KR20130060837A - A composition comprising the extract of processed panax genus plant for treating and preventing osteoporosis - Google Patents

A composition comprising the extract of processed panax genus plant for treating and preventing osteoporosis Download PDF

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KR20130060837A
KR20130060837A KR1020110127110A KR20110127110A KR20130060837A KR 20130060837 A KR20130060837 A KR 20130060837A KR 1020110127110 A KR1020110127110 A KR 1020110127110A KR 20110127110 A KR20110127110 A KR 20110127110A KR 20130060837 A KR20130060837 A KR 20130060837A
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황귀서
김복득
박정일
김진희
이혜진
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가천대학교 산학협력단
주식회사 진생사이언스
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Abstract

PURPOSE: A processed ginseng extract is provided to suppress expression of genes related to RANKL-induced osteoclast differentiation and bone resorption, and to prevent and treat metabolic bone diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating metabolic bone diseases contains a processed Panax sp. plant extract in which the content of ginsenoside Rg3, Rk1, and Rg5 is higher than the content of ginsenoside Rb1, Rb2, RC, and Rd. The Panax sp. plant is artificially processed using Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax vietnamensis, Panax japonicus, Panax elegatior, Panax wangianus, Panax bipinnatifidus, or Panax pseudoginseng. The Panax sp. plant is processed by thermal treatment of ginseng at 120-180 deg. C for 0.5-20 hours to enhance drug efficiency.

Description

가공된 인삼 추출물을 유효성분으로 함유하는 골대사성 질환 치료 및 예방용 약학 조성물{A composition comprising the extract of processed Panax genus plant for treating and preventing osteoporosis}A composition comprising the extract of processed Panax genus plant for treating and preventing osteoporosis}

본 발명의 가공된 인삼 추출물을 유효성분으로 함유하는 골대사성 질환 치료 및 예방용 약학 조성물에 관한 것이다. It relates to a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the processed ginseng extract of the present invention as an active ingredient.

1. Kumamoto H, Ooya K. ExpreASion of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) in ameloblastomas. J Oral Pathol Med. 2004 ; 33(1): 46-52.Kumamoto H, Ooya K. ExpreASion of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF) / receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF) / osteoprotegerin (OPG) in ameloblastomas. J Oral Pathol Med. 2004; 33 (1): 46-52.

2. Reddy SV. Regulatory mechanisms operative in osteoclasts. Crit Rev Eukaryot Gene Expr. 2004; 14(4): 255-70.2. Reddy SV. Regulatory mechanisms operative in osteoclasts. Crit Rev Eukaryot Gene Expr. 2004; 14 (4): 255-70.

3. Pang M, Martinez AF, Fernandez I, Balkan W, Troen BR. AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells. Gene. 2007;403(1-2):151-8.3.Pang M, Martinez AF, Fernandez I, Balkan W, Troen BR. AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells. Gene. 2007; 403 (1-2): 151-8.

4. Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L. Roles for NF-kappaB and c-Fos in osteoclasts. J Bone Miner Metab. 2005; 23 Suppl: 11-5. Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L. Roles for NF-kappa B and c-Fos in osteoclasts. J Bone Miner Metab. 2005; 23 Suppl: 11-5.

5. Khosla S. Minireview: the OPG/RANKL/RANK system. Endocrinology. 2001; 142(12):5050-5.Khosla S. Minireview: the OPG / RANKL / RANK system. Endocrinology. 2001; 142 (12): 5050-5.

6. Han SY, Lee NK, Kim KH, Jang IW, Yim M, Kim JH, Lee WJ, Lee SY. Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis. Blood. 2005;106(4):1240-5.6.Han SY, Lee NK, Kim KH, Jang IW, Yim M, Kim JH, Lee WJ, Lee SY. Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis. Blood. 2005; 106 (4): 1240-5.

7. Yamashita M, Otsuka F, Mukai T, Yamanaka R, Otani H, Matsumoto Y, Nakamura E, Takano M, Sada KE, Makino H. Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling. Regul Pept. 2010 ;162(1-3):99-1087.Yamashita M, Otsuka F, Mukai T, Yamanaka R, Otani H, Matsumoto Y, Nakamura E, Takano M, Sada KE, Makino H. Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling. Regul Pept. 2010; 162 (1-3): 99-108

8. Murakami A, Song M, Ohigashi H. Phenethyl isothiocyanate suppresses receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis by blocking activation of ERK1/2 and p38 MAPK in RAW264.7 macrophages. Biofactors. 2007;30(1):1-11.Murakami A, Song M, Ohigashi H. Phenethyl isothiocyanate suppresses receptor activator of NF-kappaB ligand (RANKL) -induced osteoclastogenesis by blocking activation of ERK1 / 2 and p38 MAPK in RAW264.7 macrophages. Biofactors. 2007; 30 (1): 1-11.

9. Choi HJ, Park YR, Nepal M, Choi BY, Cho NP, Choi SH, Heo SR, Kim HS, Yang MS, Soh Y. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways. Eur J Pharmacol. 2010 ;636(1-3):28-359.H Choi HJ, Park YR, Nepal M, Choi BY, Cho NP, Choi SH, Heo SR, Kim HS, Yang MS, Soh Y. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways. Eur J Pharmacol. 2010; 636 (1-3): 28-35

10. 고려삼의 이해, 고려인삼학회, p 9, 1995; Advances in Ginseng Research, 고려인삼학회, p 127, 1998)10. Understanding Korean Ginseng, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korea Ginseng Society, p 127, 1998)

11. 고려인삼의 이해, 고려인삼학회, pp 9-18, 2008). 11. Understanding Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

12. Kim W.Y. et al, Journal of Natural Products, vol.63, pp1702-1704, 2000; 12. Kim W.Y. et al., Journal of Natural Products, vol. 63, pp 1702-1704, 2000;

13 Kwon S W, et al., J. Chromatogr. A, 921(2), pp 335-339, 2001; 13 Kwon S W, et al., J. Chromatogr. A, 921 (2), pp 335-339, 2001;

14. Yong-Sam Keum, Kwang-Kyun Park, Jong-Min Lee, Kyung-soo Chun., Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. J. CancerLetters .150,PP.41-48,2000.14.Yong-Sam Keum, Kwang-Kyun Park, Jong-Min Lee, Kyung-soo Chun., Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. J. CancerLetters . 150, pp. 41-48,2000.

15. Young C. Kim, So R. Kim, George J. Markelonis, Tae H. Oh., Ginsenosides Rb1 and Rg3 Protect Cultured Rat Cortical Cells From Glutamate-Induced Neurodegeneration. J. NeuroscienceRes .53,PP.426-432,1998.15. Young C. Kim, So R. Kim, George J. Markelonis, Tae H. Oh., Ginsenosides Rb1 and Rg3 Protect Cultured Rat Cortical Cells From Glutamate-Induced Neurodegeneration. J. Neuroscience Res . 53, pp. 426-432, 1998.

16. Keun Young Jung, Dong seon Kim, Platelet Activating Factor Antagonist Activity of Ginsenosides. Biol . Pharm . Bull .21.PP.79-80,1998.16. Keun Young Jung, Dong seon Kim, Platelet Activating Factor Antagonist Activity of Ginsenosides. Biol . Pharm . Bull . 21.PP.79-80, 1998.

17. Kim WY, et al., J. Nat . Prod ., 63(12), pp 1702-1704, 2001 ;17. Kim WY, et al., J. Nat . Prod . , 63 (12) , pp 1702-1704, 2001;

18. Kwon SW, et al., J. Chromatogr . A., 921(2), pp 335-339, 2001 18. Kwon SW, et al., J. Chromatogr . A. , 921 (2) , pp 335-339, 2001

19. 대한민국 특허 10-0192678 약효가 증강된 가공인삼제품, 1999
19. Republic of Korea Patent 10-0192678 Processed Ginseng Product with Enhanced Drug Effect, 1999

본 발명의 가공된 인삼 추출물을 유효성분으로 함유하는 골대사성 질환 치료 및 예방용 약학 조성물에 관한 것이다. It relates to a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the processed ginseng extract of the present invention as an active ingredient.

대사는 골 형성을 담당하는 조골세포와 골 흡수를 담당하는 파골세포의 작용으로 이루어져 있으며, 이들 세포의 기능이 균형을 이루어 항상성을 유지한다. 그러나, 조골세포 활성이 저하되거나 파골세포 활성이 증가되면 골밀도가 감소하여 골다공증이 유발될 수 있다. 골다공증의 유발 요인으로는 여성의 폐경, 갑상선 기능항진, 당뇨병, 스트레스, 흡연 및 운동 부족, 신체적 노화와 glucocorticoid 계열 약물의 복용 등이 알려져 있다(1-3). Metabolism is composed of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption, and the function of these cells is balanced to maintain homeostasis. However, when osteoblast activity decreases or osteoclast activity increases, bone density decreases and osteoporosis may be induced. Factors causing osteoporosis include menopause, hyperthyroidism, diabetes, stress, lack of smoking and exercise, physical aging, and the use of glucocorticoid drugs in women (1-3).

파골세포는 대식세포 계열의 전구세포에서 다양한 분화유발인자들에 의해 분화된다(4-5). 특히, 조골세포로부터 분비되는 RANKL(receptor activator of nuclear factor kappa B ligand)은 파골전구세포 및 파골세포 표면에 존재하는 RANK(receptor activator of nuclear factor kappa B)와 결합하여 파골전구세포가 파골세포로의 분화와 활성화를 유발한다(6). 분화한 파골세포는 NF-B, c-Fos, c-jun, AP-1, NFATc1의 활성화와 MAPK, ERK, JNK, p38 활성화 과정, Src, Akt MITF 활성화등을 통하여 TRAP (tartarate resistant acid phosphatase) cathepsin K, calcitonin receptor 등 파골세포 특이 단백질 발현을 촉진한다(7-9). Osteoclasts are differentiated by various differentiation-causing factors in macrophage progenitor cells (4-5). In particular, RANKL (receptor activator of nuclear factor kappa B ligand) secreted from osteoblasts binds to osteoclast precursor cells and RANK (receptor activator of nuclear factor kappa B) present on the surface of osteoclasts. Causes differentiation and activation (6). Differentiated osteoclasts were activated by NF-B, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt MITF activation, etc. It promotes the expression of osteoclast-specific proteins such as cathepsin K and calcitonin receptor (7-9).

파낙스(Panax)속 식물은 식물 분류학상 오가피과 (Araliaceae)에 속하는 다년생 숙근초로서 지구상에 십여종이 알려져 있다. 대표적인 종으로 고려인삼 (Panax ginseng), 화기삼 (Panax quinquefolia), 전칠삼 (삼칠, Panax notoginseng), 베트남삼 (Panax vietnamensis), 죽절삼 (Panax japonicus) 등이 있으며, (고려삼의 이해, 고려인삼학회, p 9, 1995; Advances in Ginseng Research, 고려인삼학회, p 127, 1998) 기타 파낙스속 식물로는 파낙스 엘레가티오르 (Panax elegatior), 파낙스 완지아누스 (Panax wangianus), 파낙스 비핀나티푸스 (Panax bipinnatifidus), 파낙스 슈도진생 (Panax pseudoginseng) 등이 있다.Panax is a perennial herbaceous plant belonging to the genus Araliaceae in plant taxonomy. Representative species include Korean ginseng ( Panax ginseng ), flower ginseng ( Panax quinquefolia ), ginseng (samchi, Panax notoginseng ), Vietnamese ginseng ( Panax vietnamensis ) and Panax japonicus (People's Understanding, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korean Ginseng Society, p 127, 1998). Thior ( Panax elegatior ), Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), Panax pseudoinsins ( Panax pseudoginseng ).

파낙스속 식물 중 가장 유명한 인삼은 예로부터 아시아 지역에서 가장 널리 사용되어온 생약 중의 하나로, 피로회복, 자양강장, 인지기능개선, 기억력증가, 혈액순환개선, 항암, 항당뇨, 성기능개선, 노화방지, 면역력증강, 혈소판 응집억제 등 많은 약리효능이 알려져 있다 (고려인삼의 이해, 고려인삼학회, pp 9-18, 2008). Ginseng, one of the most famous Panax plants, is one of the most widely used herbal medicines in Asia. It has been widely used in Asia for many years. It has been widely used for many purposes such as fatigue, nourishment, improvement of cognitive function, memory, blood circulation improvement, And the inhibition of platelet aggregation are known. (Understanding of Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

한편, 파낙스속 식물을 가열 가공 처리하거나 또는 약산으로 처리하면 진세노사이드 구조에서 일부 당과 알콜기가 떨어져나가 약효가 더 강력한 새로운 구조의 진세노사이드, 즉, 진세노사이드 Rg3, Rg5, Rk1, Rk2, Rk3, Rs1, Rs2, Rs3, F4, Rh2, Rh3, Rh4 등이 생성되는 것이 알려져 있다(Kim W.Y. et al, Journal of Natural Products, vol.63, pp1702-1704, 2000; Kwon S W, et al., J. Chromatogr. A, 921(2), pp 335-339, 2001; Park I.H. et. al., Archives of Pharmacal Research, 25, pp.428-432 and 837-841, 2002).On the other hand, heat-processing or treating a genus of Panax plants with a weak acid removes some sugars and alcohol groups from the ginsenoside structure, and the new structure of ginsenosides having stronger potency, ie, ginsenosides Rg3, Rg5, Rk1, Rk2 , Rk3, Rs1, Rs2, Rs3, F4, Rh2, Rh3, Rh4 and the like are known to be produced (Kim WY et al, Journal of Natural Products, vol. 63, pp1702-1704, 2000; Kwon SW, et al. , J. Chromatogr.A, 921 (2), pp 335-339, 2001; Park IH et. Al., Archives of Pharmacal Research, 25, pp.428-432 and 837-841, 2002).

대한민국 등록특허 제192678호 및 미국 등록특허 제5776460호에는 진세노사이드 (Rg3+Rg5+Rk1)의 함량의 합이 (Rb1+Rb2+Rc+Rd)의 함량의 합보다도 더 커진 가공 파낙스속 식물(이하, 선삼이라 함)에 대하여 기술되어 있으며, 상기 선삼은 인삼(Panaxginseng)을 고열 고압으로 처리하여 약효가 강한 Rg3,Rg5,Rk1의 함량을 획기적으로 높인 가공 인삼이다. 선삼의 약효로는 항암작용(14), 뇌기능 개선작용(15), 혈소판 응집능 차단작용(16) 등이 보고되어있다. Republic of Korea Patent No. 192678 and U.S. Patent No. 5,76,460 include processed panax plants in which the sum of the contents of ginsenosides (Rg3 + Rg5 + Rk1) is greater than the sum of the contents of (Rb1 + Rb2 + Rc + Rd). Hereinafter, the ginseng) is described, wherein the ginseng is a processed ginseng by dramatically increasing the content of Rg 3 , Rg 5 , Rk 1 , which is highly effective by treating ginseng ( Panaxginseng ) at high temperature and high pressure. As the medicinal effect of ginseng has been reported anti-cancer effect (14), brain function improving effect (15), platelet aggregation ability blocking effect (16) and the like.

그러나, 상기 문헌들의 어디에도 아직까지 가공된 파낙스속 식물 추출물의 골다공증 치료 및 예방 효과에 대해서는 전혀 기재되거나 교시된 바가 없다.
However, none of the above documents has yet described or taught any osteoporosis treatment and prophylactic effects of the processed Panax genus extract.

본 발명자들은, 가공 인삼인 선삼의 추출물을 이용하여 RANKL 유도 파골세포 분화 및 골흡수 관련 유전자 발현을 억제하는 작용에 대해서 평가하고자 하였다. 이를 위하여 사람의 RANKL 처리하여 파골세포로 분화시킨 세포에서 가공 인삼인 선삼의 추출물이 TRAP(+) 다핵세포생성 억제 효과를 측정하였으며, 파골세포 관련 인자인 TRAP, NFATc1, JNK 등의 유전자 발현을 가공 인삼인 선삼의 추출물이 억제함을 확인하여 본 발명을 완성하였다.
The present inventors tried to evaluate the action of inhibiting RANKL induced osteoclast differentiation and bone resorption related gene expression using extracts of ginseng, processed ginseng. To this end, extracts of ginseng, processed ginseng, inhibited TRAP (+) multinucleogenesis in the cells differentiated into osteoclasts by human RANKL treatment and processed gene expressions such as TRAP, NFATc1, JNK, which are osteoclast related factors. Confirmed that the extract of ginseng ginseng inhibited to complete the present invention.

상기 목적을 해결하기 위해 본 발명은 진세노사이드 Rg3, Rk1, Rg5 함량의 합이 진세노사이드 Rb1, Rb2, Rc, Rd의 함량의 합보다 더 높도록 약효성분을 증가시킨 가공된 파낙스속 식물 추출물을 유효성분으로 함유하는 골대사성 질환 및 골질환 치료 및 예방용 약학조성물을 제공한다.
In order to solve the above object, the present invention is processed Panax genus plant extract in which the sum of the ginsenoside Rg3, Rk1, Rg5 content is higher than the sum of the content of ginsenoside Rb1, Rb2, Rc, Rd It provides a pharmaceutical composition for treating and preventing bone metabolic diseases and bone diseases containing as an active ingredient.

본원에서 정의되는 가공된 파낙스속 식물은, 바람직하게는 고려인삼 (Panax ginseng), 화기삼 (Panax quinquefolia), 전칠삼 (삼칠, Panax notoginseng), 베트남삼 (Panax vietnamensis), 죽절삼 (Panax japonicus), 파낙스 엘레가티오르 (Panax elegatior), 파낙스 완지아누스 (Panax wangianus), 파낙스 비핀나티푸스 (Panax bipinnatifidus), 또는 파낙스 슈도진생 (Panax pseudoginseng)을 인위적으로 가공시킨 식물을 포함하며, 보다 바람직하게는 고온에서 가열처리함으로써 약효가 증강된 가공인삼에 관한 것이다. 더욱 구체적으로, 인삼을 120 내지 180℃의 고온에서 0.5 내지 20시간 동안 가열처리하여 진세노사이드 (Rg3+Rg5)/(Rc+Rd+Rb₁+Rb₂)의 비율이 1.0이상이 되도록 약효성분을 증가시킨 가공인삼을 포함한다.
The processed Panax plant as defined herein is preferably Korean Ginseng (Panax ginseng), Firewood (Panax quinquefoliaHowever,Panax notoginseng), VietnamPanax vietnamensis),Panax japonicus), Panax Elegatior (Panax elegatior), Panax Wanian (Panax wangianus), Panax bisfinatipus (Panax bipinnatifidus), Or Panax Pseudogin (Panax pseudoginsengThe present invention relates to a processed ginseng which includes artificially processed plants), and more preferably, has enhanced efficacy by heating at a high temperature. More specifically, the ginseng is heated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours to increase the active ingredient so that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + RbR + Rb₂) is 1.0 or more. Contains processed ginseng.

상기 추출물은 물, 메탄올, 에탄올, 부탄올 등의 저급 알콜 또는 이들의 혼합용매, 바람직하게는, 물 또는 10 내지 100% 물 및 에탄올 혼합용매, 보다 바람직하게는, 약 50 내지 90% 물 및 에탄올 혼합용매에 가용한 추출물을 포함한다.The extract is a lower alcohol such as water, methanol, ethanol, butanol, or a mixed solvent thereof, preferably water or 10 to 100% water and ethanol mixed solvent, more preferably, about 50 to 90% water and ethanol mixed Extracts soluble in the solvent.

본원에서 정의되는 상기 골 대사성 질환은 골다공증(osteoprosis), 파제트병(paget disease), 치주질환(periodontal disease), 골전이암(metastatic cancer) 또는 류마티스 관절염(rheumatoid arthiritis), 바람직하게는 골다공증을 포함하는 것을 특징으로 한다.The bone metabolic disease as defined herein includes osteoprosis, paget disease, periodontal disease, metastatic cancer or rheumatoid arthiritis, preferably osteoporosis. Characterized in that.

이하 본 발명의 추출물을 수득하는 방법을 보다 상세하게 설명한다. Hereinafter, the method of obtaining the extract of the present invention will be described in more detail.

예를 들어, 파낙스속 식물의 열매, 줄기 또는 뿌리를 각각 세척 및 건조시킨 후, 상기 파낙스속 식물은 120 내지 180℃의 고온에서 0.5 내지 20시간 동안 가열처리한 후에 상기 개개 시료 중량의 약 1배 내지 30배, 바람직하게는 약 5배 내지 15배 (w/v) 부피의 물, C1 내지 C4의 저급 알콜 또는 이들의 혼합용매로, 바람직하게는 물 또는 에탄올, 보다 바람직하게는 물 또는 50 내지 90% 에탄올을 추출용매로 하여, 약 70 내지 120℃, 바람직하게는 80 내지 110℃의 반응온도에서 약 1 내지 6시간, 바람직하게는 3 내지 5시간 동안 가열추출법, 초음파 추출법, 환류 추출법 등의 통상적인 추출방법, 바람직하게는 환류 추출법으로 1 내지 10회, 바람직하게는 2 내지 7회 반복 추출하는 제 2단계; 상기 단계에서 수득한 추출액을 여과 및 감압 농축하는 제 3단계를 통하여 본 발명의 가공된 파낙스속 식물 추출물을 수득가능하다.
For example, after washing and drying the fruits, stems or roots of the Panax plants, respectively, the Panax plants are heat treated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours and then about 1 times the weight of the individual samples. To 30 times, preferably about 5 to 15 times (w / v) volume of water, C 1 to C 4 lower alcohols or mixed solvents thereof, preferably water or ethanol, more preferably water or Heat extraction method, ultrasonic extraction method, reflux extraction method for about 1 to 6 hours, preferably 3 to 5 hours at a reaction temperature of about 70 to 120 ℃, preferably 80 to 110 ℃ using 50 to 90% ethanol as the extraction solvent A second step of repeated extraction 1 to 10 times, preferably 2 to 7 times by a conventional extraction method such as a reflux extraction method; The processed Extract of Phanax spp. Of the present invention can be obtained through the third step of filtration and concentration under reduced pressure of the extract obtained in the above step.

또한 본 발명은 상기한 제조방법 및 상기 제조방법으로 제조된 가공된 파낙스속 식물 추출물을 유효성분으로 함유하는 골대사성 질환 및 골질환 치료 및 예방를 위한 약학 조성물을 제공한다. In another aspect, the present invention provides a pharmaceutical composition for treating and preventing bone metabolic diseases and bone diseases containing the above production method and processed Panax genus plant extract prepared by the production method as an active ingredient.

상기에서 제조된 추출물은 RANKL 유도 파골세포 분화 및 골흡수 관련 유전자 발현을 억제하는 효과가 있음을 확인하였고, 이를 연구에서 RANKL 처리하여 파골세포로 분화시킨 세포에서 가공 인삼인 선삼의 추출물이 TRAP(+) 다핵세포생성 억제 효과를 나타내었으며, 파골세포 관련 인자인 TRAP, NFATc1, JNK 등의 유전자 발현을 억제함을 확인하여 골다공증 및 골질환에 유용하게 쓰일 수 있다. 본 발명의 조성물은, 조성물 총 중량에 대하여 상기 생약 추출물을 0.1 내지 50% 중량으로 포함한다.The extract prepared above was confirmed to have an effect of inhibiting RANKL-induced osteoclast differentiation and bone resorption-related gene expression, and in this study, the extract of ginseng, which is processed ginseng, in the cells differentiated into osteoclasts by RANKL treatment was TRAP (+ ) It has been shown to inhibit the production of multinucleated cells, and to inhibit the gene expression of osteoclast-related factors such as TRAP, NFATc1, JNK, etc., which can be useful for osteoporosis and bone diseases. The composition of the present invention comprises 0.1 to 50% by weight of the herbal extract based on the total weight of the composition.

그러나 상기와 같은 조성은 반드시 이에 한정되는 것은 아니고, 환자의 상태 및 질환의 종류 및 진행 정도에 따라 변할 수 있다.However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

본 발명의 추출물을 포함하는 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

본 발명에 따른 추출물을 포함하는 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 이에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

본 발명의 추출물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 추출물은 1일 0.01 mg/kg 내지 10 g/kg으로, 바람직하게는 1 mg/kg 내지 1 g/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수 있다. 그러므로 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 및 직장, 또는 정맥 등의 방법을 통하여 투여할 수 있다. The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

상기와 같이, 본 발명의 가공된 인삼 추출물은 RANKL 유도 파골세포 분화 및 골흡수 관련 유전자 발현을 억제하는 효과가 있음을 확인하였고, 이를 연구에서 RANKL 처리하여 파골세포로 분화시킨 세포에서 가공 인삼인 선삼의 추출물이 TRAP(+) 다핵세포생성 억제 효과를 나타내었으며, 파골세포 관련 인자인 TTRAP, NFATc1, JNK 등의 유전자 발현을 탁월하게 억제함을 확인하여, 골대사성 질환치료 및 골질환 예방 및 치료에 유용하게 쓰일 수 있다.As described above, it was confirmed that the processed ginseng extract of the present invention has an effect of inhibiting RANKL-induced osteoclast differentiation and bone resorption-related gene expression, and in this study, ginseng which is processed ginseng in cells differentiated into osteoclasts by RANKL treatment. Extracts showed TRAP (+) multinucleated cell suppression effect, and inhibited gene expression of osteoclast-related factors TTRAP, NFATc1, JNK, etc., for the treatment and treatment of bone metabolic diseases and bone diseases This can be useful.

도 1은 본 발명의 가공인삼 추출물 (SG) 농도에 따른 RANKL 처리 RAW264.7 세포로부터 TRAP(+) 다핵세포 형성에 미치는 영향을 나타낸 도이며,
도 2는 본 발명의 가공인삼 추출물 (SG) 농도에 따른 TRAP 발현에 미치는 영향을 나타낸 도이며,
도 3는 본 발명의 가공인삼 추출물 (SG) 농도에 따른 NFATc1 발현에 미치는 영향을 나타낸 도이며,
도 4는 본 발명의 가공인삼 추출물 (SG) 농도에 따른 JNK 발현에 미치는 영향을 나타낸 도이다.
1 is a diagram showing the effect on the formation of TRAP (+) multinucleated cells from RANKL treated RAW264.7 cells according to the concentration of processed ginseng extract (SG) of the present invention,
Figure 2 is a view showing the effect on TRAP expression according to the processed ginseng extract (SG) concentration of the present invention,
Figure 3 is a view showing the effect on the expression of NFATc1 according to the concentration of processed ginseng extract (SG) of the present invention,
Figure 4 is a diagram showing the effect on JNK expression according to the processed ginseng extract (SG) concentration of the present invention.

이하, 본 발명을 하기 실시예 및 실험예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

단, 이는 본 발명을 예시하는 것일 뿐, 본 발명의 내용은 이에 의해 한정되는 것은 아니다.
However, it should be understood that the present invention is not limited thereto.

실시예 1. 가공 인삼 추출물의 제조Example 1 Preparation of Processed Ginseng Extract

김 등의 방법 (Kim WY, et al., J. Nat . Prod ., 63(12), pp 1702-1704, 2001; Kwon SW, et al., J. Chromatogr . A., 921(2), pp 335-339, 2001)에 의하여 특수가공인삼을 제조하였다. Methods, such as steam (Kim WY, et al, J. Nat Prod, 63 (12), pp 1702-1704, 2001;..... Kwon SW, et al, J. Chromatogr A., 921 (2), pp 335-339, 2001).

즉, 건조된 인삼(금산 인삼시장)을 잘게 자른 후, 120℃, 15 기압에서 4시간 동안 수증기를 이용하여 가열하였다. 가공된 인삼(이하 선삼이라 함) 1 kg에 80% 에탄올 2 L를 가하고 수욕상에서 4시간 이상 환류 추출하여 얻어진 추출물을 감압 농축한 다음 동결 건조하여 추출분말 약 300 g 을 수득하였다. 실험 시에는 선삼 추출물(이하 SG라 함)을 DMSO(Sigma D-2650)를 이용하여 배지에 녹인 후, pore size 0.45 ㎛의 여과지를 통과시킨 후 사용하였다. 실험에 사용한 선삼 추출물의 (Rg3 + Rg5 + Rk1)/(Rb1 + Rb2 + Rc + Rd)= 6.1 이었다.That is, dried ginseng (Gumsan ginseng market) was finely chopped, and heated using steam at 120 ° C. and 15 atm for 4 hours. 2 L of 80% ethanol was added to 1 kg of processed ginseng (hereinafter referred to as ginseng), and the extract obtained by reflux extraction for 4 hours or more in a water bath was concentrated under reduced pressure and freeze-dried to obtain about 300 g of an extract powder. In the experiment, the ginseng extract (hereinafter referred to as SG) was dissolved in a medium using DMSO (Sigma D-2650), and used after passing a filter paper having a pore size of 0.45 μm. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

실시예 2. 선삼 추출물의 사포닌 함량 분석Example 2. Analysis of Saponin Content of Sea Ginseng Extract

실시예 1에서 얻은 본 발명에 따라 제조된 가공인삼에 함유된 인삼사포닌 성분을 다음과 같은 방법에 의해 분석하였다. The ginseng saponin component contained in the processed ginseng prepared in Example 1 according to the present invention was analyzed by the following method.

40㎖ 용적의 스테인레스 스틸 용기 4개에 각각 백삼 5g과 물 5㎖를 가한 후 밀폐하여 각각 110℃에서 2시간, 120℃에서 2시간 및 3시간 및 130℃에서 2시간 동안 가열하였다. 가열이 끝난 가공인삼, 및 시판품인 백삼 및 홍삼 각 5g 씩을 취하여 메탄올 100㎖씩으로 3회 추출하고 농축시킨 후에 물에 현탁시키고 에테르 100㎖씩으로 3회 추출하였다. 남은 수층을 부탄올 100㎖씩으로 3회 추출한 후에 부탄올 분획을 농축시키고, 수득된 농축물을 메탄올에 용해시켜 HPLC(컬럼: LiChro- sorb NH₂, 이동상: CH₃CN/H₂O/i-PrOH=80/5/15→80/20/15, 검출기: ELSD(Evaporative light scattering detector))로 분석하였다. 측정된 결과는 다음 표 1에 기재된 바와 같다.Four 40 ml stainless steel vessels were added with 5 g of white ginseng and 5 ml of water, respectively, and then sealed and heated at 110 ° C. for 2 hours, 120 ° C. for 2 hours and 3 hours, and 130 ° C. for 2 hours. 5 g of each of the processed ginseng and the commercially produced white ginseng and red ginseng were taken, extracted three times with 100 ml of methanol, concentrated, suspended in water, and extracted three times with 100 ml of ether. The remaining aqueous layer was extracted three times with 100 mL of butanol, and then the butanol fraction was concentrated. → 80/20/15, detector: analyzed by ELSD (Evaporative light scattering detector). The measured results are shown in Table 1 below.

사포닌
샘플
Saponin
Sample
Rb1 Rb 1 Rb2 Rb 2 RcRc RdRd Rg3 Rg 3 Rg5 Rg 5 (Rg3+Rg5)/
(Rc+Rd+Rb1+Rb2)
(Rg 3 + Rg 5) /
(Rc + Rd + Rb 1 + Rb 2)
120/3시간120/3 hour 10.8210.82 6.916.91 8.528.52 7.647.64 28.0128.01 14.0114.01 1.241.24 120/2시간120/2 hours 10.4610.46 7.747.74 10.6610.66 5.585.58 24.1224.12 11.3511.35 1.031.03 130/2시간130/2 hours 4.064.06 3.443.44 3.983.98 3.823.82 21.0021.00 16.1716.17 2.432.43 110/2시간110/2 hours 19.0219.02 11.5511.55 10.6810.68 8.378.37 7.027.02 4.394.39 0.230.23 백삼White ginseng 22.1522.15 3.253.25 5.865.86 2.472.47 0.000.00 0.000.00 0.000.00 홍삼Red ginseng 30.1130.11 9.699.69 12.4512.45 1.761.76 1.051.05 1.051.05 0.010.01

상기 표 1에 기재된 결과로부터 알 수 있는 바와 같이, 본 발명에 따라 가열처리하여 수득한 가공인삼은 수삼, 백삼 및 홍삼 등에는 거의 또는 전혀 존재하지 않는 인삼 사포닌인 Rg 및 Rg 성분의 함량이 현저히 증가하여 우수한 약효를 나타낸다. As can be seen from the results shown in the above Table 1, the processed ginseng obtained by the heat treatment according to the present invention significantly increases the contents of Rg and Rg components, which are ginseng saponins which are almost or completely absent from fresh ginseng, white ginseng and red ginseng And exhibits excellent drug efficacy.

상기한 바와 같은 결과에 따라 인삼에 대한 가열온도의 변화에 따른 사포닌 성분, 특히 진세노사이드 Rg 및 Rg의 함량 변화를 더욱 구체적으로 확인하기 위하여 가열을 100℃, 110℃, 120℃, 130℃, 150℃, 160℃, 180℃ 및 200℃에서 2시간씩 행하여 얻어진 인삼들의 Rg 및 Rg 함량을 측정하여 가열처리하지 않은 인삼(수삼)에서의 함량과 비교하여 보았다. 그 결과는 다음 표 2에 기재한 바와 같다. According to the results as described above, in order to more specifically confirm the change in the content of saponin components, in particular ginsenoside Rg and Rg according to the change in the heating temperature for ginseng, heating is 100 ℃, 110 ℃, 120 ℃, 130 ℃, Rg and Rg content of ginseng obtained by 2 hours at 150 ° C., 160 ° C., 180 ° C. and 200 ° C. was measured and compared with the content in ginseng (salt) that was not heated. The results are shown in Table 2 below.

가열온도
(시간)
종류
Heating temperature
(time)
Kinds
비처리
(수삼)
Untreated
(Fresh ginseng)
100
2시간
100
2 hours
110
2시간
110
2 hours
120
2시간
120
2 hours
130
2시간
130
2 hours
150
2시간
150
2 hours
160
2시간
160
2 hours
180
2시간
180
2 hours
200
2시간
200
2 hours
Rg3 Rg 3 0.000.00 0.020.02 0.080.08 0.170.17 0.860.86 0.440.44 0.450.45 0.350.35 0.230.23 Rg5 Rg 5 0.000.00 0.020.02 0.050.05 0.080.08 0.440.44 0.530.53 0.560.56 0.480.48 0.380.38

주) 각각의 성분함량은 사용한 수삼의 양에 대한 함량 %로 나타낸 것이다.
Note) Each ingredient content is expressed as% of the amount of fresh ginseng used.

상기에서 보는 바와 같이 본 발명에서와 같이 120 내지 180℃에서 가열처리된 가공인삼의 경우에 Rg 및 Rg와 같은 진세노사이드의 함량이 비처리 수삼이나 홍삼(100℃ 가열)의 경우에 비해 현저히 증가하였음을 알 수 있다. 실험에 사용한 선삼 추출물의 (Rg3 + Rg5 + Rk1)/(Rb1 + Rb2 + Rc + Rd)= 6.1 이었다.As seen above, the content of ginsenosides such as Rg and Rg is significantly increased in the case of processed ginseng heated at 120 to 180 ° C as in the present invention compared to the case of untreated ginseng or red ginseng (100 ° C heating). It can be seen that. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

실험예Experimental Example 1. 세포 배양 및 약물처리 1. Cell Culture and Drug Treatment

실험에 사용한 RAW 264.7 세포는 DMEM(Dulbecco's modified eagle medium)/10% FBS(fetal bovine serum)/PC-SM 배지를 이용하여 CO2 세포배양기에서 배양하였으며, 세포수는 5x103 cells/well 로 96 well plate를 이용하여 배양하였다. 24시간 배양 후 배양액을 버린 후, 10 % FBS, 50 ng/ml RANKL, 1 ng/ml TGFb 가 첨가된 a-MEM 으로 교환하여 세포를 배양했다. 배양액에 여러 농도의 AS를 첨가해 주었다. 2일에 한번씩 동일한 배지로 교환해 주면서 6일간 배양하였다. 실험군은 (1) RANKL 처리하지 않은 대조군(N) (2) RANKL 유도 대조군(C), (2) RANKL 유도군 + 1/의 실시예1의 본 발명의 가공인삼 추출물 (SG)을 투여한 군(CM 0.1), (3) RANKL 유도군 + 5/의 실시예1의 본 발명의 가공인삼 추출물 (SG)을 투여한 군(CM1)으로 하였다.
The RAW 264.7 cells used in the experiment were cultured in a CO2 cell incubator using DMEM (Dulbecco's modified eagle medium) / 10% fetal bovine serum (FBS) / PC-SM medium, and the number of cells was 96 well plate at 5x10 3 cells / well. And cultured using. After culturing for 24 hours, the culture medium was discarded, and the cells were cultured by exchanging with a-MEM to which 10% FBS, 50 ng / ml RANKL, and 1 ng / ml TGFb were added. Different concentrations of AS were added to the culture. It was incubated for 6 days while changing to the same medium every two days. The experimental group was administered with (1) RANKL-treated control group (N) (2) RANKL-induced control group (C), (2) RANKL-induced group + 1 / processed ginseng extract of the present invention of Example 1 (SG) (CM 0.1), (3) RANKL induction group + 5 / to the group (CM1) to which the processed ginseng extract (SG) of the present invention of Example 1 was administered.

실험예 2. 파골세포 생성능 측정Experimental Example 2. Measurement of osteoclast generation capacity

RANKL(receptor activator for nuclear factor B ligand)로 RAW 264.7 cell를 파골세포로 유도한 후, 성숙한 파골세포의 발현 marker로 알려진 TRAP를 염색하여 TRAP-positive한 다핵세포(TRAP(+) MNCs)를 확인하였다. 분화시킨 세포를 PBS로 세포를 2회 세척하고, 3.7% formaldehyde -citrate-acetone 용액으로 10분 고정시키고 증류수로 2회 세척하였다. 2% TRAP fast garnet GBC base 용액과 NaNO3 용액을 같은 비율로 섞어 만든 용액과 5% naphtha AS-BI phosphoric acid, 4% acetic acid, 2% tartaric acid를 포함한 용액을 고정시킨 세포에 처리하고 상온에 30분 이상 방치하였다. 광학현미경으로 관찰하여 핵이 3개 이상인 TRAP-positive 한 다핵세포 (TRAP(+) MNCs)를 계수하여 파골세포의 생성지표로 하였다(15).
After inducing RAW 264.7 cells to osteoclasts with RANKL (receptor activator for nuclear factor B ligand), TRAP-positive multinucleated cells (TRAP (+) MNCs) were identified by staining TRAP, a marker for expression of mature osteoclasts. . Differentiated cells were washed twice with PBS, fixed with 3.7% formaldehyde-citrate-acetone solution for 10 minutes and washed twice with distilled water. Treat the cells prepared by mixing 2% TRAP fast garnet GBC base solution with NaNO3 solution in the same ratio and a solution containing 5% naphtha AS-BI phosphoric acid, 4% acetic acid and 2% tartaric acid. It was left for more than a minute. Observed by light microscopy, TRAP-positive multinucleated cells (TRAP (+) MNCs) with three or more nuclei were counted and used as an index for generating osteoclasts (15).

실험예 3. 유전자 발현에 대한 영향Experimental Example 3. Effect on Gene Expression

상기 실시예 1의 본 발명의 가공인삼 추출물 (SG)의 파골세포 분화 및 증식 관련 인자인 TRAP, NFATc1, JNK 등의 유전자 발현에 대한 영향을 알아보기 위해 하기와 같이 실험을 수행하였다.
In order to determine the effect on the gene expression of osteoclast differentiation and proliferation-related factors of TRAP, NFATc1, JNK, etc. of the processed ginseng extract of the present invention of Example 1 was performed as follows.

3-1. 총 3-1. gun RNARNA 분리  detach

파골세포로 분화한 TRAP(+) 다핵세포에 1 ml 트리졸 용액 (인비트로젠, USA)를 처리하여 총 RNA를 분리하였다. 분리한 RNA에 100 페놀과 100 클로로포름 : 아이소아밀알코올 (24:1)을 넣고 잘 섞은 후 원심분리하는 과정을 2번 반복하여 상층액을 분리한다. 0.5 ml 아이소프로필 알코올을 이용하여 RNA를 침전시킨 후 70% 에탄올로 세척하고 자연 건조시킨다. 알에네이즈 프리워터 (RNAase free water, 프로메가, 미국)에서 RNA를 녹인 후 알에네이즈-프리-디에네이즈 (RNase-free DNase, 프로메가, 미국)를 첨가하고 -70oC에 저장하였다.
Total RNA was isolated from TRAP (+) multinuclear cells differentiated into osteoclasts by treatment with 1 ml Trizol solution (Invitrogen, USA). 100 phenol and 100 chloroform: isoamyl alcohol (24: 1) are added to the isolated RNA, and the mixture is mixed well and centrifuged twice to separate the supernatant. RNA is precipitated using 0.5 ml isopropyl alcohol, washed with 70% ethanol and dried naturally. RNA was thawed in RNAase free water (RNA) free water (Promega, USA), followed by the addition of ALNAISE-free DNase (RNase-free DNase, Promega, USA) and stored at -70 ° C.

3-2. 3-2. cDNAcDNA 제조 Produce

상기 실험예 3-1에서 분리한 대조군 및 실험군 각각의 전체 RNA액 (13 ug RNA 함유)에 올리고 dT 1 을 넣은 후 조심스럽게 혼합한 다음, 70oC에서 5분간 배양하였다. 프라이머가 풀리도록 실온에서 약 10분간 방치한 다음, 사스크립트 버퍼 (cyscript buffer), 0.1 M DTT, dUTP 뉴클레오티드, dUTP 시다이-표지된 뉴클레오티드, 사스크립트 역전사효소 (Cyscript reverse transcriptase)를 첨가한 후, 아주 조심스럽게 혼합하였다. 이 후, 42oC에서 90분간 배양한 후, 얼음 상에 방치하였다. 여기에 2.5 M 수산화나트륨을 가한 후 37oC에서 15분간 배양하였으며, 2 M HEPES 버퍼를 가하여 중화시켜 cDNA를 제조하였다.Oligo dT 1 was added to the total RNA solution (containing 13 ug RNA) of each of the control and experimental groups isolated in Experimental Example 3-1, mixed carefully, and then incubated at 70 ° C. for 5 minutes. Leave primers at room temperature for about 10 minutes to release the primers, then add cyscript buffer, 0.1 M DTT, dUTP nucleotides, dUTP seeded-labeled nucleotides, Cyscript reverse transcriptase, and then add Mix carefully. Thereafter, the cells were incubated at 42 ° C. for 90 minutes and then left on ice. 2.5 M sodium hydroxide was added thereto, followed by incubation at 37 ° C. for 15 minutes, and neutralized by adding 2 M HEPES buffer to prepare cDNA.

3-3. 3-3. RealReal timetime RTRT -- PCRPCR

우선 시험관에 정량한 RNA 5 ug, 50 ng/의 랜덤 헥사머 (random hexamer) 3 , 10 mM dNTP 1 를 넣고 DEPC-H2O를 가하여 10 의 RNA/프라이머 혼합물을 만들었다. 실험용 sample을 65℃에서 5분간 배양시킨 후 1분 이상 얼음에 방치하였다. 반응 혼합물로 10배의 RT 버퍼 2 , 25 mM 염화마그네슘 4 , 0.1 M DTT 2 , RNAase(프로메가, 미국) 1 을 섞어 준비하였다. 반응 혼합물을 RNA/프라이머 혼합물에 가하여 섞고 실온에 2분간 방치한 후, SuperScript II RT (프로메가, 미국) 1 (50 units)를 가하고 25oC에 10분간 배양 시켰다. 다시 42oC에서 50분간 배양 시킨 다음, 70oC에서 15분간 가열하여 불활성 시키고 얼음 상에서 식혔다. RNase (프로메가, 미국)1 를 가하고 다시 37oC에서 20분간 배양 시킨 다음, 사용 시까지 -20oC에 보관하였다. 각각의 옵티컬 튜브 (optical tube, 깁코, 미국)에 2배의 사이버 그린믹스 (SYBR Green Mix, 다카라, 일본) 12.5 , cDNA 0.2 , 5 pmol/ 프라이머 쌍 혼합 1 , 11.3 H2O를 넣고, 50oC 2 분 1 사이클, 95oC 10분 1 사이클, 95oC 15초, 60oC 30초, 72oC 30초 40 사이클, 72oC 10분 1 사이클로 증폭시켰다. PCR을 마친 후 튜브를 꺼낸 다음, 반응액 5 ul를 사용하여 3% 아가로스 겔에서 PCR 특이성을 측정했다. SDS 7000 소프트웨어를 사용하여 리얼 타임 PCR (real time PCR) 결과를 분석하였다. First, 5 ug of RNA, 50 ng / random hexamer (random hexamer) 3, 10 mM dNTP 1, were added to the test tube, and DEPC-H 2 O was added to make an RNA / primer mixture of 10. The experimental sample was incubated at 65 ° C. for 5 minutes and then left on ice for at least 1 minute. The reaction mixture was prepared by mixing 10-fold RT buffer 2, 25 mM magnesium chloride 4, 0.1 M DTT 2, RNAase (Promega, USA) 1. The reaction mixture was added to the RNA / primer mixture, left to stand at room temperature for 2 minutes, then SuperScript II RT (Promega, USA) 1 (50 units) was added and incubated at 25 ° C. for 10 minutes. Incubated at 42 o C for 50 min, then inactivated by heating at 70 o C for 15 min and cooled on ice. RNase (Promega, USA) 1 was added and incubated again at 37 o C for 20 min, then stored at -20 o C until use. Into each optical tube (optical tube, Gibco, USA) add 2x Cyber Green Mix (SYBR Green Mix, Takara, Japan) 12.5, cDNA 0.2, 5 pmol / primer pair mix 1, 11.3 H 2 O, 50 o Amplification with C 2 min 1 cycle, 95 ° C 10 min 1 cycle, 95 ° C 15 sec, 60 ° C 30 sec, 72 ° C 30 sec 40 cycles, 72 ° C 10 min 1 cycle. After completing the PCR, the tube was taken out, and PCR specificity was measured on a 3% agarose gel using 5 ul of the reaction solution. Real time PCR results were analyzed using SDS 7000 software.

RAW264.7 세포에 RANKL (receptor activator for nuclear factor B ligand) 처리시 TRAP(+) 다핵세포(MNCs) 발현이 증가하여 파골세포 분화가 촉진되었으며, 상기 실시예에서 본 발명의 가공인삼 추출물 (SG)은 0.1 ug/ml, 1 ug/ml, 농도에서 증 RANKL (receptor activator for nuclear factor B ligand) 유도 파골세포 분화를 억제시키는 것으로 나타났다(도 1) TRAP(tartarate resistance Acid Phosphatase), JNK, NFATc1은 RANKL 처리시 발현이 증가하였으며, 상기의 본 발명의 가공인삼 추출물 (SG)은 0.1 ug/ml, 1 ug/ml, 농도에서 증가한 유전자 발현을 현저히 억제함을 확인하였다 (도 2, 도 3 및 도 4 참조). 따라서 본 발명의 가공인삼 추출물 (SG)은 TRAP, NFATc1, JNK 유전자 발현을 억제하는데 탁월한 효과가 있음을 알 수 있었다.
When RANKL (receptor activator for nuclear factor B ligand) treatment of RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation, the processed ginseng extract of the present invention (SG) Inhibited increased RANKL-induced osteoclast differentiation at concentrations of 0.1 ug / ml, 1 ug / ml (FIG. 1), tartarate resistance Acid Phosphatase (TRAP), JNK, and NFATc1 were RANKL Expression was increased during the treatment, the processed ginseng extract of the present invention (SG) was confirmed to significantly inhibit the gene expression increased in 0.1 ug / ml, 1 ug / ml, concentration (Fig. 2, Fig. 3 and 4). Reference). Therefore, the processed ginseng extract (SG) of the present invention was found to have an excellent effect in inhibiting TRAP, NFATc1, JNK gene expression.

통계처리는 스튜던트의 t-검정을 이용해 개별 비교를 하였으며, p값이 0.05 미만일 때 유의한 차이가 있는 것을 판정하였다.
Statistical treatments were compared individually using Student's t-test and determined that there was a significant difference when the p-value was less than 0.05.

하기에 본 발명의 추출물을 함유하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.
Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

제제예Formulation example 1.  One. 산제의Sanje 제조 Produce

SG 추출물 20 mgSG Extract 20 mg

유당 100 mgLactose 100 mg

탈크 10 mgTalc 10 mg

상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.
The above components are mixed and filled in airtight bags to prepare powders.

제제예Formulation example 2. 정제의 제조 2. Preparation of tablets

SG 추출물 10 mgSG Extract 10 mg

옥수수전분 100 mgCorn starch 100 mg

유당 100 mgLactose 100 mg

스테아린산 마그네슘 2 mgMagnesium stearate 2 mg

상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

제제예Formulation example 3. 캅셀제의 제조 3. Preparation of capsules

SG 추출물 10 mgSG Extract 10 mg

결정성 셀룰로오스 3 mgCrystalline cellulose 3 mg

락토오스 14.8 mgLactose 14.8 mg

마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg

통상의 캅셀제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캅셀제를 제조한다.
The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

제제예Formulation example 4. 주사제의 제조 4. Preparation of injections

SG 추출물 10 mgSG Extract 10 mg

만니톨 180 mgMannitol 180 mg

주사용 멸균 증류수 2974 mgSterile distilled water for injection 2974 mg

Na2HPO4 ,12H2O 26 mgNa 2 HPO 4 , 12H 2 O 26 mg

통상의 주사제의 제조방법에 따라 1 앰플당 (2 ㎖) 상기의 성분 함량으로 제조한다.
(2 ml) per ampoule in accordance with the usual injection method.

제제예Formulation example 5.  5. 액제의Liquid 제조 Produce

SG 추출물 20 mgSG Extract 20 mg

이성화당 10 g10 g per isomer

만니톨 5 g5 g mannitol

정제수 적량Purified water quantity

통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ml로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.
Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Claims (5)

진세노사이드 Rg3, Rk1, Rg5 함량의 합이 진세노사이드 Rb1, Rb2, Rc, Rd의 함량의 합보다 더 높도록 약효성분을 증가시킨 가공된 파낙스속 식물 추출물을 유효성분으로 함유하는 골대사성 질환의 치료 및 예방용 약학 조성물.Bone metabolic disease containing as an active ingredient a processed Panax genus plant extract with an increased active ingredient such that the sum of the ginsenosides Rg3, Rk1, and Rg5 is higher than the sum of the ginsenosides Rb1, Rb2, Rc, and Rd. Pharmaceutical compositions for the treatment and prophylaxis. 제 1항에 있어서,
상기 파낙스속 식물은 고려인삼 (Panax ginseng), 화기삼 (Panax quinquefolia), 전칠삼 (삼칠, Panax notoginseng), 베트남삼 (Panax vietnamensis), 죽절삼 (Panax japonicus), 파낙스 엘레가티오르 (Panax elegatior), 파낙스 완지아누스 (Panax wangianus), 파낙스 비핀나티푸스 (Panax bipinnatifidus), 또는 파낙스 슈도진생 (Panax pseudoginseng)을 인위적으로 가공시킨 식물을 특징으로 하는 약학조성물.
The method of claim 1,
The Panax genus plants Ginseng (Panax ginseng ), Panax ginseng ( Panax quinquefolia ), Whole Chisam (Samchi, Panax notoginseng ), Vietnamese Ginseng ( Panax vietnamensis ), Panax ginseng ( Panax japonicus ), Panax elegatior , Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), or Panax pseudoginseng ) is artificially processed.
제 1항에 있어서,
상기 파낙스속 식물은 인삼을 120 내지 180℃의 고온에서 0.5 내지 20시간 동안 가열처리하여 진세노사이드 (Rg3+Rg5)/(Rc+Rd+Rb₁+Rb₂)의 비율이 1.0이상이 되도록 약효성분을 증가시킨 가공인삼임을 특징으로 하는 약학조성물.
The method of claim 1,
The Panax genus plants are treated with ginseng at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours to provide a medicinal ingredient such that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + Rb ₁ + Rb 2) is 1.0 or more. Pharmaceutical composition characterized by increased processed ginseng.
제 1항에 있어서,
상기 추출물은 물, 메탄올, 에탄올, 부탄올 등의 저급 알콜 또는 이들의 혼합용매인 약학 조성물.
The method of claim 1,
Wherein the extract is water, a lower alcohol such as methanol, ethanol, butanol, or a mixed solvent thereof.
제 1항에 있어서, 상기 상기 골 대사성 질환은 골다공증(osteoprosis), 파제트병(paget disease), 치주질환(periodontal disease), 골전이암(metastatic cancer) 또는 류마티스 관절염(rheumatoid arthiritis)인 약학조성물.
The pharmaceutical composition of claim 1, wherein the bone metabolic disease is osteoprosis, paget disease, periodontal disease, metastatic cancer, or rheumatoid arthiritis.
KR1020110127110A 2011-11-30 2011-11-30 A composition comprising the extract of processed panax genus plant for treating and preventing osteoporosis KR20130060837A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160124007A (en) * 2015-04-17 2016-10-26 주식회사유한양행 Composition for preventing or treating inflammatory diseases or pain
KR20180065146A (en) * 2016-12-07 2018-06-18 건국대학교 글로컬산학협력단 Composition for the prevention and treatment of bone diseases containing ginseng extract
CN109295080A (en) * 2018-09-19 2019-02-01 昆明理工大学 Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes
US10646528B2 (en) 2014-08-22 2020-05-12 Wellkey Holdings Limited Method for preparing extract of genus Panax including wild ginseng or ginseng, or cambial meristematic cells derived from genus panax or extract thereof containing rare ginsenosides in high quantity

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10646528B2 (en) 2014-08-22 2020-05-12 Wellkey Holdings Limited Method for preparing extract of genus Panax including wild ginseng or ginseng, or cambial meristematic cells derived from genus panax or extract thereof containing rare ginsenosides in high quantity
KR20160124007A (en) * 2015-04-17 2016-10-26 주식회사유한양행 Composition for preventing or treating inflammatory diseases or pain
KR20180065146A (en) * 2016-12-07 2018-06-18 건국대학교 글로컬산학협력단 Composition for the prevention and treatment of bone diseases containing ginseng extract
CN109295080A (en) * 2018-09-19 2019-02-01 昆明理工大学 Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes

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