KR101153436B1 - Composition of herb medicine containing kmkkt extract for preventing or treating cytopenia - Google Patents

Abstract

The present invention relates to a composition for preventing or treating hematopenia, which comprises Ka-mi-kae-kyuk-tang (KMKKT) extract. (Ka-mi-kae-kyuk-tang, KMKKT) extracts containing the extracts of the present invention as the active ingredient. The composition of the present invention is expected to be useful in the art because it can prevent and treat hemopoietic diseases such as anemia, leukopenia, and thrombocytopenia by promoting hematopoietic activity.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating hematopenia,

The present invention relates to a composition for preventing or treating hematopenia including Ka-mi-kae-kyuk-tang (KMKKT) extract, which comprises 10 kinds of medicinal herbs such as Hwanggi, ginseng, The present invention relates to a composition for preventing or treating hemodilution by the hematopoietic activity-promoting activity of a ganoderma extract obtained from a potato-based ganoderma extract containing phosphorus, red ginseng, red ginseng, white ginseng,

Hematopoietic effects include the pathway associated with cell proliferation and differentiation in the bone marrow, the microenvironment essential for hematopoietic stem cell (HSC) growth, the formation of various types of mature functional blood cells derived from HSCs through self-renewal and differentiation (Deans, RJ and Moseley, AB, 2000. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 28, 875-884; Allen, TD and Dexter, TM 1984. The essential cells of the hemopoietic microenvironment. Exp Hematol 12, 517-521; Brugger, W., Mocklin, W., Heimfeld, S., Berenson, RJ, Mertelsmann, R. and Kanz, L., 1993. Ex vivo expansion of enriched peripheral blood CD34 + progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin Blood 81, 2579-2584). The bone marrow can be used to treat a variety of cytokines, such as stem cell factor (SCF), platelet-derived growth factor (TPO), erythropoietic growth factor (EPO), granulocyte-macrophage- colony stimulating factor (GM- IL) -1,3,5,6,7,11 and 13, respectively. Recent studies have shown that these hematopoietic cytokine receptor-mediated signals are largely mediated by tyrosine kinases. In particular, the kinase (JAK) / signal transduction and transcription activator STAT (signal transducer and activator of transcription) pathway is an essential sequence for the normal function of various cytokines in the development of hematopoietic system (Nosaka , T. and Kitamura, T., 2000. Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) in hematopoietic cells, Int J Hematol 71, 309-319). STATs phosphorylated by JAK form homodimers that separate from the receptor complex and transfer to the nucleus, which binds the interferon-activating site (GAS) and induces transcription of the target gene (Ramana, CV, Grammatikakis, N., Chernov, M., Nguyen, H., Goh, K., Williams, BR and Stark, GR, 2000. Regulation of c-myc expression by IFN-gamma through Stat1-dependent and independent pathways Embo J 19, 263-272).

Hemodopenia, such as anemia, leukopenia and thrombocytopenia, is a disease in which production of one or more types of blood cells is stopped or greatly reduced. Although chemotherapy and radiotherapy are used to treat these diseases, it causes a problem of further promoting the disease of cancer patients because it reduces the number of hematopoietic stem cells. Therefore, it is urgent to develop a natural herbal medicine agent which can prevent or treat hemodilution.

In this regard, a Chinese herbal medicine called Si-Jun-Zi-Tang has been reported to regulate hematopoietic function and immune response by controlling GM-CSF segregation, and another Chinese medicinal plant drug, 2 is known to affect hematopoiesis by modulating GM-CSF and IL-3.

East Asia, including China, Japan and Korea, used oriental herb medicines to treat various diseases after ancient times. However, the pharmaceutical preparations using oriental herbal medicines have not been widely used yet. In order for oriental herbal medicines to be a major class in therapy, more systematic studies are needed to isolate and characterize active compounds from these herbal medicines and more research is needed to understand their activation mechanisms.

Accordingly, the inventors of the present invention have been studying to develop a herbal medicine agent that can prevent hemopoietic diseases such as anemia, leukopenia, and thrombocytopenia. Among the ten herbal medicine ingredients that are reported to have anticancer activity, (CFU-GEMM) and BFU-E by inducing a variety of hematopoietic cytokines and activating the JAK / STAT signaling pathway to prevent and treat hemopoietic proliferation by performing colony forming unit granulocyte erythrocyte monocyte macrophages (CFU-GEMM) and BFU- The present invention has been completed.

An object of the present invention is to provide a composition for the prevention or treatment of hemopneumonia using herbal medicinal herbs.

The technical terms and scientific terms used in the present invention can be construed as meaning ordinary meanings understood by those of ordinary skill in the art without departing from the scope of the present invention.

The composition of the present invention for preventing or treating hematopoietic progeny comprises a ganoderma-based ganoderma extract comprising Hwanggi, Ginseng, Liliaceae, Angelica keiskei, Angelica keiskei, Dongwoo phosphorus, May exhibit a preventive or therapeutic effect on hypopnea.

Hereinafter, the present invention will be described in detail.

The present invention includes Ka-mi-kae-kyuk-tang (KMKKT) extract as an active ingredient, including hwanggi, ginseng, lily of the valley, Angelica keiskei, And a method for preventing or treating hemophilia.

In the present invention, the sweet potatoes of the present invention include Hwanggi, Ginseng, Rootanggi, Angelica gigas, Chrysanthemum, Copper and Phosphorus, Red head, White rice, Sanjogado and Glucose, preferably 4-13 weight% 4 to 12% by weight of Angelica keiskei, 4 to 12% by weight of Angelica keiskei, 4 to 12% by weight of copper, 12 to 24% by weight of copper and 12 to 24% 13 wt% and glue 4-13 wt%.

In an example of the present invention, the pottery gourd is 10 to 15 grams per pot, 10 to 15 grams of ginseng, 15 to 20 grams of ginseng root, 10 to 14 grams of Angelica gigas, 10 to 14 grams of ginseng, 25 to 30 grams of copper phosphate, 10 to 15 g of purified water, 10 to 15 g of acid water and 10 to 15 g of glue, but the scope of the present invention is not limited thereto.

In one example of the present invention, the pottery gourd is composed of 5.5 to 11.1% by weight of sulfur, 5.5 to 11.1% by weight of ginseng, 8.2 to 14.8% by weight of ginger root, 5.5 to 10.4% by weight of Angelica keiskei, 5.5 to 10.4% The present invention can be characterized in that it includes 13.7 to 22.2% by weight, 13.7 to 22.2% by weight of red head, 5.5 to 11.1% by weight of white glue, 5.5 to 11.1% by weight of acidophore and 5.5 to 11.1% by weight of glue, It is not.

Hematopoiesis is regulated by a variety of hematopoietic cytokines that stimulate the survival, proliferation and differentiation of HSCs. Group I (universal) cytokines such as IL-3, SCF and GM-CSF affect the early stages of hematopoiesis and Group II (mono-differentiative) such as EPO, G-CSF and M- It has a role in the step. The KMKKT of the present invention increases the expression of the hematopoietic cytokines IL-3, SCF, GM-CSF, EPO and TPO. Particularly proliferating and differentiating progenitor cells of various types of IL-3, such as CFU-GEMM and BFU-E, as well as by the combination of G-CSF or EPO to specific blood cells such as erythrocyte, neurotroph or macrophage Leading to the differentiation of the precursor to the precursor. EPO is a glycoprotein that regulates erythroid precursors, such as BFU-E, by regulating erythropoiesis and stimulating HSCs. TOP c-Mpl ligand is an important factor that can regulate macrophage production and platelet production and maintain the survival and expression of HSC. GM-CSF plays a role in the maintenance of hematopoietic and immune responses.

SCF is another cytokine that regulates proliferation and survival of hematopoietic progenitor cells and shows a synergistic effect with other cytokines that regulate hematopoiesis. Recent studies have demonstrated that the hematopoietic cytokines such as TPO and SCF activate their signaling pathways by binding to their receptors c-Mpl and c-Kit. In the present invention, KMKKT induces the expression of c-Kit, a receptor for SCF. These results indicate that KMKKT plays a role in promoting hematopoietic action by inducing interactions among hematopoietic cytokines and cytokine receptor-mediated signal pathways. The JAK / STAT pathway is the most common signaling pathway activated by cytokines in hematopoiesis. The JAK proteins include JAK1, JAK2, JAK3, and TYK2. STATs include STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. TPO mediates tyrosine phosphorylation of JAK2, TYK2, STAT3 and STAT5. SCF induces autophosphorylation of c-Kit and activates STAT1 by interaction with c-Kit. In the present invention, KMKKT significantly increases phosphorylation of JAK2 and STAT5a / b. The JAK2 / STAT5 pathway has been reported to be activated by EPO, TPO and G-CSF. These results suggest that the KMKKT-mediated activity of the JAK2 / STAT5 pathway can be induced by EPO and TPO, suggesting that KMKKT plays an important role in erythropoiesis or macrophage production. JAK2 is known to be required for INFγ, which is not IFNα or β, by activating STAT binding to GAS to induce a target gene associated with hematopoiesis. In addition, the KMKKT of the present invention increases the binding GAS of an electronic factor in HSC. These results indicate that KMKKT activates multiple signaling pathways associated with the various cytokines JAK2 and STAT5. In addition, the KMKKT of the present invention increases the growth of CFU-GEMM and the growth of BFU-E. All these results suggest that the KMKKT of the present invention activates hematopoietic functions including hematopoietic cytokines and receptor-mediated signaling pathways.

The present invention can be provided in health functional foods and functional beverages for the prevention and treatment of hematopoietic diseases, which comprise KMKKT extract as an active ingredient.

Foods to which this KMKKT extract can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health and health foods. Since the KMKKT extract of the present invention has little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes. The KMKKT extract of the present invention can be added to foods or beverages. At this time, the amount of KMKKT extract in the food or drink may be 0.01 to 50% by weight of the total food, and the health drink may be added in a proportion of 0.02 to 30 g, preferably 0.3 to 10 g, based on 100 ml .

The health beverage composition of the present invention has no particular limitation on the other components other than those containing the KMKKT extract as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As other flavoring agents, natural flavoring agents (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 30 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. 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 from 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention can be provided in a pharmaceutical composition for the prevention and treatment of hemodopenia, which comprises KMKKT extract as an active ingredient.

The composition of the present invention can prevent or treat hemopenia by promoting hematopoiesis.

The pharmaceutical composition for preventing or treating hematopoiesis of the present invention comprises the KMKKT extract in an amount of 0.1 to 99.99% by weight based on the total weight of the composition.

The pharmaceutical composition for the prophylaxis or treatment of cytopenia is characterized in that the KMKKT extract is present in an amount of 0.01 mg to 10 g / kg, preferably 0.1 to 1000 mg / kg, more preferably 1 to 100 mg / kg, per kg of body weight. While the dosage is appropriate, the preferred dosage depends on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

In the present invention, cytopenias include anemia, leukopenia and thrombocytopenia.

The composition containing the KMKKT extract of the present invention can be formulated as a liquid preparation by a conventional method for manufacturing a herbal medicine or can be formulated as a pharmaceutical preparation, for example, as a solution or injection according to a conventional method. Such formulation methods may be those disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, which is known in the art.

The composition of the present invention, which comprises the ginseng extract of Ganoderma lucidum, including ginseng root, ginseng root, ginseng root, ginseng root, ginseng root, ginseng root, red ginseng root, red ginseng root, ginseng root and glue, It is expected to be useful in the field because it can prevent and treat the same hemodilution.

Figure 1 shows the cytotoxicity of KMKKT in human fibroblast (a) and human peripheral blood lymphocytes.
FIG. 2 shows the effect of KMKKT on mRNA expression of (A) IL-3 (B) SCF (C) GM-SCF (D) EPO and (E) TPO in HSC.
Figure 3 shows the effect of KMKKT on secretion of (A) IL-3 (B) SCF and (C) GM-SCF in HSC.
Figure 4 shows the effect of KMKKT on c-Kit (CD117) expression in HSC.
Figure 5 shows the effect of KMKKT on the JAK / STAT pathway.
Figure 6 shows the effect of KMKKT on the growth of CFU-GEMM and BFU-E.
Figure 7 shows the modulating effect of KMKKT in HSC differentiation.
Figure 8 shows the purification of KMKKT by high performance liquid chromatography.

Hereinafter, the present invention will be described in more detail with reference to the following Production Examples and Examples. However, the following Production Examples and Examples are for illustrating the present invention, and the scope of the present invention is not limited thereto. All experiments in the present invention were performed at least three times. The results are expressed as mean ± standard deviation (SE). Statistical comparisons were performed with unpaired two-tailed student's test (p <0.05).

< Manufacturing example >

Preparation Example 1 : Preparation of a composition containing KMKKT

The herbal medicine ingredients of the composition of the present invention were obtained from Daehyung Pharmaceutical and purchased 30g of Hwanggi, 30g of Ginseng, 30g of Lentil, 30g of Angelica gigantis, 24g of Angelica gigas, (Two replicates, total 348 g) were added to an extraction container and refluxed with 2,000 ml of ethanol for 3 times to obtain a crude extract, which was then concentrated under reduced pressure The ethanol extract was lyophilized to obtain 12.91 g (12.91 x 100/348 = 3.7%) of the composition of the present invention in powder form.

Production Example 2 : Isolation of mouse hematopoietic stem cells

Four-week-old male C57BL / 6 mice were purchased from the Korea Research Institute of Chemical Technology (Daejeon, Korea) and maintained at a relative humidity of 55% at 22 ± 2 ° C under normal conditions. Hematopoietic stem cells were isolated from the bone marrow of 8-12 week old C57BL / 6 mice according to the manufacturer's instructions using HSC anti-Sca-1 microbead kit.

Preparation Example 3 : Peripheral Blood Lymphocyte (PBL) Isolation

PBL was isolated from 20 ml blood samples of healthy donors using gradient Ficoll-Hypaque (Amersham Pharmacia).

< Example >

Example 1 : Cytotoxicity analysis

Possible cytotoxicity of KMKKT was measured in normal papillary fibroblasts (mLFC) and peripheral blood cells (hPBL) isolated from mice using SRB assay. MLFC and hPBL were treated with various concentrations of KMKKT for 48 h, then fixed with 50% cold trichloroacetic acid for 1 h at 4 ° C and stained with 0.4% SRB / 1% acetic acid for 30 min at room temperature. The cells were then redissolved in 10 mM Tris-saline solution and absorbance measured at 540 nm on a microtiter plate reader (Molecular Devices E-max).

The results are shown in Fig. Here, the measured values are expressed as mean ± SE (* p <0.01). As shown in FIG. 1, KMKKT showed no cytotoxicity up to 250 μg / ml in both mLFC and hPBL, but when treated with higher concentrations (500 and 1,000 μg / ml), cell viability was significantly decreased . This means that KMKKT does not show toxicity at physiologically relevant concentrations, indicating that KMKKT is not cytotoxic in mouse papilloma cells and peripheral blood cells.

Example 2 : Real-time PCR (PCR)

Expression of EPO, GM-CSF, TPO, and the like can be controlled by various hematopoietic cytokines. In order to investigate whether KMKKT can regulate the expression of cytokines, real time PCR was performed using total RNA obtained from HSCs treated with KMKKT. Taqman probe PCR master mix and SYBR Green PCR master mix (Applied Biosystems) were used and the probes used are shown in Table 1 below. Relative mRNA expression of each gene was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The data were measured twice or three times using at least three independent test groups to determine the mean ± SE.

Gene (mouse) Probe sequence GAPDH 5'-TGCATCCTGCACCACCAACTGCTTAG-3 '(VIC) IL-3 5'-CCCTCTCTGAGGAATAAGAGCTTTC-3 '(FAM) SCF 5'-CATTACAAAACTGGTGGCAAATCTT-3 '(FAM) GM-CSF 5'-GCCCCCCAACTCCGGAAACGGACTG-3 '(FAM) EPO 5'-AGAAAATGTCACGATGGGTTGTGCA-3 '(FAM) TPO 5'-TAGGGGCACTGCACCAGATCATCAC-3 '(FAM) c-Kit 5'-ATGAACTCCTTCTCCACAAGCGC-3 '(FAM)

HSC were stimulated with rIL-3 (IL-3, 100 U / ml) and rSCF (SCF, 100 ng / ml) as a positive control, or stimulated with 100 μg / ml, 10 μg / ml and 1 μg / ml And treated with KMKKT for 4 hours. Expression of each gene was measured by real time PCR. Data were measured as mean ± SE (* p <0.01).

The results are shown in Fig. As shown in FIG. 2, rIL-3 + rSCF significantly induced the expression of IL-3, SCF, GM-CSF, EPO and TPO. KMKKT also increases mRNA levels of IL-3, SCF, GM-CSF, EPO and TPO in a dose-dependent manner. These results indicate that KMKKT induces the expression of hematopoietic cytokines in HSCs.

Example 3 : Enzyme-Linked Immunosorbent Assay (ELISA)

The effect of KMKKT on the expression of hematopoietic cytokines was confirmed by measuring the secretion levels of IL-3, SCF and GM-CSF using the R & D system and an ELISA kit purchased from Ammersham, according to the manufacturer's instructions.

The results are shown in Fig. As shown in FIG. 3, KMKKT significantly induced secretion of IL-3, SCF and GM-SCF as treated with rIL-3 + rSCF. These results indicate that KMKKT regulates the production of functional active hematopoietic cytokines.

Example 4 : Fluid cell analysis

To investigate the effect of KMKKT on the expression of c-Kit (CD117) in HSC, fluid cell analysis was performed. HSCs isolated from the bone marrow of C7bL / 6 mice were treated with KMKKT (0, 1, 10 or 100 μg / ml) for 48 hours or with rIL-3 (100 U / ml) and rSCF (100 ng / ml) (Figs. 4A and 4B). Forty-eight hours after treatment, cells were harvested and c-Kit + / Sca-1 + and c-Kit + / CD34 + positive cells were analyzed using a flow cytometer. Specifically, Sca-1 + HSCs isolated from C57BL / 6 mice were transiently transfected with picoerythrin (PE) -antomoc CD117 and fluorescein isoforms on ice. And stained with thiocyanate (FITC) - anti-mouse Sca-1 or CD34 for 30 minutes. After washing with PBS, the number of CD117 + / Sca-1 + or CD117 + / CD34 + cells was analyzed with CellQuest Software using a FACSCalibur fluid cell analyzer (Becton Dickinson).

As can be seen in Figures 4A and 4B, CD117 + / Sca-1 + and CD117 + / CD34 + cells were significantly increased by KMKKT in a dose-dependent manner, whereas c-Kit expression by rIL-3 + rSCF Judo was not reached. These results can also be confirmed by measuring the amount of c-Kit mRNA in HSC by real-time PCR 4 hours after treatment with KMKKT. Data were expressed as mean ± SE (* p <0.01).

As can be seen in Figure 4C, KMKKT induces c-Kit expression in mRNA levels in a dose-dependent manner. These results suggest that KMKKT can induce receptor expression on hematopoietic cytokines such as c-Kit in HSCs.

Example 5 : Measurement of effect of KMKKT on JAK / STAT pathway

To determine whether KMKKT could activate the JAK2 / STAT5 pathway in HSCs, immunoprecipitation and Western blotting were performed to determine the phosphorylation of JAK2 and STAT5. HSCs were treated with rIL-3 (100 U / ml) and rSCF (100 ng / ml) or with KMKKT (0, 1, 10 or 100 ug / ml) for 20 min. The treated HSC eluates were immunoprecipitated using anti-JAK2 and then immunoblotted using the indicated antibodies. In addition, the treated HSC eluates were immunoprecipitated with anti-STAT5 and then treated with anti-phosphopyrrosin (PY20) (Calbiochem), JAK2 (Santa Cruz Biotechonogies) or STAT5 Biotechnology) was immunoblotted with the antibody.

As can be seen in Figures 5A and B, KMKKT significantly increases phosphorylation of JAK2 and STAT5 without affecting expression.

In addition, since the activated STAT protein is known to bind the GAS motif of the target gene that induces the transcriptional activity, the nuclear extract obtained from the HSC treated with the radiolabeled GAS oligonucleotide and KMKKT was used for electrophoretic mobility analysis (EMSA ) Were performed. Nuclear extracts were prepared from HSCs isolated from C57BL / 6 mice. The double stranded oligonucleotide (5-CGTGGAGTTCTTGGAAATGCGCC-3) of GAS was labeled with [? - ³² P] dATP using T4 polynucleotide kinase (T4 PNK) and incubated with 20 μg of the nuclear extract in binding buffer at 4 ° C for 20 minutes Lt; / RTI &gt; The reaction mixture was electrophoresed on a 6% natural polyacrylamide gel in 0.5 x Tris / boric acid / EDTA (TBE) buffer. The gel was dried and exposed to X-ray film for autogenous recording.

The results are shown in Figure 5C. Here, * denotes a singular band. As shown in FIG. 5C, it can be seen that KMKKT increases the amount of protein bound to GAS in HSC.

All these results indicate that KMKKT activates the STAT binding to the JAK2 / STAT5 signaling pathway and the promoter of the target gene.

Example 6 : Measurement of the effect of KMKKT on the growth of CFU-GEMM and BFU-E

In order to confirm the effect of KMKKT on the growth of CFU-GEMM and BFU-E, semi-solid colony assays were performed. HSCs were plated in Isocone modified Dulbecco's semi-solid matrix culture medium (StemCell Technologies, Vancouver, Canada) containing EPO and IL-3 in 35 mm plates. 14 days after treatment with KMKKT colonies, colonies were observed with a Nikon inverted microscope and the number of colonies per 1 ^{x 10 4} HSC was calculated ( ^* p < 0.05 and ^*** p &lt; 0.05). The precursor cells generated as a result of HSC differentiation on CFU-GEMM or BFUE rolls, respectively, of a colony containing eight granulocytes and erythrocyte or three or more erythroid clusters are referred to as fission cells or granulocyte-forming units. In order to determine whether KMKKT affects progenitor cell growth, the effect of KMKKT on the CFU-GEMM phase and HSC on BFU-E colony formation was measured. The results are shown in Fig.

As shown in FIG. 6, KMKKT increased the number and growth of CFU-GEMM colonies in a dose-dependent manner. In addition, KMKKT significantly increased the number of BFU-E colonies at 100 占 퐂 / ml but not at 10 占 퐂 / ml (Fig. 6A). Further, photographs are shown after 3 days (B), 5 days (C), 10 days (D) and 14 days (E) of the CFU-GEMM colonies. These results indicate that KMKKT increases the growth of hematopoietic precursor cells such as CFU-GEMM and BFU-E.

Example 7 : Analysis of the effect of KMKKT on HSC differentiation

In order to analyze the effect of KMKKT on HSC differentiation, hematoxylin / eosin staining was performed on bone marrow tissues isolated from C57BL / 6 mice. KMKKT (25 or 250 mg / kg) was orally administered for 14 days, and benzene (2 ml / kg) was intraperitoneally injected for two days at intervals of two days to induce a mouse model of aplastic anemia.

As shown in Fig. 7, normal tissues (A) showed normal distribution of HSC and basal cells. On the other hand, the number of HSCs decreased and the number of adipocytes and macrophages increased in aplastic anemia tissue (B). In the tissues (C, D) of mice treated with KMKKT, the numbers of HSC and basal cells were increased and the number of macrophages decreased in a concentration-dependent manner, compared with aplastic anemia tissues. These results indicate that KMKKT is capable of regulating HSC differentiation.

Example 8: Purification analysis of KMKKT

For quality control (QC) of KMKKT, high performance liquid chromatography (HPLC, Beckman Coulter) was performed. Decursin and decursinol angelate were used as standard substances for the analysis.

As shown in Fig. 8, KMKKT showed two peaks of decursin and decursinol angelate. These results indicate that KMKKT is a high-efficiency bio-material.

Claims (10)

(Ka-mi-kae-kyuk-tang, KMKKT) extract which contains ginseng root, ginseng root, ginseng root, ginseng root, ginseng root, Or a pharmaceutically acceptable salt thereof. The method according to claim 1,
The Ka-mi-kae-kyuk-tang (KMKKT) comprises 4 to 13 wt% of Hwanggi, 4 to 13 wt% of ginseng, 7 to 16 wt% 12 to 24% by weight of copper and 12 to 24% by weight of red phosphorus, 4 to 13% by weight of white powder, 4 to 13% by weight of acidophore and 4 to 13% by weight of glue. &Lt; / RTI &gt; The method according to claim 1,
The Ka-mi-kae-kyuk-tang (KMKKT) contains 10 to 15 grams of ammonia per unit, 10 to 15 grams of ginseng, 15 to 20 grams of cormorant, 10 to 14 grams of Angelica keiskei, 10 to 14 grams, To 30 g, a red head of 25 to 30 g, a white gum of 10 to 15 g, a green gauze of 10 to 15 g and a glue of 10 to 15 g. (Ka-mi-kae-kyuk-tang, KMKKT) extract which comprises the extract of Panax notoginseng, Panax ginseng, Panax ginseng, Liliaceae, Angelica keiskei, Health functional food for prevention or improvement of hypopnea. (Ka-mi-kae-kyuk-tang, KMKKT) extract which comprises the extract of Panax notoginseng, Panax ginseng, Panax ginseng, Liliaceae, Angelica keiskei, Health functional drink for the prevention or improvement of hypopnea. (Ka-mi-kae-kyuk-tang, KMKKT) extract which comprises the extract of Panax notoginseng, Panax ginseng, Panax ginseng, Liliaceae, Angelica keiskei, Or a pharmaceutically acceptable salt thereof. The method according to claim 6,
Wherein the dose of the KMKKT extract is in the range of 1 to 100 mg / kg per kg of body weight. The method according to claim 6,
A pharmaceutical composition for the prevention or treatment of hematopoiesis by promoting hematopoietic action. The method according to claim 6,
Wherein the cytopenia is anemia, leukopenia, or thrombocytopenia. The method according to claim 6,
Wherein the pharmaceutical composition is formulated into a liquid or an injectable preparation.

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