KR20030083252A - Compositions Comprising Extracts of Chaga Fungus And The Use Thereof - Google Patents

Abstract

PURPOSE: Provided is a compositions comprising an extract of Inonotus Obliquus as an active ingredient and the use thereof as an anticancer agent and an antiinflammatory agent. CONSTITUTION: An extract of Inonotus Obliquus inhibits cyclooxygenase-2 and inducible nitric oxide synthase. A composition characteristically contains an effective amount of the extract, and is used as an anticancer agent and an antiinflammatory agent.

Description

Composition Comprising Extracts of Chaga Fungus And The Use Thereof}

The present invention relates to novel anti-inflammatory and cancer prevention agents. More specifically, it is derived from natural black birch mushrooms and relates to extracts that inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). .

The anti-inflammatory effects of nonsteroidal antiinflammatory drugs (NSAIDs) such as aspirin, ibuprofen, acetaminophen, etc. are mediated by inhibiting COX enzyme activity (Vane et al., Annu. Rev. Pharmacol. Toxicol. , 38, 97, 1998). COX is a major enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157, 1996).

COX has two kinds of isomers, COX-1 and COX-2, in vivo. COX-1 is expressed in steady state and is involved in protecting the gastrointestinal tract or regulating kidney function. COX-2 is transiently and rapidly expressed in cells by mitogens and cytokines during inflammation and other immune responses. NSAIDs exhibit side effects due to the lack of selectivity for the COX-2 enzyme and unnecessary inhibition of COX-1 (Masferrer et al., Proc. Natl. Acad. Sci. USA, 91, 3228, 1994; Seibert et al., Proc. Natl. Acad. Sci. USA, 91, 12013, 1994). Therefore, the current research is focused on finding substances that inhibit COX-2 more selectively in order to reduce the side effects of existing non-steroidal anti-inflammatory drugs.

First-generation COX-2 inhibitors are drugs such as nimesulide, etodolac, and meloxicam, which have strong anti-inflammatory effects in experimental animal models and at the same time have minimal side effects on the stomach. Centrally developed (Bennett et al., Drugs, 46, 1, 1993). Second generation COX-2 inhibitors have been developed through structural analysis of enzymes, such as celecoxib (celebecox), L-745,337 and the like. These second generation inhibitors have a much higher selectivity for COX-2 compared to first generation drugs, and are 100 times more selective than COX-1 (Griswold et al., Med. Res. Rev., 16, 181, 1996).

Celcoxib, which was launched in the market in the second half of 1999, has been outstanding in the pharmaceutical industry with sales of more than about $ 500 million so far, and has proven to be excellent in terms of side effects and efficacy. Recently, molecular models with strong affinity for binding sites have been designed based on accurate three-dimensional structural analysis of COX-2 proteins. The third generation COX-2 inhibitor is expected to be a substance that inhibits COX-2 gene expression itself by oxidative stress, inflammatory response, ischemia and the like.

COX-2 inhibitors, on the other hand, are expected to play an important role in the prevention of cancer as well as in the treatment of inflammation. Reportedly, the induction of COX-2 in cancer cells and malignant tumor tissues resulted in much higher levels of prostaglandin compared to normal cells, and the increase in the amount of prostaglandin in tumor tissues is largely due to COX-2 expression. Increased by (Kargman et al. Cancer Res., 55, 2556, 1995; Ristimaki et al. Cancer Res., 57, 1276, 1997). Prostaglandins, such as prostaglandin E2 (PGE2), not only promote blood vessel formation, promote cell proliferation, but also inhibit immune capacity, so their overproduction creates a good environment for cancer cell growth. Moreover, excessive expression of COX-2 inhibits apoptosis and increases the metastatic capacity of cancer cells.

In this regard, recent studies have shown a 40-50% reduction in the risk of colon cancer in people who have used NSAIDs for a long time, and colorectal polyposis in patients with familial adenomatous polyposis (FAP). It has been observed that the stage of development of this colon cancer is prevented by NSAIDs-based drugs (Peleg et al., Arch. Intern. Med., 154, 394, 1994). In addition, COX-2 expression and activity were always preceded by APC mice, a colon cancer experimental model, in the stage of modification and loss of APC gene, which is a stage of colon cancer (Prescott et al., Cell, 87, 783, 1996). In addition, various studies have shown that COX-2 significantly affects the developmental stage of colon cancer. COX-2 protein is expressed more in human stomach and breast cancer tissues than in normal tissues, and COX inhibitors are expressed in human breast cancer cells. Growth was inhibited and the incidence of cancer induced in animal models was significantly reduced by COX-2 inhibitors (Noguchi et al., Fatty Acids, 53, 325, 1995; Thompson et al., Cancer Res., 57, 267, 1997). . Therefore, it is expected that cancer can be prevented as a selective inhibitor of COX-2 in gastric and breast cancer as well as colon cancer. Therefore, COX-2 selective inhibitors are not only the main targets for the development of new anti-inflammatory drugs, but also the potential for cancer prevention.

Along with COX, attention has been paid not only to inflammation but also to cancer prevention effects, as well as inhibitors of inducible nitric oxide synthase (iNOS). NOS also has a continuous form (institute form) and inducible form, and the overproduction of nitric oxide (NO) by iNOS may cause pathological vasodilation, cytotoxicity, and tissue damage. Exhibits harmful effects. Recently, NO has been shown to cause inflammatory reactions such as increased vascular permeability, edema, or promote COX activity to promote biosynthesis of inflammatory mediators such as prostaglandins, which intensify the inflammatory response, and significantly increase iNOS in various cancer tissues. (Aeberhard et al., Biochem. Biophys. Res. Commun., 208, 1053, 1995; Barnes et al., Immunology Today, 16, 128, 1995; McCartney et al., J. Exp. Med., 178, 749, 1993).

Therefore, the present inventors have studied the anti-inflammatory and cancer prevention agents of the herbal medicines that are widely used in herbal medicine, and confirmed that the hot water extract isolated from the black birch mushroom showed excellent anti-inflammatory and cancer prevention effects and completed the present invention. Reached.

The present invention provides a pharmaceutical composition for treating inflammation, comprising a hydrothermal extract isolated from an effective amount of black birch mushroom, and a method for treating inflammation using the composition.

The present invention provides a cancer prevention or cancer prevention pharmaceutical composition comprising a hot water extract isolated from an effective amount of black birch mushroom, and a method for preventing or treating cancer by administering the pharmaceutical composition to a patient.

The present invention relates to a pharmaceutical composition containing a hot water extract of black birch mushroom (Inonotus Obliquus) as an active ingredient having a prophylactic treatment of cancer and a treatment of inflammation-related diseases. Specifically, the present invention provides a pharmaceutical composition for treating inflammation, comprising a hydrothermal extract isolated from an effective amount of black birch mushroom, and a method for treating inflammation using the composition.

Black birch mushroom refers to a mushroom that grows by growing on birch trees. Black birch mushroom is a mushroom that grows in climatically growing birch trees in the north Korea, mainly 45 degrees north latitude, and is found in Russia, Finland, Canada, etc. have.

Black birch mushrooms have long been used as a substitute for tea in the private sector, especially in Russia, but their efficacy has only recently been revealed through scientific studies. However, despite the ongoing study of extracts isolated from black birch mushrooms, no potents for COX-2 and iNOS have been isolated from this plant. As a result of intensive studies by the inventors, it was found that the extract of the black birch mushroom has a significant effect on the prophylactic treatment of cancer and the healing of inflammation related diseases.

Inflammation and tumor development are closely related. As each stage of the tumor development progresses, the enzymes involved in the inflammatory response, COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) are increased. (Kitayama et al., Carcinogenesis , 20, 2305-2310, 1999; Takahashi et al., Cancer Res ., 57, 1233, 1997). Therefore, COX-2 inhibitors are expected to play an important role not only in the treatment of inflammation but also in the prevention of cancer production.

NOS also has a constitutive form and an inducible form. The overproduction of nitric oxide (NO) by iNOS is associated with pathological vasodilation, cytotoxicity, tissue damage, etc. It is related. Recent studies have shown that NO can augment inflammatory responses by increasing vascular permeability and causing inflammatory reactions such as edema, or by promoting COX activity to promote biosynthesis of inflammatory mediators such as prostaglandins. INOS activity is greatly increased in various cancer tissues. The inventors have demonstrated that the extract of the black birch mushroom inhibits the activity of COX-2 and the activity of iNOS. Extracts of black birch mushrooms showed inhibitory activity against COX-2 in vitro in vitro and showed a high inhibitory activity with IC ₅₀ of 1.6 μg / ml (Table 2). IC ₅₀ showed significant efficacy as 4.6 μg / ml (Table 3).

Accordingly, the present invention provides an anti-inflammatory composition comprising an extract of black birch mushroom in an amount sufficient to treat inflammation, and a method for treating inflammation by administering the same. The composition of the present invention may comprise a pharmaceutically acceptable carrier. Carriers include solvents, dispersion media, absorption retardants, and the like commonly used in the pharmaceutical art. The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Thus, the compositions of the present invention can be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. can do. Preferred formulations are injections. Dosage is determined in consideration of the skill of the art depending on the type and extent of the disease, age, sex, etc. of the patient.

Furthermore, the present invention provides an anti-cancer to anticancer composition comprising an effective amount of black birch mushroom extract and a method for preventing cancer by administering the same. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Carriers include solvents, dispersion media, absorption retardants, and the like commonly used in the pharmaceutical art. The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Thus, the compositions of the present invention can be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. can do. Preferred formulations are injections. Dosage is determined in consideration of the skill of the art depending on the type and extent of the disease, age, sex, etc. of the patient.

The present invention provides a cancer prevention or anti-inflammatory food composition containing the extract of the black birch mushroom. The food used as a base material is not specifically limited, Various foods, such as drinks, confectionery, bread, noodles, seasonings, such as water, a soft drink, or fruit drink, can be based on. The food of this invention is easily made by adding the process of adding the composition of this invention mentioned above to the manufacturing process of the base food, or by adding the process of adding the composition of this invention mentioned above after manufacture of the food of base materials. You can get At this time, you may add a taste and odor correction agent as needed. The content of the above-mentioned composition in the food of the present invention varies depending on the type and preference of the intended food, but the black birch mushroom may be used to fully utilize the preventive and health treatment effects of the black birch mushroom. The plant extract containing the extract is preferably at least 0.1% by weight. Thus, the food of this invention obtained is a food which can fully utilize the health prevention effect and health treatment effect which the black birch mushroom extract originally had because it contains the composition of this invention.

The present invention will be described in more detail with reference to the following Examples. These examples are only for illustrating the present invention, and it is obvious that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1

Preparation of Samples of Black Birch Mushrooms and Preparation of Hot Water Extracts

Black birch mushrooms were collected and used near Lake Baikal in Irkutsk Autonomous Region, Siberia, Russia. The black birch mushroom used in the experiment was dried in the shade until there was no change in weight at room temperature, and then the dried sample was pulverized finely to be used as an extraction material.

The dry pulverized black birch mushroom (dry weight, 3,0 kg) was put into a 5 liter round flask, and 3.0 L of distilled water was added thereto, followed by treatment with water in a hot water at 60 ° C. for 48 hours to extract the active ingredient. The distilled water extract was filtered using filter paper (Watman 1 time, Whatman), and then the filtrate was taken and extracted twice with distilled water in the same manner. The filtrate obtained through the above process was freeze-dried and concentrated. As a result, 67 g of distilled water extract was obtained.

Example 2

Acute Toxicity Test

To examine the toxicity of the hydrothermal extract of black birch mushroom, a large amount of samples were orally administered at a time to visually observe the toxicity in experimental animals. Ten male rats (breed: ICR) weighing 20 to 30 g were treated as group 1 with oral administration of hot water extract of black birch mushrooms at 5,000 mg / kg body weight, and observed abnormal behavior. Acute toxicity was observed by measuring. The results of the acute toxicity test up to 72 hours after the hot water extract administration of black birch mushroom are shown in Table 1 below. In other words, there were no deaths and the minimum lethal dose of hot water extract of black birch mushroom was over 5,000mg / kg, and no behavioral abnormalities were observed during the observation period.

Table 1. Acute Toxicity Test Results of Hot Water Extract of Black Birch Mushrooms

Example 3

Determination of COX-2 Induced Inhibitory Activity in Vitro

COX-2 induction inhibitory activity was based on the method described in the literature (Noh et al., Journal of Pharmacy, 42, 558, 1998). Briefly, mouse macrophage RAW264.7 cells were suspended in 10% FBS-DMEM to 10 × 10 ⁵ cells / ml. Aspirin was added to the suspension to a final concentration of 250 µM, thereby irreversibly inhibiting the activity of the COX enzyme remaining in the cells. 200 μl of the cell suspension was added to each well of a 96-well cell culture plate and incubated for 2 hours at 37 ° C., 5% CO 2 to attach the cells to the well bottom. The attached cells were washed twice with PBS (phosphate buferred saline) and the cells remaining on the surface were used for the experiment.

Into each well to which cells were attached, 200 μl of 10% FBS-DMEM containing 1 μg / ml lipopolysaccharide (LPS) was added. At this time, the control group was put in the medium without LPS. Immediately after the exchange with the LPS-containing medium, the search sample was treated, and the supernatant was recovered after incubating for 16 hours at 37 ° C. and 5% CO 2. Enzyme immunoassay was used to quantify the amount of free prostaglandin-E2. The percent inhibition rate of each sample was determined based on the difference between the prostaglandin-E2 produced in the LPS-treated and non-treated groups. The results are shown in Table 2.

Table 2. COX-2 Induction Inhibitory Activities of Hot Water Extracts of Black Birch Mushrooms

Example 4

Measurement of iNOS Inhibitory Activity in Vitro

Mouse macrophages, RAW264.7 cells, were suspended at 8 × 10 ⁵ cells / ml in 10% FBS-DMEM without phenol-red. Cell suspension was added 1 ml per well of 24-well culture plate and incubated for 4 hours to attach cells to the culture plate. It was exchanged with a medium containing 1 μg / ml LPS and samples were added at the same time and incubated at 37 ° C. and 5% CO 2 for 20 hours. The supernatant was collected, 100 μl of the culture solution per well was added to a 96-well culture plate, and 150 μl of Griess reagent was added thereto, and gently shaken for 10 minutes. The absorbance was measured at 570 nm. A calibration curve was prepared using NaNO ₂ as a standard product, and the amount of nitric oxide (NO) produced in the culture solution treated with each sample was determined. The percentage inhibition of each sample was determined based on the difference between the amount of nitric oxide (NO) produced in the LPS-treated and untreated groups, based on 100% activity. The results are shown in Table 3.

Table 3. Effect of iNOS Inhibitory Activity of Hot Water Extract of Black Birch Mushrooms

The hydrothermal extract of the black birch mushroom of the present invention can reduce or alleviate inflammation and inhibit the production of cancer by inhibiting COX-2 and iNOS activity. Therefore, the composition of the present invention comprising an effective amount of the hot water extract of black birch mushroom can be usefully used as a cancer prevention composition to anti-inflammatory composition.

Claims (4)

Cancer preventive composition comprising hot water extract of black birch mushroom Cancer preventive food composition comprising a hydrothermal extract of black birch mushroom Anti-inflammatory composition comprising a hydrothermal extract of black birch mushroom Inflammation-reducing foods comprising hot water extract of black birch mushroom