PHARMACEUTICAL COMPOSITION CONTAINING EXTRACTS OF CERVUS NIPPON ANTLERS HAVING GROWTH-STIMULATING ACTIVITIES OF HEMATOPOIETIC STEM CELLS AND MEGAKARYOCYTES

Abstract: The present invention relates to a pharmaceutical composition having growth-stimulating effects on hematopoietic stem cells and megakaryocytes, containing a specific extract or Cervus nippon antlers, especially a chloroform extract of Cervus nippon antlers and to a biologically active compound isolated from the extracts of Cervus nippon antlers, to a process for preparing the biologically active compounds and to a pharmaceutical composition containing the biological active compounds. The biologically active compounds have monoacetyl diglyceride structure with various side branches as shown in formula (I) where R1 is 9-octadecenoyl and R2 is hexadecanoyl (KJ-1), or R1 is hexadecanoyl and R2 is 9-octadecenoyl (KJ-2), or R1 is hexadecanoyl and R2 is 9,12-octadecadienoyl (KJ-3), or R1 is hexadecanoyol and R2 is 9,12,15-optadecatrienoyl (KJ-4), or R1 is hexadecanoyl and R2 is 5,8,11,14-eicosatetraenoyl (KJ-5).

Description: PHARMACEUTICAL COMPOSITION CONTAINING EXTRACTS OF CERVUS NIPPON ANTLERS HAVING GROWTH-STIMULATING ACTIVITIES OF HEMATOPOIETIC STEM CELLS AND MEGAKARYOCYTES TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing extracts of Cervus nippon antlers having growth- stimulating activities of hematopoietic stem cells and megakaryocytes. More specifically, the present invention relates to a pharmaceutical composition having growth- stimulating activities of hematopoietic stem cells and megakaryocytes, containing a specific extract of Cervus nippon antlers, especially a chloroform extract of Cervus nippon antlers, to a biologically active compounds isolated from the extracts of Cervus nippon antlers, to a process for preparing the biologically active compounds and to a pharmaceutical composition containing the biologically active compounds. The biologically active compounds have monoacetyldiglyceride structure with various side branches as shown in the following formula(I) where Ri is 9-octadecenoyl and R is hexadecanoyl (hereinafter, designated as KJ-1), or R] is hexadecanoyl and R 2 is 9-octadecenoyl (hereinafter, designated as KJ-2), or Ri is hexadecanoyl and 2 is 9,12-octadecadienoyl (hereinafter, designated as KJ-3), or Ri is hexadecanoyl and R2 is 9,12,15-octadecatrienoyl (hereinafter, designated as KJ-4), or Ri is hexadecanoyl and R 2 is 5,8,11,14-eicosatetraenoyl (hereinafter, designated as KJ-5). BACKGROUND ART In traditional oriental medicine in Korea, deer antler together with ginseng have been widely used for their various acclaimed medicinal effects. The deer species from which the antlers are harvested belong to Cornii cervi parvum and include Cervus nippon Temminick var manturicus Swinhoe and Cervus elephus L. The antlers are harvested before they are cornified and dried, and uncornified testa shells are boiled in water along with other herbal medicines for use. Cervus nippon antler has been acclaimed to have numerous medicinal effects, e.g., a tonic, growth- and development-promoting effects, promoting hematopoietic function, treating nervous breakdown, beneficial to cardiac insufficiency, generally improving the function of the five viscera and six entrails (Dong-euibogam, a classical medical literature in Korea). Other literatures in traditional medicine, concerning the effects of deer antlers, also report a tonic, nourishing effect, a powerful general systemic effects including improving cardiac function, a fatigue recovery effects, enhancing systemic resistance to various diseases. Numerous previous investigation were performed to identify the active component(s) of the "mysterious" broad beneficial medicinal effects of deer antlers. Many substances were reportedly identified Cervus nippon antler and were claimed to be the active ingredient at least in part. These are : certain free amino acids, trace metallic elements, hexose, pentose, hexosamine, uronic acid, sialic acid, acid mucopolysacharides such as hyaluronic acid, chondroitin A, various fatty acids, and prostaglandins. Further, it has been reported that glycolipid, phospholipid, cholesterol hypoxanthine, cholest-5-ene-3 β ,1 a -diol, cholesterol ester, polyamine were isolated from Cervus nippon antler. Others reported the presence of estrone and estradiol receptor. These reports, however, did not specifically demonstrate biological effects of certain chemical components of Cervus nippon antlers. None of these reports have also chemically identified and synthesized them in vitro for potential pharmaceutical production. Chemotherapy and radiotherapy of malignant neoplastic diseases result not only in damages to cancer cells, which are desired result, but also normal cells which are to be preserved and protected. Especially hematopoietic cells, which provide blood cells as well as the first line of defense against infections, are very sensitive to those treatments. Therefore, myelosuppression and immunosuppression from vigorous anti-cancer chemotherapy, rather than underlying malignant disease, may lead to death of patients, and these toxic effects have been limiting factors in more aggressive anti-cancer treatment. There has been a strong need for pharmaceutical agents which can be used for protection and/or restoration of hematopoietic and immune systems during the cytotoxic anti-cancer chemotherapy and radiotherapy. Any drugs which stimulate growth and differentiation of hematopoietic stem cells would be used for such purpose. Such drugs may be used in other disease conditions where hematopoietic stem cells are abnormally suppressed or nonfunctional, as in aplastic anemia, cyclic neutropenia, congenital immune deficiency syndrome, anemia from chronic renal failure, infections due to myelosuppression and decreased immunity in elderly population. There have been a significant development in the understanding of myelopoiesis and various factors affecting several steps in hematopoiesis. Examples of the growth factors which are already developed and commercialized include granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor(G-CSF) and erythropoietin(EPO). All of these are, however, a kind of cytokines, effective only in mjectable form, they have some undesirable side reactions, their effect is very short lived, and still very expensive. There is still a strong need for a less expensive drug, and preferably effective with oral administration. The present inventors have experienced several interesting clinical cases of unexplained rapid restoration of suppressed bone marrow after cancer chemotherapy. On close questioning it was suggested that the unexplained preservation of bone marrow or rapid restoration after suppression might have been related to the fact that the patients had taken deer antler extracts, for in Korea it is not unusual that many patients combine traditional oriented medicines for the treatments given by physicians. DISCLOSURE OF INVENTION The present inventors therefore investigated the possibility of certain active ingredients being present in Cervus nippon antlers and looked for bone marrow stem cell stimulating effects of those antler extracts. As a result, we have identified that chloroform extracts of the Cervus nippon antler (hereinafter designated as "Extract CN-C") indeed has growth-stimulating effects on hematopoietic stem cells and magakaryocytes. We have then succeeded in fractionating and purifying the active compounds represented by the following formula(I): where Ri is 9-octadecenoyl and R 2 is hexadecanoyl(KJ-l), or Ri is hexadecanoyl and R 2 is 9-octadecenoyl (KJ-2), or Ri is hexadecanoyl and R 2 is 9,12-octadecadienoyl (KJ-3), or Ri is hexadecanoyl and R 2 is 9,12, 15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R 2 is 5,8, 11,14-eicosatetraenoyl (KJ-5). The present inventors have succeeded in synthesizing the active compound (I) efficiently and recognized that these purified and/or chemically synthesized active compounds from the extract CN-C or their derivatives have the biological effects of stimulating hematopoietic stem cells, megakaryocytes, monocyte, and lymphocytes and that these compounds are effective not only after parenteral administration, but uniquely effective on oral administration. It is an object of the present invention to provide a pharmaceutical composition having growth-stimulating activities of hematopoietic stem cells and megakaryocytes comprising extracts of Cervus nippon antlers as an active component. It is another object of the present invention to provide a process for preparing the biologically active compounds fractionated and purified from the extracts of Cervus nippon antler. It is the other object of the present invention to provide a compound of the formula (I) as a biologically active component of Cervus nippon antler and a process for the preparation thereof. It is a further object of the present invention to provide a pharmaceutical compostion having growth-stimulating avctivities of hematopoitic stem cells and megakaryocytes, comprising the above active compound (I) together with a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows the extraction process of Cervus nippon antler. Figure 2 shows the fractionation of the chloroform extract (CN-C) of Cervus nippon antler. Figure 3 shows a high performance thin layer chromatography (HPTLC) of the fractions obtained in each step of extraction and purification of Cervus nippon antler. a: CN-C-2-E-a-Mi of Fig. 2, b: CN-C of Fig.2, c: CN-C-2 of Fig 2, d: CN-C-2-E of Fig. 2, e: CN-C-2-E-a of Fig. 2, f: CN-C-2-E-a-Mi of Fig. 2 Figure 4 is a photomicrograph of cross section (x 100) of spleen of control mouse. Figure 5 is a photomicrograph of a cross section (x 100) of spleen of the mouse treated with GM-CSF (100U). Figure 6 is a photomicrograph of a cross secion ( x 100) of the spleen of the mouse treated with water extract of Cervus nippon antler. Figure 7 is a photomicrograph of a cross section (x 100) of the spleen of the mouse treated with compound KJ-1. Figure 8 is a photomicrograph of a cross secion ( X 200) of the spleen of the mouse treated with compound KJ-2. Figure 9 is a photomicrograph of a cross section (x 200) of the spleen of the mouse treated with compounds KJ-2 and GM-CSF. Figure 10 is a photomicrograph of a cross section (x 200) of the spleen of the mouse treated with compound KJ-3. Figure 11 is a photomicrograph of a cross section (x 200) of the spleen of the mouse treated with compound KJ-4. Figure 12 is a photomicrograph of a cross section (x 200) of the spleen of the mouse treated with compound KJ-5. Figure 13 is a photomicrograph of a cross section (x 200) of the spleen of the mouse treated with compounds KJ-5 and GM-CSF. BEST MODE FOR CARRYING OUT THE INVENTION The present invention is to provide a novel method of extratction from the Cervus nippon antler, isolation and purification of the active compounds which have growth-stimulating effects on hematopoietic stem cells and megakaryocytes. Hematopoiesis is a series of development processs in which bone marrow stem cell (pluripotent stem cell) (HSC) that are capable of both self renewal and production of progenies with potential for differentiation into various blood cell lineages (red cells, granulocytes, and megakaryocytes and lymphocytes). In blood cell system, senescent cells are continually replaced by the progeny of very small number of undifferentiated stem cells which have a capacity for self renewal and differentiation. These stem cells differentiate into and maintain cell population with specific funtional roles. The blood cells produced in the bone marrow are destroyed in a peripheral tissue after certain period of time, and such process is repeated continuously throughout the life of animal and human. HSCs have two important charateristics; a self-renewal ability to produce the same cells as temselves, and the ability to be differentaited into all systems of lymphatic and hematopoietic cells. Study of hematopoietic cells was initiated from the first discovery that bone marrow suppression can be recovered in quantitative manner by introducting normal bone marrow cells in animals after exposure to a sublethal dose of radiation. Their ability to initiate and sustain long term hematopoiesis is the basis for a successful bone marrow transplant(BMT). As flow cytometry is introduced and monoclonal antibodies on hematopoietic stem cell marker antigen and differentiation antigen are developed, selection of HSCs was possible, and biology and development of hematopoietic cells were better understood. According to the present invention, biologically active components in the Cervus nippon antler can be extracted. The present invention includes the process of their extraction, fractionation, chemical identification and synthesis. The present invention provides a pharmaceutical composition having growth-stimulating activities of hematopoietic stem cells and megakaryocytes containing as an active component a chloroform extracts of Cervus nippon antler whcih are obtained by extracting Cervus nippon antler with hexane and further extracting the residue of the hexane extract with chloroform, and if necessary, fractionating and purifying the chloroform extract of Cervus nippon antler by silica gel column chromatography and high performance thin layer chromatography (HPTLC). First, the chloroform extracts of Cervus nippon antler are obtained first by extracting Cervus nippon antler with hexane and further extracting the residue of the hexane extract with chloroform. The process of extraction is shown in Figure 1. The amount of hexane and chloroform used in this extraction process, is the amount enough to impregnate Cervus nippon antler. Generally, hexane and chloroform are used in the ratio of 4-5 i with respect to 1kg of Cervus nippon antler. The extract CN-C, a chloroform extract of Cervus nippon antler obtained from such extraction process, is fractionated and purified by a series of silica gel column chromatography and high performance thin layer chromatography(HPTLC). The subsequent steps of extract CN-C, chloroform extract of Cervus nippon antler, include: a) step of subjecting the extract CN-C to a silica gel column chromatography using chloroform/methanol as an eluent and subjecting the resulting fraction to HPTLC to give seven extracts, CN-C-1 to CN-C-7, according to the value of Rf; b) step of subjecting the extract CN-C-2 selected from the extracts of step a) to a silica gel column chromatography using hexane/ethyl acetate(10: l, v/v) as an eluent and subjecting the resulting fraction to HPTLC to give six extracts, CN-C-2-A to CN-C-2-F, according to the value of Rf; c) step of subjecting the extract CN-C-2-E selected from the extracts of step b) to a silica gel column chromatography using hexane/ethyl acetate/acetic acid (20: 1:0.5. v/v) as an eluent and subjecting the resulting fraction to HPTLC to give two extracts, CN-C-2-E-a and CN-C-2-E-b, according to the value of Rf; and d) step of dissolving the extract CN-C-2-E-a selected from the extracts of step c) in methanol to give methanol-soluble fraction CN-C-2-E-a-Ms and methanol-insoluble fraction CN-C-2-E-a-Mi (development solvent of CN-C-2-E-a-Ms and -Mi : hexane/ethyl acetate/acetic acid = 20: 1:0.5. v/v). The fractionation and purification of the chloroform extract CN-C of Cervus nippon antler are briefly shown in Figure 2. The extracts CN-C-2, CN-C-2-E and CN-C-2-E-a obtained from a series of purification processes as set forth above, and the methanol-insoluble fraction CN-C-2-E-a-Mi have growth-stimulating activities of hematopoietic stem cell and megakaryocyte as proved by the subsequent experiments. Therefore, the extract CN-C may be used as a biologically active component in the composition of the present invention. To identify the active component contained in the chloroform extract CN-C of Cervus nippon antler, the mass spectrometry analysis is carried out for the methanol-insoluble fraction CN-C-2-E-a-Mi finally obtained according to the present invention. As a result, it can be seen that this fraction contains the active compounds such as acetyldiacylglycerols or their derivatives represented by the following formula (I): H 2 C— O— Ri HC— O— R 2 H 2 C— O— C— CH 3 O where Ri is 9-octadecenoyl and R2 is hexadecanoyl(KJ-l), or Ri is hexadecanoyl and R2 is 9-octadecenoyl (KJ-2), or Ri is hexadecanoyl and R 2 is 9,12-octadecadienoyl (KJ-3), or Ri is hexadecanoyl and R 2 is 9,12, 15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R 2 is 5,8,11, 14-eicosatetraenoyl (KJ-5). The compounds of formula (I), a biologically active component contained in the chloroform extract of Cervus nippon antler or its derivatives according to the present invention, may be examplified as follows: 1) Compound KJ-1 (Rι/R 2 =9-octadecenoyl/hexadecanoyl) l-oleoyl-2-palmitoyl-3-acetyl glycerol 2) Compound KJ-2 (Rι/R 2 =9-hexadecanoyl/9-octadecenoyl) 1 -palmitoyl-2-oleoy 1-3 -acetyl gly cerol 3) Compound KJ-3 (R 1 /R 2 =hexadecanoyl/9,12-octadecadienoyl) 1 -palmitoyl-2-linoleyl-3 -acetyl glycerol 4) Compound KJ-4 (Rι/R 2 =hexadecanoyl/9,12,15-octadecatrienoyl) 1 -palmitoyl-2-linolenyl-3 -acetyl glycerol 5) Compound KJ-5 (Rι/R 2 =hexadecanoyl/5,8, l l, 14-eicosatetraenoyl) 1 -palmitoyl-2-arachidonoyl-3-acetyl glycerol Before the present invention, it has never been reported the presence of acetyldiacyl glycerols of formula (I), a biologically active component isolated from the extract of Cervus nippon antler. Among the compounds of formula (I), the compounds KJ-1, KJ-2 and KJ-3 are disclosed in Journal of High Resolution Chromatography, Vol. 19, 497-501 (1996), but there being no description concerning any pharmacological effects or their experimental data. The compounds KJ-4 and KJ-5 are novel. Accordingly, the present invention includes in its scope novel compounds KJ-4 and KJ-5 represented by formula: where Ri is hexadecanoyl and R2 is 9, 12, 15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R2 is 5,8,11, 14-eicosatetraenoyl (KJ-5). The present invention provides a pharmaceutical composition comprising as an active component one or more compound selected from the group consisting of KJ-1, KJ-2, KJ-3, KJ-4 and KJ-5 together with a pharmaceutically acceptable carrier. From the HPTLC result of Figure 3, it can be seen that monoacetyldiglycerides of the extract CN-C (chloroform extract of Cervus nippon antler) to the methanol- insoluble fraction CN-C-2-E-a-Mi obtained from the final purification step, exhibit the same band, and therefore, they can be maintained without change in the properties of material, and that their band becomes more clear as the purification is proceeded. The compound of formula (I) may be prepared by a) acetolysing phosphatidylcholine and l-R 1 -2-R 2 -3-phosphocoline derivative in the presence of acetic acid and acetic anhydride, or b) reacting glycerol with a fatty acid of Ri-OH followed by reaction with acetic acid and then reacting the product with a fatty acid of R 2 -OH in which and R 2 are as defined above. Among these methods, the step b) using glycerol as a starting material is preferred in economical viewpoint. The detailed preparation step or the reaction condition are described in the subsequent examples, but the brief principles thereof are explained as follows. The compound KJ-1, l-9-octadecenoyl-2-hexadecanoyl-3 -acetyl glycerol, may be prepared by stirring β -palmitoyl- γ -oleoylphospatidylcoline (Sigma company's reagent) in acetic acid/acetic anhydride (3:2. v/v) at a temperature of 150 °C and isolating l-9-octadecenoyl-2-hexadecanoyl-3 -acetyl glycerol by a column chlomatography in accordance with step a). In the alternative, according to the economical method for preparation, the compound KJ-1 may be prepared by reacting glycerol with oleic acid in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine, isolating the resulting monooleoyl glycerol by a column chromatography, reacting it with acetic anhydride and dimethylaminopyridine, isolating the resulting monoacetyl monooleoyl glycerol by a column chromatography, conducting the reaction with the addition of palmitic acid in the presence of dicyclohexylcarbodiimde and dimethylaminopyridine, and then isolating the resulting KJ-1 by a column chromatography. The compound KJ-2, l-hexadecanoyl-2-9-octadecenoyl-3 -acetyl glycerol, may be prepared in the same manner as the process for preparing the compound KJ-1. That is, the compound KJ-2 may be prepared by reacting glycerol with palmitic acid in the presence of dicyclohexyl carbodiimide and dimethylaminopyridine, isolating the resulting monopalmitoyl glycerol by a column chromatography, reacting it with acetic anhydride and dimethylaminopyridine, isolating the resulting monoacetyl monopalmitoyl glycerol by a column chromatography, conducting the reaction with the addition of oleic acid in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine, and then isolating the resulting KJ-2 by a column chromatography. To prepare the compound KJ-3, l-hexadecanoyl-2-9,12-octadecadieneoyl- 3 -acetyl glycerol, phospatidylcholine used as a starting material in step a) may be used by its isolation from natural material. That is, the compound KJ-3 may be obtained by introducing phospatidylcholine extracted from egg yolk in acetic acid/acetic anhydride (3:2. v/v), conducting the reaction at 150°C and isolating the resulting KJ-3 by a column chromatography. Further, according to step b), the compound KJ-3 may be prepared by reacting glycerol with palmitic acid in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine, isolating the resulting monopalmitoyl glycerol by a column chromatography, conducting the reaction with the addition of acetic anhydride and dimethylaminopyridine, and conducting the reaction with the addition of linolic acid in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine, and then isolating the product by a column chromatography. The compound KJ-4, l-hexadecanoyl-2-9,12,15-octadecatrienoyl-3-acetyl glycerol, and the compound KJ-5, l-hexanoyl-2-5,8,l l, 14-eicosa tetraenoyl-3 -acetyl glycerol, may be prepared in the similar manner to the above respective processes. The active component which exhibits growth-stimulating activities of hematopoietic stem cell and megakaryocyte in Cervus nippon antler extracts may be prepared economically in chemical method. Therefore, surprising effects equivalent to Cervus nippon extracts may be achieved by using one or more of the five materials prepared by the chemical method instead of using natural material of Cervus nippon antler which is very expensive and not readily available. The growth-stimulating factors of hematopoietic stem cell according to the present invention can be effectively utilized in clinical medicine, for example, diseases requiring improvement of lowed immunity and blood cell function by facilitating the growth of hematopoietic cells, particularly, diseases with bone marrow suppressed by chemotherapy and radiation therapy, e.g., malignant tumors malignant hematological diseases, aplastic anemia, periodic (cyclic) leukoperria immune deficiency disease immune and hematopoietic cells, anemia from end stage renal disease, infection due luekopenia, and poor marrow reserve in elderly population. The present invention is potential for manufacturing drugs which stimulate hematopoietic stem cells and megakaryocytes. The present invention porvides a pharmaceutical composition containing hematopoietic stem cell and megakaryocyte growth-stimulating substance isolated from the chloroform extracts of Cervus nippon antlers or a compound or formula (I) as an active component. When the composition of the present invention is administered for a clinical purpose, it can be formulated into a conventional preparation in pharmaceutical field, for example, preparation for oral administration or mjectable preparation. The sterile mjectable preparation, for example, water or oil suspension, may be prepared by using a dispersing agent, a wetting agent or a suspending agent in accordance with a known method. The pharmaceutically acceptable solvent which may be used includes water, Ringer's solution and isotropic NaCl solution. The sterile oil may be used as a conventional solvent or suspending medium. Any non-stimulating oil including mono- or di-glyceride may be used for such purpose. Also, the fatty acid such as oleic acid may be used in mjectable preparation. The solid preparation for oral administration may be present in the form of capsule, tablet, pill, powder and granule, particularly capsule and tablet are useful. It is preferable that the tablet and pill are prepared by encapsulation agent. The solid preparation may be prepared by mixing polymer multisugar fraction with one or more inactive diluent such as sucrose, lactose and starch, and lubricant agent such as magnesium stearate, disintegrant, binder and the like. The dosage of the pharmaceutical composition of the present invention for human being can be suitably determined depending upon kind of extract, fraction or compound to be used as an active component; body weight, diet, sex and healthy condition of subject patient; time and method of administration; amount of excretion, mixing of drug and other condition of subject patient. The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner. Example 1 1.75kg of Cervus nippon antler (Cervus nippon Temminick var. mantchuricus Swinhoe) is finely breaked and then introduced in beaker. Hexane (about 7 I ) is added in the amount enough to immerse Cervus nippon antler, heated for 2 hours at 80 °C and then extracted two times and filtered (this filtrate is referred to as extract 1). The extract 1 was distilled under reduced pressure and dried to give extract CN-H (24.6g). The extract 1 was isolated and chloroform(approximately I f) was added to the residue, heated at 70 °C for 2 hours and extracted two times and isolated (this solution is referred to as extract 2). The extract 2 was distilled under reduced pressure to give a chloroform extract of Cervus nippon antler, extract CN-C (14g). The extract 2 was further isolated and ethanol (about 6 1 ) was added to the residue, heated for 1 day at 90 °C and extracted three times and filtered. The extract 3 was distilled under reduced pressure to give extract CN-E (91g). The hematopoiesis of the extracts CN-H, CN-C and CN-E thus prepared was evaluated. As a result, the extract CN-C which is considered to have growth-stimulating effects on hematopoietic stem cell was used for the subsequent purification step. Example 2 650 ml of a solution mixture of chloroform/methanol(500: l. v/v) was added to 350g of a powder silica gel, allowed to swell and filled with an open column (bed volume = 600 ~ 650 ml). 14g of the extract CN-C obtained in Example 1 was dissolved in a minimum amount (the minium amount for dissolving extracts) of chloroform/methanol (500: 1. v/v) and subjected to a silica gel column chromatography. The chloroform/methanol (500: 1. v/v) as eluent was added to the column. 50 t of the eluent fraction (void volume: 200ml) was collected. This was developed with HPTLC (developing solvent: chloroform/ methanol=300: l. v/v), visualized with iodine and isolated according to the value of R f . The values of R f used were 0.89, 0.63, 0.42, 0.32, 0.18, 0.08 and 0.02, respectively. The seven main fractions thus prepared were isolated. Each of the isolated fractions were distilled under reduced pressure and dried to give the extracts CN-C-1 to CN-C-7. The hematopoiesis of these extracts thus prepared was evaluated as disclosed in the subsequent Experiments 1 to 5. As a result, the extract CN-C-2 which is considered to have growth-stimulating effects on hematopoietic stem cell was used for the subsequent purification step. Example 3 The fraction CN-C-2 (380mg) having excellent hematopoiesis among the fractions purified in Example 2 was taken. 50 ml of a solution mixture of hexane/ethyl acetate(50:l. v/v) was added to 20g of a powder silica gel, allowed to swell and filled with an open column (bed volume = 100ml). 380g of the extract CN-C was dissolved in a minimum amount of chloroform methanol (50: 1. v/v) and subjected to a silica gel column chromatography. The chloroform/methanol (500: 1. v/v) as eluent was added thereto in the same manner as in Example 2 and then 15 ml of the eluent fraction was collected. This fraction was developed with HPTLC (developing solvent: chloroform/methanol=50: l. v/v), visualized with iodine and isolated into six parts according to the value of Rf. The values of R f used were 0.76, 0.62, 0.43, 0.38, 0.32 and 0.11, respectively. The fractions thus prepared were isolated. Each of the isolated fractions were distilled under reduced pressure and dried to give the extracts CN-C-2-A to CN-C-2-F. The hematopoiesis of these six extracts was evaluated as disclosed in Experiments 1 to 5. As a result, the extract CN-C-2-E which is considered to have growth-stimulating effects on hematopoietic stem cell was used for the subsequent purification step. Example 4 The fraction CN-C-2-E (70mg) having excellent hematopoiesis among the fractions purified in Example 3 was taken. 10 ml of a solution mixture of hexane/ethyl acetate/acetic acid(20: 1:0.5. v/v) was added to 3.5g of a powder silica gel, allowed to swell and filled with column (bed volume = 24ml). 70g of the extract CN-C-2-E was dissolved in a minimum amount of hexane/ethyl acetate/acetic acid(20: 1:0.5. v/v) and then subjected to a silica gel column. The eluent was added thereto in the same manner as in Example 3 and then 10 ml of eluent fraction was collected. This fraction was developed with HPTLC (developing solvent: hexane/ethyl acetate/acetic acid=20: 1:0.5. v/v), visualized with iodine and then classified into two parts according to the value of Rf. The values of Rf used were 0.34 and 0.16, respectively. These two extracts were expressed as CN-C-2-E-a and CN-C-2-E-b. The hematopoiesis of the extracts thus prepared was evaluated as disclosed in Experiments 1 to 5. As a result, the extract CN-C-2-E-a which is considered to have growth-stimulating effects on hematopoietic stem cell was used in the subsequent purification step. Example 5 The first part, extract CN-C-2-E-a (62mg), among the parts isolated in Example 4 was taken. To this extract CN-C-2-E-a was added 5ml of methanol which was then isolated into methanol-soulble part (extract CN-C-2-E-a-Ms; Rf = 0.22)) and methanol-insoluble part (extract CN-C-2-E-a-Mi; R f = 0.24) to obtain 20mg of extract CN-C-2-E-a-Ms and 40mg of extract CN-C-2-E-a-Mi. The HPTLC results on respective active components obtained in Examples 1 to 5 is shown in Figure 3. It can be seen from Figure 3 that the band in extract CN-C is the same in the course of purification. Therefore, it can be seen that the active parts is not denatured or disappared in the course of purification. At first, the band hiden due to the other part was deeply develpoed as the purification was proceeded. Example 6 Structural Analysis on Hematopoiesis of Active Material The methanol-insoluble part, extract CN-C-2-E-a-Mi, among the two materials finally purified from Example 5 was analyzed with VG70-VSEQ mass spectrometer. The result of the mass spectrometer shows that the active compounds as listed in Table 1 below are present in the extracts of Cervus nippon antler: Table 1. The respective isolated compound KJ-3 shows the same results as the physico-chemical data disclosed in the following Example 7. Accordingly, identification of their chemical structure is possible. Example 7 7-1. Synthesis of l-oleoyl-2-palmitoyl-3-acetyl glycerol (KJ-1) 7-1-1. Synthesis from Glycerol Glycerol (1.0 eq) was dissolved in tetrahydrofuran (20.0eq) to which were added dicyclohexylcarbodiimide (0.7 eq) and dimethylaminopyridine (0.1 eq), and the temperature was lowered to 0°C . To the mixture was added oleic acid (0.5 eq) and reacted for 4 hours. The resulting monooleoyl glycerol was isolated with a column chromatography (n-hexane : ethyl acetate = 18 : 1. v/v), dissolved in methylene chloride (20.00 eq) to which was added acetic anhydride(l eq), and the temperature was lowered to -78 TJ . Dimethylaminopyridine (0.2 eq) was added thereto and allowed to react for 1 hour. The resulting monoacetyl monooleoyl glycerol was isolated with a column chromatography (n-hexane : ethyl acetate = 18 : 1. v/v) (yield: 23%). The isolated monoacetyl monooleoyl glycerol (0.2 eq) was further dissolved in methylene chloride (2.0 eq) to which were added dicyclohexylcarbodiimide (0.2 eq) and dimethylaminopyridine (0.2 eq), and the temperature was lowered to OTJ . Palmitic acid was added thereto and allowed to react for 2 hours. The title compound KJ-1 thus prepared was isolated with a column chlomatography (n-hexane: ethyl acetate=18:l. v/v) (yield: 3.42%). 7-1-2. Synthesis from Phosphatidylcholine (Acetolysis) 500mg of phosphatidylcholine (l-oleoyl-2-palmitoyl-3-phosphocholine) was dissolved in 50ml of acetic acid acetic anhydride (3:2. v/v) which was then allowed to react under oil bath of 150 TJ and argon gas. After completion of the reaction, the reaction solution was extracted three times with 30ml of chloroform methanol/water (8:4:3. v/v), washed with water and dried over dry sodium sulfate to obtain 300mg of the title compound KJ-1 (Yield: 71.3%). The chemical structure of the compound thus prepared was identified with IR, 1H-NMR and 13 C-NMR. IR (CH 2 C1 2 ) 3018.13 sp 2 CH peak, 17445.5 (strong) cm "1 carbonyl peak 1H NMR (CDCI3) : δ 5.32(m, 2H), vinyl proton. 5.24(m, 1H) glycerol-tert-carbon proton, 4.25(dt, 2H), 4.12(dd, 2H) glycerol-terminal carbon proton, 2.29(m, 4H) carbonyl carbon proton, 2.05(s, 3H) acetyl group proton, 1.98(br, 4H) allylic proton, 1.60(br, 4H) carbonyl- β -carbon proton, 1.23(m, 44H) hydrocarbon proton, 0.86(t, 6H) fatty acid terminal carbon proton (t : triplet, m : multiplet, dt : doublet of triplet, dd : doublet of doublet, br : broad peak). 13 C NMR (CDCI3) : δ 172.9, 172.5, 170.1 carbonyl peak, 129.8, 129.4 vinyl carbon, 68.6, 62.1, 61.8 glycerol carbon 7-2. Synthesis of l-palmitoyl-2-oleoyl-3-acetyl glycerol(KJ-2) 7-2-1. Synthesis from Glycerol The title compound KJ-2 was prepared in the same manner as in the synthesis of KJ-1 disclosed in the item 7-1-1 except that fatty acid (palmitic acid, oleic acid) was used (Yield: 3.45%). 7-2-2. Synthesis from Phosphatidylcholine The acetolysis was carried out by using phosphatidylcholine (l-palmitoyl-2-oleoyl-3-phosphocholine) in the same manner as the synthesis of KJ-1 disclosed in the item 7-1-2 to give the object compound KJ-2 (Yield: 70.2%). IR (CH2CI2) 3018.13 sp 2 CH peak, 1744.5 (strong) cm "1 carbonyl peak 1H NMR(CDC ) : δ 5.32(m, 2H) vinyl proton. 5.24(m, 1H) glycerol-tert-carbon proton, 4.25(dt, 2H), 4.12(dd, 2H) glycerol-terminal carbon proton, 2.29(m, 4H) carbonyl-side carbon proton, 2.05(s, 3H) acetyl group proton, 1.98(br, 4H) allylic proton, 1.60(br, 4H) carbonyl- ? -carbon proton, 1.23(m, 44H) hydrocarbon proton, 0.86(t, 6H) fatty acid terminal carbon proton. 13 C NMR (CDC1) : δ 172.9, 172.5, 170.1 carbonyl peak, 129.8, 129.4 vinyl carbon, 68.6, 62.1, 61.8 glycerol carbon 7-3. Synthesis of l-palmitoyi-2-linoleyl-3-acetyl glycerol(KJ-3) 7-3-1. Synthesis from Glycerol The title compound KJ-3 was prepared in the same manner as in the synthesis of KJ-1 described in the item 7-1-1 except that fatty acid (palmitic acid, linoleic acid) was used (Yield: 3.21%). 7-3-2. Synthesis from Phosphatidylcholine The acetolysis was carried out by using phosphatidylcholine (l-palmitoyl-2-linoleyl-3-phosphocholine) in the same manner as the synthesis of KJ-1 described in the item 7-1-2 to give the object compound KJ-3 (Yield: 74.5%). 7-3-3. Synthesis from Egg To nine yolks of egg (about 123.3 lg) was added 400ml of ether with stirring and then allowed to stand for about 1 hour. Only the dissloved parts was removed and then the solvent was evaporated in water bath. 300ml of acetone was added thereto with stirring and then impure phosphatidylcholine was sinked as precipitate (2.46g). This precipitate was dissolved in 20ml of chloroform/methanol/water (65:25:4. v/v) as eluent. 150g of powder silica gel was added to 400ml of chloroform/ methanol/water (65:25:4. v/v), allowed to swell and filled with an open column. The impure phosphatidylcholine precipitate was subjected to a silica gel column to collect 50ml of fraction. Only pure phosphatidylcholine (300mg) in comparison with standard material phosphatidylcholine was collected by HPTLC and the acetolysis was carried out in the same manner as in the method (item 7-1-2) for preparing compound KJ-1 from phosphatidylcholine to obtain 180mg of the object compound KJ-3. IR (CH2CI2) 3064.45 sp 2 CH peak, 1740.64 (strong) cm "1 carbonyl peak 1H NMR(CDC1 3 ) : δ 5.32(m, 4H) vinyl proton. 5.24(m, 1H) glycerol-tert-carbon proton, 4.25(dt, 2H), 4.12(dd, 2H) glycerol-terminal carbon proton, 2.72(m, 2H) olefin-carbon proton, 2.29(m, 4H) carbonyl-side carbon proton, 2.05(s, 3H) acetyl-group proton, 1.98(br, 4H) allylic proton, 1.60 (br, 4H) carbonyl- β -carbon proton, 1.23 (m, 38H) hydrocarbon proton, 0.86 (t, 6H, J=6.5Hz, 6.8Hz) fatty acid-terminal carbon proton. 13 C NMR (CDCI3) : δ 172.9, 172.5, 170.1 carbonyl peak, 129.8, 129.4 vinyl carbon, 68.6, 62.1, 61.8 glycerol-carbon 7-4. Synthesis of l-palmitoyl-2-linolenoyl-3-acetyl glycerol (KJ-4) 7-4-1. Synthesis from glycerol The title compound KJ-4 was prepared in the same manner as in the synthesis of compound KJ-1 disclosed in the item 7-1-1 except that fatty acid (palmitic acid, linoleic acid) was used (Yield: 3.17%). IR (CH 2 C1 2 ) 3018.13(weak) sp 2 CH peak, 1740.64 (strong) cm " ' carbonyl peak 1H NMR(CDC1 3 ) : δ 5.30(m, 6H) vinyl proton. 5.24(m, 1H) glycerol-tert-carbon proton, 4.25(dt, 2H), 4.12(dd, 2H) glycerol-terminal carbon proton, 2.72(t, 4H) glycerol-carbon proton, 2.29(m, 4H) carbonyl-side carbon proton, 2.05(s, 3H) acetyl-group proton, 1.98(br, 4H) allylic proton, 1.60 (br, 4H) carbonyl- β -carbon proton, 1.23 (m, 32H) hydrocarbon proton, 0.90 (t, 3H, J=7.5Hz), 0.86(t, 6H, J=6.5Hz) fatty acid-terminal carbon proton. 13 C NMR (CDCls) : δ 173.2, 172.8, 170.4 carbonyl peak, 131.8, 130.1, 128.3, 128.1, 127.7, 127.0 vinyl carbon, 68.7, 62.3, 62.0 glycerol-carbon 7-5. Synthesis of l-palmitoyl-2-arachidonoyl-3-acetyl glycerol (KJ-5) 7-5-1. Synthesis from glycerol The title compound KJ-5 was prepared in the same manner as in the synthesis of compound KJ-1 disclosed in the item 7-1-1 except that fatty acid (palmitic acid, arachidonic acid) was used (Yield: 3.31%). 7-5-2. Synthesis from Phosphatidylcholine The title compound KJ-5 was prepared in the same manner as in the synthesis of KJ-1 disclosed in the item 7-1-2 except that phosphatidylcholine ( 1 -palmitoyl-2-arachidonoy ;-3 -phosphocholine) was used (Yield: 69.2%). IR (CH 2 C1 2 ) 3064.45(weak) sp 2 CH peak, 1740.64 (strong) cm "1 carbonyl peak 1H NMR(CDC1 3 ) : δ 5.32(m, 8H) vinyl proton. 5.24(m, 1H) glycerol-tert-carbon proton, 4.25(dt, 2H), 4.12(dd, 2H) glycerol-terminal carbon proton, 2.75(t, 6H) olefin-carbon proton, 2.30(quartet, 4H) carbonyl-side-carbon proton, (t, 6H) olefin-carbon proton, 2.30(quartet, 4H) carbonyl-side carbon proton, 2.15-1.99 allylic proton, 2.05(s, 3H) acetyl-group proton, 1.65(m, 2H), 1.55(br, 2H) carbonyl- β -carbon proton, 1.23 (m, 3 OH) hydrocarbon proton, 0.86 (m, 6H) fatty acid-terminal carbon proton. 13 C NMR (CDC1 3 ) : δ 173.2, 172.8, 170.4 carbonyl peak, 130.4, 128.9, 128.7, 128.5, 128.3, 128.0, 127.5 vinyl carbon, 68.9, 62.3, 62.0 glycerol-carbon Hereinafter, hematopoiesis of Cervus nippon antler extract and its synthetic compounds of purified extract obtained in Examples 1 to 7 was evaluated in the following Experiments 1 to 5. Experiment 1 Colony-Forming Unit in Culture(CFUc assay: The ability of bone marrow stem cells to form colonies in vitro culture was evaluated in a single layer methyl cellulose culture system. 1-1. Separation of bone marrow cell of mouse Six to 8 weeks old C57B 1/6 male mice bred in specific pathogene free (SPF) condition were used. The bone marrow of mouse was separated by washing with RPMI 1640 nutrient medium (Gibco #430-1800) from mouse femora and tibiae. The separated bone marrow was filtered to obtain mononuclear cells, washed three times with RPMI 1640 medium, and then the number of cells were counted. Stem cell-rich fraction was prepared by discontinuous percol gradient centrifugation. The cell at the interface between the density of 1.063 and 1.075 solution were collected. 1-2. Preparation of Sample All extract samples of Cervus nippon antler were dissolved in dimethylsulfoxide (DMSO) to the concentration of lOOmg/ml. This solution was used as a stock solution and used for evalution of hematopoiesis. 1-3. Analysis of CFU-C (colony-forming unit in culture) The cells, collected in the method of the item 1-1 were plated with 1.2 X 10 cell/well in 1.1% methylcelluose plate which was cultured in C0 2 -incubator at 37TJ . After one week of culture, the number of colony was counted. The Cervus nippon antler extracts CN-C, CN-C-2, CN-C-2-E, CN-C-2-E-a and CN-C-2-E-a-Mi separatedly added to the stem cells and compared with control treated with 1% PBS only. The result of colony forming efficiency is shown in Table 2. The colony forming efficiency (CFE) was obtained according to the following equation. CFE =(total number of colony in sample) / (total number of colony in control group) Table 2. As can be seen from the result in Table 2, the chloroform extract of Cervus nippon antler or respective fraction purified therefrom according to the present invention exhibit excellent colony forming efficiency and thus it may be concluded that they have stimulating effects on hematopoietic stem cells. Experiment 2 Assay of in vivo CFUs (Colony-Forming Unit in Spleen) It is important to demonstrate that the KJ-1 KJ-2, and KJ-3 obtained from the Cervus nippon antler extracts to be active components of according to the present invention indeed have growth-promoting activities of hematopoiesis. For that purpose, the spleen of mouse treated with the extracted synthetic material should show higher number of nodes (hematopoietic stem cell colonies), than the spleen of mouse treated with water extract of Cervus nippon antler or untreated control. As the dose of synthetic material applied is increased, the size of spleen and the hematopoietic splenic nodules indeed increased in quantitative manner. The results are shown in the following Table 3. Table 3. As can be seen from the above results, the compounds KJ-1 to KJ-5 have growth-stimulating activities of hematopoietic cells and plateletes. Experiment 3 Assay of monocyte thymidine uptake Monocyte was taken from 100ml of whole blood of human, suspended in RPMI- 1640+2% FBS and cultured in 5% incubator at 37°C . This cell was added to well plate in the concentration of 2-3 x 10 cell/well to which was added GM-CSF and KJ-3, respectively, and cultured at 37TJ incubator. H-thymidine 1 μ Ci was added to this well, cultured for 24 hours, trypsinized, collected cells and measured thymidine uptake with beta counter. The results are shown in the following Table 4. Table 4. Experiment 4 Assay of T4/T8 thymidine uptake Monocyte was taken from the human whole blood and T cell was separated from monocyte. From this T cell, T4 and T8 cells were separated by using MACS kit column. The separated cells was suspended in IMDM+10% FBS and added to well plate in the concentration of 2-3 X 10 cell/4ml well to which was added IL-2 20/ g/ml and KJ-3 O.Olmg/ml and cultured in incubator for 7 days. H-thymidme 1 μ Ci was added to this well, cultured for 24 hours, trypsinized and measured as beta counter. The results is shown in the following Table 5. Table 5. Experiment 5 Stem cell stimulation by KJ-3, oral and intraperitoneal Colony stimulating effects (in vivo) of oral and intraperitoneal KJ-3 (0.03 mg/g/day) mouse spleens were evaluated with image analyser. The results are shown in the following Table 6. Table 6. Stem cell stimulation by KJ-3, oral and intraperioneal(i.p) As set forth above, the extracts of Cervus nippon antler isolated and purified according to the present invention, particularly the chloroform extract of Cervus nippon antler or respective fractions thereof, exhibit excellent growth-stimulating activities of hematopoietic cell and platelet. The novel biological active compounds according to the present invention exhibits growth-stimulating activities of hematopoietic stem cells and megakaryocytes. Therefore, they can be used as therapeutic agent widely in the clinical conditions requiring stimulation of I mmunological function and increase of blood cells, especially disease under condition in which bone marrow function is suppressed after chemotherapy and raiation therapy.

Claims: WHAT IS CLAIMED IS: 1. A pharmaceutical composition having growth-stimulating activities of hematopoietic stem cells and megakaryocytes containing as an active component a chloroform extracts of Cervus nippon antler (hereinafter referred to as "extract CN-C") whcih are obtained by extracting Cervus nippon antler with hexane and further extracting the residue of the hexane extract with chloroform, and if necessary, fractionating and purifying the chloroform extract of Cervus nippon antler by silica gel column chromatography and high performance thin layer chromatography (HPTLC). 2. The composition of claim 1, comprising as an active component a fraction CN-C-2 obtained by subjecting the extract CN-C to column chromatography and HPTLC using chloroform/methanol (500/1. v/v) as an eluent. 3. The composition of claim 2, comprising as an active component a fraction CN-C-2-E obtained by subjecting the extract CN-C-2 to column chromatography and HPTLC using hexan/acetaldehyde (20/1. v/v) as an eluent. 4. The composition of claim 3, containing as an active component a fraction CN-C-2-E-a obtained by subjecting the extract CN-C-2-E to column chromatography and HPTLC using hexan/ethyl acetate/acetic acid (20/1/0.5 v/v) as an eluent. 5. The composition of claim 4, comprising as an active component a fraction CN-C-2-E-a-Mi obtained by dissolving the extract CN-C-2-E-a in methanol. 6. A process for preparing a chloroform extract of Cervus nippon antler as defined in any one of claims 1 to 5 which comprises extracting Cervus nippon antler with hexane and further extracting the residue of the hexane extract with chloroform to give the chloroform extract of Cervus nippon antler, and if necessary, fractionating and purifying the chloroform extract by silica gel column chlomatography and HPTLC. 7. A pharmaceutical composition having growth-stimulating activities of hematopoietic stem cell and megakayocyte comprising as an active component one or more compound represented by the following formula (I): where Ri is 9-octadecenoyl and R 2 is hexadecanoyl(KJ-l), or Ri is hexadecanoyl and R 2 is 9-octadecenoyl (KJ-2), or Ri is hexadecanoyl and R 2 is 9,12-octadecadienoyl (KJ-3), or Ri is hexadecanoyl and R 2 is 9,12,15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R 2 is 5,8, 11,14-eicosaterraenoyl (KJ-5), together with a pharmaceutically acceptable carrier. 8. The composition of claim 6 wherein it is administrable orally or parenterally. 9. A process for preparing a compound represented by the following formaula(I): H 2 C- -o- -Ri HC- -o- -R 2 H 2 C- -o- -c- -CH 3 II O where Ri is 9-octadecenoyl and R 2 is hexadecanoyl(KJ-l), or Ri is hexadecanoyl and R 2 is 9-octadecenoyl (KJ-2), or Ri is hexadecanoyl and R 2 is 9,12-octadecadienoyl (KJ-3), or Ri is hexadecanoyl and R2 is 9,12,15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R2 is 5,8,11,14-eicosatetraenoyl (KJ-5), which comprises a) acetolysing phosphatidyl choline and l-Rι-2-R 2 -3- phosphocholine derivatives in the presence of acetic acid and anhydrous acetic acid, or b) reacting glycerol with a fatty acid of Ri-OH followed by reaction with acetic acid and then reacting the product with a fatty 10. The process of claim 9, wherein the reaction is carried out in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. 11. A compound represented by the following formula: H 2 C— O— Ri HC— O— R 2 I H 2 C— O— C— CH 3 II o where Ri is hexadecanoyl and R 2 is 9,12, 15-octadecatrienoyl (KJ-4), or Ri is hexadecanoyl and R 2 is 5,8,11, 14-eicosatetraenoyl (KJ-5).

Inventors: HAN SO YEOP, JHON GIL JA, KIM SANG HEE, KIM YANG MI, SUH GI YOUNG, YANG HYUN OK, HAN, SO, YEOP, JHON, GIL, JA, KIM, SANG, HEE, KIM, YANG, MI, SUH, GI, YOUNG, YANG, HYUN, OK
Applicants: BUKWANG PHARM IND CO LTD, HAN SO YEOP, JHON GIL JA, KIM SANG HEE, KIM YANG MI, SUH GI YOUNG, YANG HYUN OK, BUKWANG PHARM. IND. CO., LTD., HAN, SO, YEOP, JHON, GIL, JA, KIM, SANG, HEE, KIM, YANG, MI, SUH, GI, YOUNG, YANG, HYUN, OK
Application year: 1998
Main classification: A61K35
IPC codes: A61K35, C07C69