, Indonesia, and Vietnam. It is cultivated in Laos, Cambodia, Indochina and Sumatra. Bark is known by many different names in the spices trade. In the United States it is often referred to as cinnamon. Chinese cassia or cinnamon is cultivated in Kwangsi, Kweichow, Kwantung provinces. French Indochina cassia is the bark of Cinnamomum loureirii Nees. Batavia cassia is now called Korintje cinnamon, which is the bark of Cinnamomum burmannii Blume. It is cultivated in lowlands along the west coast of Sumatra. The dried, less ripe berries of these cassia are known as Cassia buds. Cassia is an evergreen with the bark of Cinnamomum. The young shoots of the Cassia tree are cut and bark twice a year. Sprout grows from stump. Thin bark is in the shape of a barrel to minimize damage. Chinese cassia quills average 30 cm in length and 0.6 mm in thickness. Cortex varies from light grayish brown to dark reddish brown. The flavor is similar to Chinese cassia. Batavia cassia quills are softer and more orderly. It is pale reddish brown, fragrant, sweet and irritating. The main ingredient is cinnamic aldehyde, with aroma and sweet spicy taste, occupying about 65 ~ 76%, essential oil, tannin, coumarin, mucus, gum, sugar, etc.

Accordingly, the present inventors have selected a substance showing anti-hepatitis B virus activity from natural products and researched to develop it as a therapeutic agent for hepatitis. trans-cinnamaldehyde or 2-methoxycinnamaldehyde inhibits the production of hepatitis B virus e and s antigens, significantly lowers DNA replication and polymerase activity of hepatitis B virus, and is cytotoxic. The present invention was completed by confirming that the low hepatitis B can be usefully used for the prevention and treatment of hepatitis B.

An object of the present invention is a Cinnamomum extract, fractions thereof, trans-cinnamaldehyde or 2-methoxycinnamaldehyde isolated from cinnamon, or pharmaceutically acceptable thereof It is to provide a pharmaceutical composition for preventing and treating hepatitis B, or a composition for health food for preventing and improving hepatitis B, which contains a possible salt as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating B-type infection containing Cinnamomum extract as an active ingredient, extracted using water, alcohol or a mixture thereof as a solvent.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating infections of type B infection, containing the ethyl acetate fraction obtained by adding water to the ethanol extract of cinnamon and suspending it, followed by the addition of ethyl acetate.

In addition, the present invention is a pharmaceutical for preventing and treating B-type infections containing butanol fraction obtained by adding butanol to the water layer formed by suspending by adding water to cinnamon ethanol extract, and then fractionated by adding ethyl acetate. To provide a composition.

In addition, the present invention is trans-cinnamaldehyde (trans-cinnamaldehyde) represented by the following formula (1), 2-methoxycinnamaldehyde (2-methoxycinnamaldehyde) represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition for preventing and treating B-type infection containing.

In addition, the present invention provides a composition for preventing and improving type B infection, which contains cinnamon or extract as an active ingredient, extracted using water, ethanol or a mixture thereof as a solvent.

In addition, the present invention is a trans-cinnamaldehyde (trans-cinnamaldehyde) represented by the formula (1), 2-methoxycinnamaldehyde (2-methoxycinnamaldehyde) represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient It provides a composition for preventing and improving the health food containing B-type infection.

[Formula 1]

[Formula 2]

Cinnamomum extract, fractions thereof, or trans-cinnamaldehyde or 2-methoxycinnamaldehyde isolated from cinnamon according to the present invention are used to produce hepatitis B virus e antigen and s antigen. It can be used as a pharmaceutical composition for inhibiting, significantly reducing DNA replication and polymerase activity of hepatitis B virus, and having low cytotoxicity.

1 is a diagram showing the effect of natural plant separation formula TCA (trans-cinnamaldehyde) and MCA (2-methyxy-cinnamaldehyde) to suppress hepatitis B virus e antigen secretion in hepatitis B virus-infected hepatocytes.
2 is a diagram showing the effect of inhibiting the secretion of hepatitis B virus s antigen in hepatocytes infected with natural plant isolates TCA and MCA hepatitis B virus.
Figure 3 is a diagram showing the effect of the natural plant separation formula TCA affects the DNA replication of hepatitis B virus;
Lane 1: DMSO;
Lane 2: 1.25 μm TCA;
Lane 3: 2.5 μm TCA; And
Lane 4: 5 μm TCA.
Figure 4 is a diagram showing the effect of the natural plant separation formula MCA affects the DNA replication of hepatitis B virus;
Lane 1: DMSO;
Lane 2: 1.25 MCA;
Lane 3: 2.5 MCA; And
Lane 4: 5 MCA.

Hereinafter, the present invention will be described in detail.

The present invention is a Cinnamomum extract of the present invention, a fraction thereof, or a trans-cinnamaldehyde (TCA) represented by the following Chemical Formula 1 isolated from cinnamon or 2-methoxycinnaaldehyde described by the following Chemical Formula 2 It provides a pharmaceutical composition for the prevention and treatment of hepatitis B containing (2-methoxycinnamaldehyde; MCA) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

[Formula 2]

The cinnamon extract, its fractions and active compounds

1) extracting cinnamon with water, alcohol or a mixture thereof;

2) adding an organic solvent to the extract of step 1) to prepare an organic solvent fraction; And

3) Preferably, the fraction of step 2) is prepared by a separation purification method comprising column chromatography to obtain an active compound, but is not limited thereto.

In this method, cinnamon in step 1) can be used both without limitation, whether grown or commercially available.

The cinnamon is preferably used alone or in combination with a branch or a peel, but is not limited thereto.

In the above method, the alcohol of step 1) is preferably C ₁ to C ₂ lower alcohol, it is preferable to use ethanol or methanol as the lower alcohol, but is not limited thereto. The organic material is better eluted in 100% alcohol and the glycoside is better eluted in an aqueous alcohol solution, so it can be used as an alcohol or an aqueous alcohol solution as needed. During the extraction, the solvent is preferably extracted by adding 5 to 15 times the amount of dried and ground cinnamon, and the extraction is preferably performed by adding 7 to 10 times, but is not limited thereto. The extraction time is preferably 2 hours to 10 hours, more preferably 3 hours to 5 hours, but is not limited thereto. The number of extraction is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto. The extraction method may be hot water extraction, reflux extraction, shaking extraction or Soxhlet extraction, etc., but is not limited to a specific method.

In the above method, the organic solvent of step 2) is preferably ethyl acetate or butanol, but is not limited thereto. In the present invention, an aqueous ethanol extract of cinnamon was suspended in water, and then solvent fractions were sequentially fractionated with ethyl acetate and butanol to concentrate each fraction.

In the above method, the column chromatography of step 3) is performed by column chromatography using a column using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin. The active compound can be isolated and purified. Column chromatography may be carried out several times by selecting an appropriate filler as needed, and may be used as a solvent, but not limited to chloroform-methanol, ethyl acetate-methanol or dichloromethane-methanol.

The trans-cinnaaldehyde of Formula 1 and 2-methoxycinnaaldehyde of Formula 2 were isolated from cinnamon extract using column chromatography or purchased from Sigma-Aldrich (St. Louis, MO, USA). Both can be used.

As the pharmaceutically acceptable salt of the present invention, addition salts formed by free acid are useful. Suitable free acids may be organic and inorganic acids, inorganic acids may be hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, and organic acids may be citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Etc. may be used, but the present invention is not limited thereto.

In a specific embodiment of the present invention, the inventors investigated the effects on hepatitis B virus e antigen and s antigen production using TCA and MCA, resulting in decreased activity of e and s antigens even at low concentrations. Confirmation was made (see FIGS. 1 and 2).

In a specific embodiment of the present invention, the inventors of the present invention measured the hepatitis B virus DNA replication activity of TCA and MCA and found that the TCA and MCA of the present invention significantly lowered the DNA replication of hepatitis B virus ( 3 and 4).

In a specific embodiment of the present invention, the present inventors examined the cytotoxicity of TCA and MCA, it was confirmed that the TCA and MCA of the present invention can be useful in the prevention and treatment of hepatitis B is low cytotoxicity (See Table 2).

In a specific embodiment of the present invention, the present inventors orally administered TCA and MCA to rats, and as a result of the toxicity test, 50% lethal dose (LD ₅₀ ) by oral toxicity test is a stable substance of at least 5g / kg or more It was confirmed.

Thus, the cinnamon extract, fractions thereof, or trans-cinnaaldehyde or 2-methoxycinnaaldehyde isolated from cinnamon or pharmaceutically acceptable salts thereof, inhibit the production of hepatitis B virus e antigen and s antigen. In addition, the DNA replication activity of hepatitis B virus was significantly lowered, and the cytotoxicity was low, and thus it was confirmed that it can be usefully used as an active ingredient for the prevention and treatment of hepatitis B pharmaceutical composition.

To the total weight of the composition of the present invention contains 0.1 to 99.9% by weight of the cinnamon extract of the present invention, fractions thereof, or trans-cinnaaldehyde or 2-methoxycinnaaldehyde isolated from cinnamon as an active ingredient, It may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select externally or intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterine epidural or cerebrovascular injection methods for parenteral administration. For skin use externally.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is the present invention 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably based on the amount of trans-cinnaaldehyde or 2-methoxycinnaaldehyde isolated from cinnamon extract, fractions thereof, or cinnamon Is 50 to 300 mg / kg and may be administered 1 to 6 times a day.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In another aspect, the present invention provides a composition for preventing and improving B-type infections containing a cinnamon extract as an active ingredient, extracted using water, ethanol or a mixture thereof as a solvent.

In addition, the present invention for the prevention and improvement of type B infection containing a trans-cinna aldehyde represented by the formula (1), 2-methoxycinna aldehyde represented by the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient Provides a composition for health food.

The cinnamon extract of the present invention, fractions thereof, or trans-cinnaaldehyde or 2-methoxycinnaaldehyde isolated from cinnamon or pharmaceutically acceptable salts thereof can produce hepatitis B virus e antigen and s antigen. It inhibits, significantly lowers DNA replication and polymerase activity of hepatitis B virus, and is low in cytotoxicity, and thus can be usefully used for health food for prevention and improvement of hepatitis B virus.

The health functional food of the present invention may be added with cinnamon extract, fractions thereof, trans-cinnaaldehyde or 2-methoxycinnaaldehyde as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the pharmaceutical compositions of the present invention are added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice beverage and flesh for the production of vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Production Examples.

However, the following Examples, Experimental Examples and Preparation Examples illustrate the present invention concretely, and the contents of the present invention are not limited by Examples, Experimental Examples and Production Examples.

< Example  1> cinnamon Cinnamomum ) Preparation of extract

<1-1> Cinnamon Ethanol Aqueous Extract

Cinnamon, which was purchased at Gyeongdong Market, was dried and pulverized, 200 g of cinnamon was placed in a 3 L flask, and 1.5 L of an 80% ethanol aqueous solution was added for 3 hours. This process was repeated three times to collect the supernatant, filtered through a filter paper and concentrated under vacuum to give 7 g of an aqueous cinnamon ethanol extract.

<1-2> Cinnamon Water Extract

In Example <1-1>, 10.2 g of cinnamon water extract was obtained in the same manner except distilled water was added instead of an aqueous 80% ethanol solution.

<1-3> Cinnamon 100% Methanol Extract

8 g of cinnamon methanol extract was obtained in the same manner as in Example <1-1> except that 100% methanol was added instead of an 80% ethanol aqueous solution.

< Example  2> from cinnamon extract Fraction  Produce

5.5 g of the cinnamon ethanol aqueous solution extract obtained in Example <1-1> was suspended in 1 L of water, and then solvent fractionated sequentially with ethyl acetate and butanol. First, 1 liter of ethyl acetate was added to a cinnamon ethanol extract suspended in 1 liter of water, and then dissolved. Then, only the components dissolved in the ethyl acetate layer were separated and dried in a vacuum to dry in vacuum. This process was repeated three times to obtain an ethyl acetate fraction (0.7 g). 1 liter of butanol was added to the remaining aqueous layer, and only the components dissolved in the butanol layer were separated and dried under vacuum. This process was repeated three times to obtain a butanol fraction (3.5 g).

< Example  3> trans- Shin Nam Aldehyde (trans- cinnamaldehyde ; TCA ) And 2-methoxycinnaaldehyde (2- methoxycinnamaldehyde ; MCA) Preparation

Cinnamon methanol extract extracted in Example <1-3> was dissolved in methylene chloride and added to silica gel (Merck, Art No. 9385) to adsorb the active material, and then the ratio of ethyl acetate and nucleic acid was 95: 5 to 80: The active fractions were separated by silica gel column chromatography changing to 20. The obtained fractions were adsorbed on a C18 column and eluted with methanol and water to partially purify to obtain trans-cinnamaldehyde of [Formula 1] and 2-methoxycinnamaldehyde of [Formula 2].

< Experimental Example  1> Trans- Shin Nam Aldehyde ( TCA ) And 2- Of methoxycinnaaldehyde (MCA)  Anti-type B virus activity measurement

In order to investigate the effect of the TCA and MCA of the present invention on the anti-hepatitis virus activity against hepatitis B virus, animal cells derived from human liver tissue (HepG2.2.2.15) were treated with TCA and MCA of the present invention by concentration. Afterwards, the inhibitory effect of e antigen and s antigen production was examined.

<1-1> 2.2.15. Cell culture

In order to investigate the activity, the gene of hepatitis B virus is inserted into HepG2 cells (human hepatoblastoma cells) derived from human liver tissue, thereby discharging the hepatitis B virus e antigen (HBeAg) into the medium. . Cell line (Keui Ueda et al., Virology 169, 213-21, 1989) was used. 2.2.15. Cells were treated with MEM medium (Gibco, USA) supplemented with 4 μg / ml gentamicin (Gentamicin; Sigma, USA) and 10% heat treated fetal bovine serum (FBS; Gibco) in a 10 cm diameter petri dish. In cultivation using, 37 ° C, 5% CO ₂ incubator was grown to form a cell monolayer and trypsin treatment at 3-4 days intervals were passaged.

<1-2> Measurement of the inhibitory effect of hepatitis B virus e antigen production

In the case of hepatitis B virus e antigen, enzyme immunoassay was performed using a reagent for diagnosing hepatitis B antigen (Enzygnost HBeAg monoclonal; Behring). 2.2.15 cells cultured in Experimental Example <1-1> were added to a 24-well plate with 100 μl of a MEM medium culture medium without FBS at a concentration of 2 × 10 ⁴ per well and 37 ° C., 5% CO. Incubated for 24 hours in ₂ incubator and then treated with TCA and MCA of the present invention by concentration. After 48 hours of treatment with TCA and MCA, only the culture was taken and the culture was centrifuged at 1,500 rpm for 5 minutes to remove the cell isolate. Only the supernatant obtained at this time was dispensed to a diagnostic reagent plate coated with monoclonal antibody (HBeAb) against hepatitis B antigen (HBeAg) by a predetermined amount, and then left in a 37 ° C. incubator for 1 hour. The diagnostic reagent plate was washed three times with phosphate buffer solution, and then a predetermined amount of HBeAb labeled peroxidase was dispensed into each well and left in a 37 ° C. incubator for 1 hour to allow secondary binding with HBeAg. It was done. The excess unbound antibody was washed three times with phosphate buffer solution and the color reaction was induced by dispensing a certain amount of the solution containing TMB (tetramethyl benzidine dihydrochloride) substrate which causes color reaction by peroxidase. The diagnostic reagent plate that completed the reaction was evaluated for the change of e antigen by reading the degree of absorption at 450 nm.

By the above method, the degree of reducing the e antigen of the hepatitis B virus of the TCA and MCA of the present invention was measured at various concentrations, and the hepatitis antigen reduction ability of the TCA and MCA of the present invention was shown.

As a result, as shown in Figure 1, it was found that TCA exhibits anti-hepatitis virus activity by lowering the production of e antigen secreted to the outside of the cell even at a concentration of 20 μM, MCA is secreted to the outside of the cell even at a concentration of 20 μM It was found that the anti-hepatitis virus activity was shown by decreasing the production of e antigen (FIG. 1). At this time, the drug concentration (IC ₅₀ ) that prevents 50% of e antigen generation in HepG2 2.2.15 cells was determined and shown in Table 1 below (Table 1).

division IC ₅₀ (Μm) Trans-cinnamaldehyde (TCA) 15 2-methoxycinnamaldehyde (MCA) 17

<1-3> Analysis of effects on hepatitis B virus s antigen

The present inventors examined whether the TCA and MCA of the present invention affect the production of hepatitis B virus S antigen in the same manner as in Experimental Example <1-2>.

As a result, as shown in Fig. 2, TCA and MCA of the present invention showed the effect of inhibiting the production of hepatitis B virus S antigen in cells and the production of S antigen secreted outside the cell (Fig. 2).

< Experimental Example  2> Hepatitis B Virus DNA Replication Measurement

Infected with hepatitis B virus 2.2.15. Natural Plant Separation in Cell Lines Treatment of the formulas TCA and MCA by concentration, and after 9 days of drug treatment, hepatitis B virus was isolated from the cells to proceed with Southern blot (southern blot). Specifically, α- ³² P-dCTP labeled probe and the membrane were reacted for 16 hours. After washing with 2 x SSC / 0.1% SDS, 1 X SSC / 0.1% SDS, 0.5 X SSC / 0.1% SDS, the amount of bound radiation was measured by exposing to an X-ray film.

As a result, as shown in Figure 3, the natural plant separation formula TCA and MCA of the present invention was confirmed to reduce the DNA replication of hepatitis B virus (Fig. 3 and 4).

< Experimental Example  3> Cytotoxicity test

Known methods (Korba, BE et al., Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication, Antiviral) to confirm the toxicity to cells by verifying the activity of TCA and MCA of the present invention research, 19, 55-70, 1992) and HepG2 cells of TCA and MCA of the present invention and 2.2.15. The cytotoxicity expressed against the cells was investigated.

Specifically, 100 μl of 2 × 10 ⁴ cells per well was added to a 24-well plate, incubated for 24 hours in a 37 ° C. incubator, and then treated with various concentrations of TCA and MCA samples of the present invention for 3 days. Afterwards, the culture solution was removed and 20 μl of staining solution (tetrazolium compound, inner salt and phenazine ethosulfate) was dispensed and placed in the incubator for 1 hour. Wash twice with buffer to remove excess staining solution and add 20 μl of fixation solution (50% ethanol, 1% cold acetic acid) and stir for 30 min. The concentration of the extract, which measured in meters and caused 50% decrease in absorbance compared to the absorbance of cells without TCA and MCA, was determined as CC ₅₀ (50% cytotoxic concentration). The results are shown in Table 2 below.

division cc ₅₀ (Μm) Trans-cinnamaldehyde (TCA) 75.7 2-methoxycinnamaldehyde (MCA) 77.1

< Experimental Example  4> Mixed extract of the present invention Rat  Acute Toxicity Experiment

In order to determine the acute toxicity of TCA and MCA of the present invention, the following experiment was performed.

Specifically, acute toxicity test was performed using 6 week-old SPF rats. TCA and MCA of the present invention were each suspended in 0.5% methylcellulose solution and administered once orally at a dose of 5 g / kg / 15 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, all animals treated with TCA and MCA of the present invention had no significant clinical symptoms or dead animals, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, autopsy findings, and the like. TCA described by formula (1) and MCA described by formula (2) of the present invention showed no change in toxicity up to 5 g / kg in rats and the minimum lethal dose (LD ₅₀ ) for oral administration was determined to be a safe substance of 5 g / kg or more.

< Manufacturing example  1>: Preparation of pharmaceutical preparation

<1-1> Sanje  Produce

2 g of extract of Example <1-1>

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of extract of Example <1-1>

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of extract of Example <1-2>

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of extract of Example <1-2>

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of extract of Example <1-3>

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Formulation example  2>: Manufacture of food

<2-1> Preparation of Cooking Seasoning

20 to 95 parts by weight of the ethyl acetate fraction of <Example 2> of the present invention was prepared for cooking spices for health promotion.

<2-2> Preparation of Flour Food

0.5 to 5.0 parts by weight of the ethyl acetate fraction of <Example 2> of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare foods for health promotion.

<2-3> soup  And preparation of gravy

0.1 ~ 5.0 parts by weight of the extract of Example <1-1> of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles, and broths.

<2-4> ground Beef  Produce

10 parts by weight of the extract of Example <1-1> of the present invention was added to ground beef to prepare a ground beef for health promotion.

<2-5> Production of Dairy Products

5 to 10 parts by weight of the extract of Example <1-2> of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-6> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The extract of Example <1-2> of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

It was prepared by combining the dry powder of the grains, seeds and cinnamon extract prepared in the following ratio.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

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

Example <1-2> dry powder of the extract (3 parts by weight),

(0.5 part by weight),

(0.5 parts by weight)

< Formulation example  3>: Manufacture of beverage

<3-1> Health drink  Produce

       1000 mg of extract of Example <1-2>

       Citric acid 1000 mg

       100 g oligosaccharides

       Plum concentrate 2 g

       1 g of taurine

       Purified water was added to a total of 900 ml

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

<3-2> Vegetable juice  Produce

5 g of the extract of Example <1-2> of the present invention was added to 1,000 ml of tomato or carrot juice to prepare a vegetable juice for health promotion.

<3-3> Fruit juice  Produce

1 g of the extract of Example <1-2> of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

Claims (7)

A pharmaceutical composition for preventing and treating infections of type B infection containing cinnamomum extract, which is extracted using water, alcohol or a mixture thereof as a solvent, as an active ingredient.
The method of claim 1, wherein the cinnamon extract is water, C ₁ A pharmaceutical composition for preventing and treating type B infections, which is extracted with a lower alcohol of C ₂ or a mixed solvent thereof.
Suspension by adding water to the cinnamon ethanol extract suspended, ethyl acetate acetate obtained by adding the ethyl acetate fraction as an active ingredient pharmaceutical composition for preventing and treating infection.
A pharmaceutical composition for preventing and treating type B infection, comprising butanol fraction obtained by adding butanol to the water layer formed by adding water to a cinnamon ethanol extract and then suspending it by adding ethyl acetate.
Trans-cinnamaldehyde represented by the following formula (1), 2-methoxycinnamaldehyde (2-methoxycinnamaldehyde) represented by the following formula (2) or a pharmaceutically acceptable salt thereof type B containing Pharmaceutical compositions for the prevention and treatment of infections:

[Formula 1]

; And
(2)

.
A health food composition for preventing and improving type B infection, which contains cinnamon or an extract, which is extracted using water, ethanol or a mixture thereof as a solvent, as an active ingredient.
Trans-cinnamaldehyde represented by the following formula (1), 2-methoxycinnamaldehyde (2-methoxycinnamaldehyde) represented by the following formula (2) or a pharmaceutically acceptable salt thereof type B containing Health food compositions for preventing and improving infections:

[Formula 1]

; And
(2)

.

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