JP2005272441A - Dioscoreas sp. extract and its medical use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an oral composition which enhances the proliferation and differentiation of cells and mitigates side effects caused by chemotherapy. <P>SOLUTION: A method of treating osteoporosis comprises administering a pharmaceutical composition containing a fraction of the extract of Dioscorea sp. as an active ingredient to a patient, and a method of mitigating side effects caused by chemotherapy is provided. The extract is prepared by the process comprising a step of extracting the tuber of Dioscorea sp. with an alcohol base solvent and a step of purifying the obtained extract. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to Dioscorea sp. In particular, the present invention relates to oral compositions that can enhance cell proliferation and differentiation, as well as alleviate side effects induced by chemotherapy. The present invention provides a method for treating osteoporosis and a method for alleviating side effects caused by chemotherapy.

  Dioscorea, also known as “wild yam”, is a member of the monocotyledonous family of yams that are distributed in the tropics and subtropics. There are approximately 650 species worldwide, of which 93 and 9 variants are found in China, and 14 and 5 variants are found in Taiwan.

  Dioscorea is one of the most important medicinal plants used in traditional Chinese medicine, and its medical effect has been studied for many years. In 1936, Tsukamoto et al. Isolated diosgenin, a steroid saponin of Dioscorea, from a plant of the Dioscorea family and then used it as a rapid synthetic raw material for medical steroids. Aradhana, Rao Ar. Kale RK. In this study, diosgenin was shown to promote the proliferation of rat mammary epithelial cells. In Non-Patent Document 1, J. Org. L. Beneytout et al., When diosgenin was added to human erythroleukemia (HEL) cell culture, diosgenin induced changes in the morphological and biochemical characteristics of megakaryocytes, and thus diosgenin induced megakaryocyte differentiation of HEL cells. It has been reported that it can be used as a substance. Non-Patent Document 2 suggests that Dioscorea steroid extract has significant activity as an antioxidant to regulate serum lipid levels.

  Dehydroepiandrosterone (DHEA) has a chemical structure similar to that of diosgenin and is known to have anticancer, antioxidant and antidiabetic effects as well as bone mass control effects. Serum DHEA levels gradually decrease with age and are associated with aging. From various studies, it is speculated that the Dioscorea diosgenin extract may be converted to DHEA in the human body and thus compensate for DHEA, which decreases with age. However, these studies show that diosgenin can increase HDL levels while reducing serum lipid over-oxidation and reducing serum triglyceride loss and LDL hyperoxidation damage. This was only done for older people who took the diosgenin present in Dioscorea.

Regarding the effect of DHEA in controlling the bone mass, Ben A. et al. A. Scheven et al., Although DHEA and its sulfate derivative (DHEA-S) did not, by themselves, exert significant effects directly and independently on human osteoblast proliferation and differentiation. Treatment with a bone cell modulating agent, 1,25 (OH) ₂ D ₃ resulted in enhanced specific alkaline phosphatase (ALP) activity, a specific marker of osteoblast maturation Reported that. This study shows that the effect of DHEA / DHEA-S on osteoblast proliferation and differentiation can be established through effects on 1,25 (OH) ₂ D ₃ -induced changes in bone cells. Indicated.

In accordance with the present invention, Dioscorea sp. It has been found that the methanol extract and further extract fractions have biological activity in cell regeneration. In particular, without the presence of any bone cell regulator, Dioscorea sp. Methanol extract of and the further extract fraction itself stimulates osteogenic cell proliferation and differentiation to complement osteoprogenitor cells in the bone, so that osteoblast maturation and osteoblast It has been found to promote calcification, thereby achieving bone repair, restoration and regeneration, in other words, preventing and treating osteoporosis. Further, the Dioscorea sp. The extract not only stimulates the proliferation and differentiation of hematopoietic stem cells in the bone marrow in the presence of GM-CSF, but also of patients suffering from white blood cell and red blood cell deficiencies caused by anti-cancer drug treatment. It promotes recovery and can therefore be used in combination with anticancer drugs as a chemotherapeutic adjuvant.
Biochemical & Biophysical Research Communications 207 (1): 398-404, Feb / 1995. Life Sciences 59 (11): 147-57, 1996 Life Sciences 62 (1): 59-68, 1998

  Accordingly, the object of the present invention is to achieve Dioscorea sp. It is to provide an oral composition for enhancing the proliferation and differentiation of osteogenic cells, comprising an extract of

  Another object of the present invention is Dioscorea sp. An oral composition for use as an anticancer drug adjuvant is provided.

  A further object of the present invention is to provide an oral composition useful for the prevention and treatment of osteoporosis.

  A further object of the present invention is to provide an oral composition useful as an adjuvant to alleviate the side effects caused by anticancer drugs.

  In yet another object, the present invention relates to Dioscorea sp. Pharmaceutically active in patients in need of treatment. A method of treating osteoporosis is provided which comprises orally administering an effective amount of an extract of.

  In yet a further object, the present invention provides a method for treating pharmaceutically active Dioscorea sp. A method for alleviating side effects caused by chemotherapy using a chemotherapeutic adjuvant comprising administering an effective amount of an extract of orally.

  Other features and advantages of the present invention will become apparent in the following detailed description of the preferred embodiments with reference to the accompanying drawings.

The present invention relates to Dioscorea sp. Based on the discovery of the biological activity of the extract, in particular its activity on cell regeneration. Dioscorea sp prepared according to the method of the present invention
. Experiments have confirmed that the methanol or ethanol extracts and further extract fractions of the present contain active substances that enhance the proliferation and differentiation of mouse bone marrow progenitor cells. In particular, Dioscorea sp. The methanol or ethanol extract and further extract fraction of itself enhances cell proliferation of functional osteogenic cells and induces extensive differentiation of osteogenic cells into osteoblasts and osteoblasts It also enhances cell mineralization. Furthermore, Dioscorea sp. The methanol extract of can alleviate the side effects caused by anticancer drugs. In particular, the methanol extract restores white blood cells and red blood cells present in the peripheral blood of mice treated with cyclophosphamide (CY.). Therefore, Dioscorea sp. This methanol extract is useful in the prevention and treatment of osteoporosis, a common disease in the aging process, and could be used in combination with anticancer drugs as a chemotherapeutic adjuvant.

  Cells capable of self-renewal and differentiation are called stem cells. Stem cells are present at maximal levels during the fetal period and gradually decrease in number with age. Thus, it has been speculated that there is an important correlation / association between stem cells and senescence. Stem cells in adults can produce specific responses to specific messages that are transmitted through changes in the microenvironment where new stem cells are produced or differentiate into specific cells. When the stem cell receives a differentiation message, the stem cell regenerates quickly and in large quantities and then finally proceeds to differentiation. These stem cells are used to maintain cell balance in adults and to replenish the number of cells that die due to natural or injury.

  Stem cells in the bone marrow are divided into two types of cells: hematopoietic stem cells (which are two more specialized types of stem cells: lymphoid progenitor cells (which produce T and B lymphocytes) and myeloid progenitor cells ( This produces white blood cells, red blood cells and megakaryocytes)}, and stromal cells, which are the source of cells that form support structures in the bone marrow. The stromal cells have the property of adhering to the bottom of plastic culture plates and even myoblasts during culture. Stromal cells are required for the proliferation and differentiation of hematopoietic stem cells.

  Stem cell production and number are greatly reduced as aging occurs, resulting in various problems of aging, with osteoporosis being the most common. Causes of osteoporosis include a loss of balance between bone formation and resorption. Osteoblasts derived from osteogenic cells contribute to bone formation consisting of bone matrix and skeletal mineralization. Osteogenic cells are derived from bone marrow stromal cells. Dexamethasone and ascorbic acid can promote the proliferation and growth of osteogenic cells and allow the cells to differentiate into mature osteoblasts. During the differentiation process, different markers of osteoblasts are expressed: first there is collagen matrix deposition and after 10-14 days alkaline phosphatase (ALP) is expressed. Alkaline phosphatase is widely used as a biochemical marker for discrimination of osteoblast activity and its actual function is not yet known, but it is recently thought to be involved in the skeletal calcification process. After 21 days of continuous culture, the cells secrete osteocalcin and eventually mineralize to form bone nodules.

  In the present invention, the inventor unexpectedly found that Dioscorea sp. Oral compositions comprising an extract of methanol or ethanol, or a further extract fraction, can be used to enhance the growth and differentiation of osteogenic cells in the absence of an osteocyte modulating agent, and thus the active extract It has been found that a composition comprising can be used for the treatment of osteoporosis.

In the present invention, an oral composition for enhancing cell proliferation and differentiation is Dioscorea sp. As an active ingredient. The extract was obtained from Dioscorea sp. From a root tuber part and obtained using an alcohol-based solvent as an extraction solution. This preparation process involves (a) an alcohol-based solvent (wherein the alcohol-based solvent is methanol, an ethanol-based solvent, or a mixture thereof) in the presence of an acid, preferably in the presence of 1% acetic acid. . Extraction of the tuber of the tuber.

In addition, the resulting extract can be further extracted based on polarity so as to obtain a pharmacologically active fraction. In one preferred embodiment of the present invention, the methanol extract is further subjected to partition chromatography comprising the following steps:
(B) extracting the methanol extract obtained from step (a) using a solvent mixture of ethyl acetate and water and separating the ethyl acetate extract from the aqueous extract present in the aqueous phase. ;
(C) adding n-butanol solvent to the aqueous phase for further extraction and separating the butanol extract from the remainder of the aqueous extract remaining in the aqueous phase; and (d) step Adding 75% alcohol solvent to the aqueous phase obtained from (c) to extract and further remove the polysaccharide to obtain a purified water extract.

  Dioscorea sp. In order to confirm the biological activity of the components of Dioscorea tuber, the biological activity of the Dioscorea tuber methanol extract and cells obtained from healthy mice and human patients suffering from glucocorticoid-induced osteoporosis Was analyzed.

  From the experimental results shown in FIG. 1, the present inventor unexpectedly found that Dioscorea sp. It has been found that the methanol extract and further extract fractions enhance the growth of osteogenic cells without the aid of bone cell modulating agents. Under the same concentration, DHEA showed no potentiating effect on the proliferation of osteogenic cells. In FIG. 2, this result is shown in Dioscorea sp. The extract significantly increased the amount of alkaline phosphatase expressed in normal cells, ie the extract prepared according to the present invention was able to stimulate differentiation of osteogenic cells into mature osteoblasts .

  The inventor further confirmed the effect on abnormal bone marrow cells obtained from a human patient suffering from glucocorticoid-induced osteoporosis. Glucocorticoids are an essential therapy for various inflammatory and autoimmune diseases. However, prolonged use of glucocorticoids is one of the most common iatrogenic causes of osteoporosis. Glucocorticoids can increase bone loss through various effects on osteoblasts: inhibition of osteoblast lineage replication, reduced production of new osteoblasts, and induction of osteoblast death. From the results shown in FIG. 3, the inventor of the present invention extracts a patient suffering from glucocorticoid-induced osteoporosis because the active extract of the present invention increases the expression level of alkaline phosphatase in such cells. It has been found that the function of osteoblasts can be restored by treatment with an object.

  Furthermore, in the treatment of osteoporosis in vivo, Dioscorea sp. In order to further confirm the effectiveness of the extract of the present invention, the present inventor conducted an experiment on normal mice and mice that had been ovariectomized to induce osteoporosis that were orally administered the extract of the present invention. .

  In FIG. 4-5, this result is shown in vivo by Dioscorea sp. Was demonstrated to increase the expression level of alkaline phosphatase and calcification of osteoblasts obtained from normal and ovariectomized mice. Therefore, Dioscorea sp. Since this extract not only controls the growth and differentiation of osteogenic cells, but also controls bone formation and remodeling, the active extract can prevent and treat osteoporosis.

  On the other hand, proliferation and differentiation of bone marrow cells are governed by specific factors such as BMP-2 (bone morphogenetic protein-2), TGF-β, IL-4, EGF, and GM-CSF. It was known that there would be. When this factor changes in the culture environment, stem cells differentiate into different cells according to the specificity of the factor. For example, BMP-2, TGF-β, IL-4 and EGF are positively involved in the proliferation and differentiation of bone marrow stromal cells into osteoblast lineages. In this study, the inventor has identified Dioscorea sp. Of methanol extract of BMP-2, TGF-β, and IL-4 on gene expression and Dioscorea sp. Experiments were carried out to confirm the effect on the proliferation and differentiation of bone marrow stem cells in the presence of EGF and GM-CSF of the methanol extract and additional extract fractions.

  In FIG. 6, this data is based on Dioscorea sp. Of methanol extract of BMP-2, TGF-β, and IL-4 gene expression, especially that of BMP-2 and TGF-β was found to increase. Furthermore, from the experimental results shown in FIG. 7 and Table 2, the present inventor has obtained Dioscorea sp. Was found to stimulate the differentiation of mouse bone marrow cells in the presence of EGF. A further extract fraction of the methanol extract, DioMPw, enhances the proliferation of mouse bone marrow cells.

  With respect to GM-CSF, GM-CSF can act on specific receptor complexes present on hematopoietic progenitors to stimulate bone marrow hematopoiesis, so that hematopoietic progenitor monocytes, neutrophils in the bone marrow In addition, proliferation and differentiation into macrophages can be promoted. Therefore, GM-CSF appears to be a potential treatment for restoring macrophages in patients treated with chemotherapy. In this study, the inventor found that Dioscorea sp. In the presence of GM-CSF. It was found that proliferation of bone marrow cells was enhanced under stimulation of the methanol extract and additional extract fractions (see Table 3). In addition, this result indicates that stem cell differentiation is similar to that of Dioscorea sp. It was shown that it was enhanced by a further extract fraction. Under the same conditions, DHEA showed an effect of enhancing cell differentiation but failed to enhance cell proliferation. Therefore, this study is based on Dioscorea sp. The methanol extract and additional extract fractions of GM-CSF may aid GM-CSF in restoring macrophage counts reduced by chemotherapy throughout proliferation and in the growth of bone marrow cells and can be used as chemotherapy adjuvants.

  Furthermore, the inventor has reported that in vivo Dioscorea sp. Against chemotherapy as a chemotherapeutic adjuvant. The application of the extract was confirmed. Cyclophosphamide (CY) is a drug used in the treatment of many cancers; however, it destroys bone marrow function, reduces blood cells such as white blood cells, macrophages, and red blood cells, and many other Causes side effects. In the present invention, cyclophosphamide was used to cause leukopenia in mice to create an animal model for use in measuring the function of the active extract of the present invention as a chemotherapeutic adjuvant. The results obtained show that the active extract of the present invention prevents the decrease in white blood cell count and maintains the red blood cell count and hemoglobin content at normal levels and promotes recovery from leukopenia in CY-treated mice did. Thus, the active extract of the present invention can be used as a chemotherapeutic adjuvant to alleviate side effects induced by anticancer drugs.

Experiments conducted in accordance with the present invention have clearly been shown by Dioscorea sp. The present invention and its further extract fractions have been shown to enhance osteogenic cell proliferation and differentiation in the absence of osteocyte modulating agents, the present invention is directed to Dioscorea sp. In the treatment of osteoporosis. Provide application of. Furthermore, the extract prepared according to the present invention can be used as a chemotherapeutic adjuvant because it increases and restores the number of macrophages, leukocytes, and red blood cells that have been reduced by chemotherapy.

  The following examples are provided to illustrate the invention. This example does not limit the invention and should be construed as such.

Preliminary step 1: Dioscorea sp. Preparation of methanol extract of Dioscorea sp. Taken from Yang-Ming Mountain in Taiwan 4 kg of peeled tubers were immersed in a 1% acetic acid solution overnight. The resulting solid portion was then frozen at -70 ° C and lyophilized. The lyophilized part was immersed in a methanol solution in the presence of 1% acetic acid. After stirring and adjusting the methanol concentration to 40% by volume, the mixed solution was allowed to stand overnight and then separated by centrifugation. The resulting soluble fraction is lyophilized and referred to as DioMs.

  In addition, DioMs was subjected to partition chromatography comprising the following steps: Extracting DioMs using a solvent mixture (1: 1) of ethyl acetate and water and acetic acid from the water extract present in the aqueous phase. Separating the ethyl extract (referred to as DioMPe); performing further extraction by adding n-butanol solvent to the aqueous phase and extracting the butanol extract (DioMPb and DIOMPb) from the remaining water extract in the aqueous phase. As well as adding 75% alcohol solvent to the aqueous phase to extract and further remove the polysaccharide to obtain a purified water extract (referred to as DioMPw).

Preliminary step 2: Dioscorea sp. Preparation of Mouse Feed Containing Methanol Extract of Purina Chow 5001, which is a commercially available mouse feed, was ground into a powder. Dioscorea sp. Of the lyophilized methanol extract was added to the ground feed in an amount to replace the same amount from the ground feed to form a feed mixture. The feed mixture was mixed uniformly with distilled water, reshaped by extrusion, baked in a microwave oven at a suitable output for 2 minutes, cooled to room temperature and then frozen at -70 ° C. After lyophilization, the feed mixture was formed into pellets very similar to the characteristics of Purina Chow feed. The formed pellets are stored in a −20 ° C. refrigerator. The pellet is warmed to room temperature on the day of feeding and sterilized by UV lamp irradiation on a sterilization work table. Feed mixes with different concentrations of methanol extract were prepared.

Preliminary step 3: Bone marrow cell isolation and culture Under sterile conditions, SPF grade C3H / HeN mice were sacrificed and the femur was injected with a liquid culture of DMEM / F12 to remove bone marrow cells (flush out ). The cells were sterilized no. Filter through 53 nylon mesh. To the single cell suspension thus obtained, DMEM / F12 culture medium containing N2 was added and adjusted to a suitable concentration.

Preliminary step 4: Preparation of mouse-derived osteogenic cells Under sterile conditions, the femurs of SPF grade C3H / HeN mice were obtained and injected with DMEM / F12. Bone marrow cells are removed and no. Filter through 53 sterile nylon mesh. To the resulting single cell suspension, DMEM / F12 culture medium containing 15% FCS was added to adjust the cell concentration.

Cells were treated with DME containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone in a T flask for 6 days.
The cells were cultured in M / F12 medium, and the culture medium was changed every 3 days. The cell concentration was 10 ⁶ cells / cm ² . On day 6, suspended cells and culture medium were removed (drawn
out). The adhering cell layer was washed with 1 × PBS warmed to room temperature and then treated with 0.01% EDTA at 37 ° C. for 5-10 minutes. EDTA was removed and the reaction was stopped in culture medium containing FCS. Subsequently, it was treated with TEG for 5 minutes at 37 ° C. The reaction was similarly stopped in culture medium containing FCS. All cells were harvested and centrifuged at 1000 rpm for 5 minutes.

Example 1: Dioscorea sp. Proliferative Response of Mouse Osteogenic Cells Treated with Methanol Extract of and the Extracted Fraction The cells obtained in Preparatory Step 4 were dispersed using a 22G gauge needle and 15% FCS, 50 μg / ml Suspended in DMEM / F12 medium containing ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone, 4.5 × 10 ⁴
Cell / ml concentration. 225 μl cell suspension was added to each well of a 96-well microplate. After 3 hours, 25 μl methanol extract, each of the extracted fractions and DHEA were added to the cell suspension in each well and incubated for 72 hours. An MTT assay was then performed. A 1 mg / ml MTT solution was added to each well and allowed to react for 4 hours. MTT lysis buffer (20% SDS-50% DMF) is added to each well in an amount of 150 μl / well and allowed to react for 16 hours, and the absorbance is obtained for the cell suspension obtained in each well. D. Measurements were taken at 570 nm.

  As shown in FIG. 1, Dioscorea sp. Under the stimulation of a methanol extract, osteogenic cell proliferation is enhanced, where concentrations of 0.01 and 0.1 μg / ml show a significant enhancing effect. Under the same concentration, DHEA has no effect on enhancing osteogenic cell proliferation. The extracted fractions, DioMPe and DioMPb, enhanced cell proliferation at 10-400 μg / ml, where DioMPe shows excellent effects.

Example 2: Dioscorea sp. Against murine osteoblast differentiation as measured by in vitro alkaline phosphatase activity (ALP). Effect of Methanol Extracts and Various Extracted Fractions Osteogenic cells collected in Preparatory Step 4 were incubated in T-flasks for 6 days. Cells were dispersed with a 22G gauge needle and the cell concentration was adjusted to 5 × 10 ³ cells / cm ² . The cells were then incubated in a 6-well plate (where 4.5 ml of cell culture medium was added to each well), and 0.5 ml methanol extract and each of the extracted fractions were added the next day. did. After 14 days of incubation, an alkaline phosphatase activity assay was then performed as described below.

The culture medium was extracted and the cell layer was washed several times with PBS. 0.5% Triton in PBS
X-100 was added to each well. The resulting suspension is subjected to a freezing and thawing process at temperatures of -70 ° C and 37 ° C, respectively. The process is performed twice to obtain a test sample. 50 μl of test sample was transferred from each well to an ELISA plate. Then 50 μl AMP-substrate buffer (2-amino-2-methyl-1-propanol (AMP, 0.5M) in distilled water, pH 10; 2 mM magnesium chloride and 9 mM p-nitrophenyl phosphate) was added to the ELISA plate. For 10 to 20 minutes at room temperature. Immediately after measuring the absorbance at 410 nm wavelength using an ELISA reader, the protein concentration in each well was measured quantitatively. The measured alkaline phosphatase activity is expressed in units / μg.

  As shown in FIG. 2, expression of alkaline phosphatase (which is a specific expressed marker for mature osteoblasts) was shown after 14 days incubation of bone marrow progenitor cells. Dioscorea sp. Each of the methanol extract and a further extracted fraction of the methanol extract significantly increased the amount of expressed alkaline phosphatase, wherein 0.1 μg / ml methanol extract, 0.1 μg / ml ml DioMPb, 0.01-0.1 [mu] g / ml DioMPe, and 0.1 [mu] g / ml DioMPw showed the maximum enhancing effect.

Example 3: Methanol extract Dioscorea sp. Against alkaline phosphatase (ALP) activity of bone marrow cells derived from patients suffering from glucocorticoid-induced osteoporosis. Effect The Taipei Veterans General Hospital patients bone marrow cells obtained from a, 7 days, 15% FCS, 50μg / ml ascorbic acid, were cultured in DMEM / F12 medium containing 10 mM beta-sodium glycerophosphate, and 10nM dexamethasone. An alkaline phosphatase activity assay was then performed.

  As shown in FIG. 3, the expression of alkaline phosphatase was shown. Compared to the control group and the positive group (1 nM estrogen, which is a known treatment for osteoporosis), Dioscorea sp. The methanol extract increased the amount of alkaline phosphatase expressed, where 10 μg / ml methanol extract showed the greatest enhancement effect.

Example 4: Dioscorea sp. Against bone marrow cell alkaline phosphatase (ALP) activity and mineralization in vivo. Effect of different methanol extracts Methanol extracts at different concentrations (0, 40, 200, and 1000 mg / ml) were prepared for oral administration. After oral administration of different doses (volumes) of methanol extract for 5 days, the mice were sacrificed to obtain their bone marrow cells.

(1) Alkaline Phosphatase Activity Assay The resulting bone marrow cells were transferred to 96-well microplates at 2 × 10 ⁵ cells / well [where 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium glycerophosphate and 10 nM dexamethasone. Was added] and incubated for 2 days at 37 ° C. in a 5% CO ₂ incubator. 125 μl / well culture medium was withdrawn and replaced with fresh medium (125 μl / well) containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone. After 4 days incubation, an alkaline phosphatase activity assay was performed.

  As shown in FIG. 4 (A), the expression of alkaline phosphatase was shown. In FIG. 4A, Dioscorea sp. Of methanol extract increases the amount of alkaline phosphatase expressed, where 1000 mg / kg of methanol extract can reach up to a 3-fold enhancement compared to the control group.

(2) Nodule formation assay This assay is used to analyze bone mass mineralization. Bone marrow cells obtained from mice orally dosed with different concentrations (0, 40, 200, and 1000 mg / ml) of methanol extract were seeded in 24-well plates at 1 × 10 ⁶ cells / well and 15% The cells were cultured in α-MEM medium containing FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone, and incubated for 24 hours at 37 ° C. in a 5% CO ₂ incubator.
500 μl / well culture medium was withdrawn and replaced with fresh medium (500 μl / well) containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone. To analyze bone mineralization, cells were incubated for an additional 15 days and the culture medium was changed every 4 days. A nodule formation assay was then performed as shown below.

The culture medium is withdrawn and the cells are placed in a 5% CO ₂ incubator for 30 minutes at 37 ° C. for 50 minutes.
Fix by reacting with 0 μl / well formalin. Formalin is removed and the cells are rinsed three times with sterile water, 200 μl / well of 2% Alizarin Red S solution (which reacts with calcium) is added into the wells, and The cells were further incubated for 10 minutes at 37 ° C. in a 5% CO ₂ incubator. The alizarin solution was then removed and the cells were rinsed 3 times with absolute alcohol. The calcified area of bone mass was measured by Meta image.

  As shown in FIG. 4B, when compared with the control group, Dioscorea sp. The methanol extract promoted bone mineralization, where the 1000 mg / kg concentration methanol extract showed a maximum enhancement effect up to 3.5 times.

Example 5: Dioscorea sp. Against alkaline phosphatase (ALP) activity and calcification of bone marrow cells in an ovariectomized mouse model. In effect sterile conditions of methanol extract, a group of SPF grade C57BL / 6j mice, by removing the ovaries subjected to surgery to induce occurrence of osteoporosis, and another group, use as a control group Therefore, only surgery was performed without removing the ovaries (referred to as sham-operated mice).

  Different concentrations (0, 40, 200, and 1000 mg / ml) of methanol extract were prepared for oral administration. After oral administration of different doses (volumes) of methanol extract for 42 days, the mice were sacrificed to obtain their bone marrow cells.

(1) Alkaline phosphatase activity assay Bone marrow cells obtained from mice orally sham-operated with different concentrations (0, 40, 200, and 1000 mg / ml) of methanol extract and 2 × 10 ⁵ cells / well In 96-well microplates [wherein 250 μl of α-MEM medium containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone was added] and incubated for 2 days at 37 ° C. Incubated in a 5% CO ₂ incubator. 125 μl / well culture medium was withdrawn and replaced with fresh medium (125 μl / well) containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone. After 4 days incubation, an alkaline phosphatase activity assay was performed.

As shown in FIG. 5 (A), the expression of alkaline phosphatase was shown. In FIG. 5A, Dioscorea sp. Of methanol extract was shown to increase the amount of expressed alkaline phosphatase, where 1000 mg / kg of methanol extract showed the greatest enhancing effect.

(2) Nodule formation assay Bone marrow cells obtained from mice orally sham-operated with different concentrations (0, 40, 200, and 1000 mg / ml) of methanol extract were collected at 1 × 10 ⁶ cells / well. Inoculate in 24-well plates, incubate in α-MEM medium containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone and 24 hours at 37 ° C. with 5% CO ₂ incubator Incubated in 500 μl / well culture medium was withdrawn and replaced with fresh medium (500 μl / well) containing 15% FCS, 50 μg / ml ascorbic acid, 10 mM sodium β-glycerophosphate and 10 nM dexamethasone. To analyze bone mineralization, cells were incubated for an additional 15 days and the culture medium was changed every 4 days. A nodule formation assay was performed.

  As shown in FIG. 5B, when compared with the control group, Dioscorea sp. The methanol extract promoted bone mineralization, where 1000 mg / kg methanol extract showed the greatest enhancement effect.

Example 6: Effect of methanol extract Dioscorea sp on cytokine gene expression in bone marrow cells Total RNA was obtained from Example 4 using the Ultraspec ^™ RNA isolation kit (Biotex laboratories INC, USA). Extracted from the resulting bone marrow cells. 5 μg total RNA and 2.5 μg oligo dT were heated at 70 ° C. for 10 minutes, cooled to room temperature for 10 minutes, then 4 μl 10 mM dNTPs, 0.5 μl rRNasin, 1 μl (10 units) AMV (Avian Myeloblastosis virus) reverse transcriptase and The buffer was added and the final reaction volume was 26.5 μl. CDNA was obtained by reacting the reaction solution at 42 ° C. for 60 minutes and then at 90 ° C. for 5 minutes. To 2.5 μl of the resulting cDNA, 0.5 μl 10 mM dNTP, 0.5 μl polymerase (2 units) and its buffer, 1 μl of 10 μM targeting primer were added and the final volume of the reaction mixture was 25 μl . PCR was performed on suitable cycles, each cycle consisting of 45 seconds of denaturation at 94 ° C, 45 seconds of annealing at a suitable annealing temperature, and 1 minute extension at 72 ° C. The reaction product was visualized by electrophoresis on a 2% agarose gel. The PCR primer sequences are shown in Table 1. The experimental results are shown in FIG.

  As shown in FIG. 6, gene expression of BMP-2, TGF-β, and IL-4 is increased (particularly BMP-2 and TGF-β).

Example 7: Dioscorea sp. Against morphological changes in bone marrow cells of C3H mice in the presence of epidermal growth factor (EGF). Effect of Methanol Extract of Mice Bone marrow cells (1 × 10 ⁴ cells / well) obtained in Preparatory Step 3 in 96-well microplates containing DMEM / F12 culture medium with N 2 and 10 ng / ml EGF And 24 hours at 37 ° C. in a 5% CO ₂ incubator. Methanol extract and DHEA were each added to different wells. Wells containing pure culture medium with no methanol extract and DHEA added and subjected to the same incubation procedure were used as controls. In addition, for use as a positive control, the same procedure as the control was repeated except that 50 ng / ml EGF was used. Proliferative response was measured by MTT assay. According to the MTT assay, 1 mg / ml MTT solution was added to each well, and after 4 hours of reaction, MTT lysis buffer (20% SDS-50% DMF) was added thereto in an amount of 150 μg / well. The resulting mixture was reacted for 16 hours. Absorbance was measured in O.D. D. Measurements were taken at 570 nm.

  As shown in FIG. 7, in the event that progenitor cells in the bone marrow induced by EGF proliferate, Dioscorea sp. Of methanol extract was found to further stimulate and significantly differentiate bone marrow progenitor cells. It can be noted that more desirable optimal results were obtained when the concentration of methanol extract used was 10 μg / ml. It is further shown in Table 2 that the cells showed a significantly enhanced proliferative effect when 10 μg / ml DioMPw was used for stimulation. In the event that 100 μg / ml DioMPw is used, as shown in Table 2, cell growth rates can reach 1.9 times that of controls.

Example 8: Dioscorea sp. In the presence of GM-CSF. Of 1 × 10 ⁴ cells obtained in proliferative responses preliminary step 3 of methanol extract and further bone marrow cells of mice treated with the extracted fraction was inoculated into each well of 96-well microplates, N2 and 4ng Cultured in DMEM / F12 medium containing / ml mGM-CSF. Dioscorea sp. With different concentrations. Of methanol extract was added to each of the cultured cells obtained in different wells. The resulting methanol extract-containing cultured cells in different wells were then incubated for 14 days at 37 ° C. in a 5% CO ₂ incubator. Cells cultured in DMEM / F12 medium containing N2 and 20 ng / ml mGM-CSF are used as a positive control. An MTT assay was performed. At this point, a 1 mg / ml MTT solution was added to each well and allowed to react with the cultured cells there for 4 hours. MTT lysis buffer (20% SDS-50% DMF) was added in an amount of 150 μl / well. The resulting mixture was reacted for 16 hours. Absorbance was measured in O.D. D. Measurements were taken at 570 nm.

As shown in Table 3, Dioscorea sp. At concentrations ranging from 0.001 μg / ml to 1000 μg / ml. The bone marrow cell proliferation rate was significantly increased under the stimulation of the methanol extract. It can be noted that the most preferred range of concentration for the methanol extract is 0.01-1000 μg / ml in terms of growth enhancement. All fractions obtained from further extraction of the methanol extract are found to have the ability to enhance cell growth. Among them, DioMPb and DioMPe were found to have particularly excellent effects on growth enhancement. In addition, 20-week-old adult mouse bone marrow cells were treated with Dioscorea sp. It was found that differentiation was possible upon stimulation with a methanol extract.
Extracted fractions of methanol extract [these were extracted by a solvent mixture of water and ethyl acetate or subsequently by extracting the water extract in turn using butanol and 75% alcohol] Among other things, both the ethyl acetate extracted fraction and the 75% alcohol extract fraction obtained from further extraction of the water extract with the 75% alcohol both differentiated 20-week-old adult mouse bone marrow cells. I understand that it promotes. However, under the same conditions, DHEA, in contrast, only shows the effect of enhancing cell differentiation and does not enhance cell proliferation. Thus, Dioscorea sp., Which exhibits excellent effects on both stem cell regeneration and differentiation. Extract can assist GM-CSF in restoring the number of macrophages reduced by chemotherapy and can be used as a chemotherapy adjuvant.

Example 9: Effect of methanol extract on the number of leukocytes and erythrocytes and hemoglobin content in peripheral blood of leukopenia mice induced by cyclophosphamide Different concentrations (0, 20, 100, and 500 mg / ml) ) Methanol extract was prepared for oral administration. Mice were injected intraperitoneally with 200 and 100 mg / kg of cyclophosphamide (CY) on days 0 and 5 to cause leukopenia, and blood was collected regularly for anemia. ) Caused the condition. Mice were orally dosed with different dosages (volumes) of methanol extract on day 1 until the mice were sacrificed. Peripheral blood collected from retro-orbital sinus was sampled on days 0, 4, 8, and 12.

  To the blood obtained on days 0, 4, 8, and 12 (0.1 ml), 25 μl EDTA solution (72 mg / ml) was added to prevent blood clotting and Turku's solution (0.01% crystal violet) (2% acetic acid containing) and diluted 10 × or 20 ×. The number of white blood cells was counted with a microscope.

To the blood obtained on day 8 (0.1 ml), 25 μl EDTA solution (72 mg / ml) was added to prevent blood clotting and diluted 2000 × with saline. The number of red blood cells was counted. Hemoglobin (Hbg) content was measured by Worthington RE et al., Ex
Peripheral Hematology , 15: 85-92, 1987.

  From the results shown in FIGS. 8-10, the active extract of the present invention alleviates the decrease in white blood cell count in peripheral blood and promotes leukocyte recovery and maintains red blood cell and Hbg content to normal levels. Is shown.

  While particular forms of the invention have been illustrated and described, it will be apparent from the foregoing that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention not be limited except as by the appended claims.

(A) Dioscorea sp methanol extract and DHEA, and (B) Each additional extract fraction is a bar graph illustrating the effect on differentiation of osteogenic cells in C3H mice. (A) Methanol extract of Dioscorea sp, and (B) of each additional extract fraction to mature osteoblasts of mouse osteogenic cells in vitro as measured by alkaline phosphatase (ALP) activity. Includes a bar graph showing the effect on differentiation. 1 is a bar graph showing the in vitro effect of a methanol extract of Dioscorea sp on alkaline phosphatase (ALP) activity of bone marrow cells obtained from patients suffering from glucocorticoid-induced osteoporosis. (A) differentiation of mouse osteogenic cells into mature osteoblasts as measured by alkaline phosphatase (ALP) activity, and (B) bone mass mineralization in bone marrow cells of healthy mice as measured by nodule formation. , Including a bar graph showing the in vivo effect of the methanol extract of Dioscorea sp (the vertical axis of FIG. 4 (B) represents a pixel area related index). FIG. 5 includes a bar graph showing the in vivo effect of a methanol extract of Dioscorea sp on (A) alkaline phosphatase (ALP) activity and (B) bone mineralization in bone marrow cells of ovariectomized mice (FIG. 5B The vertical axis of) indicates a pixel area related index). Dioscorea sp. Shows the results of RT-PCR of cytokines in bone marrow cells isolated from mice orally administered with a methanol extract of. Dioscorea sp. Against morphological changes in primary cultures of mouse bone marrow cells in the presence of epidermal growth factor (EGF). It consists of a phase contrast micrograph showing the effect of a methanol extract of. Dioscorea sp. Against leukocyte count in leukopenia mice induced by cyclophosphamide (CY). The in vivo effect of the methanol extract of is shown. Dioscorea sp. Against the red blood cell (RBC) count in peripheral blood of cyclophosphamide (CP) -induced leukopenia mice suffering from severe anemia. It is a bar graph which shows the in vivo effect of the methanol extract of. For the hemoglobin content in the peripheral blood of cyclophosphamide (CP) -induced leukopenia mice suffering from severe anemia, Dioscorea sp. It is a bar graph which shows the in vivo effect of the methanol extract of.

SEQ ID NOs: 1 and 2 are IL-4 amplification primers.
SEQ ID NOs: 3 and 4 are TGF-β amplification primers.
SEQ ID NOs: 5 and 6 are BMP-2 amplification primers.
SEQ ID NOs: 7 and 8 are β-actin amplification primers.

Claims (14)

Dioscorea sp. A method of treating osteoporosis, comprising orally administering an effective amount of a pharmaceutical composition comprising an extract of the above as an active ingredient to a patient in need of such treatment. The extract was washed with Dioscorea sp. In an alcohol-based solvent in the presence of acid. The method of claim 1, wherein the method is prepared by a process comprising the step of extracting a tuber of tuber. The method of claim 2, wherein the alcohol-based solvent used in the preparation process is methanol, an ethanol-based solvent, or a mixture thereof. The method of claim 2, wherein the acid used in the preparation process is 1% acetic acid. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. And (b) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water from the aqueous extract present in the aqueous phase. The method of claim 1, prepared by a process comprising the step of separating the ethyl acetate extract. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. (B) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water, and extracting the acetic acid from the water extract present in the aqueous phase Separating the ethyl extract, and (c) adding the n-butanol solvent to the aqueous phase obtained from step (b) for further extraction and remaining water extract in the aqueous phase The process according to claim 1, prepared by a process comprising the step of separating butanol extract from the process. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. (B) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water, and extracting the acetic acid from the water extract present in the aqueous phase Separating the ethyl extract, (c) adding further n-butanol solvent to the aqueous phase obtained from step (b) for further extraction, from the water extract remaining in the aqueous phase Separating the butanol extract, and (d) adding 75% alcohol solvent to the aqueous phase obtained from step (c) to extract and further remove the polysaccharide to obtain a purified water extract. The method of claim 1, prepared by a process comprising. For patients being treated with chemotherapy, Dioscorea sp. A method of alleviating the side effects caused by chemotherapy comprising orally administering an effective amount of a pharmaceutical composition comprising an extract of. The extract was washed with Dioscorea sp. In an alcohol-based solvent in the presence of acid. 9. The method of claim 8, wherein the method is prepared by a process comprising the step of extracting tubers. 10. The method of claim 9, wherein the alcohol based solvent used in the preparation process is methanol, an ethanol based solvent, or a mixture thereof. The method according to claim 9, wherein the acid used in the preparation process is 1% acetic acid. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. And (b) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water, and extracting the extract from the water extract present in the aqueous phase. 9. A method according to claim 8 prepared by a process comprising the step of separating the ethyl acetate extract. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. (B) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water, and extracting the acetic acid from the water extract present in the aqueous phase Separating the ethyl extract, and (c) adding the n-butanol solvent to the aqueous phase obtained from step (b) for further extraction, the water extraction remaining in the aqueous phase 9. A method according to claim 8 prepared by a process comprising the step of separating butanol extract from the product. The extract is (a) Dioscorea in an alcohol-based solvent in the presence of an acid.
sp. (B) extracting the extract obtained from step (a) using a solvent mixture of ethyl acetate and water, and extracting the acetic acid from the water extract present in the aqueous phase Separating the ethyl extract, (c) adding the n-butanol solvent to the aqueous phase obtained from step (b) for further extraction and remaining the water extract in the aqueous phase Separating the butanol extract from the mixture, and (d) adding 75% alcohol solvent to the aqueous phase obtained from step (c) to extract and further remove the polysaccharide to obtain a purified water extract. 9. The method of claim 8, wherein the method is prepared by a process comprising:

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