KR100625102B1 - A Pharmaceutical Composition Comprising Bamboo Leaf Extract - Google Patents

Abstract

The present invention relates to a bamboo leaf extract obtained by treating organic solvents and activated carbon from bamboo leaves, a method for producing an electrical extract, a promyelocytic leukemia therapeutic agent and an autoimmune therapeutic agent comprising the electrical extract as an active ingredient. Bamboo leaf extract of the present invention is prepared by extracting the bamboo leaves with a mixed solvent of water and an organic solvent, and then treated with activated carbon to obtain a fraction that is adsorbed on the activated carbon, and again extracted with a mixed solvent of water and an organic solvent. Bamboo leaf extract of the present invention can suppress the induction of promyelocytic leukemia, and can inhibit the induction of autoimmune diseases by overexpression of IL-12 p40 gene, it can be widely used in the treatment of various immune diseases will be.

Bamboo leaf, leukemia cure, autoimmune cure

Description

A pharmaceutical composition comprising bamboo leaf extract             

1 is a graph showing the correlation between the addition of bamboo leaf extract and the differentiation of cells.

2A is a graph showing the expression level of CD11b expressed on the surface of differentiated cells in the state in which nothing is added.

Figure 2b is a graph showing the expression level of CD11b expressed on the surface of the cell in the state of adding only bamboo leaf extract.

2C is a graph showing the expression level of CD11b expressed on the surface of cells in the state in which only 1,25-dihydroxy vitamin D ₃ is added.

FIG. 2D is a graph showing the expression level of CD11b expressed on the surface of cells in the state in which bamboo leaf extract and 1,25-dihydroxy vitamin D ₃ are simultaneously added.

2E is a graph showing the expression level of CD11b expressed on the surface of cells in the state in which only ATRA is added.

Figure 2f is a graph showing the expression level of CD11b expressed on the surface of the cell in the state of adding the bamboo leaf extract and ATRA at the same time.

Figure 3 is a graph showing the change in the expression level of IL-12, depending on the amount of bamboo leaf extract added.

Figure 4 is a graph showing the change in the expression level of IL-12 p40 mRNA, depending on the addition of bamboo leaf extract.

The present invention relates to a bamboo leaf extract and a pharmaceutical composition comprising the same. More specifically, the present invention relates to a bamboo leaf extract obtained by treating organic solvents and activated carbon from bamboo leaves, a method for producing an electrical extract, an promyelocytic leukemia therapeutic agent and an autoimmune therapeutic agent comprising the electrical extract as an active ingredient. will be.

About 280 kinds of bamboo are known around the world as flowers and plants. About 70 kinds of bamboo are grown or grown in Korea. There are 11 varieties of varieties, including daedae, wangdae (seam porridge), jongjongju (juksundae), ojuk, dough, island, haejangjuk (shinwoodae), gatdae, walnut, mountain porridge, and daedae. According to Dongbobogam, herbaceous cortex, new agricultural plant, etc., bamboo is known to be effective in treating stroke, hypertension, and bamboo leaves are used for pneumonia, bronchitis, etc. for the purpose of fever, expectoration, and refreshing.

Recent studies have shown that bamboo leaves have anti-cancer, anti-inflammatory and antibiotic effects, and lignin extracted from bamboo leaves has excellent anti-cancer effects. Methanol extract of bamboo leaves has apoptosis in cell lines. It is known to exhibit anticancer activity induced by (Kim et al., Anticancer Res., 23: 2355-2361, 2003). In addition, Korean Patent Publication No. 2001-0069130 discloses a bamboo leaf extract that can be used as a food preservative by exhibiting antimicrobial activity and a method for preparing the same, and WO 98/57545 discloses a composition for lowering cholesterol containing phytosterol extracted from bamboo shoots. US Patent No. 3,418,311 discloses a polysaccharide exhibiting anticancer activity extracted from bamboo.

The reason why the bamboo leaf extract exhibits the effects as an anticancer, anti-inflammatory and antibiotic is expected to be that the bamboo leaf extract plays a role in regulating the immune system. If bamboo leaf extract acts as an immunomodulator, it is expected to be used for the prevention and treatment of new immune system diseases other than the known effects. The relationship with is not known.

Therefore, the necessity to identify the correlation between the bamboo leaf extract and immune-related diseases has emerged constantly.

Accordingly, the present inventors have made intensive studies to investigate the correlation between the bamboo leaf extract and immune-related diseases. As a result, the bamboo leaf extract obtained by treating the organic solvent and activated carbon from the bamboo leaf differentiates undifferentiated promyeloid cells. We could reduce the production of abnormally increased interleukin-12 (IL-12) in macrophages, confirming that bamboo leaf extract could be used as a treatment for promyelocytic leukemia leukemia and autoimmune diseases. The present invention has been completed.

The first object of the present invention is to provide a bamboo leaf extract.

It is a second object of the present invention to provide a method for producing an electrical extract.

A third object of the present invention is to provide a promyelocytic leukemia therapeutic agent comprising the electric bamboo leaf extract as an active ingredient.

It is a fourth object of the present invention to provide an autoimmune disease therapeutic agent comprising an electric bamboo leaf extract as an active ingredient.

In the method for preparing the bamboo leaf extract of the present invention, the bamboo leaves are immersed in a solvent in which water and acetone are mixed in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v), and are left at room temperature for 1 to 3 months. Filtering to obtain a liquid component; The obtained liquid component was dried to obtain a solid component, dissolved in water of pH 4.0, and then filtered to obtain a liquid component and a solid component, respectively. The solid component was dissolved again in water of pH 8.0, and then filtered. Obtaining a component, and mixing the respective liquid components; The mixed liquid components were titrated to pH 8.0, stirred with activated carbon, filtered to obtain a solid, and a solvent mixed with water and acetone in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v) was added thereto. Stirring and filtering to obtain a liquid component; And drying the previously obtained liquid component to obtain a solid content. In this case, the organic solvent is not particularly limited thereto, and preferably, acetone or the like is used.

The inventors of the present invention, while studying the effect on the biological activity of the material extracted from the bamboo leaves, it has been noted that the electrical extract can affect the immune system diseases, so far known, bamboo leaves have anti-inflammatory activity This is because it was expected to be able to assist the immune system activity in the body. While studying the effects of bamboo leaf extract on various immune activities, while focusing on promyeloid leukemia caused by non-differentiation of promyelocytes, bamboo leaf extract was dedifferentiated. It has been found that they can differentiate promyeloid cells. That is, the promyeloblasts are normally differentiated, and differentiated into single cells, macrophages, granulocytes, etc., if the promyeloblasts are not differentiated, can cause promyelocytic leukemia. Thus, the present inventors prepared a bamboo leaf extract obtained by treating organic solvents and activated carbon from bamboo leaves, and treated the human promyelocytic leukemia cell line HL-60. As a result, the bamboo leaf extract alone did not induce differentiation of the electric cell line. However, 1,25-dihydroxy vitamin D ₃ (1,25- (OH) ₂ D ₃ ) or all-trans retinoic acid (ATRA) and bamboo leaf extract, which are known to induce differentiation of electric cell lines, When used in combination, it was confirmed that the differentiation-inducing effect of 1,25- (OH) ₂ D ₃ or ATRA can be amplified. That is, when the bamboo leaf extract was treated with HL-60 together with 1,25- (OH) ₂ D ₃ or ATRA, the ratio of differentiated cells increased rapidly, and the CD11b cell surface antigen expressed in the differentiated cells was increased. It can be seen that the expression level is rapidly increased. Therefore, the bamboo leaf extract of the present invention was expected to be used as a therapeutic agent for promyelocytic leukemia.

Accordingly, the therapeutic agent for promyelocytic leukemia of the present invention comprises bamboo leaf extract as an active ingredient, and includes a pharmaceutically acceptable carrier.

On the other hand, while studying other effects of the electric bamboo leaf extract, it was found that the electric bamboo leaf extract can reduce the production of abnormally increased interleukin-12 (IL-12) in macrophages. Previous studies have shown that macrophages produce a certain level of IL-12. When LPS (lipopolysaccharide) is treated on macrophages, the production of IL-6 and IL-12 increases rapidly, and excessive amounts of IL -12 is known to cause autoimmune diseases. However, when the LPS-treated macrophages treated with bamboo leaf extract, the production of IL-6 was not reduced, but only the production of IL-12 was drastically reduced, and the activity of the cells was not reduced. Therefore, the bamboo leaf extract of the present invention was expected to be used as a therapeutic agent for autoimmune diseases caused by excessive IL-12.

Accordingly, the therapeutic agent for autoimmune diseases of the present invention includes bamboo leaf extract as an active ingredient, and includes a pharmaceutically acceptable carrier.

Bamboo leaf extract contained as an active ingredient of the promyelocytic leukemia and autoimmune diseases of the present invention is a pharmaceutically acceptable binder (eg polyvinylpyrrolidone, hydroxypropyl cellulose), disintegrant (eg carboxymethyl Cellulose calcium, sodium starch glycolate), diluents (e.g. corn starch, lactose, soybean oil, crystalline cellulose, mannitol), glidants (e.g. magnesium stearate, talc), sweeteners (e.g. white sugar, fructose, sorbitol, aspartame), Stabilizers (sodium carboxymethylcellulose, alpha or beta cyclodextrins, vitamin C, citric acid, white lead), preservatives (e.g. methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate) and flavorings (e.g. ethyl vanillin, masking flavor, Menthol flavono, herbal flavor) can be prepared into pharmaceutical preparations such as tablets, capsules, soft capsules, solutions, ointments or injections.

Acute Toxicity Test of Bamboo Leaf Extract

Oral administration of bamboo leaf extract of the present invention to 6 to 7 week old non-rodent beagle dogs was performed to investigate the mortality rate within 24 hours, wherein the females were 6 to 8 kg and the males were 7 to 9 kg. 8 each was used. As a result, dead individuals do not occur up to 5g / kg, the bamboo leaf extract of the present invention is safe enough to observe acute toxicity even up to 5g per kg, safe in vivo as a treatment for promyelocytic leukemia and autoimmune diseases May be administered.

Effective amount

The dosage of the bamboo leaf extract, which is the active ingredient in the pharmaceutical composition of the present invention, may be divided into 6 to 30 mg / kg body weight once or three times per day according to the age, sex, symptoms, administration method or prevention purpose of the patient. Can be. Dosage levels for patients with specific symptoms may vary by those skilled in the art depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 : Preparation of Bamboo Leaf Extract

2 kg of bamboo leaves were immersed in 3 L of 80% (v / v) acetone, left at room temperature for 2 months, filtered to remove bamboo leaves, to obtain a liquid component, and then using a rotary evaporator. Dry completely to obtain a solid. Then, the solid was dissolved in 1 L of water (pH 4.0) and filtered to obtain a liquid component, and then the filtered solid was dissolved in 1 L of water (pH 8.0) and filtered to obtain a liquid component. The two liquid components were mixed, titrated to pH 8.0, 100 g of activated carbon was added, stirred for 1 hour, and then filtered to remove the liquid components. 0.5 L of 80% (v / v) acetone was added to the electrofiltered activated carbon, stirred at room temperature for 1 hour, and then filtered to obtain a liquid component. The obtained liquid component was dried using a rotary evaporator to obtain a solid of 10g, which was used as a bamboo leaf extract.

Example 2 Effects of Bamboo Leaf Extract on Differentiation of HL-60 Cells

Promyelocytic differentiation is normally differentiated into single cells, macrophages, granulocytes, etc. If the promyeloblasts do not differentiate, it is known that can cause leukemia, human promyelocytic leukemia cell line HL -60 is not differentiated on its own and is known to differentiate when 1,25-dihydroxy vitamin D ₃ (1,25- (OH) ₂ D ₃ ) or retinoic acid (ATRA) is treated. Thus, the HL-60 cells were treated with bamboo leaf extract alone, mixed with bamboo leaf extract and 1,25- (OH) ₂ D ₃ , or mixed with bamboo leaf extract and ATRA, followed by differentiated HL-60 cells. The ratio of 60 cells was measured using a nitroblue tetrazolium reduction assay (NBT assay), and the expression of cell surface antigen expressed on the surface of the differentiated cells was confirmed by cell fluorescence analysis.

Example 2-1 Determination of Proportion of Differentiated HL-60 Cells Using NBT Assay

The degree of differentiation of HL-60 cells was carried out by nitroblue tetrazolium reduction assay, as known (Collins et al., J. Exp. Ned., 149: 969-974, 1979): HL- 60 cell lines were cultured in RPMI 1640 medium (Gibco BRL, USA) containing 10% (v / v) fetal calf serum, and electrophoresis examples at concentrations of 0, 50, 100, 200 or 400 μg / ml in electrical medium, respectively. Bamboo leaf extract prepared in 1 was added, and 5nM 1,25- (OH) ₂ D ₃ or 50nM ATRA was added to the medium to which the bamboo leaf extract of each concentration was added, and then incubated at 37 ° C. for 72 hours. It was. Then, each cell (2 × 10 ⁵ ) obtained by centrifuging each culture was suspended in PBS in which 0.2% (w / v) nitroblue tetrazolium and 1 ng / ml plasminogen activator were dissolved. After reacting at 37 ° C. for 0.5 hour under dark conditions, the production frequency of the generated blue-black spots was confirmed to determine the ratio of differentiated cells (see FIG. 1). 1 is a graph showing the correlation between the addition of bamboo leaf extract and the differentiation of cells, (□) shows the cells treated only with bamboo leaf extract, (▨) is the bamboo leaf extract and 1,25- (OH) ₂ D ₃ shows the cells treated together, and (■) shows the cells treated with the bamboo leaf extract and ATRA. As shown in FIG. 1, HL-60 cells were hardly differentiated when treated with bamboo leaf extract alone, but differentiated HL-60 when mixed with 1,25- (OH) ₂ D ₃ or ATRA. The ratio of cells can be sharply improved, and the ratio of differentiated cells increased in proportion to the amount of bamboo extract treated.

Example 2-2 : Confirmation of Expression of Cell Surface Antigen by Cell Fluorescence Analysis

Using a known method, the expression level of CD11b, a cell surface antigen of differentiated HL-60 cells, was analyzed using cell fluorescence (see Kim NJ et al., Kor. J. Pharmacogn., 26: 368). -376, 1995).

That is, the HL-60 cell line was cultured in RPMI 1640 medium (Gibco BRL, USA) containing 10% (v / v) fetal bovine serum, and 400 μg / ml bamboo leaf extract, 5 nM 1,25- (OH ) ₂ D ₃ , 50 nM ATRA, 400 μg / ml bamboo leaf extract + 5 nM 1,25- (OH) ₂ D ₃ and 400 μg / ml bamboo leaf extract + 50 nM ATRA, respectively, were added at 37 ° C. for 72 hours Incubated. Then, single cell suspensions were obtained from each culture, washed with PBS, followed by addition of fluorescent (fluorescein isothiocyanate, FITC) labeled anti-human CD14 monoclonal antibody or anti-human CD11b monoclonal antibody (Becton Dickinson, USA). The reaction was carried out at 4 ° C. for 1 hour. Then, the cells were washed with PBS, fixed with PBS containing 1% (v / v) formalin, and then the number of cells and the degree of fluorescence developed in each cell were measured using an Epic V Flow Cytofluorograph, Coulter Electronics, USA; excitation 486-575 nm: emission 568-690 nm) and a multi-parameter data acquisition and display system (MDADS) were used for quantitative measurements. At this time, a HL-60 cell line treated with nothing was used as a control (see FIGS. 2A to 2F). Figure 2a to 2f is a graph comparing the expression level of the CD11b expressed on the surface of the differentiated cells according to the addition of bamboo leaf extract, Figure 2a is nothing added control, Figure 2b added only bamboo leaf extract Experimental group, Figure 2c is the experimental group added only 1,25- (OH) ₂ D ₃ , Figure 2d is the experimental group added simultaneously with bamboo leaf extract and 1,25- (OH) ₂ D ₃ , Figure 2e is added only ATRA Experimental group and Figure 2f represents the experimental group to which the bamboo leaf extract and ATRA added simultaneously, the part of (■) represents the state before the treatment of each experimental group, the part of (□) is the state after the treatment of each experimental group Indicates. As shown in Figure 2a to 2f, it was found that the expression level of CD11b was significantly increased when bamboo leaf extracts were added at the same time than when only 1,25- (OH) ₂ D ₃ or ATRA was added.

Example 2-3 : Confirmation of the effect of bamboo leaf extract on promyelocytic leukemia disease animals

In Examples 2-1 and 2-2, the bamboo leaf extract of the present invention was found to play a role in promoting the differentiation of undifferentiated myeloid cells. Thus, whether or not bamboo leaf extract can have a similar effect in vivo was confirmed using a myeloid leukemia disease animal.

After dissolving the bamboo leaf extract prepared in Example 1 in physiological saline, 20 μg in 120 three-week-old male promyelocytic leukemia disease model mice (AKR / N Slc, Central Experimental Animal Co., Ltd., Korea) Intraperitoneal injection with 2 μg / weight (g) / hour (day) ATRA for 7 days at a dose of body weight (g) / hour (day), followed by collecting 20 bone marrow every 7 days, Bone marrow was collected for 5 weeks). Some mice received only 2 μg / weight (g) / hour (day) ATRA as a control. The number of promyelocytic cells in the bone marrow collected electrically under the microscope was counted and converted into the number of promyelocytic cells per 1 µl of bone marrow, and compared with the increase and decrease of promyelocytic cells over time (see Table 1). At this time, the disease-controlled mouse intraperitoneally injected with physiological saline in which bamboo leaf extract was not dissolved was used as a positive control, and the normal mouse intraperitoneally injected with physiological saline in which bamboo leaf extract was not dissolved was used as a negative control.

In animals treated with bamboo leaf extract, changes in promyelocytic cells over time (unit: cell count / bone marrow) Elapsed time (days) Positive control Negative control ATRA administration group ATRA and bamboo leaf extract group 0 20 11 20 20 7 42 12 30 28 14 58 13 45 24 21 79 11 55 21 28 95 12 62 17 35 - ^* 11 80 17

*: Unable to count promyelocytic cells due to mouse death

As shown in Table 1, it was found that in the mice administered with the bamboo leaf extract in parallel, the number of promyelocytic cells decreased over time to maintain a constant level. Promyelocytic leukemia mice (positive control) had an increase in proliferating myeloid cells over time and all 20 animals died at 30 days, which could no longer count promyelocytic cells, Considering that normal mice (negative control) maintained a certain level of promyelocytic cell number, it was confirmed that bamboo leaf extract could inhibit proliferation of promyeloid cells caused by dedifferentiation of promyeloid cells.

Incorporating the results of the above Examples 2-1 to 2-3, bamboo leaf extract can reduce the number of undifferentiated promyeloid cells in vitro or in vivo, which is the non-differentiated promyeloid cells in bamboo leaf extract It was confirmed that this is because it promotes the differentiation, it can be seen that the bamboo leaf extract of the present invention can be used as a therapeutic agent for promyelocytic leukemia caused by undifferentiated promyelocytic.

Example 3 Effect of Bamboo Leaf Extract on the Production of IL-12

Macrophage cells produce a certain level of IL-12, but when LPS (lipopolysaccharide) is added, it is known to produce too much IL-12. What effect does bamboo leaf extract have on the overproduction of IL-12? It was confirmed in the protein level and mRNA level of IL-12 expressed.

Example 3-1 Measurement of Changes in IL-12 Protein Expression According to Treatment of Bamboo Leaf Extract

RAW26437 cells were incubated for 24 hours at 37 ° C., 5% (v / v) CO ₂ in RPMI1640 medium (Gibco BRL, USA) containing 10% (v / v) fetal bovine serum, and then treated with nothing. Non-negative control, positive control treated with 5 μg / ml LPS only, experimental group 1, 5 μg / ml LPS and 10 μg / ml bamboo leaf extract simultaneously treated with 5 μg / ml LPS and 1 μg / ml bamboo leaf extract simultaneously Experimental group 2, 5µg / ml LPS and 50µg / ml bamboo leaf extract treated simultaneously Experimental group 3, 5µg / ml LPS and experimental group 4 and 5µg / ml bamboo leaf extract treated simultaneously And Experimental Group 5, which were treated with 200 μg / ml bamboo leaf extract at the same time, were prepared and incubated for another 24 hours. Then, the amount of IL-12 expressed in each cell was quantified. At this time, the amount of IL-12 was determined by ELISA method using a monoclonal antibody specific for IL-12 p40 (Kang BY et al., Biochem. Pharmacol., 63: 1901-1910, 2002) (see Figure 3). Figure 3 is a graph showing the change in the expression level of IL-12, depending on the amount of bamboo leaf extract added. As shown in FIG. 3, when only LPS was treated (positive control), IL-12 was expressed up to a level of about 3.5 ng / ml, but when bamboo leaf extract was simultaneously treated, the amount of IL-12 was decreased. When bamboo leaf extract was treated in an amount of ㎍ / ㎖ or more it can be seen that the expression level of IL-12 is no longer reduced.

Example 3-2 Measurement of Changes in IL-12 mRNA Expression According to Treatment of Bamboo Leaf Extract

RAW26437 cells were incubated for 24 hours at 37 ° C., 5% (v / v) CO ₂ in RPMI1640 medium (Gibco BRL, USA) containing 10% (v / v) fetal bovine serum, and then treated with nothing. Negative control group, positive control group treated with 5 µg / ml LPS only, and experimental groups treated with 5 µg / ml LPS and 200 µg / ml bamboo leaf extract at the same time were prepared, and further incubated for 24 hours.

Subsequently, total mRNA was obtained from each cell, which was used as a template and RT-PCT using oligo (dT) as a primer was performed to obtain cDNA for each mRNA. Then, the obtained cDNA was amplified by a PCR method to obtain genes of IL-12 p40, IL-12 p35, IL-6 and β-actin, respectively. At this time, the following were used as primers:

IL-12 p40-Sense: 5'-agaagctaaccatctcctggtttg-3 '(SEQ ID NO: 1)

IL-12 p40-antisense: 5'-ccggagtaatttggtgcttcacac-3 '(SEQ ID NO: 2)

IL-12 p35-Sense: 5'-cagcgttccaacagcctc-3 '(SEQ ID NO: 3)

IL-12 p35-antisense: 5'-gcagagtctcgccattatg-3 '(SEQ ID NO: 4)

IL-6-Sense: 5'-gaacaacgatgatgcactt-3 '(SEQ ID NO: 5)

IL-6-antisense: 5'-gtagagaacaacataagtc-3 '(SEQ ID NO: 6)

β-actin-sense: 5'-ggaatcctgtggcatccatgaaac-3 '(SEQ ID NO: 7)

β-actin--antisense: 5'-aaaacgcagctcagtaacagtccg-3 '(SEQ ID NO: 8)

Each obtained gene was then compared by electrophoresis (see FIG. 4). Figure 4 is a graph showing the change in the expression level of IL-12 p40 mRNA, depending on the addition of bamboo leaf extract. As shown in Figure 4, when the LPS was treated alone, the expression levels of IL-12 p40, IL-12 p35 and IL-6 mRNA increased rapidly, but when LPS and bamboo leaf extracts were treated simultaneously IL-12 p40 The expression level of mRNA was reduced to the level of no LPS treatment. However, p35, another subunit of IL-12, did not decrease mRNA expression level even when bamboo leaf extract was treated, and even bamboo leaf extract was treated even with IL-6, another interleukin induced by LPS. It was found that the expression level of mRNA was not reduced.

As such, the unchanged amount of mRNA of p35, another subunit of IL-12, means that the bamboo leaf extract of the present invention specifically acts on the expression of IL-12 p40, and the amount of mRNA of IL-6 This change does not mean that the decrease in the amount of mRNA of IL-12 p40 is not due to a decrease in cellular function due to cytotoxicity, the bamboo leaf extract of the present invention is specific for the expression of IL-12 p40 only It can be seen that the suppression.

Example 3-3 Confirmation of the Effect of Bamboo Leaf Extract on Animals Overexpressing IL-12

Summarizing the results of Examples 3-1 and 3-2 described above, the bamboo leaf extract of the present invention specifically inhibits the expression of IL-12 p40 induced by the treatment of LPS, which is an IL-12 p40 gene. The level of transcription was regulated at. Thus, it was confirmed by measuring the concentration of interleukin-12 (IL-12) in the spleen of mice fed LPS whether bamboo leaf extract can have a similar effect in vivo. In other words, macrophages present in the spleen of mice produce IL-12, and mice that have excessively fed LPS are known to produce excessive IL-12 in electric macrophages (see Patent Publication 2002-14435). Ho), bamboo leaf extracts were treated in mice overproducing IL-12, and it was confirmed whether IL-12 production decreased.

Feeding with LPS in 120 male DBA / 2 mice at 2 weeks of age was fed during the experiment. On the other hand, after dissolving the bamboo leaf extract prepared in Example 1 in physiological saline, every 7 days after 7 days at a dose of 20 ㎍ / body weight (g) / hour (day) in the male male DBA / 2 mice Intraperitoneally once every other day, 20 spleens were taken every 7 days after the start of extract injection, and the spleens were collected for 35 days (5 weeks) (see Kang BY et al., Br. J. Pharmacol). , 128: 380-384, 1999). Each collected spleen was homogenized in 10 ml of physiological saline, incubated for 48 hours in a CO ₂ incubator, and the concentration of IL-12 in the supernatant was measured by ELISA (see Kang BY et al. , Br. J. Pharmacol., 128: 380-384, 1999 (see Table 2). At this time, as a positive control, mice fed with LPS intraperitoneally injected with physiological saline in which bamboo leaf extract was not dissolved, and as a negative control, did not eat LPS intraperitoneally injected with physiological saline in which bamboo leaf extract was not dissolved. Mice were used.

Changes in IL-12 Concentration Over Time in Mice Administered Bamboo Leaf Extract (Unit: ng / ml) Elapsed time (days) Positive control Negative control Bamboo leaf extract administration group 0 111 23 112 7 167 25 100 14 256 31 80 21 213 28 90 28 380 29 64 35 327 27 52

As shown in Table 2, it was found that the concentration of IL-12 decreases with time in mice administered with bamboo leaf extract. Given that the concentration of IL-12 increases over time in the positive control and maintains a certain level of IL-12 in the negative control, the bamboo leaf extract is a macrophage of the spleen It was confirmed that abnormal overexpression of IL-12 produced in

Incorporating the results of the above Examples 3-1 to 3-3, it was confirmed that the bamboo leaf extract can suppress the overexpression of IL-12 induced by abnormal factors in vitro or in vivo, the present invention It was confirmed that the bamboo leaf extract of can be used as a therapeutic agent for autoimmune diseases caused by overexpression of IL-12.

As described and demonstrated in detail above, the present invention is a bamboo leaf extract obtained by treating organic solvents and activated carbon from bamboo leaves, a method for producing an electrical extract, a promyelocytic leukemia therapeutic agent and an autoimmune comprising the electrical extract as an active ingredient Provide a cure. Bamboo leaf extract of the present invention can suppress the induction of promyelocytic leukemia, and can inhibit the induction of autoimmune diseases by overexpression of IL-12 p40 gene, it can be widely used in the treatment of various immune diseases will be.

Attach sequence list electronic file  

Claims (5)

delete delete delete The bamboo leaves were immersed in a solvent mixed with water and acetone in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v), left at room temperature for 1 to 3 months, and then dried by drying the liquid component obtained by filtration. To obtain a liquid component by dissolving in water of pH 4.0, and then filtered to obtain a liquid component and a solid, respectively, and then dissolving the electrical solid in water of pH 8.0, and then filtered to obtain a liquid component. The mixture was titrated to pH 8.0, stirred with addition of activated charcoal, and filtered to obtain a solid. The mixture was mixed with water and acetone in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v), followed by stirring. Bamboo leaf extract obtained by drying the liquid component obtained by filtration again as an active ingredient, comprising a pharmaceutically acceptable carrier, a promyelocytic leukemia therapeutic agent. The bamboo leaves were immersed in a solvent mixed with water and acetone in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v), left at room temperature for 1 to 3 months, and then dried by drying the liquid component obtained by filtration. To obtain a liquid component by dissolving in water of pH 4.0, and then filtered to obtain a liquid component and a solid, respectively, and then dissolving the electrical solid in water of pH 8.0, and then filtered to obtain a liquid component. The mixture was titrated to pH 8.0, stirred with addition of activated charcoal, and filtered to obtain a solid. The mixture was mixed with water and acetone in a ratio of 0.3: 0.7 to 0.1: 0.9 (v / v), followed by stirring. Bamboo leaf extract obtained by drying the liquid component obtained by filtration again as an active ingredient, comprising an pharmaceutically acceptable carrier, autoimmune disease therapeutic agent.

Images