KR100415826B1 - Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients - Google Patents

Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients Download PDF

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KR100415826B1
KR100415826B1 KR10-2000-0071397A KR20000071397A KR100415826B1 KR 100415826 B1 KR100415826 B1 KR 100415826B1 KR 20000071397 A KR20000071397 A KR 20000071397A KR 100415826 B1 KR100415826 B1 KR 100415826B1
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신준식
김상태
한용남
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

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Abstract

본 발명은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것)과 천수근(Harpagophytum procumbensDC.)의 분말이나 또는 물, 저급알콜, 초산에틸, 방향족 탄화수소, 염소화탄화수소에서 선택된 용매로 추출한 엑스에서 선택된 1종 이상의 성분을 주성분으로 함유하고, 여기에 맥아 (HORDEI FRUCTUS GERMINIATUS), 우슬[ACHYRANTHIS BIDENTATAE RADIX), 당귀 (ANGELICAE GIGANTIS RADIX), 숙지황(REHMANNIAE RADIX PREPARAT), 두충(EUCOMMIAE CORTEX), 육계(CINNAMOMI CORTEX), 홍화자(CARTHAMI FRUCTUS), 구판(또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), 오공(SCOLOPENDRA), 오가피 (ACANTHOPANACIS CORTEX), 방풍(LEDEBOURIELLAE RADIX), 감초(GLYCYRRHIZAE RADIX), 구기자(LYCII FRUCTUS), 속단(DIPSACI RADIX), 녹각(CERVI CORNU), 단삼 (SALVIAE MILTIORRHIZAE RADIX), 산사(CRATAEGII FRUCTUS), 현삼(SCROPHULARIAE RADIX), 익모초(LEONURI HERBA), 신곡(MASSA MEDICATA FERMENTATA), 약콩(SOJAE SEMEN), 적두(PHASEOLI SEMEN)에서 선택된 1종 이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸에서 선택된 용매로 추출한 엑스로 조성된 조성물이며 본 조성물은 골다공증, 류마티스 관절염, 디스크증상의 예방과 치료에 탁월한 효과를 가진다.The present invention is a powder of oral water, lower alcohol, ethyl acetate, aromatic hydrocarbon, chlorination of CIBOTII RHIZOMA (dried roots of Cibotium barometz (L.) and Harpagophytum procumbens DC.) It contains at least one component selected from X extracted with a solvent selected from hydrocarbons, and contains malt (HORDEI FRUCTUS GERMINIATUS), Ussel (ACHYRANTHIS BIDENTATAE RADIX), Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT, (EUCOMMIAE CORTEX), broiler (CINNAMOMI CORTEX), safflower (CARTHAMI FRUCTUS), old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), SCOLOPENDRA, OGAPI (ACANTHOPANACIS CORTEX), windproof (AEHH RADIC) LYCII FRUCTUS, DIPSACI RADIX, CERVI CORNU, GREEN GREEN, SALVIAE MILTIORRHIZAE RADIX, CRATAEGII FRUCTUS, HYUNDAI GREEN, SCROPHULARIAE RADIX, LONURED HERMASS ICATA FERMENTATA), SOJAE SEMEN, PHASEOLI SEMEN is a composition consisting of X extracted with a solvent selected from water, lower alcohol, ethyl acetate or at least one supplementary herbal medicine, and the composition is osteoporosis, rheumatoid arthritis, It has an excellent effect on the prevention and treatment of disc symptoms.

Description

구척 및 천수근을 주성분으로 함유하는 약학적 제제 {Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients}Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients}

본 발명은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 및/또는 천수근(Harpagophytum procumbens DC.)을 주성분으로 함유하는 약학적 제제에 관한 것이다.The present invention relates to a pharmaceutical formulation containing CIBOTII RHIZOMA (dried root-shaped stem of Cibotium barometz (L.)) and / or Cinnamon Root (Harpagophytum procumbens DC.) As a main component.

현재 우리나라에서 다양한 골질환으로 호소하는 경우가 매년 증가 추세인 것으로 알려져있다. 그러나 골질환 치료를 목적으로 사용하는 화학요법, 수술요법 등은 아직 완벽한 성과를 거두지 못한 형편이다. 이러한 골질환은 노화로 인한 만성 내지 퇴행성으로 진행되고 그에 따르는 고통뿐만 아니라 의료부담이 많이 차지하고 있어 임상 분야에 전반적으로 활용할 수 있는 신물질의 개발이 절실히 요청되고 있는 실정이다.At present, the cases of complaints with various bone diseases in Korea are known to increase every year. However, chemotherapy and surgery, which are used for the treatment of bone diseases, have not yet achieved perfect results. These bone diseases are progressing from chronic to degenerative due to aging and the medical burdens as well as the pain accompanying them are required to develop new materials that can be utilized in the clinical field as a whole.

성인병 및 노인성 질환으로 알려진 류마티스성 관절염과 퇴행성 관절염은 난치성질환으로 알려져 있으며(도 2 a), 이 질환들은 관절의 활액세포의 활성화와 그로 인한 노화 및 자가면역에 의한 요인에 기인한 발병기전이 알려져있다(도2 b). 관절염 세포를 사멸 또는 억제하거나 또는 이 세포가 생성하는 효소인 사이클로옥시젠에이스 Ⅱ(cyclooxygenaseⅡ, COX-II)(도2 c)의 발현을 억제 또는 저해하는 약물을 개발하므로서 류마치스성과 퇴행성 관절염을 치유할 수 있다고 예측된다.Rheumatoid arthritis and degenerative arthritis, known as adult disease and senile disease, are known as refractory diseases (Fig. 2a), and these diseases are known to have pathogenesis due to the activation of synovial cells of the joints and the factors caused by aging and autoimmunity. (FIG. 2 b). Rheumatoid and degenerative arthritis can be cured by developing drugs that kill or inhibit arthritis cells or inhibit or inhibit the expression of cyclooxygenase II (COX-II) (Fig. 2c), an enzyme produced by these cells. It is expected.

본 발명자들은 류마치스성 관절염, 퇴행성 관절염, 허리·목의 디스크질환에 사용될 수 있는 한방제제에 관하여 오랜 연구를 행하여 왔다. 그 결과 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 및/또The present inventors have conducted a long study on herbal medicines that can be used for rheumatoid arthritis, degenerative arthritis, and disc diseases of the waist and neck. As a result (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)) and / or

는 천수근(Harpagophytum procumbens DC.)의 분말이나 또는 엑스를 주성분으로 함유하고, 필요하면 맥아(HORDEI FRUCTUS GERMINIATUS), 우슬[ACHYRANTHIS BIDENTATAE RADIX), 당귀(ANGELICAE GIGANTIS RADIX), 숙지황(REHMANNIAE RADIX PREPARAT), 두충(EUCOMMIAE CORTEX), 육계(CINNAMOMI CORTEX), 홍화자(CARTHAMI FRUCTUS), 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), 오공(SCOLOPENDRA), 오가피(ACANTHOPANACIS CORTEX), 방풍(LEDEBOURIELLAE RADIX), 감초(GLYCYRRHIZAE RADIX), 구기자(LYCII FRUCTUS), 속단(DIPSACI RADIX), 녹각(CERVI CORNU), 단삼(SALVIAE MILTIORRHIZAE RADIX), 산사(CRATAEGII FRUCTUS), 현삼(SCROPHULARIAE RADIX), 익모초(LEONURI HERBA), 신곡(MASSA MEDICATA FERMENTATA), 약콩(SOJAE SEMEN), 적두(PHASEOLI SEMEN)에서 선택된 1종 이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소 또는 염소화탄화수소에서 선택된 용매로 추출한 엑스가 분자생물학적 실험과 동물실험에서 관절염과 염증을 억제하는 놀라운 사실을 발견하여 본 발명을 완성하였다.It contains powder or X as the main ingredient of Harpagophytum procumbens DC. (EUCOMMIAE CORTEX), broiler (CINNAMOMI CORTEX), safflower (CARTHAMI FRUCTUS), old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), SCOLOPENDRA, OGAPI (ACANTHOPANACIS CORTEX), windproof (AEHH RADIC) LYCII FRUCTUS, DIPSACI RADIX, CERVI CORNU, GREEN GREEN, SALVIAE MILTIORRHIZAE RADIX, CRATAEGII FRUCTUS, HYUNDAI (SCROPHULARIAE RADIX) ), X-rays extracted with one or more auxiliary herbs selected from SOJAE SEMEN and PHASEOLI SEMEN or solvents selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons. In the experiments and animal experiments to discover the amazing facts to suppress arthritis and inflammation, the present invention was completed.

전세계적으로 골질환에 관한 연구가 활발히 진행되고, 이에 따라 골수내 일련의 면역지식이 급증함에 따라 골질환의 병인과 관절염의 발병기전이 밝혀져 최근에 몇가지 골다공증 치료제가 개발되고 알렌드레이드(allendrate), 타목시펜(Tamoxifen), 비타민D3(Vitamin D3), 부갑상선 호르몬(parathyroidhormone: PTH) 그리고 류마티스 관절염 치료제로 COX-II 저해제가 이미 상품화에 성공하였는데 항소염제인 설파살라진(sulfasalazine)(Becker K, Gromer S, Schirmer RH, Muller S. Thioredoxin reductase as a pathophysiological factor and drug target. Eur. J. Biochem., 2000 Oct 15;267(20):6118-6125), thioredoxin reductase(a pathophysiological factor and drug target. Eur. J. Biochem. 2000 Oct 15;267(20):6118-6125 등이 알려져서 임상 내지 연구개발중에 있는 실정이며, 한편 골다공증 치료제로서는 알렌드로네이트(alendronate), 랄록시펜(raloxifene), 칼시토닌(calcitonin)(Moraghan TJ, Perez EA. Mayo Clin Proc. 2000 Aug;75(8):821-9.), 에스트라디올(estradiol)(Andersson TL, Stehle B, Davidsson B, Hoglund P. Maturitas. 2000 Jun 30;35(3):245-52), genistein(Mazurek AP. Polkowski K.Acta Pol Pharm. 2000 Mar-Apr;57(2):135-55., 1,25-dihydroxyvitaminD(3)(Am. J. Physiol. Endocrinol. Metab. 2000 Jul;279(1):E213-20), parathyroid hormone(Hunziker J, Wronski TJ, Miller SC. J. Dent. Res. 2000 Jun;79(6):1431-8), alendronate(Kashyap AS, Kashyap S. Postgrad Med J. 2000 Jul;76(897):417-8), estrogen receptor modulators, calcitonin, and bisphosphonates( Wimal-awansa SJ. J. Clin. Densitom. 2000 Summer;3(2):187-201)에 의해 알려져 있다.As a result of active research on bone disease worldwide, as a result of the rapid increase in the bone marrow immunity, the pathogenesis of bone disease and the pathogenesis of arthritis have been revealed. Recently, several osteoporosis treatments have been developed and allenradates. , Tamoxifen, Vitamin D 3 (Vitamin D 3 ), Parathyroidhormone (PTH), and COX-II inhibitors have already been commercialized to treat rheumatoid arthritis. Sulfasalazine, an anti-inflammatory drug (Becker K, Gromer S) , Schirmer RH, Muller S. Thioredoxin reductase as a pathophysiological factor and drug target.Eur.J. Biochem., 2000 Oct 15; 267 (20): 6118-6125), thioredoxin reductase (a pathophysiological factor and drug target.Eur. J. Biochem. 2000 Oct 15; 267 (20): 6118-6125 and the like are known and are in clinical or research and development, and as the treatment for osteoporosis, alendronate and raloxifene Calcitonin (Moraghan TJ, Perez EA. Mayo Clin Proc. 2000 Aug; 75 (8): 821-9.), Estradiol (Andersson TL, Stehle B, Davidsson B, Hoglund P. Maturitas. 2000 Jun 30; 35 (3): 245-52), genistein (Mazurek AP. Polkowski K. Acta Pol Pharm. 2000 Mar-Apr; 57 (2): 135-55., 1,25-dihydroxyvitamin D (3) ( Am. J. Physiol.Endocrinol. Metab. 2000 Jul; 279 (1): E213-20), parathyroid hormone (Hunziker J, Wronski TJ, Miller SC. J. Dent. Res. 2000 Jun; 79 (6): 1431-8), alendronate (Kashyap AS, Kashyap S. Postgrad Med J. 2000 Jul; 76 (897): 417-8), estrogen receptor modulators, calcitonin, and bisphosphonates (Wimal-awansa SJ. J. Clin. Densitom. 2000 Summer; 3 (2): 187-201).

구척은 열대지방에서 서식하는 금모구척(Cibotium barometzJ. Smith)으로, 뿌리줄기를 약용으로 사용하며 [대한약전외 한약(생약) 규격집 주해서, 지형준, 이상인 편저, 한국메디칼인덱스사, 79쪽, 1988], 구척과(Dicksoniaceae)에 속한다. 한방 문헌에 근거하여 민간에서는 구척이 근골을 강하게 해주는 효능이 있는 것으로 기록되어 있으며 구전되어 전해지는데 금모구척의 성분으로는 오니틴(onitin), 오니틴 4-O-베타-디-알로피라노시드(onitin 4-O-β-D-allopyranoside), 오니틴 4-O-베타-디-글루고피라노시드(onitin 4-O-β-D-glucopyranoside), 프테로신 알(pterosin R ; 4-deoxy, 4-chloro-onitin)이 알려져 있으며 오니틴은 평활근 이완작용이 있는 것으로 밝혀져 있다[Murakami, Takao ; Satake, Toshiko ; Ninomiya, Katsumi ; Iida, Hideki ; Yamauchi, Kazuhiko ; Tanaka, Nobotoshi ; Saiki, Yasuhisa ; Chen, Chiu-Ming, Pterosin-derivate aus der Famile Pteridaceae.Phytochemistry, 19, 1743(1980). Yang, Meei-Shieu, Studies on the Twian fork medicine Ⅵ. Studies on onitin.Planta Medica, p25 (1986)].Gucheok is a citrusium barometz J. Smith that lives in the tropics. It uses root stems for medicinal purposes. 1988], belonging to the Dicksoniaceae. According to the Chinese literature, it is recorded that the vertebrae are effective in strengthening the musculoskeletons in the private sector, and they are orally transmitted. The components of the umbilical cord are onitin and onitine 4-O-beta-di-alolopranoside. (onitin 4-O-β-D-allopyranoside), onitin 4-O-beta-di-glucopyranoside, pterosin R; 4- deoxy, 4-chloro-onitin) is known and onitin has been shown to have smooth muscle relaxation [Murakami, Takao; Satake, Toshiko; Ninomiya, Katsumi; Iida, Hideki; Yamauchi, Kazuhiko; Tanaka, Nobotoshi; Saiki, Yasuhisa; Chen, Chiu-Ming, Pterosin-derivate aus der Famile Pteridaceae. Phytochemistry , 19, 1743 (1980). Yang, Meei-Shieu, Studies on the Twian fork medicine Ⅵ. Studies on onitin. Planta Medica , p 25 (1986)].

천수근은 아프리카 칼라하리사막에 서식하는 일명 '악마의 발톱'(Harpagophytum procumbensDC.)이라는 별명을 가지고 있으며 근경을 아프리카와 유럽에서 관절염에 사용하고 있는 [Schmidt, S. ; Rothenfelde, B., The great significance of Harpagophytum root. Zeitschrift fur Naturheilkunde. 22, 48(1978). Seeger, P. C., Harpagophytum, an effective plant remedy.Erfahrungsheilunde, vol. 8, 1978. Soulimani, R. ; Younos, C. ; Mortier, F. ; Derrieu, C., The role of stomachal activity of plant extracts, using as an example extracts ofHarpagophytum procumbens. Canad. J. Physiol. Pharm.72(12), 1532] Pelaliaceae과 식물이다. 천수근의 성분으로는 하르파지드(harpagide), 하르파고시드(harpagoside), 프로쿰비드(procumbide)[Kampf, R.,Schweiz. Apoth.-Ztg.,114, 377(1976). Sticher, O.,Dtsch. Apoth.-Ztg.,117. 1279(1977). Caprasse, M.,J. Pharm. Belg.,35, 143(1980).], 8-O-(파라-쿠마노일)-하르파지드[8-O-(p-coumaroyl)-harpagide], 6'-O-(파라-쿠마노일)-프로쿰비드[6'-O-(p-coumaroyl)-procumbide], 프로쿰보시드(procumboside)가 알려져 있으며[Kikuch, Tohru ; Matsuda, Satoko ; Kubo, Yoko ; Namba, Tsuneo, New iridoid glucosides fromHarpagophytum procumbensDC.Chem. Pharm. Bull.,31(7), 2296(1983)] 이들 성분의 약리작용은 알려져 있지 않다.Chun Soo- Keun , nicknamed the Harpagophytum procumbens DC, inhabiting the Kalahari desert in Africa, uses rhizome for arthritis in Africa and Europe [Schmidt, S.; Rothenfelde, B., The great significance of Harpagophytum root. Zeitschrift fur Naturheilkunde. 22, 48 (1978). Seeger, PC, Harpagophytum, an effective plant remedy. Erfahrungsheilunde , vol. 8, 1978. Soulimani, R .; Younos, C .; Mortier, F .; Derrieu, C., The role of stomachal activity of plant extracts, using as an example extracts of Harpagophytum procumbens. Canad. J. Physiol. Pharm. 72 (12), 1532] Pelaliaceae family. As a component of Chun Kun-geun (harpagide), harpagoside (harpagoside), procumbide (procumbide) [Kampf, R., Schweiz. Apoth.-Ztg., 114, 377 (1976). Sticher, O., Dtsch. Apoth.-Ztg., 117. 1279 (1977). Caprasse, M., J. Pharm. Belg., 35, 143 (1980).], 8-O- (Para-Cumanoyl) -Harphazide [8-O- (p-coumaroyl) -harpagide], 6'-O- (Para-Cumanoyl) ) -Procumbide [6'-O- (p-coumaroyl) -procumbide], procumboside is known [Kikuch, Tohru; Matsuda, Satoko; Kubo, Yoko; Namba, Tsuneo, New iridoid glucosides from Harpagophytum procumbens DC. Chem. Pharm. Bull., 31 (7), 2296 (1983)] The pharmacological action of these components is unknown.

본 발명자들은 구척의 유효분획인 부탄올 분획(CBB)을 세파덱스 LH-20에서 크로마토그래피를 실시하여 3종의 소분획 CBB-10, CBB-20과 CBB-30으로 나누어 동물실험을 실시한 결과 이 3종의 소분획 모두가 유효하였다. CBB-20은 설탕이 함유된 분획으로 유효성분을 분리하지 못하였고, CBB-30은 폴리페놀성 화합물이 함유되어 있어 성분을 분리하지 못하였다. 그러나 CBB-10 소분획으로부터 3종의 단일물질 CBB-11, CBB-12 및 CBB-13을 분리 정제할 수 있었다. 화학구조를 규명한 결과 CBB-11은 오니틴(onitin), CBB-12는 다우코스테롤(daucosterol)로서 이미 알려진 화합물이었으나 CBB-13은 신규의 화합물이므로 신바로메틴(shinbarometzin)이라 명명하였다. 이 화합물의 구조는 다음과 같으며 그 화학구조는 2- O-(9z,12z-octadecadienyl)-3-O-[α-galactopyranosyl-(1"-6')-O-β-D-galactopyranosyl]glycerol이었다.The present inventors chromatographed an effective fraction of butanol fraction (CBB) in Sephadex LH-20 and divided the three small fractions CBB-10, CBB-20 and CBB-30 into animal experiments. All small fractions of the species were valid. CBB-20 did not separate the active ingredient into the fraction containing sugar, and CBB-30 did not separate the component because it contained a polyphenolic compound. However, three single substances CBB-11, CBB-12 and CBB-13 could be separated and purified from the CBB-10 subfraction. As a result of the chemical structure, CBB-11 was known as onitin and CBB-12 was known as daucosterol, but since CBB-13 is a novel compound, it was named shinbarometzin. The structure of this compound is as follows and its chemical structure is 2-O- (9z, 12z-octadecadienyl) -3-O- [α-galactopyranosyl- (1 "-6 ')-O-β-D-galactopyranosyl] It was glycerol.

CBB-13(신바로메틴)과 유사한 구조식을 가진 화합물들이 천남성(Arisaema amurenseMax.)으로부터 분리·보고된 바 있으나[Jung, Jee H. ; Lee, Hongkun ; Kang, Sam Sik, Diacyglycerylgalactosides fromArisaema amurense. Phytochemistry, 42(2), 447(1996)], 이들 보고된 화합물들은 1번과 2번 위치에 여러 가지 포화지방산 또는 불포화지방산이 에스테르 결합을 하고 있으나, CBB-13(신바로메틴)은 글리세롤의 1번 위치에 포화 또는 불포화지방산의 에스테르 결합이 없는 물질로서 신규 물질임을 입증하였다.Compounds having a structural formula similar to CBB-13 ( sincbarmethine ) have been isolated and reported from Arisaema amurense Max. [Jung, Jee H .; Lee, Hongkun; Kang, Sam Sik, Diacyglycerylgalactosides from Arisaema amurense. Phytochemistry , 42 (2), 447 (1996)], but these reported compounds have ester linkages of various saturated or unsaturated fatty acids at positions 1 and 2, but CBB-13 (sincbarmethine) is a derivative of glycerol. It was proved to be a novel material with no ester bond of saturated or unsaturated fatty acids at position 1.

한편 천수근의 유효분획인 에틸아세테이트 분획(LNE분획)와 부탄올 분획(LNB분획)을 합치고 실리카겔에서 크로마토그래피를 실시하여 LN-1, LN-2, LN-3를 각각 분리·정제하였다. 화학구조를 규명한 결과 LN-1은 하르파고시드(harpagoside), LN-2는 스타키오스(starchyose), LN-3는 하르파지드(harpagide)임을 동정하였다. 하르파고시드와 하르파지드는 천수근에서 이미 분리·보고된 화합물이며[von H. L.ichti:; A. von Wartburg, Die struktur des Harpagosides. Helvetica ChinicaActa, 49, Fasciculus 5, 1552(1996)], 스타키오스는 면실과 대두에 그 함량이 비교적 많은 사당류의 일종으로(김동훈 저, 식품화학, 185쪽, 1978, 탐구당), 금회 천수근에서는 본 발명자들이 처음으로 분리한 것이다.On the other hand, ethyl acetate fraction (LNE fraction) and butanol fraction (LNB fraction), which are effective fractions of Chunsu Geun, were combined and chromatographed on silica gel to separate and purify LN-1, LN-2 and LN-3, respectively. As a result of the chemical structure, it was identified that LN-1 is harpagoside, LN-2 is starchyose, and LN-3 is harpagide. Harpagoside and harpazide are compounds that have already been isolated and reported in the shallow water [von H. L.ichti :; A. von Wartburg, Die struktur des Harpagosides. Helvetica Chinica Acta, 49, Fasciculus 5, 1552 (1996)], Stachyose is a type of tetrasaccharide that has a relatively high content in cotton and soybeans (Kim Dong-hoon, Food Chem., P. 185, 1978). The inventors separated for the first time.

본 발명자들은 구척과 천수근에서 분리·정제한 화합물에 대하여 관절염에 유효성을 검정하였다. 그 결과 CBB-13(신바로메틴, 신규)와 LN-3(하르파지드)가 가장 약효가 뛰어났고 CBB-11(오니틴)과 LN-1(하르파고시드)는 중정도의 약효가 있음을 입증하였다. 또한 하르파고시드를 알카리성에서 가열하면 하르파지드가 얻어진다는 사실은 이미 알려져 있었으나[Kikuchi, Tohru ; Matsuda, Satoko ; Kubo Yoko ; Namba, Tsuneo, New iridoid glucosides fromHarpagophytum procumbensDC.Chem. Pharm. Bull.,31(7), 2296(1983)], 금회 본 발명자들은 알카리성에서 가열하지 않고 실온에서도 하르파고시드로부터 하르파지드가 생성된다는 결과를 얻었다.The present inventors assayed the effectiveness of arthritis for the compounds isolated and purified from the umbilical cord and the medullary muscle. As a result, CBB-13 (sinbarometin, new) and LN-3 (harpazide) had the highest efficacy, while CBB-11 (onitine) and LN-1 (harpagoside) had moderate efficacy. Proved. It has also been known that the heating of the hapagosides in alkaline yields the hapazide [Kikuchi, Tohru; Matsuda, Satoko; Kubo Yoko; Namba, Tsuneo, New iridoid glucosides from Harpagophytum procumbens DC. Chem. Pharm. Bull., 31 (7), 2296 (1983)], the present inventors have obtained that the hapazides are produced from harpagoside even at room temperature without heating in alkaline.

본 발명의 목적은 구척의 항관절염 효과가 있는 성분인 신바로메틴(신규)와 오니틴이 유효성분이라는 점, 천수근의 성분인 하르파지드와 하르파고시드가 항관절염 효과가 있는 유효성분이라는 점, 항관절염 효과가 비교적 낮은 하르파고시드로부터 항관절염 효과가 매우 높은 하르파지드를 얻을 수 있다는 점에 있다.It is an object of the present invention that cinnabarmethine (new) and onitine, which are components of the anti-arthritis effect of the nasolabial fold, are active ingredients, and that the ingredients of harpazide and harpagoside, which are components of the myelium muscle, are effective ingredients having an anti-arthritis effect. Therefore, it is possible to obtain harpazide having a very high anti-arthritis effect from harpagoside having a relatively low anti-arthritis effect.

본 발명에서는 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부, 및/또는 천수근(Harpagophytum procumbensDC.) 10 내지 300중량부 또는 그의 엑스를 주성분으로 함유하고, 필요하면 맥아(HORDEI FRUCTUS GERMINIATUS) 10 내지 200중량부, 우슬[ACHYRANTHIS BIDENTATAE RADIX) 50 내지 500중량부, 당귀(ANGELICAE GIGANTIS RADIX) 50 내지500중량부, 숙지황(REHMANNIAE RADIX PREPARAT) 50 내지 500중량부, 두충(EUCOMMIAE CORTEX) 50 내지 500중량부, 육계(CINNAMOMI CORTEX) 50 내지 500중량부, 홍화자(CARTHAMI FRUCTUS) 50 내지 500중량부, 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 내지 500중량부, 오공(SCOLOPENDRA) 10 내지 200중량부, 오가피(ACANTHOPANACIS CORTEX) 10 내지 200중량부, 방풍(LEDEBOURIELLAE RADIX) 10 내지 200중량부, 감초(GLYCYRRHIZAE RADIX) 10 내지 200중량부, 구기자(LYCII FRUCTUS) 50 내지 500중량부, 속단(DIPSACI RADIX) 100 내지 500중량부, 녹각(CERVI CORNU) 100 내지 500중량부, 단삼(SALVIAE MILTIORRHIZAE RADIX) 20 내지 200중량부, 산사(CRATAEGII FRUCTUS) 10 내지 200중량부, 현삼(SCROPHULARIAE RADIX) 10 내지 200중량부, 익모초(LEONURI HERBA) 100 내지 500중량부, 신곡(MASSA MEDICATA FERMENTATA) 50 내지 500중량부, 약콩(SOJAE SEMEN) 50 내지 500중량부, 적두(PHASEOLI SEMEN) 10 내지 200중량부에서 선택된 1종 이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소 또는 염소화탄화수소에서 선택된 용매로 추출한 엑스가 첨가된 조성물이 분자생물학적 실험과 동물실험에서 관절염과 염증을 억제하는 놀라운 사실을 발견하여 본 발명을 완성하였다.In the present invention, 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. It contains as a main ingredient, if necessary, 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of cowl (ACHYRANTHIS BIDENTATAE RADIX), 50 to 500 parts by weight of Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of liquorice (GLYCYRRHIZAE RADIX) (LYCII FRUCTUS) within 50 500 parts by weight, DIPSACI RADIX 100 to 500 parts by weight, CERVI CORNU 100 to 500 parts by weight, 20 to 200 parts by weight of SALVIAE MILTIORRHIZAE RADIX, 10 to 200 parts by weight of Sansa (CRATAEGII FRUCTUS), 10 to 200 parts by weight of scorching radish, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of soybean (SOJAE SEMEN), PHASEOLI SEMEN 10 to 200 parts by weight of one or more supplements selected from supplementary medicines or solvents selected from water, lower alcohols, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons are added to the composition to inhibit arthritis and inflammation in molecular biological experiments and animal experiments The present invention was completed by discovering surprising facts.

따라서 본 발명의 목적은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부, 및/또는 천수근(Harpagophytum procumbensDC.) 10 내지 300중량 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소 또는 염소화탄화수소에서 선택된 용매로 추출한 엑스를제공하는 것이다.Accordingly, an object of the present invention is 10 to 200 parts by weight of the spine (CIBOTII RHIZOMA: dried roots of the Cibotium barometz (L.)), and / or 10 to 300 parts by weight or / or Harpagophytum procumbens DC. It provides X extracted with a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbon or chlorinated hydrocarbon.

본 발명의 다른 목적은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부, 및/또는 천수근(Harpagophytum procumbensDC.) 10 내지 300중량부 또는 이의 엑스를 주성분으로 함유하고, 여기에 맥아(HORDEI FRUCTUS GERMINIATUS) 10 내지 200중량부, 우슬[ACHYRANTHIS BIDENTATAE RADIX) 50 내지 500중량부, 당귀(ANGELICAE GIGANTIS RADIX) 50 내지 500중량부, 숙지황(REHMANNIAE RADIX PREPARAT) 50 내지 500중량부, 두충(EUCOMMIAE CORTEX) 50 내지 500중량부, 육계(CINNAMOMI CORTEX) 50 내지 500중량부, 홍화자(CARTHAMI FRUCTUS) 50 내지 500중량부, 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 내지 500중량부, 오공(SCOLOPENDRA) 10 내지 200중량부, 오가피(ACANTHOPANACIS CORTEX) 10 내지 200중량부, 방풍(LEDEBOURIELLAE RADIX) 10 내지 200중량부, 감초(GLYCYRRHIZAE RADIX) 10 내지 200중량부, 구기자(LYCII FRUCTUS) 50 내지 500중량부, 속단(DIPSACI RADIX) 100 내지 500중량부, 녹각(CERVI CORNU) 100 내지 500중량부, 단삼(SALVIAE MILTIORRHIZAE RADIX) 20 내지 200중량부, 산사(CRATAEGII FRUCTUS) 10 내지 200중량부, 현삼(SCROPHULARIAE RADIX) 10 내지 200중량부, 익모초(LEONURI HERBA) 100 내지 500중량부, 신곡(MASSA MEDICATA FERMENTATA) 50 내지 500중량부, 약콩(SOJAE SEMEN) 50 내지 500중량부, 적두(PHASEOLI SEMEN) 10 내지 200중량부에서 선택된 1종 이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소 또는 염소화탄화수소에서 선택된 용매로 추출한 엑스가 첨가된 조성물을 제공하는 것이다.Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of vertebrae (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Or it contains X as a main ingredient, here 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of ACHYRANTHIS BIDENTATAE RADIX, 50 to 500 parts by weight of Angelicae GIGANTIS RADIX, Suhji sulfur (REHMANNIAE) RADIX PREPARAT) 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of licorice (GLYCYRRHIZAE RADIX) Department, Goji (LYCII FRUCTU) S) 50 to 500 parts by weight, DIPSACI RADIX 100 to 500 parts by weight, CERVI CORNU 100 to 500 parts by weight, Salvia Militare (SALVIAE MILTIORRHIZAE RADIX) 20 to 200 parts by weight, Sansa (CRATAEGII FRUCTUS) 10 to 200 Parts by weight, 10-200 parts by weight of scorphoulea radix, 100-500 parts by weight of LEONURI HERBA, 50-500 parts by weight of MASSA MEDICATA FERMENTATA, 50-500 parts by weight of soybean (SOJAE SEMEN), red beans ( PHASEOLI SEMEN) to provide a composition to which the extract extracted with a solvent selected from one or more auxiliary herbs selected from 10 to 200 parts by weight or water, lower alcohol, ethyl acetate, aromatic hydrocarbon or chlorinated hydrocarbon.

본 발명의 또다른 목적은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부, 및/또는 천수근(Harpagophytum procumbensDC.) 10 내지 300중량 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소 또는 염소화탄화수소에서 선택된 용매로 추출한 엑스를 함유한 조성물에 약제학적으로 통상으로 허용되는 부형제, 보조제, 희석제, 보존제, 등장화제, 활탁제 등에서 선택된 성분을 혼합하고 통상의 약학적 제제를 제조하는 방법으로 통상의 약학적 제제로 제제화한 약학적 제제를 제공하는 것이다.Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae ( CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Or a component selected from excipients, adjuvants, diluents, preservatives, isotonic agents, suspending agents, etc., which are pharmaceutically acceptable in a composition containing X extracted with a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons thereof. It is to provide a pharmaceutical formulation formulated into a conventional pharmaceutical formulation by mixing and preparing a conventional pharmaceutical formulation.

본 발명의 또다른 목적은 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부, 및/또는 천수근(Harpagophytum procumbensDC.) 10 내지 300중량부 또는 이의 엑스를 주성분으로 함유하고, 여기에 맥아(HORDEI FRUCTUS GERMINIATUS) 10 내지 200중량부, 우슬[ACHYRANTHIS BIDENTATAE RADIX) 50 내지 500중량부, 당귀(ANGELICAE GIGANTIS RADIX) 50 내지 500중량부, 숙지황(REHMANNIAE RADIX PREPARAT) 50 내지 500중량부, 두충(EUCOMMIAE CORTEX) 50 내지 500중량부, 육계(CINNAMOMI CORTEX) 50 내지 500중량부, 홍화자(CARTHAMI FRUCTUS) 50 내지 500중량부, 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 내지 500중량부, 오공(SCOLOPENDRA) 10 내지 200중량부, 오가피(ACANTHOPANACIS CORTEX) 10 내지 200중량부, 방풍(LEDEBOURIELLAE RADIX) 10 내지 200중량부, 감초(GLYCYRRHIZAE RADIX) 10 내지 200중량부, 구기자(LYCII FRUCTUS) 50 내지 500중량부, 속단(DIPSACI RADIX) 100 내지 500중량부, 녹각(CERVI CORNU) 100 내지 500중량부, 단삼(SALVIAEMILTIORRHIZAE RADIX) 20 내지 200중량부, 산사(CRATAEGII FRUCTUS) 10 내지 200중량부, 현삼(SCROPHULARIAE RADIX) 10 내지 200중량부, 익모초(LEONURI HERBA) 100 내지 500중량부, 신곡(MASSA MEDICATA FERMENTATA) 50 내지 500중량부, 약콩(SOJAE SEMEN) 50 내지 500중량부, 적두(PHASEOLI SEMEN) 10 내지 200중량부에서 선택된 1종 이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸에서 선택된 용매로 추출한 엑스를 함유하고 여기에 약제학적으로 통상으로 허용되는 부형제, 보조제, 희석제, 보존제, 등장화제, 활탁제 등에서 선택된 성분을 혼합하고 통상의 약학적 제제를 제조하는 방법으로 통상의 약학적 제제로 제제화한 약학적 제제를 제공하는 것이다.Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae ( CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Part or its extract as a main component, and here 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of ACHYRANTHIS BIDENTATAE RADIX, 50 to 500 parts by weight of Angelica GIGANTIS RADIX REHMANNIAE RADIX PREPARAT) 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing) TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50-500 parts by weight, 10-200 parts by weight of SCOLOPENDRA, 10-200 parts by weight of ACANTHOPANACIS CORTEX, 10-200 parts by weight of windproof (LEDEBOURIELLAE RADIX), GLYCYRRHIZAE RADIX 10-200 Parts by weight, wolfberry (LYCII FRU) CTUS) 50 to 500 parts by weight, 100 to 500 parts by weight of DIPSACI RADIX, 100 to 500 parts by weight of CERVI CORNU, 20 to 200 parts by weight of SALVIAEMILTIORRHIZAE RADIX, 10 to 200 parts by weight of Sansa (CRATAEGII FRUCTUS) Tofu, 10 to 200 parts by weight of scalloped radish, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of soya beans (SOJAE SEMEN), red beans (PHASEOLI) SEMEN) containing from 10 to 200 parts by weight of one or more auxiliary herbs or extracts thereof selected from solvents selected from water, lower alcohols and ethyl acetate, and excipients, adjuvants, diluents, preservatives, isotonics which are pharmaceutically acceptable herein. It is to provide a pharmaceutical formulation formulated into a conventional pharmaceutical preparation by mixing the ingredients selected from a topical agent, a suspending agent and the like and preparing a conventional pharmaceutical preparation.

도 1은 구성약제로부터 증류수 및 유기 용매로 추출, 감압 농축, 동결 건조한 의약 조성물의 건조중량을 회수하는 공정에 관한 것이며(a), 유효약물의 추출 과정에 따르는 유기용매 분획 추출방법을 나타낸 것이다(b, c).Figure 1 relates to a process for recovering the dry weight of the pharmaceutical composition extracted from the constituents with distilled water and organic solvents, concentrated under reduced pressure, freeze-dried (a), shows an organic solvent fraction extraction method according to the extraction process of the active drug ( b, c).

도 2는 골질환에서 대표적인 관절염의 유발부위인 관절에서의 발병원인기전(a), 부위(b), 발병과정의 분자생물학적 신호전달 경로에 관한 것이다(c).Figure 2 relates to the pathogenesis factors (a), site (b), molecular biological signaling pathways of the pathogenesis in the joint, a representative site of arthritis in bone disease (c).

도 3은 환자로부터 만성 및 퇴행성 골질환인 대표적인 관절염의 유발부위인 관절에서의 활액세포의 생존 및 세포형태학적 양상을 나타낸 것이다.Figure 3 shows the survival and cytomorphic appearance of synovial cells in the joints, which are representative sites of representative arthritis, chronic and degenerative bone diseases from patients.

도 4은 의약 조성물의 유기용매 분획인 각 분획물을 75㎍/ml이 되게 2주간 경구투여하여 처리하여 관절염의 만성 및 퇴행성 골질환 증상의 한 부분인 부종을 유도 후 70일간 부종억제 현상을 나타낸 것이다.Figure 4 shows the treatment of each fraction of the organic solvent fraction of the pharmaceutical composition to oral administration to 75 ㎍ / ml for 2 weeks to show the phenomenon of edema inhibition 70 days after induction of edema which is a part of the symptoms of chronic and degenerative bone disease of arthritis .

도 5은 유효약물인 유기용매를 10 ㎍/ml의 농도에서 처리하여 수정란에 2일간 37℃ 항온배양기에 배양한 후 활액세포를 5x 103세포수로 CAM상에 조심해서 주사기로 주입하여 3일간 관찰하여 나타난 CAM의 수와 분포양상을 나타낸 것이다5 is treated with an organic solvent as an effective drug at a concentration of 10 ㎍ / ml and cultured in a fertilized egg at 37 ℃ incubator for 2 days and then carefully injected syringe with 5 × 10 3 cell number on the CAM with a syringe for 3 days It shows the number and distribution of CAMs observed.

도 6은 의약 조성물인 물추출물을 관절염의 유도쥐에 처리하여 70일 경과후, 연골조직의 파괴정도를 H/E 조직염색을 나타낸 것이다.Figure 6 shows the treatment of water extracts of the pharmaceutical composition in the arthritis induced rats after 70 days, the degree of cartilage tissue destruction H / E tissue staining.

도 7은 의약 조성물인 단방약제의 유기용매 분획물을 10 ㎍/ml 농도에서 24시간 처리하여 Raw264.7 세포주에 LPS를 자극하여 NO생성을 억제 하는지를 나타낸 막대 그래프이며(a) 상기방법에 의해 유기 용매 분획물인 CBB분획분과 LNE분획분에 대하여 LPS를 자극하여 NO생성을 억제 하는지를 나타낸 막대 그래프이다(b).7 is a bar graph showing whether the organic solvent fraction of the mono-drug, which is a pharmaceutical composition, is treated for 24 hours at a concentration of 10 ㎍ / ml for 24 hours to inhibit NO production by stimulating LPS in a Raw264.7 cell line. It is a bar graph showing whether NO inhibits NO production by stimulating LPS for fractions CBB and LNE.

도 8은 유효약물인 유기 분획물을 약 20㎍/ml정도 처리후 활액 세포주에 LPS를 자극하여 세포사를 유도하는지의 현상을 나타낸 것이며 동 방법에 근거하여 활액세포상에서도 동일한 양상이 나타나는지를 관찰한 것이다.Figure 8 shows the phenomenon of inducing cell death by stimulating LPS to synovial cell lines after treatment with an organic fraction of about 20 ㎍ / ml, and observed whether the same pattern appears on the synovial cells based on the same method.

도 9은 유효약물인 유기 분획물을 약 20㎍/ml정도 처리후 Raw 264.7 세포주에 LPS를 자극하여 세포주기에 미치는 영향을 flow cytometry 분석을 실시한 것이다(a), 동 방법에 근거하여 활액막 세포상에서도 동일한 양상이 나타나는지를 관찰한 것이다(b).9 is a flow cytometry analysis of the effects on the cell cycle by stimulating LPS in Raw 264.7 cell line after treatment with an organic fraction of about 20㎍ / ml (a), the same on synovial membrane cells based on the same method (B) to observe the appearance of the pattern.

도 10은 유효약물인 유기용매를 통해 활액세포주에 LPS를 자극하여 SDS-PAGE electrophoresis을 실시하여 COX-II 효소 단백질의 발현을 억제하는 것인지를 나타낸 것이다.10 shows whether SPS-PAGE electrophoresis is performed to inhibit the expression of COX-II enzyme protein by stimulating LPS to synovial cell lines through an organic solvent, an effective drug.

도 11은 유효약물인 유기용매를 통해 활액세포주에 LPS를 자극하여 RT-PCR을 실시하여 iNOS와 COX-II 효소 합성을 억제하는 것을 나타낸 것이며(a). 동일 방법으로 각 분획에서 iNOS(b)와 COX-II 효소 합성을 억제하는 것을 나타낸 것이다(c).Figure 11 shows that stimulating LPS to the synovial cell line through the organic solvent as an effective drug to inhibit the synthesis of iNOS and COX-II enzyme by RT-PCR (a). In the same way it is shown to inhibit the synthesis of iNOS (b) and COX-II enzyme in each fraction (c).

도 12은 의약 조성물의 활성 성분으로 확인된 상기의 화합물이 관절내 활액막세포의 COX-II를 억제효과를 유도하는지 조사하기 위해 세포를 2차 항체인 FITC를 표지하여 호일로 빛을 차광하여 이를 1시간정도 방치한 다음 이를 형광현미경에서 관찰한 것이다.FIG. 12 shows that the compound identified as the active ingredient of the pharmaceutical composition induces the inhibitory effect of COX-II of intramucosal synovial cells. After standing for some time, it was observed under a fluorescence microscope.

도 13은 의약 조성물의 유기용매 분획인 각 분획물을 75㎍/ml이 되게 2주간 경구투여하여 처리하여 관절염의 만성 및 퇴행성 골질환 증상의 한 부분인 부종을 유도 후 70일간 부종억제 현상을 나타낸 것을 x-ray로 사진촬영한 결과이다.FIG. 13 shows the treatment of edema, which is a part of chronic and degenerative bone disease symptoms of arthritis, after 70 days of oral administration of each fraction, which is an organic solvent fraction of the pharmaceutical composition, to 75 µg / ml, for 70 days. The result is a photo taken with x-ray.

도 14은 의약 조성물의 물추출물을 내원환자의 disc 환자를 대상으로 임상적인 개선 효과를 CT 촬영한 결과이다.14 is a CT scan of the water extract of the pharmaceutical composition of the clinical improvement effect in the disc patients of the hospital patient.

도 15은 의약 조성물을 내원환자의 disc 환자를 대상으로 임상적인 개선효과를 MRI 촬영한 결과이다.15 is a result of MRI imaging the clinical improvement effect of the pharmaceutical composition in the disc patients of the inpatients.

도 16은 내원환자의 disc 환자를 대상으로 척추신경 마비현상의 기전에 대한 nogo-A의 존재부위에 대한 그림이다.16 is a diagram illustrating the presence of nogo-A on the mechanism of spinal nerve palsy in disc patients of hospital patients.

도 17은 신경전달 마비에 관한 신경부위인 axon 주위에 존재하는 oligodendricyte에서 injury가 야기되고 nogo-A가 분비하므로 신경전달이 차단되는 경로를 나타낸 것이다.FIG. 17 illustrates a pathway in which neurotransmission is blocked because injury is caused in oligodendricyte existing around axon, which is related to neurotransmission paralysis, and nogo-A is secreted.

도 18은 신경전달 마비에 관한 신경부위인 axon 주위에 존재하는 oligodendricyte에서 injury가 야기되는 것처럼 nogo-A를 발현시킨 뇌세포에 신경전달이 차단되는 경로를 나타낸 것이고 NGF나 CBB-13/LNE-2를 처리시 신경전달이 원상태로 복귀되는 neutrite가 재생되어 신경전달이 복귀되는 양상을 나타낸 것이다.FIG. 18 shows a pathway in which neurotransmission is blocked in nogo-A-expressing brain cells as if injury is caused in oligodendricyte around axon, a neuronal paralysis associated with neurotransmission, and NGF or CBB-13 / LNE-2 Neutrite regenerates when neurotransmission returns to its original state during the treatment.

상기 생약성분은 각각 분말화하거나, 또는 각각 단독으로 추출하여 엑스를 얻고 이들 엑스를 혼합하거나 또는, 필요한 생약성분을 합하고 함께 추출하여도 좋다.The herbal ingredients may be powdered, or each may be extracted alone to obtain an extract, and these extracts may be mixed, or the necessary herbal ingredients may be combined and extracted together.

다음에 실시예 및 실험예로서 본 발명을 더욱 상세히 기재하며, 이들이 본 발명의 범위를 한정하는 것은 아니다.Next, the present invention will be described in more detail with reference to Examples and Experimental Examples, which do not limit the scope of the present invention.

실시예 1Example 1

1. 방풍, 우슬 오가피, 두충, 당귀을 주원료로 구판(자라), 구기자, 숙지황, 육계(계피), 속단, 녹각, 단삼, , 산사, 현삼, 익모초, 마발, 구척, 오공등 한방약제를 부 원료로 가감해서 총건조중량이 2 kg되게 중량하여 3차 증류수(또는 정제수)로 2 ∼ 5배의 부피로 배합하여 150℃ 조건하에 6시간동안 서서히 열을 가하여 최종적인 부피가 1,000 ml이 되게 한 다음, 이를 감압 농축하여 200 ml이 되게 한후 이를 동결시켜 300g의 동결건조중량을 얻었다(도1 a).1. The main ingredients of windproof, hyssop scabies, tofu, and dong mulberry as raw materials such as gupan (Growa), gojija, hungjihwang, broiler (cinnamon), soybean paste, green tea, sweet ginseng,, Sansa, Hyunsam, motherwort, walnut, gukcheok, goku, etc. The total dry weight is added to 2 kg and mixed with 2 to 5 times the volume of tertiary distilled water (or purified water). After concentrating it under reduced pressure to 200 ml and freezing it, a freeze-dried weight of 300 g was obtained (FIG. 1 a).

2. 물 추출물에서 추출, 분리 정제되는 조성물이다. 본 발명에서는 이러한 추출방법에서 건조중량을 각각 2 kg을 증류수 5,000 ml에 섞어서 150℃ 끊는 수조에, 6시간 중탕한 후 가제로 여과, 추출하여 나온 여과액을 끓는 수욕상에서 8시간 환류 추출하였고 같은 방법으로 1회 더 추출하였다. 이를 다시 감압농축하여 동결건조하여 350g정도의 건조중량을 획득하였다. 본 발명의 물추출물 엑스를 분리, 정제하는 공정을 제공하는 것은 또 다른 목적으로 한다.2. A composition that is extracted and separated from the water extract. In the present invention, 2 kg of dry weight in each extraction method was mixed with 5,000 ml of distilled water, and then heated in a 150 ° C water bath for 6 hours, filtered with gauze, and extracted from the filtrate under reflux for 8 hours in a boiling water bath. Extracted once more. It was concentrated under reduced pressure again and lyophilized to obtain a dry weight of about 350 g. It is another object of the present invention to provide a process for separating and purifying water extracts X.

상기와 같은 일련의 건조 과정에 수득되는 물엑스는 각각 후술하는 실시예에 의해 입증하는 바와 같이 골다공증, 류마티스 관절염, 항 신경전달마비 및 항소염 활성효과를 가지고 있기 때문에 물엑스를 비롯한 각 유기용매는 본 발명의 조성물에서 유효성분으로 사용될수 있다(도 1).The water extract obtained in the above-described drying process has osteoporosis, rheumatoid arthritis, anti-neurotransmission paralysis and anti-inflammatory activity, as demonstrated by the examples described below. Can be used as an active ingredient in the composition of the present invention (Fig. 1).

실시예 2Example 2

생약의 추출 및 분획Extraction and fractionation of herbal medicine

1) 구판(CR) : 50 g의 구판 분말에 물 250 ml와 부탄올 250 ml를 가하여 끓는 수욕상에서 8시간 환류추출하였고 같은 방법으로 1회 더 추출하였다. 수층은 여과하고 동결건조하여(CRW) 1.88g을 얻었다. 부탄올층은 감압후에 건조시켜(CRB) 1.46 g을 얻었다.1) Sphere (CR): 250 g of water and 250 ml of butanol were added to 50 g of sphere powder, and refluxed for 8 hours in a boiling water bath. The aqueous layer was filtered and lyophilized (CRW) to give 1.88 g. The butanol layer was dried under reduced pressure (CRB) to give 1.46 g.

2) 오공(SS) : 오공분말 350 g에 2 L의 70 % 에탄올을 가해 실온에서 7일간 냉침추출을 하였다. 냉침추출을 2회 더 실시하여 70 % 에탄올 엑스 98 g을 얻었다. 이 엑스를 500 ml의 물에 현탁시키고 헥산(500 ml×3회) 및 부탄올(500 ml×3회)으로 차례로 추출하여 헥산분획(SSH) 70 g, 부탄올분획(SSB) 12.87 g을 각각 얻었다. 수층은 동결건조하여(SSW) 11.48 g을 얻었다.2) Pore (SS): To 350 g of pore powder, 2 L of 70% ethanol was added, followed by cold extraction for 7 days at room temperature. Cold extraction was carried out two more times to obtain 98% of 70% ethanol. The extract was suspended in 500 ml of water and extracted sequentially with hexane (500 ml x 3) and butanol (500 ml x 3) to obtain 70 g of hexane fraction (SSH) and 12.87 g of butanol fraction (SSB), respectively. The aqueous layer was lyophilized (SSW) to give 11.48 g.

3) 우슬(AB), 구척(CB), 천수근(LN) : 우슬 분말 1,507 g, 구척 분말 1,848 g 및 천수근 분말 1,205 g에 70 % 에탄올 8 L씩을 각각 가하여 수욕상에서 6시간씩 4회 추출하여 우슬 엑스 408 g, 구척 엑스 404 g, 천수근 엑스 598 g을 각각 얻었다. 각각 엑스를 물 1 L에 현탁시키고 헥산(1 L×3회) 및 부탄올(1 L×3회)으로 차례로 추출하였다. 각각의 추출액을 감압농축하였다. 각각의 70 % 에탄올 엑스 및 용매 분획의 수율은 다음과 같다(도 1 b).3) Emulsion (AB), Gucheok (CB), Cheon Soo-Geun (LN): 1,507 g of Emulsion Powder, 1,848 g of Gucheok Powder, and 1,205 g Cheonsu-Geun Powder, respectively, add 8 L of 70% ethanol and extract 4 times in water bath for 6 hours. X 408 g, Guol X 404 g, and Cheon Geun X 598 g were obtained, respectively. Each was suspended in 1 L of water and extracted sequentially with hexane (1 L × 3 times) and butanol (1 L × 3 times). Each extract was concentrated under reduced pressure. Yields of each 70% ethanol extract and solvent fractions are as follows (FIG. 1 b).

우슬(AB) 엑스 : 70 % 에탄올 408 g, ABE 10.3 g, ABB 44 g, ABW 342 g ;Wedge (AB) x: 70% ethanol 408 g, ABE 10.3 g, ABB 44 g, ABW 342 g;

구척(CB) 엑스 : 70 % 에탄올 404 g, CBE 52 g, CBB 93 g, CBW 263 g ;CB extract: 70% ethanol 404 g, CBE 52 g, CBB 93 g, CBW 263 g;

천수근(LN) 엑스 : 70 % 에탄올 598 g, LNE 49 g, LNB 85 g, LNW 456 g.LN X: 70% Ethanol 598 g, LNE 49 g, LNB 85 g, LNW 456 g.

실시예 3Example 3

구척 CBB분획으로부터 소분획 CBB-10, CBB-20 및 CBB-30의 제조Preparation of Small Fractions CBB-10, CBB-20, and CBB-30 from Old CBB Fractions

실시예 2에서 분리된 CBB분획 90 g을 80 % 메탄올 300 ml에 녹이고, 이 용액을 3등분하여 3회에 걸쳐 세파덱스 LH-20 칼럼(6×90 cm, 80 % 메탄올로 평형화)에서 크로마토그래피를 실시하였다. 첫 번째 분획(CBB-10)은 백탁액으로 용출되는 것을 모은 것으며, 두 번째 분획(CBB-20)은 설탕이 주로 용출되는 분획을 모은 것이다. 세 번째 분획(CBB-30)은 칼럼을 60 % acetone으로 세척하여 모은 것이다. 각 소분획의 수율은 CBB-10, 2.1 g ; CBB-20, 58 g ; CBB-30, 23 g이었다.90 g of the CBB fraction isolated in Example 2 was dissolved in 300 ml of 80% methanol, and the solution was triturated and chromatographed on a Sephadex LH-20 column (6 × 90 cm, equilibrated with 80% methanol) in three times. Was carried out. The first fraction (CBB-10) is a collection of eluted as a white turbidity, and the second fraction (CBB-20) is a collection of mainly eluted sugars. The third fraction (CBB-30) was collected by washing the column with 60% acetone. The yield of each small fraction was CBB-10, 2.1 g; CBB-20, 58 g; 23 g of CBB-30.

실시예 4Example 4

구척의 소분획 CBB-10으로부터 화합물 CBB-11, CBB-12 및 CBB-13의 분리Isolation of Compounds CBB-11, CBB-12, and CBB-13 from Small Fractional CBB-10

구척의 소분획 CBB-10(2 g)을 실리카겔 칼럼에서 클로로포름/메탄올(CM으로 약칭) 20:1 용매를 출발로 하여 CM 10:1→5:1→3:1→2:1 용매를 차례로 용출시켰다. CM 10:1 용출액에서 화합물 CBB-11(오니틴) 25 mg(메탄올에서 무정형분말)을, CM 5:1 용출액에서 CBB-12(다우코스테롤) 95 mg(메탄올에서 무색 침상결정)을 얻었다. CM 2:1 용출액을 모아 세파덱스 LH 20(용매는 메탄올)에서 정제하여 화합물 CBB-13(신바로메틴, 신규) 420 mg(클로로포름/메탄올 용매에서 무정형)을 얻었다.Old fractional CBB-10 (2 g) was purified by silica gel column starting with chloroform / methanol (abbreviated as CM) 20: 1 solvent, followed by CM 10: 1 → 5: 1 → 3: 1 → 2: 1 solvent. Eluted. 25 mg (Amorphous Powder in Methanol) of Compound CBB-11 (onitine) were obtained in a CM 10: 1 eluent, and 95 mg (Colorless needles in methanol) were obtained in CM 5: 1 Eluent. The CM 2: 1 eluate was collected and purified in Sephadex LH 20 (solvent as methanol) to give 420 mg (amorphous in chloroform / methanol solvent) of compound CBB-13 (sincbarmethine, novel).

1) CBB-11(오니틴)1) CBB-11 (onitine)

융점 : 212℃Melting Point: 212 ℃

1H-NMR(CDCl3)δ : 1.19(6H, s, 10, 11-H3), 2.34(3H, s, 12-H3), 2.59(3H, s, 15-H2), 2.83 (2H, s, 3-H2), 3.00(2H, m, 13-H2), 3.61(2H, m, 14-H2) 1 H-NMR (CDCl 3 ) δ: 1.19 (6H, s, 10, 11-H 3 ), 2.34 (3H, s, 12-H 3 ), 2.59 (3H, s, 15-H 2 ), 2.83 ( 2H, s, 3-H 2 ), 3.00 (2H, m, 13-H 2 ), 3.61 (2H, m, 14-H 2 )

2) CBB-12(다우코스테롤)2) CBB-12 (Dow Costolol)

융점 : 295℃Melting Point: 295 ℃

IRυ(cm-1) : 3409(OH), 2934, 1464, 1379, 1165, 1074, 1024, 801IRυ (cm -1 ): 3409 (OH), 2934, 1464, 1379, 1165, 1074, 1024, 801

3) CBB-13(제이스바로메틴, 신규)3) CBB-13 (Jace Barrometin, New)

융점 : 143℃Melting Point: 143 ℃

[α]: +0.642(c=1.25.%, 메탄올)[α] : +0.642 (c = 1.25.%, Methanol)

IRυ(cm-1) : 3400(OH), 3010(C=CH), 1735, 1721(에스테르), 1650, 1638(C=C), 1144, 1074(C-O), 669(CH=CH, cis)IRυ (cm -1 ): 3400 (OH), 3010 (C = CH), 1735, 1721 (ester), 1650, 1638 (C = C), 1144, 1074 (CO), 669 (CH = CH, cis)

UV 한계흡수(메탄올)UV limit absorption (methanol)

1H-NMR(CD3OD) : 표1 1 H-NMR (CD 3 OD): Table 1

13C-NMR(CD3OD) : 표1 13 C-NMR (CD 3 OD): Table 1

LC/MS m/z : 677.5[(M-1)-]LC / MS m / z: 677.5 [(M-1) - ]

EI/MSm/z(%): 354(C21H38O4 +, 3.5)(glycerol linolate), 299([354-C4H7]+, 8.3), 262(C17H30CO+, 18.5), 163(C12H19 +, 5.6), 149(C11H17 +, 7.4), 134(C10H16 +, 29.6), 123(C9H15 +, 7.4), 109(C8H13 +, 18.5), 95(C7H11 +, 37.0), 81(C6H9 +, 54.6), 67(C5H7 +, 57.4), 55(C4H7 +, 74.1)EI / MS m / z (%) : 354 (C 21 H 38 O 4 + , 3.5) (glycerol linolate), 299 ([354-C 4 H 7 ] + , 8.3), 262 (C 17 H 30 CO + , 18.5), 163 (C 12 H 19 + , 5.6), 149 (C 11 H 17 + , 7.4), 134 (C 10 H 16 + , 29.6), 123 (C 9 H 15 + , 7.4), 109 ( C 8 H 13 + , 18.5), 95 (C 7 H 11 + , 37.0), 81 (C 6 H 9 + , 54.6), 67 (C 5 H 7 + , 57.4), 55 (C 4 H 7 + , 74.1)

실시예 5Example 5

화합물 CBB-13 아세테이트의 제조Preparation of Compound CBB-13 Acetate

화합물 CBB-13 분말 40 mg을 피리딘 0.5 ml에 녹이고 무수초산 1 ml를 가하여 잘 혼합한 다음 실온에서 하룻밤 방치하여 상법에 따라 아세칠화 하였다. 반응액을 감압농축한 다음 실리카겔에서 클로로포름/아세칠아세테이트 3:1 용매로 칼럼크로마토그래피를 실시하여 CBB-13의 아세테이트 45 mg(무정형)을 얻었다.40 mg of compound CBB-13 powder was dissolved in 0.5 ml of pyridine, 1 ml of anhydrous acetic acid was added, mixed well, and the mixture was left overnight at room temperature to be acetonitrile according to a conventional method. The reaction solution was concentrated under reduced pressure, and then subjected to column chromatography on silica gel with chloroform / acetate acetate 3: 1 solvent to obtain 45 mg (amorphous) of acetate of CBB-13.

융점 :Melting Point:

IRυ(cm-1) : 2930, 2857, 1752, 1638, 1373, 1229, 1138, 1067, 910, 602IRυ (cm -1 ): 2930, 2857, 1752, 1638, 1373, 1229, 1138, 1067, 910, 602

1H-NMR(CDCl3) : 표1 1 H-NMR (CDCl 3 ): Table 1

13C-NMR(CDCl3) : 표1 13 C-NMR (CDCl 3 ): Table 1

표 1. CBB-13과 그 아세테이트의 핵자기공명테이타Table 1. Nuclear magnetic resonance data of CBB-13 and its acetates

실시예 6Example 6

천수근의 LNE 및 LNB 분획으로부터 화합물 LN-1, LN-2 및 LN-3의분리Isolation of Compounds LN-1, LN-2, and LN-3 from the LNE and LNB Fractions of Chun Soo-Keun

LNE 46 g 및 LNB 81 g을 합하고 여기에 물 500 ml에 현탁시키고 클로로포름/메탄올(CM) 2:1 용액 600ml를 가하여 혼합시킨 후 클로로포름층을 취하였다. 다시 CM 2:1용매로 2회 더 추출하여 클로로포름층을 취하였다. 클로로포름층을 모아 농축하여 38 g의 엑스(Fr.1)를 얻었고 수층을 농축하여 약 80 g의 엑스(Fr.2)를 얻었다. 클로로포름 엑스(Fr.1)를 실리카겔 칼럼에서 정제하고 세파덱스 LH-20(메탄올 용매)에서 재정제하여 화합물(12 g)을 얻었다. 수층 엑스(Fr.2)를 실리카겔 칼럼에서 CM 20:1→10:1→8:1→4:1→2:1 및 메탄올 용매로 크로마토그래피를 실시하였다. CM 10:1 용출액에서 화합물 LN-1(5.1g)을 다시 얻었다. 메탄올 용출액을 모아(6.5 g) 세파덱스 LH-20 칼럼에 50 % 아세톤 수용액으로 3회, 물을 용매로 크로마토그래피를 실시하여 화합물 LN-2(4.5 g)을 얻었다.46 g of LNE and 81 g of LNB were combined and suspended in 500 ml of water, 600 ml of chloroform / methanol (CM) 2: 1 solution was added, mixed, and the chloroform layer was taken. The mixture was extracted two more times with a CM 2: 1 solvent to obtain a chloroform layer. The chloroform layer was collected and concentrated to give 38 g of X (Fr. 1), and the aqueous layer was concentrated to obtain about 80 g of X (Fr. 2). Chloroform X (Fr. 1) was purified on a silica gel column and recrystallized in Sephadex LH-20 (methanol solvent) to obtain a compound (12 g). Aqueous layers (Fr. 2) were chromatographed on a silica gel column with CM 20: 1 → 10: 1 → 8: 1 → 4: 1 → 2: 1 and methanol solvent. Compound LN-1 (5.1 g) was obtained again in a CM 10: 1 eluate. The methanol eluate was collected (6.5 g) and chromatographed with a 50% acetone aqueous solution on a Sephadex LH-20 column three times, and water was chromatographed with a solvent to obtain compound LN-2 (4.5 g).

위의 CM 4:1 용출액을 모아 다시 실리카겔 칼럼에서 CM 4:1로 크로마토그래피를 실시하여 LN-3(0.36 g)을 얻었다. 화합물 LN-1, LN-2 및 LN-3는 각각 하르파고시드, 스타키오스 및 하르파지드였다.The above CM 4: 1 eluate was collected and chromatographed with CM 4: 1 on a silica gel column to obtain LN-3 (0.36 g). Compounds LN-1, LN-2 and LN-3 were harpagoside, starchiose and harfazide, respectively.

1) LN-1(하르파고시드)1) LN-1 (Harpagoside)

융점 : 120℃(무정형)Melting Point: 120 ℃ (Amorphous)

MSm/z(%) : 148([C9H8O2]+, 74.8), 147(79.1), 77(100)MS m / z (%): 148 ([C 9 H 8 O 2 ] + , 74.8), 147 (79.1), 77 (100)

IRυ(cm-1) : 3430(OH), 1690(C=O), 1638(C=C), 1578, 1561, 1543, 1508,1499(benzen), 1186, 1119, 1074, 1015(C-O), 990, 955(CH=CH, trans), 770, 716, 685(CH=CH, cis)IRυ (cm -1 ): 3430 (OH), 1690 (C = O), 1638 (C = C), 1578, 1561, 1543, 1508,1499 (benzen), 1186, 1119, 1074, 1015 (CO), 990 , 955 (CH = CH, trans), 770, 716, 685 (CH = CH, cis)

1H-NMR(CD3OD)δ : 1.53(3H, s, 10-H3), 2.02(1H, dd, J=4.2, 15 Hz, 7-H), 2.27(1H, dt, J=15, 1.2 Hz, 7-H), 2.94(1H, br.s, 9-H), 3.22(1H, dd, J=7.8, 9.0 Hz glc-2), 3.35(1H, d-like, J=9.9 Hz, glc-4), 3.36(2H, ddd, J=2.1, 5.1, 10 Hz, glc-5), 3.41(1H, dd, J=8.7, 9.3 Hz, glc-3), 3.72(1H, dd, J=5.7, 12 Hz, glc-6), 3.76(1H, dd, J=1.2, 4.2 Hz, 6-H), 3.93(1H, dd, J=2.4, 12 Hz, glc-6), 4.62(1H, dd, J=8.1Hz, glc-1), 4.94(1H, dd, J=1.5, 6.3 Hz, 4-H), 6.18(1H, d, J=1.2 Hz, 1-H), 6.41(1H, d, J=6.3 Hz, 3-H), 6.51(1H, d, J=15.9 Hz, C=CH-C=O), 7.38-7.42(3H, m, benzen-3H), 7.6-7.61(2H, m, benzen-2H), 7.67(1H, d, J=15.9 Hz, CH=C-C=O) 1 H-NMR (CD 3 OD) δ: 1.53 (3H, s, 10-H 3 ), 2.02 (1H, dd, J = 4.2, 15 Hz, 7-H), 2.27 (1H, dt, J = 15 , 1.2 Hz, 7-H), 2.94 (1H, br.s, 9-H), 3.22 (1H, dd, J = 7.8, 9.0 Hz glc-2), 3.35 (1H, d-like, J = 9.9 Hz, glc-4), 3.36 (2H, ddd, J = 2.1, 5.1, 10 Hz, glc-5), 3.41 (1H, dd, J = 8.7, 9.3 Hz, glc-3), 3.72 (1H, dd , J = 5.7, 12 Hz, glc-6), 3.76 (1H, dd, J = 1.2, 4.2 Hz, 6-H), 3.93 (1H, dd, J = 2.4, 12 Hz, glc-6), 4.62 (1H, dd, J = 8.1 Hz, glc-1), 4.94 (1H, dd, J = 1.5, 6.3 Hz, 4-H), 6.18 (1H, d, J = 1.2 Hz, 1-H), 6.41 (1H, d, J = 6.3 Hz, 3-H), 6.51 (1H, d, J = 15.9 Hz, C = CH-C = O), 7.38-7.42 (3H, m, benzen-3H), 7.6- 7.61 (2H, m, benzen-2H), 7.67 (1H, d, J = 15.9 Hz, CH = CC = O)

2) LN-2(스타키오스)2) LN-2 (Starchios)

융점 : 148℃Melting Point: 148 ℃

[α]: +138.6[α] : +138.6

1H-NMR(D2O)δ : 3.33(2H, s, β-Fru(f)-1-H2), 4.20(1H, d, J=9.0 Hz, β-Fru(f)-3H), 4.97(2H, d, J=2.4 Hz, 2×α-Gal(p)-anomeric proton), 5.41(1H, d, J=3.9 Hz, α-Glu(p)-anomeric proton) (약어 : β-Fru(f), β-fructofuranosyl; α-Gal(p), α-glactopyranosyl; α-Glu(p), α-glucopyranosyl) 1 H-NMR (D 2 O) δ: 3.33 (2H, s, β-Fru (f) -1-H 2 ), 4.20 (1H, d, J = 9.0 Hz, β-Fru (f) -3H) , 4.97 (2H, d, J = 2.4 Hz, 2 × α-Gal (p) -anomeric proton), 5.41 (1H, d, J = 3.9 Hz, α-Glu (p) -anomeric proton) (abbreviation: β -Fru (f), β-fructofuranosyl; α-Gal (p), α-glactopyranosyl; α-Glu (p), α-glucopyranosyl)

13C-NMR(D2O)δ : 표 2 13 C-NMR (D 2 O) δ: Table 2

표 2. LN-2(스타키오스)의 핵자기공명데이타Table 2. Nuclear Magnetic Resonance Data for LN-2 (Starchios)

위치location LN-2LN-2 표준품Standard 위치location LN-2LN-2 표준품Standard sucrosesucrose α-Gla(p)α-Gla (p) α-Glu(p)α-Glu (p) α-Gal(p)α-Gal (p) 1'One' 92.8692.86 92.8992.89 1"One" 99.1399.13 93.793.7 2'2' 70.2570.25 71.7971.79 2"2" 69.2069.20 69.969.9 3'3 ' 73.4873.48 73.3073.30 3"3 " 70.2570.25 70.270.2 4'4' 69.5669.56 69.9569.95 4"4" 70.1070.10 70.270.2 5'5 ' 72.0472.04 73.1273.12 5"5 " 71.7571.75 71.771.7 6'6 ' 66.6366.63 62.0962.09 6"6 " 67.2467.24 62.562.5 β-Fru(f)β-Fru (f) α-Gal(p)α-Gal (p) 1One 63.2263.22 63.0863.08 1'"One'" 98.7998.79 93.793.7 22 104.56104.56 104.41104.41 2'"2'" 69.0469.04 69.969.9 33 82.1082.10 82.0982.09 3'"3 '" 70.1370.13 70.270.2 44 74.7674.76 74.7374.73 4'"4'" 70.0070.00 70.270.2 55 77.1277.12 77.1677.16 5'"5 '" 71.4471.44 71.771.7 66 61.9061.90 60.8560.85 6'"6 '" 62.2062.20 62.562.5

실시예 7Example 7

LN-2의 완전 산가수분해Complete Acid Hydrolysis of LN-2

LN-2 (20 mg)에 4N HCl/다이옥산/벤젠(3:1:2) 혼합용액 2.4 ml를 가하여 100℃에서 한시간 가열하였다. 냉각 후 수층을 클로로포름/메탄올/물(30:20:5) 용맬 실리카겔 판에서 TLC를 수행하였다. 그결과 과당/포도당/칼락토오스가 1:1:2비율임을 동정할 수 있었다.2.4 ml of 4N HCl / dioxane / benzene (3: 1: 2) mixed solution was added to LN-2 (20 mg), followed by heating at 100 ° C. for one hour. After cooling, the aqueous layer was subjected to TLC on a silica gel plate for chloroform / methanol / water (30: 20: 5). As a result, it could be identified that fructose / glucose / calactose was 1: 1: 1 ratio.

실시예 8Example 8

LN-2의 부분 산가수분해Partial Acid Hydrolysis of LN-2

LN-2 (500 mg)에 2N HCl/다이옥산/벤젠(3:1:2) 혼합용액 5.0 ml를 가하여 93℃에서 40분간 가열하였다. 냉각 후 반응액을 농축한 다음 세파덱스 G-25(용매 : 5 % 에탄올)로 분자여과를 하여 LN-21 (120 mg)을 동결건조하여 얻었다. LN-21을 위의 방법과 같이 완전 산가수분해를 시켜 TLC를 실시한 결과 과당은 검출되지 않고 포도당과 갈락토오스만 검출되었다.5.0 ml of 2N HCl / dioxane / benzene (3: 1: 2) mixed solution was added to LN-2 (500 mg) and heated at 93 ° C. for 40 minutes. After cooling, the reaction solution was concentrated and then molecularly filtered with Sephadex G-25 (solvent: 5% ethanol) to obtain LN-21 (120 mg) by lyophilization. LLC-21 was subjected to TLC with complete acid hydrolysis as in the above method. As a result, only fructose and galactose were detected without fructose.

LN-21(α-디-갈락토피라노실(1"→6')-α-디-갈락토피라노실(1'→6)글루코피라노시드LN-21 (α-di-galactopyranosyl (1 "→ 6 ')-α-di-galactopyranosyl (1' → 6) glucopyranoside

1H-NMR(D2O)δ : 4.66(d, J=8.1 Hz, β-Glu(p)-anomeric proton), 4.98(2H, m, 2×α-Gal(p) -anomeric proton), 5.23(d, J=3.6 Hz, α-Gal(p)-anomeric proton)(4.66ppm면적/5.23ppm면적 비율 = 2.14) 1 H-NMR (D 2 O) δ: 4.66 (d, J = 8.1 Hz, β-Glu (p) -anomeric proton), 4.98 (2H, m, 2 × α-Gal (p) -anomeric proton), 5.23 (d, J = 3.6 Hz, α-Gal (p) -anomeric proton) (4.66 ppm area / 5.23 ppm area ratio = 2.14)

3) LN-33) LN-3

융점 : 120℃(무정형)Melting Point: 120 ℃ (Amorphous)

1H-NMR(CD3OD)δ : 1.24(3H, s, 10-H3), 1.79(1h, ddd, J=0.9, 4.2, 13.8 Hz, 7β-H), 1.90(1H, dd, J=4.8, 13.8 Hz, 7α-H), 2.54(1H, br.s, 9-H), 3.21(1H, dd, J=7.8, 9.0 Hz, 2'-H), 3.31(1H, 4'-H), 3.34(1H, m, 5'-H), 3.38(1H, t, J=9.0 Hz, 3'-H), 3.66(1H, dd, J=5.7, 12.0 Hz, 6'-H), 3.70(1H, t-like, J=∼4.5 Hz,6-H), 3.90(1H, dd, J=1.5, 12.0 Hz, 6'-H), 4.57(1H, d, J=7.8 Hz, 1'-H), 4.94(1H, dd, J=1.5, 6.3 Hz, 4-H), 5.73(1H, d, J=0.9 Hz, 1-H), 6.31(1H, d, J=6.6 Hz, 3-H) 1 H-NMR (CD 3 OD) δ: 1.24 (3H, s, 10-H 3 ), 1.79 (1h, ddd, J = 0.9, 4.2, 13.8 Hz, 7β-H), 1.90 (1H, dd, J = 4.8, 13.8 Hz, 7α-H), 2.54 (1H, br.s, 9-H), 3.21 (1H, dd, J = 7.8, 9.0 Hz, 2'-H), 3.31 (1H, 4'-) H), 3.34 (1H, m, 5'-H), 3.38 (1H, t, J = 9.0 Hz, 3'-H), 3.66 (1H, dd, J = 5.7, 12.0 Hz, 6'-H) , 3.70 (1H, t-like, J = 4.5 Hz, 6-H), 3.90 (1H, dd, J = 1.5, 12.0 Hz, 6′-H), 4.57 (1H, d, J = 7.8 Hz, 1'-H), 4.94 (1H, dd, J = 1.5, 6.3 Hz, 4-H), 5.73 (1H, d, J = 0.9 Hz, 1-H), 6.31 (1H, d, J = 6.6 Hz , 3-H)

실시예 9Example 9

하르파고시드(LN-1)으로부터 하르파지드(LN-3)의 제조Preparation of Harpazide (LN-3) from Harpagoside (LN-1)

하르파고시드(LN-1) 560 mg을 메탄올 20 ml에 녹이고 1N 가성소다 1 ml를 가하여 잘 섞은 다음 하룻밤 실온에 방치하고 세파덱스 LH-20(용매 : 80 % 메탄올)칼럼을 통과시켰다. 용출액으로부터 하르파지드 400 mg과 신나민산 소다(sodium cinnamate) 125 mg을 얻었다.560 mg of harpagoside (LN-1) was dissolved in 20 ml of methanol, 1 ml of 1N caustic soda was added well, left to stand at room temperature overnight, and passed through a Sephadex LH-20 (solvent: 80% methanol) column. From the eluate, 400 mg of harpazide and 125 mg of sodium cinnamate were obtained.

실시예 10Example 10

실시예 6과 유사한 방법으로 1N 가성소다 대신에 2 % 탄산소다 수용액을 가하고 실시예 6과 같이 실시하여 하르파지드와 신나민산소다를 얻었다.In a similar manner to Example 6, 2% aqueous sodium carbonate solution was added instead of 1N caustic soda, and the same procedure as in Example 6 was conducted to obtain harpazide and sodium cinnamic acid.

실시예 11Example 11

천수근으로부터 하르파지드의 다량제조Manufacturing of large quantities of harpajid from Tenshu

천수근 분말 1 Kg을 메탄올 20 L로 실온에서 4회 침출하고 침출액을 모아 감압하에 농축하여 얻은 메탄올 엑스 475 g을 정제수 1.5 L에 녹이고, 헥산 1.5 L로 3회 추출한 다음 수층에 5 % 가성소다 수용액 0.1 L 를 가하여(pH 11.5∼12.5) 잘 혼합한 후 실온에서 하룻밤 방치하였다. 이 반응액을 이소프로필알콜 2L씩 2회 추출하였다. 이소프로필알콜분획을 모아 정제수 1 L로 세척한 다음 이소프로필알콜층을 감압하에 농축하여 15 g의 분말을 얻었다. 이 분말을 95 % 에탄올로 평형화시킨세파덱스 LH-20 칼럼(크기 10×100 cm)에서 크로마토그래피를 실시하였다. 하르파지드가 용출되는 분획(실리카겔 박층크로마토그래피, 용매 : 클로로포름/메탄올/물 70:30:4, Rf값=0.31)을 모아 농축하여 하르파지드 12 g을 얻었다.1 Kg of Chun-Geun Powder was leached 4 times at room temperature with 20 L of methanol, and the leachate was collected and concentrated under reduced pressure. 475 g of methanol was dissolved in 1.5 L of purified water, extracted three times with 1.5 L of hexane, and then the aqueous solution of 5% caustic soda in water was 0.1 L was added (pH 11.5-12.5), mixed well, and it was left to stand overnight at room temperature. The reaction solution was extracted twice with 2 L of isopropyl alcohol. Isopropyl alcohol fractions were collected, washed with 1 L of purified water, and the isopropyl alcohol layer was concentrated under reduced pressure to obtain 15 g of powder. The powder was chromatographed on a Sephadex LH-20 column (size 10 × 100 cm) equilibrated with 95% ethanol. The fractions in which harpazide elutes (silica gel thin layer chromatography, solvent: chloroform / methanol / water 70: 30: 4, Rf value = 0.31) were collected and concentrated to obtain 12 g of harfazide.

실시예 12Example 12

천수근 75g , 당귀150g, 구기자150g, 두충120g, 우슬240g, 육계100g, 홍화자100g, 구판 140g을 실시예 1과 같은 방법으로 물로 추출하여 엑스를 얻는다.Chunsu Geun 75g, Angelica 150g, Gugija 150g, Tofu 120g, 240g, Broiler 100g, Safflower 100g, Old plate 140g in the same manner as in Example 1 to obtain an X.

실시예 13Example 13

천수근 75g , 당귀150g, 구기자150g, 두충120g, 우슬240g, 육계100g, 홍화자100g, 구판 140g을 합하고 분말화한다.75g of Cheonsu-geun, Angelica 150g, Gugija 150g, Tofu 120g, Emulsion 240g, Broiler 100g, Safflower 100g, Old plate 140g are combined and powdered.

실시예 14Example 14

천수근 75g, 당귀150g, 오가피50g, 구기자150g, 두충120g, 우슬240g, 육계100g, 홍화자 100g, 구판140g을 실시예 1과 같은 방법으로 물로 추출하여 엑스를 얻는다.Chunsu Geun 75g, Angelica 150g, Ogapi 50g, Gugijaja 150g, Tofu 120g, Ulsan 240g, Broiler 100g, Safflower 100g, Old plate 140g in the same manner as in Example 1 to obtain X.

실시예 15Example 15

구척50g, 구판140g, 오공50g, 오가피50g, 두충120g, 우슬240g, 방풍50g을 물로 추출하여 엑스를 얻는다.Gucheok 50g, Gupan 140g, Goku 50g, Ogapi 50g, Tofu 120g, 240g dew, 50g windproof is extracted with water to get X.

실시예 16Example 16

맥아 50g, 우슬240g, 당귀150g, 숙지황100g, 두충120g, 육계100g, 홍화자100g, 구판(자라)140g, 오공50g, 오가피50g, 방풍50g, 구척50g, 감초75g, 구기자150g, 속단150g, 녹각150g, 단삼70g, 산사25.5g, 현삼45g, 익모초150g, 신곡 100g,약콩80g, 적두 50g, 천수근75g을 70% 에탄올로 추출하고 에탄올을 제거하여 엑스를 얻는다.Malt 50g, Walnut 240g, Angelica 150g, Sookjihwang 100g, Tofu 120g, Broiler 100g, Safflower 100g, Gupan 140g, Goku 50g, Ogapi 50g, Windproof 50g, Gucheok 50g, Licorice 75g, Gugija 150g, Sokcho 150g, Green 150g , 70g of red ginseng, 25.5g of Sansa, 45g of Hyunsam, 150g of motherwort, 100g of ginseng, 80g of red beans, 50g of red beans, 75g of Chunsu root with 70% ethanol and ethanol is removed to obtain X.

실험 예 1Experimental Example 1

관절염의 부종억제 효과Edema Inhibitory Effect of Arthritis

본 발명의 의약 조성물이 가지는 부종 억제효과를 관찰하기 위해 체중 200g 이내의 흰쥐를 실험군당 6마리를 대상으로 부종을 유도하기 위해 ZYMOSAN-A(20 mg/ml/KG) 0.5ml과 Freund's Adjuvant 0.5ml을 혼합하여 좌측 족삼리에 주사한 후 부종의 진행상태를 70일간 관찰하였는데 부종의 유도 전/후를 사진 촬영하였고 또한 물추출물 및 유기용매 분획을 각각 0.6mg/ml이 되게 조제한 후 흰쥐 체중 1KG당 1ml씩 1일 1회동안 14일간 경구 투여하여 부종 억제 여부를 조사하였다. 시료로는 의약 조성물에서 분리된 유기용매 추출물(실험 예2 참조)인 ABE, ABB, ABW, CBE, CBB, CBW, LNE, LNB, LNW, CRB, CRW, SSH, SSB, SSW를 각각 사용하였다. 관찰된 결과는 부종은 4일부터 유도되었으며 최고 정점에 도달하는데는 약 45일정도 경과하였으며 70일 경과할때까지를 본 연구 기간으로 설정하여 추출물의 부종 억제효과를 관찰하였다. 측정된 결과는 정밀한 게이지를 사용하여 부종 크기를 조사하였다.In order to observe the edema inhibitory effect of the pharmaceutical composition of the present invention in order to induce edema in 6 rats per experimental group in the body of 200g body weight ZYMOSAN-A (20 mg / ml / KG) 0.5ml and Freund's Adjuvant 0.5ml Was mixed and injected into the left foot, and the progress of edema was observed for 70 days. Photographs were taken before and after induction of edema, and water extracts and organic solvent fractions were prepared to be 0.6 mg / ml, respectively. Oral administration for 14 days once a day to investigate whether the edema is suppressed. As a sample, ABE, ABB, ABW, CBE, CBB, CBW, LNE, LNB, LNW, CRB, CRW, SSH, SSB, and SSW, which are organic solvent extracts separated from the pharmaceutical composition, were used, respectively. The observed results showed that the edema was induced from day 4, and it reached about 45 days to reach the highest peak, and the edema inhibitory effect of the extract was observed by setting the period up to 70 days. The measured results were examined for edema size using a precision gauge.

실험예 2Experimental Example 2

류마티스 관절염의 활액막세포의 생존 유무Survival of Synovial Cells in Rheumatoid Arthritis

내원한 류마티스성 관절염환자(rheumatoid artheritis)로부터 분리된 관절 활액막세포를 5% DMEM medium에 penicilin/ streptomycin이 함유한 배지에 104세포를 6 well culture dish에 분주하고 37℃에서 24시간 배양하고 생존하는 세포를 관찰하였으며 세포 존재 유무 및 세포증식 상태를 관찰하기 위해 위상차 현미경의 200배율에서 세포사가 유도된 세포를 촬영하여 조사하였다.Arthroscopy of synovial synovial cells isolated from patients with rheumatoid artheritis was performed by dispensing 10 4 cells in a 6 well culture dish in a medium containing penicilin / streptomycin in 5% DMEM medium and incubating at 37 ° C for 24 hours. The cells were observed, and the cell death induced cells were examined at 200 times magnification of the phase contrast microscope to observe the presence or absence and cell proliferation state of the cells.

실험예 3Experimental Example 3

수정란 장뇨막에서의 활액막세포 혈관신생 유도Induction of Synovial Cell Angiogenesis in Fertilized Egg Uterus

실험 예4에서 분리된 세포를 대상으로 실험 예 2에서 수득된 유기용매 분획물에 대해서 활액세포의 혈관신생의 정도에 미치는 영향을 알아보기 위해 수정란에 활액세포를 주입하고 37℃ incubator에 3일간 배양하여 장뇨막(chorioallantoic membrane: CAM)의 형성을 관찰하였으며 이 상태에서 상기 방법에 의해 분리된 조성물을 각각 10㎍/ml이 되게 CAM위에 분주하여 CAM의 감소정도를 관찰하였다.To investigate the effect of the organic solvent fraction obtained in Experimental Example 2 on the cells isolated in Experimental Example 4 on the degree of angiogenesis of synovial cells, synovial cells were injected into fertilized eggs and cultured in an incubator for 3 days. The formation of chorioallantoic membrane (CAM) was observed, and in this state, the composition separated by the above method was dispensed on the CAM to be 10 µg / ml, respectively.

실험예 4Experimental Example 4

관절염 조직의 연골조직 파괴 정도Cartilage destruction of arthritis tissue

실험 예3에서 의약 조성물인 양근탕과 청파전을 경구투여하여 관절부위에 존재하는 연골의 마모상태 내지 meltalloprotease의 분비로 인한 연골조직 및 연골세포의 파괴정도를 확인하기 위해 hematoxylin/eosin 염색을 통해 위상차 현미경 200 배율로 관찰한 후 사진 촬영을 하였다.In Experimental Example 3, the phase difference was determined by hematoxylin / eosin staining to determine the degree of destruction of cartilage tissue and cartilage cells due to oral administration of the pharmaceutical composition Yang Geun-tang and Cheongpajeon to the secretion of cartilage present in the joint area and secretion of meltalloprotease. After observing with a microscope 200 magnification, the picture was taken.

실험예 5Experimental Example 5

난소 적출흰쥐로부터 골다공증 억제실험Osteoporosis Suppression in Ovarian Extracted Rats

본 발명의 의약 조성물이 가지는 골다공증 억제효과를 관찰하기 위해 체중 200g 암컷의 흰쥐를 대상으로 난소를 제거하여 일일 투여 농도를 5 mg/ml으로 해서 경구투여하여 4주 경과를 확인한 후 이를 대퇴부를 기준으로 위 아래부위 약 1cm 절단후 탈회와 parrafin embeded된 블록을 제작하여 microtome에서 5㎛ 되게 절단 후 hematoxylin/eosin 염색을 통해 위상차 현미경 100 배율로 관찰한 후 사진 촬영을 하였다.In order to observe the osteoporosis inhibitory effect of the pharmaceutical composition of the present invention to remove the ovaries in the female body of 200g body weight of the daily dose of 5 mg / ml oral administration to confirm the course of 4 weeks and then based on the thigh After cutting about 1cm of the upper and lower parts, demineralized and parrafin-embedded blocks were prepared, cut to 5㎛ in the microtome, and observed with a phase contrast microscope at 100 magnification through hematoxylin / eosin staining.

실험예 6Experimental Example 6

리피드폴리사카라이드로 자극한 마이크로파아지 세포내의 NO형성도NO Formation in Microporous Cells Stimulated with Lipidpolysaccharide

본 발명의 방법에 따라 수득된 추출물에 대하여 상기한 바와 같이 부종을 인한 혈액내의 강력한 염증 유발원인 나이트리옥사이드(nitric oxide:NO)의 합성억제를 확인하기 위해 마이크로파아지(macrophage)인 RAW264.7를 96 microplate에 각 well마다 103세포수가 되게 분주한 다음 12시간 배양하여 리피드폴리사카라이드 (lipidpolysachrride: LPS)를 50 ng/well이 되게 각 well에 분주하고 2시간동안 자극을 가한 다음 유기용매 및 각 분획물을 최종농도가 10㎍/ml이 되도록 가한 다음 Greiss reagent 용액 50㎕을 첨가하고 실온에서 반응을 시킨 다음 엘라이저(enzyme-linked immunosorbent assay(ELISA) reader의 540 nm에서 흡광도를 측정하였다. 표준용액인 0.1, 1, 10, 20, 50, 100, 150μM sodium nitrite를 sodium nitroprusside dihydrate(SNP)으로 발색반응을 실시하여 비교 분석하였다.For the extract obtained according to the method of the present invention RAW264.7 which is a macrophage (macrophage) to confirm the inhibition of the synthesis of nitric oxide (NO), a potent source of inflammation in the blood due to edema as described above Dispense 10 3 cells per well into 96 microplates, incubate for 12 hours, and dispense lipidid polysaccharides (lipidpolysachrride (LPS)) into each well at 50 ng / well, stimulate for 2 hours, and then add organic solvent and each Fractions were added to a final concentration of 10 μg / ml, 50 μl of Greiss reagent solution was added, the reaction was performed at room temperature, and the absorbance was measured at 540 nm of an enzyme-linked immunosorbent assay (ELISA) reader. Phosphorus 0.1, 1, 10, 20, 50, 100, 150μM sodium nitrite was analyzed by color reaction with sodium nitroprusside dihydrate (SNP).

실험예 7Experimental Example 7

마크로파아지세포와 활액막 세포주의 세포사 유도Induction of Cell Death of Macrophage and Synovial Cell Lines

본 발명에 따라 분리된 의약 조성물의 활성 성분으로 확인된 상기의 화합물이 염증반응에 관여하는 macrophage인 raw 264.7세포와 활액막 세포의 세포사를 유도하는지 조사하기 위해 세포를 5% DMEM medium에 penicilin/streptomycin이 함유한 배지상에 104세포를 6 well culture dish에 분주하고 37℃에서 24시간 배양하는데 상기 추출물을 10㎍/ml이 되게 첨가하여 반응을 관찰하였다. 세포사 관찰은 편광 현미경의 200배율에서 세포사가 유도된 세포를 촬영하여 조사하였다.In order to investigate whether the compound identified as an active ingredient of the pharmaceutical composition isolated according to the present invention induces cell death of raw 264.7 cells, which are macrophages involved in the inflammatory response, and synovial membrane cells, penicilin / streptomycin was added to 5% DMEM medium. 10 4 cells were dispensed in 6 well culture dish on the containing medium and incubated at 37 ° C. for 24 hours. The extract was added to 10 μg / ml to observe the reaction. Cell death observation was examined by photographing cells induced cell death at 200-fold magnification of the polarization microscope.

실험예 8Experimental Example 8

마크로파아지세포와 활액막 세포주의 세포주기에 미치는 영향Effects of Macrophage and Synovial Cell Lines on Cell Cycle

실험 예7에서처럼 2종의 세포를 배양한 다음 피비에스(phosphate-buffered saline:PBS)로 세척하여 LPS를 50 ng/ml이 되게 각 dish에 분주하고 2시간동안 자극을 가한다음 추출물 최종농도가 20㎍/ml이 되도록 2시간동안 가한 다음 상등액을 제거하고 PBS로 세척한 후 이를 trypsin을 처리하고 세포를 모아서 1.5ml eppendorf tube에 넣은 다음 1,000 rpm에서 5분간 원심분리하여 상등액을 제거한 후 100% EtOH를 1ml 첨가하여 고정한다. 이때 propidium iodide 5㎍/ml와 RNAse을 혼합해서 준비하고 상기 고정된 세포를 원심분리하여 상등액을 제거한 다음 PBS로 한번 세척한다. 동시에 상기 고정된 DNA에 염색시약을 첨가하여 37℃에서 30분간 항온조에서 가온시킨 다음 propidium iodide로 염색한 세포는 호일에 밀봉하여 4℃에 보관하고 유세포 검색분석기(Flow cytometry analysis)를 실시하였는데 분석기기는 FACscan ( Becton Dicknson, CA)이다.Incubate two cells as in Experiment 7, wash them with phosphate-buffered saline (PBS), dispense LPS into each dish at 50 ng / ml, and stimulate for 2 hours. Extract final concentration is 20 After adding the solution to ㎍ / ml for 2 hours, the supernatant was removed, washed with PBS, treated with trypsin, the cells were collected and placed in a 1.5ml eppendorf tube, centrifuged at 1,000 rpm for 5 minutes to remove the supernatant, followed by 100% EtOH. Add 1 ml and fix. At this time, 5 μg / ml propidium iodide and RNAse were mixed and prepared, and the fixed cells were centrifuged to remove the supernatant and then washed once with PBS. At the same time, the dye was added to the immobilized DNA and warmed in an incubator at 37 ° C. for 30 minutes, and the cells stained with propidium iodide were sealed in foil and stored at 4 ° C. and subjected to flow cytometry analysis. Is FACscan (Becton Dicknson, CA).

실험예 9Experimental Example 9

RT-PCR을 통해 COX-II와 iNOS 단백질의 전사수준에서 발현억제Inhibition of expression at the transcription level of COX-II and iNOS proteins via RT-PCR

상기 실험 7예에서처럼 본 발명의 의약 조성물에서 유효성분으로 사용되는 추출물에 류마티스 관절염의 원인인 COX-II와 유도성 NO합성효소(inducible nitric oxide synthetase: iNOS)의 저해활성 성분을 규명하기 위하여 세포를 T75 flask에 105세포수가 되게 분주한 다음 7일간 배양하고 LPS를 50 ng/ml이 되게 각 flask에 분주하고 2시간동안 자극을 가한다음 추출물 최종농도가 50㎍/ml이 되도록 가한 다음 세포를 1.5ml eppendorf tube에 모아서 15,000 rpm에서 5분간 원심분리하여 상등액을 제거하고 RNAzol 용액을 200㎕를 첨가한 다음 chloroform 50㎕를 가하고 조심스럽게 pippeting하여 세포를 lysis하고 이를 15,000 rpm에서 4℃하에 15분간 원심분리하여 total RNA를 회수한 다음 isopropanol 동량을 넣고 4℃에서 15분간 침전시켜 75% EtOH로 한번 세척하여 건조시킨 다음 RNase free dH20를 20㎕을 넣고 60℃ 에서 30분간 가하여 녹인 다음 total RNA 5㎕에 10 mM dNTP 5㎕, 25mM MgCl26㎕, 10x RNA PCR buffer 5㎕, RNase inhibitor 1㎕, AMV-Optimized Taq 1㎕, AMV reverse Transcriptase XL 1㎕, 50 pM specific primer (sense/ antisense) 1㎕, RNase free dH20 26㎕을 첨가하여 50℃에서 20분간 역전사 반응을 실시하고, 94℃에서 2분간 반응을 정지시켜서 DNA합성반응(polymerase chain reaction: PCR)를 실시하였는데 반응조건은 94℃ 1min, 55℃ 45sec, 70℃ 60sec에서 35 cycles를 진행시켜 70℃에서 최종적으로 5분간 elogation 반응을 실시하여 종결한 후 이 PCR 산물을 1% agarose gel에 elute시켜 사이즈 마커를 기준으로 395 bp와 278 bp 부근의 band유무를 확인하였다.In order to identify inhibitory components of COX-II and inducible nitric oxide synthetase (iNOS), which are the causes of rheumatoid arthritis, extracts used as active ingredients in the pharmaceutical composition of the present invention as in the seventh experiment. Dispense to 10 5 cells in a T75 flask, incubate for 7 days, dispense LPS to 50 ng / ml in each flask, add stimulus for 2 hours, and add the final concentration to 50 µg / ml. Collected in ml eppendorf tubes and centrifuged at 15,000 rpm for 5 minutes to remove supernatant, add 200 µl of RNAzol solution, add 50 µl of chloroform and carefully pippet lyse the cells and centrifuge at 15,000 rpm for 15 minutes at 4 ° C. by a number of times, total RNA, and then put into the same volume isopropanol to precipitate at 4 ℃ 15 bungan was washed once with 75% EtOH and dried and then put 20㎕ the RNase free dH 2 0 60 For 30 minutes and then dissolved by adding total RNA 10 mM dNTP 5㎕, 25mM MgCl in 5㎕ 2 6㎕, 10x RNA PCR buffer 5㎕, RNase inhibitor 1㎕, AMV-Optimized Taq 1㎕, AMV reverse Transcriptase XL 1㎕, 50 1 μl of pM specific primer (sense / antisense) and 26 μl of RNase free dH 2 0 were added to perform reverse transcription reaction at 50 ° C. for 20 minutes, and the reaction was stopped at 94 ° C. for 2 minutes to polymerase chain reaction (PCR). The reaction conditions were 35 cycles at 94 ° C 1min, 55 ° C 45sec, 70 ° C 60sec, and finally the elogation reaction at 70 ° C for 5 minutes, followed by elute the PCR product on 1% agarose gel. Based on the size marker, the presence or absence of band around 395 bp and 278 bp was confirmed.

실험예 10Experimental Example 10

SDS-PAGE에 의한 COX-II의 단백질 발현 확인Confirmation of Protein Expression of COX-II by SDS-PAGE

COX-2의 활성을 저해하는지를 규명하기 위하여 본 발명의 방법에 따라 수득된 상기 기재된 추출물에 대하여 만성 류마티스 관절염 환자에서 분리된 synovial cell(활액막세포)를 T75 flask에 105세포수가 되게 분주한 다음 7일간 배양하고 LPS를 50 ng/ml이 되게 각 flask에 분주하고 2시간동안 자극을 가한다음 추출물 최종농도가 50㎍/ml이 되도록 가한 다음 세포를 lysis buffer(0.1M SDS, sodium azide, PMSF, pepstatin, leupstatin, pH8.0) 0.5ml으로 녹여 1.5ml eppendorf tube에 모아서 100℃ 끊은 물에 열을 가해 불활성시킨 다음 5분간 실온에서 식힌 다음 10% polyacrylamide gel(SDS-PAGE)에서 전기영동한 다음 염색하여 destaining 용액에서 탈색시켜 관찰한다.In order to determine whether COX-2 activity is inhibited, the synovial cells (synovial membrane cells) isolated from chronic rheumatoid arthritis patients were dispensed to 10 5 cells in a T75 flask to the above-described extract obtained according to the method of the present invention. Incubate daily and dispense LPS into each flask at 50 ng / ml, add stimulus for 2 hours, add extract final concentration to 50 µg / ml, and then lysis buffer (0.1M SDS, sodium azide, PMSF, pepstatin). , leupstatin, pH8.0), dissolved in 0.5ml, collected in 1.5ml eppendorf tube, heated to 100 ° C, inactivated by water, inactivated, cooled at room temperature for 5 minutes, electrophoresed on 10% polyacrylamide gel (SDS-PAGE), and then dyed. Observe by decoloring in destaining solution.

실험예 11Experimental Example 11

면역세포화학분석법에 의한 COX-II 단백질 발현확인Confirmation of COX-II Protein Expression by Immunocytochemical Analysis

본 발명에 따라 분리된 의약 조성물의 활성 성분으로 확인된 상기의 화합물이 관절내 활액막세포의 COX-II를 억제효과를 유도하는지 조사하기 위해 세포를 5% DMEM medium에 penicilin/streptomycin이 함유한 배지상에 104세포를 둥근유리판(slimb cover glass)가 들어 있는 6 well culture dish에 분주하고 37℃에서 24시간 배양하는데 상기 추출물을 10㎍/ml이 되게 첨가하여 반응을 관찰하였으며 폐액을 제거 한 후 세포를 한번 PBS로 세척한 다음 세포를 metanol로 세포 위에 떨어지게 고정 시킨다음 PBS로 세척을 실시하고 1차 항체인 COX-II를 표지하여 4℃에서 1시간정도 방치하고 2차 항체인 형광체(fluorescein isothiocyanate:FITC)를 표지하여 호일로 빛을 차광하여 이를 1시간정도 방치한 다음 이를 형광현미경에서 관찰한다.In order to investigate whether the compound identified as the active ingredient of the pharmaceutical composition isolated according to the present invention induces the inhibitory effect of COX-II of intramucosal synovial membrane cells, the cells were cultured on a medium containing penicilin / streptomycin in 5% DMEM medium. 10 4 cells were dispensed in a 6 well culture dish containing a slim cover glass and incubated at 37 ° C. for 24 hours. The extract was added to 10 ㎍ / ml and the reaction was observed. After washing with PBS, the cells were fixed on the cells with metanol, and then washed with PBS. After washing with PBS, labeled with the primary antibody COX-II, it was left at 4 ° C. for about 1 hour and the secondary antibody phosphor (fluorescein isothiocyanate: FITC) is labeled and shaded with foil and left for about 1 hour and observed under a fluorescence microscope.

실험예 12Experimental Example 12

X-RAY로 부종억제 흰쥐의 관절부위 분해유무Decomposition of Joint Site in Edema-inhibited Rats with X-ray

상기 실험예 1에서처럼 물추출물 및 유기용매 분획을 각각 75㎍/ml이 되게 14일간 경구 투여하여 부종 억제 여부를 조사하였다. 촬영시 부종유도 흰쥐를 마취시켜 X-RAY사진 촬영하였다.As in Experiment 1, the water extract and the organic solvent fractions were orally administered for 14 days to be 75 µg / ml, respectively, to investigate whether edema was suppressed. X-ray photographs were taken of anesthetized rats during edema.

실험예 13Experimental Example 13

CT촬영으로 ruptured disc 치료전/후 효과Pre / post effect of ruptured disc treatment by CT scan

상기 실시예 2에서처럼 분리 정제된 의약 조성물을 통해 임상적인 약리 효과를 조사하기 위해 물추출물을 1일 복용량 500 mg이 되게 ruptured disc 환자를 대상으로 디스크가 용출되어 나온 실례를 통해 2개월간 복용한 다음 치료전후 CT 촬영을 실시하여 보았다.In order to investigate the clinical pharmacological effect through the separated and purified pharmaceutical composition as in Example 2, the patient was taken for two months through an example in which the disc was eluted in a patient with a ruptured disc such that the daily dose was 500 mg. Before and after CT scans were performed.

실험예 14Experimental Example 14

MRI 촬영으로 ruptured disc 치료전/후 효과MRI scan before / after ruptured disc treatment

상기 실시예 2에서처럼 분리 정제된 의약 조성물을 통해 임상적인 약리 효과를 조사하기 위해 디스크가 용출되어 나온 실례의 환자를 대상으로 2개월간 복용한 다음 치료전후 MRI 촬영을 실시하여 보았다.As in Example 2, an example patient was taken for two months after the disc was eluted to examine the clinical pharmacological effect through the separated and purified pharmaceutical composition, and then pre and post-treatment MRI was performed.

실험예 15Experimental Example 15

척추 신경마비 차단에 대한 치료효과Therapeutic Effect on Spinal Nerve Palsy Block

상기의 실시예 2에서 분리된 화합물이 골질환으로 nogo-A에 의해서 신경마비현상이 유도되어 그로 인한 신경마비 현상에 억제효과를 유도하는지 조사하기 위해 한국세포주 은행에서 분양받은 brain에서 기원한 글리아 아세포종(glioblastoma세포: U-373 MG)를 10% DMEM medium에 penicilin /streptomycin이 함유한 배지상에 104세포를 6 well culture dish에 분주하고 37℃에서 24시간 배양하고 nogo-A가 잘 발현되는 vector를 stable transfection를 실시하여 신경의 neurite가 감소 내지 신장억제되는지를 확인하여 관찰하였으며 동시에 neuronal growth factor(NGF)를 처리시 재생되는지 혹은 상기 분획물을 처리시 동일한 현상이 유도되는지를 확인하여 위상차 현미경의 200배율에서 사진 촬영하여 관찰하였다.In order to investigate whether the compound isolated in Example 2 is a bone disease induced neuronal phenomena by nogo-A and induces an inhibitory effect on the resulting neuronal palsy, glia originated from brains sold by the Bank of Korea Cell Line Glioblastoma cells (U-373 MG) were dispensed in 6 well culture dishes with 10 4 cells on a medium containing penicilin / streptomycin in 10% DMEM medium, incubated at 37 ° C for 24 hours and well expressed in nogo-A. Stable transfection of the vector was performed to determine whether neuronal neurites were reduced or renal suppressed.At the same time, reconstitution of neuronal growth factor (NGF) or whether the same phenomena were induced in the treatment of the fractions were performed. It was observed by photographing at 200 magnification.

실험예 16Experimental Example 16

실험예 2에서처럼 배양한 용기에서 관찰한 결과 1일 배양한 퇴행성 관절염인 환자경우세포가 자라지 않았으며 육안상 부착세포가 존재하지 않고 현탁상태로 부유하고 있었으며 만성 관절염환자에서 분리된 경우는 세포가 자라고 7일 경과후 증식이 이루어지고 있음을 위상차 현미경의 200배율에서 세포를 관찰할 수 있었다. 따라서 퇴행성 관절염 환자는 활액막 전체가 연골조직을 둘러싸고 있지 않고 마모되어 활액막 세포의 부재를 통해 연골조직이 파괴를 더욱 촉진하는 것을 알 수 있었으며 반대로 만성 관절염 경우 활액막 세포가 염증이나 자가면역으로 인한 세포증식이 병적으로 진행되어진다(도 3).As a result of observation in the cultured container as in Experimental Example 2, the cells did not grow in patients with degenerative arthritis cultured on a daily basis. After 7 days, the cells were observed at 200 times magnification of the phase contrast microscope. Therefore, in patients with degenerative arthritis, the entire synovial membrane was not surrounded by the cartilage tissue and was worn out, and the absence of the synovial membrane cells promoted the destruction of the cartilage tissue. Progress pathologically (FIG. 3).

실험예 17Experimental Example 17

상기 실시예 2로부터 분리된 각각 분획을 부종 유도 실험에 적용시 CBB-12, 13, 과 LNE-1, 3에서 강하게 부종 억제효과를 나타 내었는데 특히 CBB-12와 LNE-3가 유의성이 가장 강하였다. 아래와 같이 70일 경과한 상태에서 본 화합물의 효과에대한 부종 억제효과에 대하여 관찰되어진 결과는 표 3, 4 와 도 4 .에 기재하였다.When the fractions isolated from Example 2 were applied to the edema induction experiment, CBB-12, 13, and LNE-1, 3 showed strong edema inhibitory effect, especially CBB-12 and LNE-3 had the strongest significance. It was. The results observed for the edema inhibitory effect on the effect of the compound in the elapsed 70 days as described below are shown in Tables 3, 4 and 4.

표 3 . 유기 용매 추출별 부종 유도 억제정도(단위: mm )Table 3. Degree of inhibition of edema induction by organic solvent extraction (unit: mm)

샘 플Sample 약물처리전Before drug treatment 약물처리후After drug treatment 샘 플Sample 약물처리전Before drug treatment 약물처리후After drug treatment ABEABE 15.5±215.5 ± 2 8.5±28.5 ± 2 LNBLNB 15.6±115.6 ± 1 5±15 ± 1 ABBABB 14.6±314.6 ± 3 10.1±110.1 ± 1 LNWLNW 14.5±214.5 ± 2 8±18 ± 1 ABWABW 14.4±114.4 ± 1 12.2±212.2 ± 2 CRBCRB 15.5±115.5 ± 1 9±29 ± 2 CBECBE 16.3±216.3 ± 2 4.1±14.1 ± 1 CRWCRW 15.6±115.6 ± 1 9±19 ± 1 CBBCBB 14.6±114.6 ± 1 3.6±13.6 ± 1 SSHSSH 14.5±214.5 ± 2 12±212 ± 2 CBWCBW 15.5±215.5 ± 2 7.2±27.2 ± 2 SSBSSB 13.3±113.3 ± 1 13±113 ± 1 LNELNE 14.5±314.5 ± 3 5.3±15.3 ± 1 SSWSSW 14.4±214.4 ± 2 13±113 ± 1

표 4. 부종 억제효과가 가장 우수한 유기용매 분획Table 4. Organic Solvent Fractions with the Best Suppression of Edema

분획 시료Fraction sample 약물처리 전Before drug treatment 약물 처리 후After drug treatment CBBCBB- 13CBB- 20CBB- 30CBBCBB- 13CBB- 20CBB-30 14.6±214.4±214.5±114.3±114.6 ± 214.4 ± 214.5 ± 114.3 ± 1 3.6 ±12.5 ±13.1 ±23.6 ±13.6 ± 12.5 ± 13.1 ± 23.6 ± 1 LNELNE- 1LNE-3LNELNE-1 LNE-3 15.6±115.1±215.2±115.6 ± 115.1 ± 215.2 ± 1 5.2 ±15.1 ±23.8 ±15.2 ± 15.1 ± 23.8 ± 1

실험예 18Experimental Example 18

상기 실험예 3에서처럼 수득된 추출물에 대하여 활액세포의 혈관신생의 정도에 미치는 영향을 CAM의 감소정도로 관찰하였는데 그 결과로는 정상적인 수정란에서는 CAM의 형성이 다소 느리지만 synovial cell을 주입한 세포에서는 강력하게 CAM의 형성이 유도되었으며 이에 비해 CBB-13와 LNE-3분획군에서는 대조군보다 더 강하게 유도가 억제됨을 알 수 있었다. 따라서 상기 조성물에서 synovial 세포의 증식 억제뿐만 아니라 활액막의 세포에 의한 angiogenesis(혈관신생)의 CAM 유도도 억제시키는 양상을 추측해 볼 때 상기 약물은 관절염의 치료에 우수한 효과를 가짐을 알 수 있다(도 5).The effect of the extract obtained as in Experimental Example 3 on the degree of angiogenesis of synovial cells was observed as a decrease in CAM. As a result, the formation of CAM was slightly slow in normal fertilized eggs, but strongly in the cells injected with synovial cells. The formation of CAM was induced and compared with the control group in the CBB-13 and LNE-3 fractions. Therefore, when the composition of the composition inhibits the proliferation of synovial cells as well as inhibits CAM induction of angiogenesis (angiogenesis) by the cells of the synovial membrane, it can be seen that the drug has an excellent effect in the treatment of arthritis (Fig. 5).

실험예 19Experimental Example 19

상기 실험예 4에서처럼 관절부위에 존재하는 연골의 마모상태 내지 meltalloprotease의 분비로 인한 연골조직 및 연골세포의 파괴정도를 확인하기 위해 hematoxylin/eosin 염색을 통해 위상차 현미경 100 배율로 관찰한 후 사진 촬영을 실시한 결과 본 약물인 정상군경우 연골조직의 분포 양상은 균일하게 골조직의 분포나 거물상이 골고루 존재하나 대조군인 경우 골파괴가 많이 진행되고 골조직이 분해된 흔적이 보인 반면 물추출물에서는 정상군과 유사한 골조직 형태를 보임을 알 수 있는데 이는 본 물추출물이 연골조직의 파괴 내지 연골 재생기능이 있음을 알았다(도 6).In order to confirm the degree of destruction of cartilage tissue and cartilage cells due to the wear state of cartilage present in the joint area or secretion of meltalloprotease as in Experimental Example 4, the result was photographed after observation with a phase contrast microscope of 100 magnification through hematoxylin / eosin staining. Results In the normal group of the drug, the distribution of cartilage was uniformly distributed evenly in the distribution of bone tissues, but in the control group, bone destruction progressed and bone tissues were disintegrated. It can be seen that this water extract was found to have cartilage tissue destruction to cartilage regeneration (Fig. 6).

실험예 20Experimental Example 20

상기 실험예 6에서처럼 비교 분석하여 상기 약물에 대한 수치를 상대 평가로 유추한 결과는 막대 그래프에서 나타난 것처럼 대조군은 약 98μM정도로 높은 반면 CBB, CBE, LNE, LNB 분획물에서 유의성 있는 NO형성 저해효과를 보임을 알 수 있고(도 7a), 특히 CBB와 LNE 분획계열에서 CBB-13과 LNE-3에서 더 강하게 저해효과를 보임을 알 수 있었다. 이는 본 조성물들이 NO형성을 유도하는 iNOS의 합성을 억제함으로 기인한 결과라 사료된다(도 7b).The results of inferring the relative values of the drug by comparative analysis as in Experimental Example 6 showed that the control group was about 98 μM high as shown in the bar graph, while showing significant NO inhibitory effect in the CBB, CBE, LNE, and LNB fractions. It can be seen (Fig. 7a), especially in the CBB and LNE fractional series showed a stronger inhibitory effect in CBB-13 and LNE-3. This is believed to be a result of the present composition by inhibiting the synthesis of iNOS inducing NO formation (Fig. 7b).

실험예 21Experimental Example 21

실험예 7에서처럼 상기의 화합물이 염증반응에 관여하는 macrophage인 raw 264.7세포와 synovial cell의 세포사를 유도하는지 조사하였는데 그 결과 대조군경우 세포 증식이 활발하게 진행하고 있는데 비해 약물 처리군경우 CBB와 LNE 분획계열인 CBB-13, 20과 LNE-1, 3에서 더 강하게 세포사를 유도하는 효과를 보임을 알 수 있었다 따라서 본 조성물이 두종의 세포에서 세포사를 유도하고 결론적으로 염증 및 부종을 억제하는데 관여한다고 사료된다(도 8a, b).As in Experiment 7, it was examined whether the compound induces cell death of raw 264.7 cells and synovial cells, which are macrophages involved in the inflammatory response. As a result, the cell proliferation was actively progressed in the control group, whereas the CBB and LNE fractions were used in the drug treatment group. Phosphorylation of CBB-13, 20, and LNE-1, 3 showed a strong effect on inducing cell death. Therefore, this composition is thought to be involved in inducing cell death in two cells and consequently inhibiting inflammation and edema. (Figures 8a, b).

실험예 22Experimental Example 22

상기 실험예 8에서 본 의약 조성물인 유효성분의 분획물이 세포 주기에 미치는 영향을 관찰해 본 결과 synovial fibroblast 세포주와 macrophage인 raw 264.7 세포주를 분석한 결과 macrophage 세포주에서는 대조군 경우 전형적으로 G0/G1phase가 상대적으로 제일 높은 반면 S phase는 제일 낮은 peak를 보이고 G2/M phase 경우 중간 peak를 나타내고 있다. 특히 약물 처리군에서 CBB-13 경우 apoptosis(세포사)를LNE-3에서 G0/G1arrest를 유도하는 것으로 보아 상기 약물들은 세포사를 유도하는 약물인 것을 예측되어진다(도 9a). 한편 synovial cell 세포주에서는 대조군경우 일반적으로 증식이 왕성한 세포주와 달리 세포의 G0/G1phase가 상대적으로 G2/M phase보다 낮고 S phase가 대체로 높은 peak를 보이고 있는데 이는 세포분열보다 핵분열이 많이 일어나거나 증식 속도가 느린 전이성 암세포나 angiogenesis가 일어나는 세포의 양상으로 본 약물인 CBB-13은 G0/G1arrest양상을 나타내고 있으며. LNE-3에서 apoptosis를 유도하는 것으로 보아 상기 약물들은 synovial cell에서는 세포사를 유도하는 약물인 것을 예측되어진다(도 9b). 따라서 본 분획물인 CBB-13과 LNE-3 경우 세포사를 유도함으로 골질환의 원인인 세포의 증식을 억제함으로 골질환 치료에 유의성이 있음을 알 수 있다.As a result of observing the effect of the fraction of the active ingredient of the pharmaceutical composition in Experimental Example 8 on the cell cycle, the synovial fibroblast cell line and the macrophage raw 264.7 cell line were analyzed. In the macrophage cell line, the control group was typically G 0 / G 1 phase. Is relatively highest while S phase has the lowest peak and G 2 / M phase shows the middle peak. In particular, in the case of CBB-13 in the drug treatment group, apoptosis (cell death) induces G 0 / G 1 arrest in LNE-3, and thus, it is predicted that the drugs are drugs that induce cell death (FIG. 9A). On the other hand, in the synovial cell line, the G 0 / G 1 phase of the cell is relatively lower than the G 2 / M phase and the S phase is generally higher than the cell line in which the proliferation is generally strong. Or CBB-13, which is a metastatic cancer cell with slow growth rate or angiogenesis cell, exhibits G 0 / G 1 arrest. Inducing apoptosis in LNE-3, it is expected that these drugs are drugs that induce cell death in synovial cells (FIG. 9B). Therefore, it can be seen that the CBB-13 and LNE-3 fractions of the present invention are significant in the treatment of bone diseases by inhibiting the proliferation of cells that cause bone diseases by inducing cell death.

실험예 23Experimental Example 23

상기 실험예 12에서처럼 본 발명의 의약 조성물에서 유효성분이 synovial cells내 류마티스 관절염의 원인인 COX-2의 발현을 저해하는지를 10% polyacrylamide gel(SDS PAGE)에서 전기영동한 다음 염색하여 destaining 용액에서 탈색시켜 관찰한 결과는 COX-II단백질의 분자량은 약 70KD정도이고 대조군 경우 강한 band를 보인데 반해 CBB-13과 LNE-3 경우 약한 band를 보이는 것으로 보아 본 약물은 단백질의 발현을 억제하는 약리작용을 가짐을 알 수 있다(도 10).As in Experiment 12, whether the active ingredient inhibits the expression of COX-2, which is the cause of rheumatoid arthritis in synovial cells, was electrophoresed on a 10% polyacrylamide gel (SDS PAGE) and stained to decolorize in a destaining solution. The results showed that COX-II protein had a molecular weight of about 70KD and a strong band in the control group, but a weak band in the CBB-13 and LNE-3 groups, indicating that the drug has a pharmacological effect of inhibiting protein expression. It can be seen (Fig. 10).

실험예 24Experimental Example 24

상기 실험 예9에서처럼 PCR를 실시하여 전기영동을 실시한 바 iNOS 경우 PCR 산물은 278 bp 부근의 band유무를 확인하였는데 lane 1, 2, 3, 4는 각각 대조군, ABB, SSB, CRB이고 발현에 영향이 없었으며 이에 비해 lane 5, 6, 7, 8경우는 CBB, CBB-13, LNE, LNE-3으로 단백질 발현에 영향을 주고 있음을 확인할 수 있었고 COX-II경우는 lane 1, 2, 3에서는 발현에 영향이 없는 반면 lane 4, 5, 6, 7, 8에서는 전사 수준에서 억제효과를 보였다(11 a). 실시 예15에서처럼 본 발명의 의약 조성물에서 유효성분으로 사용되는 추출물에 류마티스 관절염의 원인인 COX-2와 iNOS의 발현을 조사한 결과 iNOS 단백질경우 각 분획물의 elute lane에 해당되는 lane 1, 3, 4는 각각 대조군, ABB, CRB이고 발현에 전혀 영향이 없었으며 이에 비해 lane 2, 5, 6, 7, 8경우는 각각 CBB, LNE, SD, DS, YM이고 단백질 발현을 강하게 억제하는 것을 알 수 있다(도 11b), COX-2경우는 또한 lane 1, 2, 3, 5에서는 각각 대조군, CBW, ABB, LNW이며 발현에 전혀 영향이 없었으며 lane4, 6, 7, 8은 각각 CBB-13, 20과 LNE-1, 3 이며 COX-II의 발현을 강하게 억제하는 것을 알 수 있다 (도 11c).PCR was performed by electrophoresis as in Experiment 9, and in case of iNOS, PCR product was confirmed to have a band around 278 bp. Lanes 1, 2, 3, and 4 were control, ABB, SSB, and CRB, respectively. In contrast, lanes 5, 6, 7, and 8 were found to affect protein expression with CBB, CBB-13, LNE, and LNE-3, whereas COX-II was expressed in lanes 1, 2, and 3 In contrast, lanes 4, 5, 6, 7, and 8 showed inhibitory effects on transcriptional levels (11 a). As in Example 15, the extracts used as active ingredients in the pharmaceutical composition of the present invention were examined for the expression of COX-2 and iNOS, which are the causes of rheumatoid arthritis, and lanes 1, 3, and 4 corresponding to the elute lanes of each fraction of the iNOS protein. The control group, ABB and CRB, respectively, had no effect on expression, whereas lanes 2, 5, 6, 7, and 8 were CBB, LNE, SD, DS, and YM, respectively. 11B), COX-2 was also a control group, CBW, ABB and LNW in lanes 1, 2, 3, and 5, respectively, and had no effect on expression. Lanes 4, 6, 7, and 8 were CBB-13, 20 and It can be seen that LNE-1, 3 and COX-II expression is strongly inhibited (Fig. 11c).

실험예 25Experimental Example 25

본 발명에 따라 분리된 의약 조성물이 관절내 활액막 세포 COX-II의 억제효과를 유도하는지 조사하기 위해 실험 예11에서처럼 세포를 metanol로 세포 위에 떨어지게 고정 시킨다음 PBS로 세척을 실시하고 1차 항체인 COX-II를 표지하여 4℃에서 1시간정도 방치하고 2차 항체인 FITC를 표지하여 호일로 빛을 차광하여 이를 1시간정도 방치한 다음 이를 형광현미경에서 관찰한 결과 대조군은 정상군에 비해 COX-II의 단백질이 강하게 발현이 되고 CBB-13와 LNE-3경우는 발현정도가 크게 감소하는 경향을 보임을 알 수 있었다. 이는 COX-II의 단백질 발현이 상기 실시예 16에서처럼 전사수준에서 전사를 억제하기 때문에 기인한 결과라 사료된다(도 12).In order to investigate whether the pharmaceutical composition isolated according to the present invention induces the inhibitory effect of intraarticular synovial cell COX-II, the cells were fixed on the cells with metanol and then washed with PBS, as in Experiment 11, and washed with PBS. After labeling -II and leaving it at 4 ℃ for 1 hour and labeling the secondary antibody FITC, it was shaded with foil and left for 1 hour, and observed under a fluorescence microscope. The protein was strongly expressed and the expression level of CBB-13 and LNE-3 tended to decrease significantly. This may be due to the fact that protein expression of COX-II inhibits transcription at the transcription level as in Example 16 (FIG. 12).

실험예 26Experimental Example 26

실시예 2에서처럼 의약 조성물이 가지는 부종 억제효과를 관찰하였는데 분리된 유기용매 추출물 CBB-12, 13, 20, 30와 LNE-1, 3를 각각 사용하여 부종을 유도한 흰쥐를 마취시켜 X-RAY로 사진촬영하였는데 관찰된 결과는 대조군경우 류마티스 관절염의 연골부위가 심하게 파괴되고 골조직의 분해정도가 강하게 손상을 입었으며 약 70일 경과까지 손상 정도가 최고 정점에 도달하였다. 한편 대조군과 달리 CBB-13과 LNE-3에서 관절염 부위의 연골이 파괴되는 것을 억제하고 골 합성을 촉진하는 효과를 가짐을 알 수 있었다(도 13).As in Example 2, the edema inhibitory effect of the pharmaceutical composition was observed. Using the isolated organic solvent extracts CBB-12, 13, 20, 30 and LNE-1, 3, anesthetized rats were anesthetized with X-RAY. Photographs showed that the cartilage of rheumatoid arthritis was severely damaged and the degree of degradation of bone tissue was severely damaged in the control group, and the damage reached its peak until about 70 days. On the other hand, unlike the control group, it was found that the cartilage at the arthritis site was inhibited and the bone synthesis was promoted in CBB-13 and LNE-3 (FIG. 13).

실험예 27Experimental Example 27

상기 실험예 13에서처럼 임상적인 약리효과를 조사하기 위해 본 발명의 방법에 따라 수득된 물추출물군에 대하여 디스크 환자를 대상으로 디스크가 용출되어 나온 실례를 통해 2개월간 복용한 다음 치료전후로 CT 촬영을 실시하여 보았는데 치료전 대조군에서는 ruptured disc가 파열되어 나오는데 비해 치료후에는 disc가 소실되고 신경염이 소멸되고 그로 인한 신경마비가 제거되었다(도 15).In order to investigate the clinical pharmacological effect as in Experimental Example 13, the water extract group obtained according to the method of the present invention was taken for 2 months through an example in which the disk was eluted from the disk patient, and then CT scans were performed before and after treatment. In the control group before treatment, the ruptured disc was ruptured, but after treatment, the disc was lost, neuritis was eliminated, and the resulting nerve palsy was removed (FIG. 15).

실험예 28Experimental Example 28

실험예 1에서 수득된 물추출물군에 대하여 디스크 환자를 MRI 촬영을 실시하여 보았는데 치료전 대조군에서는 ruptured disc가 파열되어 나오는데 비해 치료후에는 물추출물군에서 disc가 소실되고 그로 인한 신경마비가 제거되었다(도 16).MRI was performed on the patients with the water extract group obtained in Experimental Example 1, but the ruptured disc was ruptured in the control group before the treatment, but after the treatment, the disc was lost in the water extract group and the resulting nerve paralysis was removed. 16).

실험예 29Experimental Example 29

상기 실험예 17에서처럼 신경의 neurite가 감소 내지 억제되는지를 확인하여 관찰하였으며 동시에 neuronal growth factor(NGF)를 처리시 neurite가 재생되거나 혹은 상기 분획물을 처리시 동일한 현상이 유도되는지를 확인하여 편광 현미경의 200배율에서 사진 촬영하여 관찰하였는데 nogo-A가 발현된 U-373세포주에서는 원래의 길게 뻗어 나온 neurite가 줄어들거나 거의 소실되는 양상을 보이며 NGF, CBB-13 및 LNE-3를 각각 처리시 세포에서 neurite가 길어지는 양상을 볼 수 있었다. 이로써 본 약물에 의하여 NGF와 비교시 신경이 재생하도록 neurite의 재생을 촉진하는 것임을 알 수 있었다(도 18a, b, c).As shown in Experiment 17, it was confirmed whether the neuronal neurite was reduced or suppressed, and at the same time, when the neuronal growth factor (NGF) was treated, it was confirmed whether the neurite was regenerated or the same phenomenon was induced when the fraction was treated. The photographs were observed at magnification. Nogo-A-expressing U-373 cell line showed the decreased or almost disappeared original elongated neurite. When NGF, CBB-13 and LNE-3 were treated, I could see the longer side. As a result, it was found that the drug promotes the regeneration of neurite so that the nerve regenerates as compared with NGF (FIGS. 18A, B, and C).

실험예 30Experimental Example 30

본 발명에 따라 분리된 의약 조성물의 활성 성분으로 확인된 상기의 화합물은 골질환에서 각각 효과를 보이는 화합물을 화학적 구조결정을 통해 확인을 실시한 결과 본 약물의 신규물질로 CBB-13은 shinbarometin으로 명명하였고, LNE-1, 3은 각각 하르파고시드, 하르파지드로 이미 화학적 동정이 알려진 구조이나 약리효과가 아직 미확인된 상태에서 효능에 대한 결과를 처음 밝혀지게 되었다.The compound identified as the active ingredient of the pharmaceutical composition isolated according to the present invention was identified by chemical structural determination of each compound having an effect on bone disease as a novel substance of the drug CBB-13 was named shinbarometin , LNE-1, and 3 are harpagoside and harpazide, respectively, and the results of efficacy are known for the first time when the structure and pharmacological effects are known.

이상의 실험결과로부터 확인되는 바와 같이, 본 발명의 신규 조성물은 탁월한 약리효과를 가진다.As confirmed from the above experimental results, the novel composition of the present invention has excellent pharmacological effect.

따라서, 본 발명의 조성물은 의약품으로서 유용하게 사용될 수 있다.Therefore, the composition of the present invention can be usefully used as a pharmaceutical.

본 발명의 조성물은 환자의 성별, 나이, 질병의 정도등에 의하여 그 사용량이 달라질 수 있으나, 본 발명의 조성물을 물이나 유기용매로 추출하지 않고, 생약 자체를 사용하는 경우에는 일일 1.0g 내지 50g의 양을 사용할 수 있으며, 엑스의 형태로 사용될 경우에는 일일 10mg 내지 1000mg을 일일 1회 내지 수회 투여할 수 있다.The composition of the present invention may vary depending on the sex, age, degree of disease, etc. of the patient, but when using the herbal medicine itself without extracting the composition of the present invention with water or an organic solvent, 1.0g to 50g per day Amounts may be used, and when used in the form of X, 10 mg to 1000 mg per day may be administered once to several times a day.

본 발명의 화합물은 골다공증, 관절염 및 파열된 디스크에 효능을 가지는 기존에 이미 약제로 사용되고 있는 알렌드레이트(allendrate), 타목시펜(tamoxifen), 비타민 B3, 부갑상선 호르몬(parathyroid hormone: PTH), 설파살라진(sulfasalazine), 티오레독신 리덕타제(thioredoxin reductase), 알렌드로네이트(alendronate), 랄록시펜(raloxifene), 칼시토닌(calcitonin), 에스타라디올(estradiol), 게니스테인(genistein), 1,25-디하이드록시바이타민 D3, 알렌드로네이트(alendronate), 에스트로젠 수용체 조절제(estrogen receptor modulator), 비포스포네이트(biphosphonates), 하르파지드(harpagide: 본 발명자가 그 용도를 개발한 약물임)등과 같은 약물과 병용하여 사용될 수도 있다.Compounds of the present invention are already used as an agent for osteoporosis, arthritis and ruptured discs, allenrate, tamoxifen, vitamin B 3 , parathyroid hormone (PTH), sulfasalazine (sulfasalazine) ), Thioredoxin reductase, alendronate, raloxifene, calcitonin, calcitonin, estradiol, genistein, 1,25-dihydroxyvitamin D 3 , may be used in combination with drugs such as alendronate, estrogen receptor modulators, biphosphonates, harpagide (the drug for which the inventors have developed their use), and the like.

본 발명의 화합물과 상기의 약물을 병용하면, 기존 약물의 상용량을 줄일 수 있고, 따라서 기존 약물들이 가지는 문제점들을 경감시킬 수 있다.By using the compound of the present invention and the above drug, it is possible to reduce the normal dose of the existing drug, thereby reducing the problems with the existing drugs.

본 발명의 화합물은 약제학적으로 통상으로 사용되는 부형제, 보조제, 무통화제, 등장화제, 보존제, 및 기타 약제학적으로 통상으로 허용되는 보조제와 혼합하고 약제학적으로 통상으로 허용되는 제제형태로 제제화하여 약학적 제제를 제조할 수 있다. 이러한 약제학적 제제형태로는 주사제, 액제, 정제, 캡슐제, 산제, 시럽제 등으로 제제화 할 수 있다.The compounds of the present invention may be mixed with excipients, adjuvants, analgesics, isotonic agents, preservatives, and other pharmaceutically acceptable adjuvants, which are commonly used pharmaceutically, and formulated into pharmaceutically acceptable formulations for pharmaceutical purposes. Suitable formulations may be prepared. Such pharmaceutical preparations may be formulated into injections, solutions, tablets, capsules, powders, syrups and the like.

다음에 제제실시예로서 본 발명을 더욱 상세히 설명한다.Next, the present invention will be described in more detail as formulation examples.

제제실시예 1Formulation Example 1

실시예 2의 구척엑스 CBB분획 50mg50 mg of Guchu extract CBB of Example 2

주사용 멸균증류수 적량Appropriate sterile distilled water for injection

pH 조절제 적량pH adjuster

실시예 2의 구척엑스 CBB분획 50mg을 주사용 증류수에 용해하고 pH 조절제로 pH약 7.6로 조절한 다음 전체를 2ml로 한 후 2ml용량의 앰플에 충진하고 멸균하여 주사제를 제조한다.50 mg of Gucheol X CBB fractions of Example 2 were dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

제제실시예 2Formulation Example 2

실시예 2의 천수근 엑스 50mgTenthmus root X 50mg of Example 2

주사용 멸균증류수 적량Appropriate sterile distilled water for injection

pH조절제 적량pH adjuster

실시예 2의 천수근엑스 50mg을 주사용 멸균증류수에 용해하고 pH조절제로 pH약 7.2로 조절하고 전체를 2ml로 한 다음 2ml용량의 앰플에 충진하여 주사제를 제조한다.50 mg of Chun-Geun-X of Example 2 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, and the whole was made into 2 ml, and then filled in an ampoule of 2 ml to prepare an injection.

제제실시예 3Formulation Example 3

실시예 12의 엑스 200mgX 200mg of Example 12

유당 100mgLactose 100mg

전분 100mgStarch 100mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

제제실시예 4Formulation Example 4

실시예 11의 하르파지드 10mgHarpazide 10mg of Example 11

유당 100mgLactose 100mg

전분 50mgStarch 50mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

제제실시예 5Formulation Example 5

실시예 13의 분말 1000mg1000 mg of powder of Example 13

유당 100mgLactose 100mg

스테아린산 마그네슘 2mg2 mg magnesium stearate

상기의 성분을 혼합한 후 내부가 폴리에틸렌클로라이드로 코팅된 지포에 충진하고 씰링하여 산제를 제조한다.After mixing the above components, the inside is filled with a polyethylene coated coated chloride and sealed to prepare a powder.

제제실시예 6Formulation Example 6

실시예 14의 엑스 500mgX 500mg of Example 14

유당 50mgLactose 50mg

전분 50mgStarch 50mg

탈크 2mgTalc 2mg

스테아린산마그네슘 적량Magnesium stearate appropriate amount

상기의 성분을 혼합하고 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충진하여 캡슐제를 제조한다.The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

제제실시예 6Formulation Example 6

실시예 15의 엑스 400mgX 400mg of Example 15

유당 100mgLactose 100mg

전분 93mgStarch 93mg

탈클 2mgTackle 2mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충진하여 캡슐제를 제조한다.The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

제제실시예 7Formulation Example 7

실시예 14의 엑스 5gX 5g of Example 14

설탕 20g20 g of sugar

이성화당 20g20 g of isomerized sugar

레몬향 적량Lemon flavor

정제수를 가하여 전체 100mlAdd 100 ml of purified water

상기의 성분을 통상의 액제의 제조방법에 따라서 혼합하고 100ml 의 갈색병에 충진하고 멸균시켜서 액제를 제조한다.The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

제제실시예 8Formulation Example 8

실시예 16의 엑스 5gX 5g of Example 16

설탕 20g20 g of sugar

이성화당 20g20 g of isomerized sugar

레몬향 적량Lemon flavor

정제수를 가하여 전체 100mlAdd 100 ml of purified water

상기의 성분을 통상의 액제의 제조방법에 따라서 혼합하고 100ml 의 갈색병에 충진하고 멸균시켜서 액제를 제조한다.The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

제제실시예 9Formulation Example 9

실시예 2의 천수근엑스 50mgTensile root extract 50mg of Example 2

알렌드레이트 10mgAlenate 10mg

주사용 멸균증류수 적량Proper sterile distilled water for injection

pH조절제 적량pH adjuster

실시예 2의 천수근엑스 50mg을 주사용 증류수에 용해하고 pH 조절제로 pH약 7.6로 조절한 다음 전체를 2ml로 한 후 2ml용량의 앰플에 충진하고 멸균하여 주사제를 제조한다.50 mg of Chun Geun-X extract of Example 2 was dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

제제 실시예 10Formulation Example 10

실시예 12의 엑스 50mgX 50mg of Example 12

타목시펜 10mgTamoxifen 10mg

주사용 멸균증류수 적량Appropriate sterile distilled water for injection

pH조절제 적량pH adjuster

실시예 12의 엑스 50mg을 주사용 멸균증류수에 용해하고 pH조절제로 pH약 7.2로 조절하고 전체를 2ml로 한 다음 2ml용량의 앰플에 충진하여 주사제를 제조한다.X 50 mg of Example 12 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, and the whole was made to 2 ml, followed by filling into a 2 ml ampoule to prepare an injection.

제제실시예 11Formulation Example 11

실시예 14의 엑스 200mgX 200mg of Example 14

설파살라진 20mg20 mg sulfasalin

유당 100mgLactose 100mg

전분 100mgStarch 100mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

제제실시예 12Formulation Example 12

실시예 15의 엑스 200mgExample 200 x 200mg

알렌드로네이트 50mgAlendronate 50mg

유당 100mgLactose 100mg

전분 50mgStarch 50mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

제제실시예 13Formulation Example 13

실시예 16의 엑스 500mgX 500mg of Example 16

유당 50mgLactose 50mg

전분 50mgStarch 50mg

탈크 2mgTalc 2mg

스테아린산마그네슘 적량Magnesium stearate appropriate amount

상기의 성분을 혼합하고 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충진하여 캡슐제를 제조한다.The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

제제실시예 14Formulation Example 14

실시예 2의 구척엑스 50mgGuolgi extract 50mg of Example 2

랄록시펜 10mgRaloxifene 10mg

유당 50mgLactose 50mg

전분 50mgStarch 50mg

탈크 2mgTalc 2mg

스테아린산마그네슘 적량Magnesium stearate appropriate amount

상기의 성분을 혼합하고 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충진하여 캡슐제를 제조한다.The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

제제실시예 14Formulation Example 14

실시예 2의 천수근엑스 50mgTensile root extract 50mg of Example 2

파미드론산 디소디움 20mgPamideronic disodium 20mg

유당 100mgLactose 100mg

전분 93mgStarch 93mg

탈클 2mgTackle 2mg

스테아린산 마그네슘 적량Magnesium stearate proper amount

상기의 성분을 혼합하고 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충진하여 캡슐제를 제조한다.The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

제제실시예 16Formulation Example 16

실시예 16의 엑스 2000mgX 2000mg of Example 16

파미드로네이트 디소디움 1000mgPamideronide Disodium 1000mg

설탕 20g20 g of sugar

이성화당 20g20 g of isomerized sugar

레몬향 적량Lemon flavor

정제수를 가하여 전체 100mlAdd 100 ml of purified water

상기의 성분을 통상의 액제의 제조방법에 따라서 혼합하고 100ml 의 갈색병에 충진하고 멸균시켜서 액제를 제조한다.The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

이상에서 상세히 설명한 바와 같이, 본 발명인 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 및/또는 천수근(Harpagophytum procumbensDC.)의 분말이나 또는 엑스를 주성분으로 함유하고, 필요하면 맥아(HORDEI FRUCTUS GERMINIATUS), 우슬[ACHYRANTHIS BIDENTATAE RADIX), 당귀(ANGELICAE GIGANTIS RADIX), 숙지황(REHMANNIAE RADIX PREPARAT), 두충(EUCOMMIAE CORTEX), 육계(CINNAMOMI CORTEX), 홍화자(CARTHAMI FRUCTUS), 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), 오공(SCOLOPENDRA), 오가피(ACANTHOPANACIS CORTEX), 방풍(LEDEBOURIELLAE RADIX), 감초(GLYCYRRHIZAE RADIX), 구기자(LYCII FRUCTUS), 속단(DIPSACI RADIX), 녹각(CERVI CORNU), 단삼(SALVIAE MILTIORRHIZAE RADIX), 산사(CRATAEGII FRUCTUS), 현삼(SCROPHULARIAE RADIX), 익모초(LEONURI HERBA), 신곡(MASSA MEDICATA FERMENTATA), 약콩(SOJAE SEMEN), 적두(PHASEOLI SEMEN)에서 선택된 1종이상의 보조생약 또는 이의 물, 저급알콜, 초산에틸, 방향족탄화수소, 염소화탄화수소에서 선택된 용매로 추출한 엑스를 함유하는 조성물은 골다공증, 류마티스 관절염, 디스크증상의 예방과 치료에 탁월한 효과가 있다.As described in detail above, the main component of the present invention is CIBOTII RHIZOMA: dried hair root stem of Cibotium barometz (L.) and / or powder or X of the myelium root ( Harpagophytum procumbens DC.). If necessary, Malt (HORDEI FRUCTUS GERMINIATUS), Umbrella (ACHYRANTHIS BIDENTATAE RADIX), Angelica (ANGELICAE GIGANTIS RADIX), SUNGJIANG (REHMANNIAE RADIX PREPARAT), CHOAP (EUCOMMIAE CORTEX), Broiler (CINNAMOAMI CORT) , Gupan (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), Goku (SCOLOPENDRA), Ogapi (ACANTHOPANACIS CORTEX), Windproof (LEDEBOURIELLAE RADIX), Licorice (GLYCYRRHIZAE RADIX) CERVI CORNU, Salvia (SALVIAE MILTIORRHIZAE RADIX), Sansa (CRATAEGII FRUCTUS), Hyunsam (SCROPHULARIAE RADIX), Motherwort (LEONURI HERBA), Singok (MASSA MEDICATA FERMENTATA), Medicinal Beans (SOJAE SEMEN) 1 SEMIPH Paper boy Composition containing X and extracted with herbal medicines or their water, lower alcohols, ethyl acetate, aromatic hydrocarbon, solvents selected from chlorinated hydrocarbon is an excellent effect in the prevention and treatment of osteoporosis, rheumatoid arthritis, disk symptoms.

Claims (5)

구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것)의 분말 또는 이의 물, 탄소수 1-4의 저급알콜 또는 이들의 혼합용매중에서 추출하여 얻어진 골다공증, 류마티스 관절염 또는 디스크 증상의 예방과 치료에 효능이 있는 엑스.Osteoporosis, rheumatoid arthritis, or the like obtained by extracting from powder of citric acid (CIBOTII RHIZOMA: dried root of Cibotium barometz (L.)) or water thereof, lower alcohol having 1-4 carbon atoms or mixed solvents thereof X is effective for the prevention and treatment of disc symptoms. 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것)의 분말이나 또는 물, 탄소수 1-4의 저급알콜 또는 이들의 혼합용매중에서 추출하여 얻어진 엑스를 주성분으로 함유하고, 여기에 천수근(Harpagophytum procumbens DC.), 맥아(HORDEI FRUCTUS GERMINIATUS), 우슬[ACHYRANTHIS BIDENTATAE RADIX), 당귀(ANGELICAE GIGANTIS RADIX), 숙지황(REHMANNIAE RADIX PREPARAT), 두충(EUCOMMIAE CORTEX), 육계(CINNAMOMI CORTEX), 홍화자(CARTHAMI FRUCTUS), 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), 오공(SCOLOPENDRA), 오가피(ACANTHOPANACIS CORTEX), 방풍(LEDEBOURIELLAE RADIX), 감초(GLYCYRRHIZAE RADIX), 구기자(LYCII FRUCTUS), 속단(DIPSACI RADIX), 녹각(CERVI CORNU), 단삼(SALVIAE MILTIORRHIZAE RADIX), 산사(CRATAEGII FRUCTUS), 현삼(SCROPHULARIAE RADIX), 익모초(LEONURI HERBA), 신곡(MASSA MEDICATA FERMENTATA), 약콩(SOJAE SEMEN), 적두(PHASEOLI SEMEN)에서 선택된 1종 이상의 보조생약 또는 이의 물, 탄소수 1내지 4의 저급알콜 또는 이의 혼합용매로 엑스 1종이상을 함유한 골다공증, 류마티스 관절염, 디스크 증상의 예방과 치료에 효능이 있는 조성물.CIBOTII RHIZOMA (extracted from dried roots of Cibotium barometz (L.)) Powder or water, lower alcohols having 1-4 carbon atoms or mixed solvents thereof. And here, Hargogophytum procumbens DC., Malt (HORDEI FRUCTUS GERMINIATUS), Umbrella (ACHYRANTHIS BIDENTATAE RADIX), Angelica (ANGELICAE GIGANTIS RADIX), REHMANNIAE RADIX PREPARAT, Chickpeas (ORTEXMIMI) Caf (DIPSACI RADIX), CERVI CORNU, Salvia (SALVIAE MILTIORRHIZAE RADIX), Sansa (CRATAEGII FRUCTUS), Hyunsam (SCROPHULARIAE RADIX), Motherwort (LEONURI HERBA), Shingok (MASSA MEDICATA Medicinal Soybeans) Choose from (PHASEOLI SEMEN) At least one auxiliary crude drugs or the water thereof, which composition is effective in the prevention and treatment of lower alcohols or contains at least 1 x species to a mixed solvent thereof osteoporosis, rheumatoid arthritis, the symptoms of the disk 1 to 4 carbon atoms. 제 1항 또는 제 2항에 있어서, 구척(CIBOTII RHIZOMA: 금모구척(Cibotium barometz(L.)의 뿌리모양의 줄기를 건조한 것) 10 내지 200중량부를 주성분으로 함유하고, 여기에 천수근(Harpagophytum procumbens DC.) 10 내지 300중량부, 맥아(HORDEI FRUCTUS GERMINIATUS) 10 내지 200중량부, 우슬[ACHYRANTHIS BIDENTATAE RADIX) 50 내지 500중량부, 당귀(ANGELICAE GIGANTIS RADIX) 50 내지 500중량부, 숙지황(REHMANNIAE RADIX PREPARAT) 50 내지 500중량부, 두충(EUCOMMIAE CORTEX) 50 내지 500중량부, 육계(CINNAMOMI CORTEX) 50 내지 500중량부, 홍화자(CARTHAMI FRUCTUS) 50 내지 500중량부, 구판( 또는 자라: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 내지 500중량부, 오공(SCOLOPENDRA) 10 내지 200중량부, 오가피(ACANTHOPANACIS CORTEX) 10 내지 200중량부, 방풍(LEDEBOURIELLAE RADIX) 10 내지 200중량부, 감초(GLYCYRRHIZAE RADIX) 10 내지 200중량부, 구기자(LYCII FRUCTUS) 50 내지 500중량부, 속단(DIPSACIRADIX) 100 내지 500중량부, 녹각(CERVI CORNU) 100 내지 500중량부, 단삼(SALVIAE MILTIORRHIZAE RADIX) 20 내지 200중량부, 산사(CRATAEGII FRUCTUS) 10 내지 200중량부, 현삼(SCROPHULARIAE RADIX) 10 내지 200중량부, 익모초(LEONURI HERBA) 100 내지 500중량부, 신곡(MASSA MEDICATA FERMENTATA) 50 내지 500중량부,약콩(SOJAE SEMEN) 50 내지 500중량부, 적두(PHASEOLI SEMEN) 10 내지 200중량부에서 선택된 1종 이상의 보조생약 또는 이의 물, 탄소수 1-4의 저급알콜 또는 이들의 혼합용매에서 선택된 용매로 추출한 엑스를 함유한 골다공증, 류마티스 관절염, 디스크증상의 예방과 치료에 효능이 있는 조성물.The method according to claim 1 or 2, wherein 10 to 200 parts by weight of CIBOTII RHIZOMA (dried stems of Cibotium barometz (L.) dried) is contained as a main component, and here, Harpagophytum procumbens DC .) 10 to 300 parts by weight, 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of cowl (ACHYRANTHIS BIDENTATAE RADIX), 50 to 500 parts by weight of Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of liquorice (GLYCYRRHIZAE RADIX) (LYCII FRUCTUS) 50 to 500 Part, 100-500 parts by weight of DIPSACIRADIX, 100-500 parts by weight of CERVI CORNU, 20-200 parts by weight of SALVIAE MILTIORRHIZAE RADIX, 10-200 parts by weight of CRATAEGII FRUCTUS, Hyunsam (SCROPHULARIAE RADIX) ) 10 to 200 parts by weight, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of SOJAE SEMEN, 10 to 200 parts by weight of PHASEOLI SEMEN A composition effective in the prevention and treatment of osteoporosis, rheumatoid arthritis, disc symptoms, containing X extracted with a solvent selected from at least one auxiliary medicine selected from the above or water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. 제 1항 내지 제 3항의 조성물을 약제학적으로 통상으로 허용되는 부형제, 보조제, 희석제, 등장화제, 보존제, 활탁제, 용해보조제와 함께 약제학적으로 통상으로 허용되는 약학적 제제형태로 제제화한 골다공증, 류마티스성 관절염 및 디스크증상의 예방과 치료에 효능이 있는 약학적 제제.Osteoporosis formulated in the form of a pharmaceutically acceptable pharmaceutical formulation of the composition of claim 1 together with pharmaceutically conventionally acceptable excipients, adjuvants, diluents, isotonic agents, preservatives, suspending agents, dissolution aids, Pharmaceutical preparations effective for the prevention and treatment of rheumatoid arthritis and disc symptoms. 제 4항에 있어서, 기존에 이미 약제로 사용되고 있는 알렌드레이트(allendrate), 타목시펜(tamoxifen), 비타민 B3, 부갑상선 호르몬(parathyroid hormone: PTH), 설파살라진(sulfasalazine), 티오레독신 리덕타제(thioredoxin reductase), 알렌드로네이트(alendronate), 랄록시펜(raloxifene), 칼시토닌(calcitonin), 에스타라디올(estradiol), 게니스테인(genistein), 1,25-디하이드록시바이타민 D3, 알렌드로네이트(alendronate), 에스트로젠 수용체 조절제(estrogen receptor modulator), 비포스포네이트 (biphosphonates), 하르파지드(harpagide: 본 발명자가 그 용도를 개발한 약물임)에서 선택된 1종 이상의 약물을 더 함유하고, 여기에 약제학적으로 통상으로 허용되는 부형제, 보조제, 희석제, 등장화제, 보존제, 활탁제, 용해보조제와 함께 약제학적으로 통상으로 허용되는 약학적 제제형태로 제제화한 골다공증, 류마티스성 관절염 및 디스크증상의 예방과 치료에 효능이 있는 약학적 제제.The method of claim 4, wherein the drug is already used as an allenrate, tamoxifen, vitamin B 3 , parathyroid hormone (PTH), sulfasalazine, thioredoxin reductase, or thioredoxin reductase. ), Alendronate, raloxifene, calcitonin, calcitonin, estradiol, genistein, 1,25-dihydroxyvitamin D 3 , alendronate, estrogen receptor modulator (etrogen receptor modulator), biphosphonates, harpagide (harpagide is a drug that the inventors developed the use) further contains one or more pharmaceutically acceptable Osteoporosis, which is formulated in the form of a pharmaceutically acceptable pharmaceutical formulation with excipients, adjuvants, diluents, isotonic agents, preservatives, suspending agents, and dissolution aids. The pharmaceutical formulation with efficacy in the treatment and prevention of arthritis and tooth disc symptoms.
KR10-2000-0071397A 2000-11-28 2000-11-28 Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients KR100415826B1 (en)

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KR20160004684A (en) 2014-07-04 2016-01-13 원광대학교산학협력단 A method for making gel of scolopendra subspinipes, the gel, a method for making medicine acupuncture liquid comprising the gel, and the medicine acupuncture liquid

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KR101152753B1 (en) * 2011-12-30 2012-06-18 신준식 Pharmaceutical composition for preventing and treating of metabolic bone disease comprising the harpagophytum
KR20160004684A (en) 2014-07-04 2016-01-13 원광대학교산학협력단 A method for making gel of scolopendra subspinipes, the gel, a method for making medicine acupuncture liquid comprising the gel, and the medicine acupuncture liquid

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