KR20030030312A - Composition for radio-sensitizer - Google Patents

Abstract

PURPOSE: A composition of a radiation sensitizing agent which contains an extract of Fomitella fraxinea(Bull.: Fr.) Ryv. as an active ingredient is provided to minimize adverse effect of radiation and to allow the composition to exhibit excellent sensitizing effect on radiation. CONSTITUTION: The composition of a radiation sensitizing agent contains 0.01 to 50% by weight of an extract of Fomitella fraxinea obtained by extraction with a solvent selected from ethanol, methanol, butanol and propanol. For an example, Fomitella fraxinea is dried and ground. 50g of Fomitella fraxinea powder is extracted in 10L aqueous methanol solution, followed by reaction at 40deg.C for 3 days, filtration and freeze- drying to produce 150g of Fomitella fraxinea extract. The composition has an LD50 value of 400mg/1kg(body weight).

Description

Radiation sensitizer composition {COMPOSITION FOR RADIO-SENSITIZER}

The present invention relates to a radiation sensitizer composition, and more particularly to a radiation sensitizer composition excellent in radiation sensitizing effect.

Tumors can be classified into malignant tumors and benign tumors. Cancer, a kind of malignant tumor, is one of the major diseases to be solved by modern medicine. In Korea, the incidence of cancer is increasing year by year, and it is reported that it is the highest cause of death. Accordingly, the development of multiple treatment methods for cancer treatment is required.

At present, in the treatment of malignant tumors, local therapies such as surgery, radiation therapy, and systemic therapy such as chemotherapy and immunotherapy are used. These therapies are rarely used as monotherapy, and are carried out in various combinations of therapies.

In the treatment of tumors, radiation therapy is a therapy that can destroy tumor tissue without causing pain by irradiating the tumor tissue site of the patient, and is not used alone but in combination with various chemotherapeutic agents. In particular, when combined with radiation therapy and chemotherapy, the therapeutic effect is higher than when treated with chemotherapeutic agents alone, and the treatment side effects may be minimized when used at a much lower concentration than when treated with chemotherapeutic agents alone. In recent years, studies have shown that esophageal cancer and some limited carcinomas have improved survival by an average of 5 years or more, using chemotherapy and radiation therapy prior to surgery (Arch. Surg. 2001, 136: 737-743).

As such, a chemotherapeutic agent used in combination with radiation therapy is called a radio-sensitizer as a drug that increases the sensitivity of tumors to radiation, and research on radiation sensitizers is actively underway. Radiation sensitizers developed so far include taxol, 5-fluorouracil, cisplatin, and the like.

However, when treating such radiation sensitizers alone or in combination, there is a side effect that can damage normal cells in addition to cancer cells, radiation sensitizers using natural products that are considered safe for the human body has not yet been developed.

The present invention has been made to solve the above problems, and an object thereof is to provide a radiation sensitizer composition having excellent radiation sensitization effect while minimizing side effects.

Another object of the present invention to provide a radiation sensitizer composition excellent in the safety to the human body using natural products.

In order to achieve the above object, the present invention provides a radiation sensitizer composition comprising a longevity mushroom extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, to develop a radiation sensitizer excellent anti-cancer effect in the native flora and fauna, research and research on traditional herbal ancient books and folk remedies, the antitumor promoting effect on radiation therapy in longevity mushroom It has been found and completed the present invention.

The radiation sensitizer composition of the present invention comprises an extract of longevity mushroom having excellent antitumor enhancement effect.

Longevity mushroom ( Fomitella fraxinea ) used in the present invention is a mushroom belonging to the Democratic Pleurotus eryngii family, also called in the country black sash butterfly , acacia ganoderma , longevity mushroom and the like. Longevity mushrooms are generally known as medicinal mushrooms that are effective against adult diseases such as high blood pressure and diabetes.

Longevity mushroom extract used in the present invention can be obtained by a conventional extraction method, the representative method will be described in detail as follows.

After the extraction solvent is added to the longevity mushroom, it is left at room temperature or warmed for a certain time to extract the active ingredient. At this time, the longevity mushroom may be naturally dried or forcedly dried using a machine and then finely used as a grinder to increase extraction efficiency.

In the extraction process, the amount of the extraction solvent is suitable if the amount of the longevity mushroom can be sufficiently extracted, it can be adjusted according to the dry state of the longevity mushroom. For example, when using less dried longevity mushrooms, 1 L of extraction solvent is suitable. As the extraction solvent, a polar solvent can be used, and representative examples thereof include one or more selected from the group consisting of ethanol, methanol, butanol, and propanol. When 70% methanol is used, a large amount of the extract containing the radiation sensitizing component can be ensured, which is more preferable.

Subsequently, the extract is filtered to remove the residue, and the supernatant may be concentrated under reduced pressure or spray dried to prepare a longevity mushroom extract powder.

Longevity mushroom extract obtained by the above method is excellent as an anti-tumor enhancing effect on radiation therapy, it is useful as an active ingredient in a radiation sensitizer composition.

In the sensitizer composition of the present invention comprising a longevity mushroom extract, the content of the longevity mushroom extract is preferably 0.01 to 50% by weight based on the total radiation sensitizer composition. When the content of the longevity mushroom extract is less than 0.01% by weight, the radiation sensitization effect may hardly appear. When the content of the longevity mushroom extract is greater than 50% by weight, the increase of the radiation sensitization effect is not increased by increasing the content of the longevity mushroom extract. The sensitizer composition may include additives such as purified water, excipients, stabilizers or preservatives, in addition to the longevity mushroom extract, the additives have no radiation sensitizing effect.

The radiation sensitizer composition containing the longevity mushroom extract of the present invention may be prepared in various formulations such as oral, injectable, topical, or applied as tablets, capsules, infusions, granules, or pills.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, an Example is for illustrating this invention and this invention is not limited only to these.

Example 1 Preparation of a Radiation Sensitizer Composition

Longevity mushrooms were forcibly dried in a dryer and chopped with a grinder to obtain a pulverized product of longevity mushrooms. 500 g of the longevity mushroom pulverized product was mixed with 10 L of an aqueous 70% methanol solution as an extraction solvent, and heated at 40 ° C. for 3 days. The mixed solution was filtered to separate the supernatant from the residue. The supernatant was concentrated under reduced pressure and lyophilized to obtain 150 g of longevity mushroom extract. The longevity mushroom extract extracted with methanol as an extraction solvent was a dark brown powder and had excellent solubility.

Experimental Example 1 Measurement of Radiation Sensitization Effect

The longevity mushroom extract obtained by the method of Example 1 was used to measure the radiosensitizing effect on cancer cells of humans and mice. At this time, human cancer cells were tested with skin cancer cells KB, lung cancer cells SM-MES-1 and A549, and uterine cancer cells HeLa, mice were tested with S180 (sarcoma).

10% fetal calf serum was added to each of the human and mouse cancer cells in the logarithmic phase, and the cancer cells were dispensed in MEM medium to the number of 5 × 10 ³ cells per well of a 4-well plate, 37 ° C., 5% Pre-cultured for 24 hours in a carbon dioxide thermohygrostat. After culturing for 48 hours by adding 150 g of the longevity mushroom extract of Example 1 to the pre-cultured cancer cells, using a radiation irradiator (Gammacell 3000 Elan, Nordion, USA, Cs 137) to the cancer cells to which the longevity mushroom extract was added 3.6 10 Gy was investigated by Gy / min. The cancer cells were cultured for 4 days by replacing with fresh medium immediately after irradiation. The inhibition of cancer cell proliferation of longevity mushroom extracts was determined using the SRB assay (Sulforhodamine B assay) (J. Natl. Cancer Inst. 1990, 82: 1107-1112; Anti-Cancer Drugs 1995, 6: 115-123). After the incubation was completed, according to the SRB method, the culture solution of each well was removed, and the remaining cells were treated with 10% trichloroacetic acid (TCA; trichloroacetic acid) for 1 hour to fix the cells. Trichloroacetic acid was then removed, washed with water and dried at room temperature. To this, a dye solution in which 0.4% SRB was dissolved in 1% acetic acid solution was added, and the cells were stained by standing at room temperature for 30 minutes, and then washed with 1% acetic acid solution to remove all SRBs not bound to the cultured cells. The cells stained with SRB were eluted with the cancer cells in combination with Tris buffer, and the absorbance of each well was measured at a wavelength of 520 nm.

In addition, cancer cells of humans and mice not added with the longevity mushroom extract of Example 1 were treated with physiological saline instead of the longevity mushroom extract of Example 1 as a longevity mushroom extract untreated control (radiation alone treatment group). Was carried out in the same manner as the above method. The untreated control group was carried out in the same manner as above except that 10 gray gray were not irradiated. To calculate the growth inhibition rate of cancer cells according to the formula 1 shown in Table 1 below.

[Calculation 1]

(Wherein

T: absorbance of radiation and longevity mushroom extracts alone or in combination

C: absorbance of radiation and longevity mushroom extract untreated group)

Classification KB SK-MES-1 A549 HeLa S180 Untreated longevity mushroom extract (radiation alone) 9% 12% 20% 12% 18% No radiation (only longevity mushroom extract) 32% 11% 28% 37% 30% Longevity Mushroom Extract and Radiation Treatment 80% 32% 56% 76% 69%

Table 1 shows the proliferation inhibition rate for each cancer cell. As shown in Table 1, it can be seen that cancer cells treated with longevity mushroom extract have a greater effect on inhibiting cancer cell proliferation than cancer cells treated only with radiation. In addition, the proliferation inhibition rate of cancer cells treated with longevity mushroom extract and irradiated with radiation was up to 8 times higher than that of cancer cells not treated with longevity mushroom extract.

As in the above results, longevity mushroom extract itself can be seen that not only has an anti-tumor effect, but also shows a synergistic effect on tumor treatment using radiation.

Experimental Example 2 Safety Test of Longevity Mushroom Extract

In order to confirm the safety of the longevity mushroom extract of Example 1, the LD ₅₀ (amount that kills 50% of experimental animals), which is an indicator of drug toxicity, was determined. In order to obtain the LD ₅₀ represented by the longevity mushroom extract of Example 1, cytotoxicity experiments were performed on normal cells in vitro and on mice in vitro.

(1) in vitro cytotoxicity measurement

Renal cells MDBK of the young calf and fibroblast cells of human lung tissues were isolated and primary cells cultured in primary culture were used as toxicology cells. 0.1 mg, 1 mg, and 10 mg of the longevity mushroom extract of Example 1 were treated with physiological saline, respectively, to prepare 1 ml of sample. The sample was administered to fibroblast cells of MDBK and human lung tissue, and the degree of inhibition of cell proliferation was measured using the same method as the SRB assay of Example 1, and the cells were treated in the same manner as in Formula 1 of Experimental Example 1. The inhibition of proliferation was calculated and shown in Table 2 below.

Cell types Sample processing concentration (mg / mL) 10 One 0.1 Kidney Calf Cells (MDBK) 29% 10% 3% Fibroblasts of Human Lung Tissue 19% 12% 0 %

In general, in the SRB assay cytotoxicity measurement, if the inhibition rate of proliferation of cells is 50% or more, it is determined that the treatment material is toxic at the concentration treated to the cells, and thus the inhibition rate of cell proliferation shown in Table 2 is within the range of 0 to 30%. As shown, it can be confirmed that the longevity mushroom extract of Example 1 is safe for human and animal cells.

(2) in vitro cytotoxicity test

Using the longevity mushroom extract of Example 1 as a sample, for each of 10 normal ICR mice (23 ± 2 g) females and males in each group, a sample of 4 mg to 400 mg per 1 kg of body weight was added to 1 ml of physiological saline, respectively. The cells were treated orally by concentration for 30 days. The group to which the sample without the longevity mushroom extract of Example 1 was administered was performed in the same manner as the treated group.

Sample processing concentration (mg / kg) % Mortality LD ₅₀ (mg / kg) 0 0 400mg / kg or more 4 0 40 0 400 0

Table 3 shows the LD ₅₀ for the longevity mushroom extract of Example 1. As shown in Table 3, the LD ₅₀ value of the longevity mushroom extract of Example 1 was constant regardless of the concentration of the sample.

In this experiment, it is difficult to prepare at a concentration of 400 mg / kg or more due to the solubility characteristics of the sample, and thus it cannot be administered to animals at a concentration of 400 mg / kg or more. Accordingly, the LD ₅₀ value is 400 mg or more per kg of body weight, it can be confirmed that it can be safely administered to the human body.

In addition, no clinical symptoms such as abnormal behavior of the experimental animals were observed during the experiment using the longevity mushroom extract. At 30 days after the administration of the sample, there was no mortality in the experimental animals, and the weight change of the treated group was not significantly different from the weight change of the control group. No unusual abnormalities of major long term were observed.

In the above observations, the average feed intake per group and the change in the average negative amount per group were not recognized as significant differences between the groups administered to both the female and the male.

From the above results, it can be seen that the longevity mushroom extract of Example 1 is safe for humans.

The pharmaceutical composition comprising the longevity mushroom extract of the present invention can minimize side effects to radiation, and excellent radiation sensitizing effect.

Claims (3)

Radiation sensitizer composition comprising a longevity mushroom extract. The radiation sensitizer composition of claim 1, wherein the radiation sensitizer composition comprises a longevity mushroom extract in an amount of 0.01 to 50% by weight. The radiation sensitizer composition of claim 1, wherein the longevity mushroom extract is obtained by extracting a longevity mushroom with a solvent selected from one or more selected from the group consisting of ethanol, methanol, butanol, and propanol.