KR20130071216A - Food additives comprising extract of skg showing an anti-osteoporosis - Google Patents

Food additives comprising extract of skg showing an anti-osteoporosis Download PDF

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KR20130071216A
KR20130071216A KR1020110138596A KR20110138596A KR20130071216A KR 20130071216 A KR20130071216 A KR 20130071216A KR 1020110138596 A KR1020110138596 A KR 1020110138596A KR 20110138596 A KR20110138596 A KR 20110138596A KR 20130071216 A KR20130071216 A KR 20130071216A
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extract
osteoporosis
disease
suppressing
radix
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김복득
황귀서
박정일
김진희
유동열
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주식회사 진생사이언스
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/14—Extraction

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  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Medicines Containing Plant Substances (AREA)
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Abstract

PURPOSE: A food additive is provided to reduce bone diseases including a osteoporosis through suppressing osteoclast differentiation and suppressing activity of osteoclast including suppressing expression of TRAP of oasteoclas and suppressing of expression of MMP-9 and Cathepsin K. CONSTITUTION: A food additive for a metabolic bone disease improvement and prevention is an extract from complex crude drug are contained as an active ingredient. A complex herb medicine includesrehmanniae Radix Preparata, houttuynia cordata, eucommia ulmoides, achyranthes, angelica gigas, chinese matrimony vine, hoelen, paeonia japonica, cinnamomi Cortex spissus, asiasari Radix, angelicae dahuricae Radix, aconiti Tuber, glycyrrhizae radix and ginger at a combination (w/w) ratio of 1~10;1~10:1~10:1~10:1~10:1~10:1~10:1~10:1~10: 1~10:1~10:1~10:1~10: 1~10. The extract is available in a solvent selected from water including purified water, a lower alcohol, and their mixed solvent of a carbon number 1 to 4. The metabolic bone disease is osteoporosis, Paget disease, periodontal disease, metastatic cancer, or rheumatoid arthritis.

Description

항골다공증 활성을 나타내는 ＳＫＧ 추출물을 함유하는 식품첨가물 {Food additives comprising extract of SKG showing an anti-osteoporosis}Food additives containing extract of SKG showing an anti-osteoporosis}

본 발명은 항골다공증 활성을 나타내는 SKG 추출물을 함유하는 식품첨가물에 관한 것이다.The present invention relates to a food additive containing SKG extract exhibiting anti-osteoporosis activity.

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. 정보섭외, 도해향약대사전, 영림사, 1998.
10. Information Interview, Dohae Hyangdae, Yeonglimsa, 1998.

골 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). 이들은 대부분 조골세포 기능을 억제하거나 파골세포 활성을 증가시켜 골다공증이 나타난다. 따라서, 파골세포 기능을 억제하면 골다공증을 치료하거나 예방할 수 있다.Bone 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). Most of them show osteoporosis by inhibiting osteoblast function or increasing osteoclast activity. Therefore, inhibiting osteoclast function can treat or prevent osteoporosis.

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, MITF, Src, Akt 활성화등을 통하여 TRAP (tartarate resistant acid phosphatase) cathepsin K, MMP-9 등 파골세포 특이 단백질 발현을 촉진하여 이들이 골조직을 파괴하는 골흡수를 유발한다.(7-9). 파골세포의 분화와 기능이 활성화되어 흡수가 증가하면, 골다공증을 비롯한 다양한 골질환이 발생할 수 있다.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, MITF, Src, Akt activation, etc. ) Promotes the expression of osteoclast specific proteins such as cathepsin K and MMP-9, which induces bone resorption that destroys bone tissue (7-9). When osteoclast differentiation and function are activated and absorption is increased, various bone diseases including osteoporosis can occur.

숙지황은 중국원산의, 현삼과 (Scrophulariaceae)에 속하는 지황(Rehmannia glutinosa) 근경을 잘 쪄낸 것을 지칭하며, 그 성분으로서는 시토스테롤(sitosterol), 만니톨(mannitol), 캄페스테롤(campesterol), 레마닌(rehmanin), 카탈폴(catapol) 등의 성분들을 함유하는 것으로 알려져 있으며, 혈압강하작용, 간보호작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p906-909 참조)Sukjihwang refers to the botanical root of Rehmannia glutinosa belonging to the Scrophulariaceae, which is native to China. , Catapol, etc. It is known to contain ingredients such as blood pressure lowering effect, liver protection effect is known (information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p906-909).

어성초는 제주도 및 울릉도 등에 분포하는, 삼백초과 (Saururaceae)에 속하는 약모밀(Houttuynia cordata) 뿌리를 포함하는 전초를 지칭하며, 그 성분으로서는 데카노일 아세트알데히드 (decanoyl acetaldehyde), 메틸-n-노닐(nonyl)-케톤(ketone), 라날룰(linalool), 카리오필렌(caryophyllene), 미르센(myrcene), 퀘르시트린(quercitrin), 퀘르세틴(quercetin) 등의 성분들을 함유하는 것으로 알려져 있으며, 항균작용, 항바이러스 작용, 이뇨작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p812-813 참조)Eoseongcho refers to an outpost including the root of Houttuynia cordata belonging to the Saururaceae, distributed in Jeju Island and Ulleungdo, etc., and its components include decanoyl acetaldehyde, methyl-n-nonyl- It is known to contain ingredients such as ketone, linalool, caryophyllene, myrcene, myrcene, quercitrin, and quercetin. Efficacy of diuretic effect is known (see Information Disclosure, Dohae Hyangdae Dictionary, Younglimsa, 1998, p812-813)

두충은 중국원산의, 돌나물과 (Crassulaceae)에 속하는 두충나무(Eucommia ulmoides)의 근피를 지칭하며, 그 성분으로서는 구타(gutta)-페르카(percha), 알칼로이드(alkaloid), 케토스(ketose), 리놀렌산(linolenic acid) 등의 성분들을 함유하는 것으로 알려져 있으며, 혈압강하작용, 이뇨작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p605-606 참조).Toothworm refers to the root of Eucommia ulmoides belonging to Crassulaceae of Chinese origin, and its components include gutta-percha, alkaloid, ketose, It is known to contain ingredients such as linolenic acid, and it is known to have an effect of lowering blood pressure and diuretic effect (see information disclosure, Dohae Hyangjeomsa, Yeonglimsa, 1998, p605-606).

우슬은 한국 전국에 분포하는, 비름과 (Amaranthaceae)에 속하는 쇠무릎 (Achyranthes japonica) 또는 당우슬(Achyranthes bidentata)의 뿌리를 지칭하며, 그 성분으로서는 트리테르페노이드 사포닌(triterpenoid saponoin), 엑디스테론(ecdysterone), 이노코스테론(inokosterone) 등의 성분들을 함유하는 것으로 알려져 있으며, 자궁수축작용, 혈압강하작용, 진통작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p340-342 참조).The dew is the root of Achyranthes japonica or Achyranthes bidentata belonging to the Amaranthaceae, which is distributed throughout Korea, and its components include triterpenoid saponoin and ecsterone ( It is known to contain ecdysterone) and inokosterone, etc., and it is known to have effects such as uterine contraction, blood pressure lowering and analgesic. 342).

당귀는 한국에서 재배되는, 미나리과 (Umbelliferae)에 속하는 왜당귀 (Angelica acutiloba) 또는 당귀(Angelica sinensis)의 뿌리를 지칭하며, 그 성분으로서는 프탈릭 산(phtalic acid), 리구스틸리드(ligustilide), 부틸리덴 프탈리드(butylidene phthalide), 크니딜리드 (cnidilide), 세스퀴테르펜(sesqtiterpene) 등의 성분들을 함유하는 것으로 알려져 있으며, 자궁수축작용, 항균효과 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p407-408 참조).Angelica refers to the root of Angelica acutiloba or Angelica sinensis belonging to the Umbelliferae cultivated in Korea, and its components include phthalic acid, ligustilide, boo It is known to contain components such as butylidene phthalide, cnidilide, sesquitifenpenes, and is known for its effects such as uterine contraction and antimicrobial effects. See Metabolism, Young Lim, 1998, pp. 407-408.

구기자는 한국 전국에 분포하는, 가지과 (Solanaceae)에 속하는 구기자나무 (Lycium chinensis)의 성숙한 과실을 지칭하며, 그 성분으로서는 카로텐(carotene), 시토스테롤(sitosterol), 리놀렌산(linolenic acid) 등의 성분들을 함유하는 것으로 알려져 있으며, 혈당 및 콜레스테롤 강하작용, 부교감신경 흥분작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p826-828 참조)Goji refers to the mature fruit of Lycium chinensis belonging to the Solanaceae, distributed throughout the country, and contains carotene, sitosterol, and linolenic acid. It is known to have effects such as hypoglycemia and cholesterol lowering effect, parasympathetic nerve stimulation effect (see information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p826-828).

백복령은 전국에 분포하는 구멍쟁이버섯과(Polyporaceae)에 속하는 복령(Poria cocos)의 균핵 내부의 백색부분을 얇은 조각이나 작은 각 모양으로 자른 것을 백복령이라 지칭하며, 그 균핵 성분으로서는 파키만(pachyman), 파키민산 (pachymic acid), 튜물로식산(tumulosic acid), 키틴질, 단백질, 스테롤 등의 성분들을 함유하는 것으로 알려져 있으며, 이뇨작용, 항균작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p40-42 참조).Baekbokryeong refers to the white slices of the inside of the Poria cocos belonging to Polyporaceae, which are distributed throughout the country, in thin slices or in small angular shapes, and is called Pachyman. It is known to contain ingredients such as pachymic acid, tumulosic acid, chitin, protein, and sterols.It is known for its diuretic and antimicrobial effects. See Yeonglim, 1998, pp. 40-42).

백작약은 전국에 분포하는 작약과 (Paeoniaceae)에 속하는 백작약 (Paeonia japonica) 및 이의 동속식물의 뿌리를 지칭하며, 그 성분으로서는 파에오니플로린(paeoniflorin), 정유, 지방유, 수지, 탄닌(tannin), 안식향산 등의 성분들을 함유하는 것으로 알려져 있으며, 진경작용, 혈압강하작용, 진경 및 진통작용, 항균작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p524-525 참조).Earl of Peony refers to the roots of Paeonia japonica and its related plants, which belong to the National Peony and Paeoniaceae.The ingredients include paeoniflorin, essential oils, fatty oils, resins, tannins, and benzoic acid. It is known to contain such ingredients, and the effect of jingyeong, blood pressure lowering, jingyeong and analgesic, antibacterial action is known (information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p524-525).

육계는 한국 전국에 분포하는, 가지과 (Solanaceae)에 속하는 구기자나무 (Lycium chinensis)의 성숙한 과실을 지칭하며, 그 성분으로서는 카로텐(carotene), 시토스테롤(sitosterol), 리놀렌산(linolenic acid) 등의 성분들을 함유하는 것으로 알려져 있으며, 혈당 및 콜레스테롤 강하작용, 부교감신경 흥분작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p826-828 참조).Broiler refers to the mature fruit of Lycium chinensis belonging to Solanaceae, which is distributed throughout Korea, and contains components such as carotene, sitosterol, and linolenic acid. It is known to have effects such as hypoglycemia and cholesterol lowering effect, parasympathetic nerve stimulation effect (refer to information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p826-828).

세신은 한국 제주도 및 전국에 분포하는, 쥐방울과 (Aristolochiaceae)에 속하는 족도리풀 (Asarum heterotropoides) 및 동속식물의 뿌리 달린 전초를 지칭하며, 그 성분으로서는 메틸유게놀(methyl eugenol), 사프롤(safrol), 피넨(pinene), 유카본(eucarvone) 등의 성분들을 함유하는 것으로 알려져 있으며, 해열, 진통 및 진해작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p746-747 참조).Sessin refers to rooted outposts of Asarum heterotropoides belonging to Aristolochiaceae, which are distributed in Jeju Island and throughout Korea, and include methyl eugenol and safrol. , Pinene, eucarvone and other ingredients are known to be effective, such as antipyretic, analgesic and anti-tussive effect is known (information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p746-747) ).

백지는 한국 전국에 분포하는, 미나리과 (Umbelliferae)에 속하는 구릿대 (Angelica dahurica) 및 동속식물의 뿌리를 지칭하며, 그 성분으로서는 백-안젤리신(byak-angelicin), 백-안젤리놀(byak-angelicol), 옥시퓨세다닌(oxypeucedanin), 입페라토린(imperatorin), 이소임페라토린(isoimperatorin), 펠로프테린(phellopterin) 등의 성분들을 함유하는 것으로 알려져 있으며, 항균작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p409-411 참조).White paper refers to the roots of Angelica dahurica and the same plants belonging to the Umbelliferae, which are distributed all over Korea, and its components include byak-angelicin and byak-angelicol. It is known to contain components such as oxypeucedanin, oxyperucein, imperatorin, isoimperatorin and isopopterin and is known for its antimicrobial effects. Doha Hyangje Dictionary, Yeonglimsa, 1998, pp. 409-411).

부자는 한국 전국에 분포하는, 미나리아재비과 (Ranunculacaea)에 속하는 바꽃 (오두; Aconitum carmichaeli)의 신생 자근(子根을)을 지칭하며, 그 성분으로서는 히게나민(higenamine), 코리네인(coryneine), 아코니틴(aconitine), 메사코니틴(mesaconitine), 히파코니틴(hypaconitine), 제사코니틴(jesaconitine) 등의 성분들을 함유하는 것으로 알려져 있으며, 항균작용 등의 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p473-476 참조).The rich refers to the new roots of Aconitum carmichaeli, a genus of Ranunculuca, which is distributed throughout the country in Korea, and its components include higenamine, coryneine, It is known to contain ingredients such as aconitine, mesaconitine, hypaconitine, and jesaconitine, and the effects of antimicrobial activity are known. Pharmacological Dictionary, see Yeonglimsa, 1998, p473-476).

감초는 중국, 몽고 등지에서 분포 및 재배되는 콩과(Leguminosae)에 속하는 감초(Glycyrrhiza uralensis)의 뿌리 및 근경을 지칭하며, 그 성분으로서는 글리시리진(gylcyrrhizin), 리퀴리티게닌(liquiritigenin), 리퀴리틴(liquiritin), 네오리퀴리틴(neoliquirititin) 등의 성분들을 함유하는 것으로 알려져 있으며, 해독작용, 항염, 항알레르기작용, 간보호 작용 등에 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p685-687 참조).Licorice refers to the root and rhizome of Glycyrrhiza uralensis belonging to the legumes (Leguminosae) distributed and cultivated in China, Mongolia, etc., and its components include glycylrrhizin, liquiritigenin and liquiritin. It is known to contain ingredients such as liquiritin and neooliquirititin, and is known for its detoxification, anti-inflammatory, anti-allergic and hepatoprotective effects. , p685-687).

생강은 한국 남부지역에서 재배되는 생강과(Zingiberaceae)에 속하는 생강(Zingiber officinale)의 신선한 근경을 지칭하며, 그 성분으로서는 징기베롤(zingiberol), 징기베렌(zingiberene), 펠란드렌(phellandrene), 캄펜(camphene) 등의 성분들을 함유하는 것으로 알려져 있으며, 소화촉진작용, 식욕증진작용, 항균작용, 항원충작용 등에 효능이 알려져 있다(정보섭외, 도해향약대사전, 영림사, 1998, p260-262 참조). Ginger refers to the fresh root of the ginger (Zingiber officinale) belonging to the Zingiberaceae family, which is cultivated in southern Korea. Its ingredients include zingiberol, zingiberene, phellandrene, It is known to contain ingredients such as camphene, and it is known to be effective in promoting digestion, appetite promotion, antimicrobial action, and antiperspirant action (information disclosure, Dohae Hyangdae, Yeonglimsa, 1998, p260-262). ).

그러나 상기 문헌 어디에도 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강으로 구성된 복합생약 혼합물(본원에서, SKG이라 함) 추출물의 골다공증에 대한 효과에 대한 어떠한 개시 또는 교시된 바가 없다. However, none of the above-mentioned literatures has an effect on the osteoporosis of the extract of the complex herbal mixture (herein referred to as SKG) consisting of Sukjihwang, Eoseongcho, Toothworm, E. There is no disclosure or teaching of.

이에 본 발명자들은 SKG 추출물의 파골세포 분화 억제 효과, 파골세포 분화 억제 및 파골세포 활성 관련 인자인 TRAP, MMP-9, Cathepsin K등의 유전자 발현을 억제하여, 골다공증 억제 효과를 확인함으로써 본 발명을 완성하게 되었다.
Accordingly, the present inventors have completed the present invention by confirming the osteoporosis inhibitory effect by inhibiting the gene expression of the osteoclast differentiation, osteoclast differentiation and osteoclast activity-related factors such as TRAP, MMP-9, Cathepsin K, etc. Was done.

상기 목적을 달성하기 위하여, 본 발명은 파골세포 억제효능을 나타내는 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강으로 구성된 복합생약 추출물을 유효성분으로 함유하는 골대사성 질환 개선 및 예방용 식품첨가물을 제공한다.In order to achieve the above object, the present invention is effective in the complex herbal extract consisting of Sukjihwang, Eoseongcho, Tobacco, Esoteric, Angelica, Gugija, Baekbokryeong, Baekjak, Broiler, Sessin, Baekji, Rich, Licorice and Ginger showing the osteoclast inhibition effect. Provided are food additives for improving and preventing bone metabolic diseases.

구체적으로, 상기 복합생약은 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강의 배합(w/w)비가 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10 : 1∼10, 바람직하게는, 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5 : 1∼5, 보다 바람직하게는, 1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2:1∼2의 배합비로 배합됨을 특징으로 한다.Specifically, the compound herbal medicine is a mixture (w / w) ratio of Sukji Hwang, Eoseongcho, Toesok, Woojeol, Dongguk, Gojija, Baekbokyeong, Baekjak, Broiler, Sessin, White Paper, Rich, Licorice and Ginger 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10, preferably 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5: 1 to 5, more preferably 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 to 2: 1 It is characterized by mix | blending in the compounding ratio of -2: 1-2: 1-2: 1-2: 1-2.

본원에서 정의되는 추출물은 정제수를 포함한 물, 탄소수 1 내지 4의 저급 알코올 또는 이들의 혼합용매로부터 선택된 용매, 바람직하게는 물에 가용한 추출물을 의미한다. An extract as defined herein means an extract available in water, including purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water.

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 추출물은 상기 한약재 복합생약재로부터 추출가능하다.The extract of the present invention is extractable from the herbal medicine complex herbal medicine.

예를 들어, 본 발명의 추출물은 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강의 구성 한약재를 건조시킨 후, 시료 중량의 약 1배 내지 100배, 바람직하게는 약 1배 내지 50배 (w/v) 부피의 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합용매로, 바람직하게는 물 또는 물 및 에탄올을 추출용매로 하여, 약 10 내지 120, 바람직하게는 30 내지 90의 반응온도에서 약 2시간 내지 7일간, 바람직하게는 12 시간 내지 26시간 동안 가열추출법, 초음파 추출법, 환류 추출법, 초고압추출법 등의 통상적인 추출방법, 바람직하게는 환류 추출법으로 1 내지 10회, 바람직하게는 1 내지 5회 반복 추출하는 제 2단계; 상기 단계에서 수득한 추출액을 여과하여 감압 농축하는 제 3단계; 상기 농축된 추출물을 동결 건조하는 제 4단계의 제조방법을 포함하는 단계를 통하여 본 발명의 추출물을 얻을 수 있다.For example, the extract of the present invention, dried dried medicinal herb of Sukjihwang, Eoseongcho, Tobacco, Ussae, Angelica, Goji, Baekbokyeong, Baekjak, Broiler, Sessin, Baekji, Rich, Licorice and Ginger, about 1 times the weight of the sample. To 100 times, preferably about 1 to 50 times (w / v) volume of water, C 1 to C 4 lower alcohols or mixed solvents thereof, preferably water or water and ethanol as extractant Conventional extraction methods such as heat extraction, ultrasonic extraction, reflux extraction, ultrahigh pressure extraction, and the like for about 2 hours to 7 days, preferably 12 hours to 26 hours at a reaction temperature of about 10 to 120, preferably 30 to 90, Preferably a second step of repeating extraction 1 to 10 times, preferably 1 to 5 times by reflux extraction; A third step of filtering and extracting the extract obtained in the above step under reduced pressure; The extract of the present invention may be obtained through a step including a method of preparing a fourth step of freeze-drying the concentrated extract.

또한 본 발명은 상기 제조방법 및 상기 제조방법으로 제조된 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강으로 구성된 복합생약 추출물을 함유하는 골 대사성 질환의 개선 및 예방을 위한 식품첨가물을 제공한다. In another aspect, the present invention is a bone containing a compound herbal extract consisting of Sukjiwang, Eoseongcho, Tofu, Emulja, Angelica, Gugija, Baekbokyeong, Baekjak, Broiler, Sessin, Baekji, Rich, Licorice and Ginger prepared by the above method. Provided are food additives for the improvement and prevention of metabolic diseases.

본 발명에 의한 추출물은 파골세포 분화 억제 효과, 파골세포 분화 및 증식 관련 인자인 TRAP, MMP-9, Cathepsin K등의 유전자 발현 억제 효과를 나타내므로, 골다공증 억제용 식품첨가물로서 유용함을 확인하였다.The extract according to the present invention exhibits an inhibitory effect on osteoclast differentiation, osteoclast differentiation and proliferation-related factors, such as TRAP, MMP-9, Cathepsin K, etc., and thus is useful as a food additive for inhibiting osteoporosis.

본 발명의 추출물은, 추출물 총 중량에 대하여 상기 생약복합추출물을 0.01 내지 99% 중량으로 포함한다.Extract of the present invention, the total drug extract comprises from 0.01 to 99% by weight of the herbal extracts.

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 건강보조식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다.Examples of the health supplement food of the present invention include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

또한, 본 발명의 추출물은 목적 질환의 예방 및 개선을 위한 목적으로 식품 또는 음료에 첨가될 수 있다. 이때, 식품 또는 음료 중의 상기 추출물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5g, 바람직하게는 0.3 내지 1g의 비율로 가할 수 있다. In addition, the extract of the present invention may be added to food or beverage for the purpose of preventing and ameliorating the desired disease. At this time, the amount of the extract in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5g, preferably 0.3 to 1g based on 100ml.

본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기추출물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다.The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

상기 외에 본 발명의 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 시료는 천연 과일 주스 및 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 시료 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the sample of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the sample of the present invention.

본 발명의 식품 조성물의 양은 일반적으로 전체 식품 중량의 0.01 내지 30 중량 %로 가할 수 있으며, 음료 조성물에는 100 ㎖를 기준으로 0.02 내지 30 g, 바람직하게는 0.3 내지 1 g의 비율로 본 발명의 식품조성물을 가할 수 있다.The amount of the food composition of the present invention may generally be added in an amount of 0.01 to 30% by weight of the total food weight, and the beverage composition may contain 0.02 to 30 g, preferably 0.3 to 1 g, based on 100 ml. The composition can be added.

또한, 본 발명의 상기 skg추출물을 유효성분으로 함유하는 식품 조성물은 제과제빵류, 면류, 두부, 육가공류, 김치를 포함한 발효식품 등의 식품형태가 가능하다.In addition, the food composition containing the skg extract of the present invention as an active ingredient is possible in the form of food, such as confectionery breads, noodles, tofu, processed meats, fermented foods including kimchi.

본 발명의 식품첨가제는 식품에 대한 살균제, 향신료, 조미제, 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등 또는 식품소재의 필수원료를 포함할 수 있다.Food additives of the present invention, flavoring agents such as fungicides, spices, seasonings, various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like or essential ingredients of food materials.

이 때 식품첨가제는 식품을 침지, 분무 또는 혼합하여 상기 식품에 첨가할 수 있으며, 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.At this time, the food additive may be added to the food by dipping, spraying or mixing the food, and the ratio of such additive is not so important, but it is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention. to be.

본 발명의 식품은 과일, 야채, 과일이나 야채의 건조제품이나 절단제품, 과일주스, 야채주스, 이들의 혼합주스이거나 산성음료수를 포함하는 음료류, 쿠키, 사탕, 카라멜, 껌 등과 같은 제과류, 제빵류, 아이스크림 제품류, 다(茶)류, 요구르트와 같은 발효유식품, 유가공식품, 양념류, 주류, 통조림 또는 병조림류, 면류, 축산 가공식품, 수산가공식품, 미생물발효식품, 두류식품, 곡류식품, 육가공류, 감초류, 허브류 중 어느 하나 또는 하나 이상이다.The food of the present invention is a fruit, vegetable, dried or cut product of fruit or vegetable, fruit juice, vegetable juice, mixed juice or beverages containing acidic beverages, cookies, candy, caramel, gum, etc. , Ice cream products, teas, fermented milk foods such as yogurt, dairy foods, condiments, alcoholic beverages, canned or bottled foods, noodles, livestock processed foods, fish processed foods, microbial fermented foods, soybean foods, cereals, meat processed meats , Licorice, herbs, or any one or more.

본 발명에 의한 SKG 추출물은 파골세포 분화 억제 효과(TRAP(+) 다핵세포생성 억제), 파골세포 분화 및 증식 관련 인자인 TRAP, MMP-9, Cathepsin K등 등의 유전자 발현 억제 효과를 나타내므로, 골다공증 억제용 식품첨가물로 유용하게 이용될 수 있다.SKG extract according to the present invention exhibits the effect of inhibiting osteoclast differentiation (TRAP (+) multinucleated cell production), gene expression inhibitory factors such as TRAP, MMP-9, Cathepsin K, etc. It can be usefully used as a food additive for inhibiting osteoporosis.

도 1은 SKG 추출물 농도에 따른 RANKL 처리 RAW264.7 세포로부터 TRAP(+) 다핵세포 형성에 미치는 영향을 나타낸 도이며
도 2는 SKG 추출물 농도에 따른 TRAP 발현에 미치는 영향을 나타낸 도이며,
도 3는 SKG 추출물 농도에 따른 MMP-9 발현에 미치는 영향을 나타낸 도이며,
도 4는 SKG 추출물 농도에 따른 Cathepsin K 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 SKG extract concentration
Figure 2 is a diagram showing the effect on the expression of TRAP according to the SKG extract concentration,
Figure 3 is a diagram showing the effect on the expression of MMP-9 according to the SKG extract concentration,
4 is a diagram showing the effect on Cathepsin K expression according to the SKG extract concentration.

이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.
However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

실시예 1. SKG 추출물의 제조Example 1.Preparation of SKG Extract

건조시킨 지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강을 동량으로 배합한(w/w) 복합생약에 증류수를 중량의 10배를 가한 후에 4시간 가온 추출하였으며, 추출물(본원에서 "SKG" 라 함)을 농축 및 냉동건조한 후에 하기 실험예의 시료로 사용하였다.
(W / w) same amount of dried kohwang, eoseongcho, tofu, hyssop, Angelica, goji berry, baekbokyeong, baekjak, broiler, sessin, white paper, rich, licorice and ginger (w / w) After adding 10 times the weight of distilled water to the complex herbal extract was warmed for 4 hours, the extract (hereafter referred to as "SKG") was concentrated and freeze-dried and used as a sample of the following experimental example.

실험예 1. 세포 배양 및 약물처리Experimental Example 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), (3) RANKL 유도군 + 실시예1의 10ug/ml SKG 추출물을 투여한 군(SKG10), (4) RANKL 유도군 + 실시예1의 50ug/ml SKG 추출물을 투여한 군(SKG50), (5) RANKL 유도군 + 실시예1의 100ug/ml SKG 추출물을 투여한 군(SKG100)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 (1) RANKL-treated control group (N) (2) RANKL-induced control group (C), (3) RANKL-induced group + group administered the 10ug / ml SKG extract of Example 1 (SKG10), (4) RANKL induction group + group administered the 50ug / ml SKG extract of Example 1 (SKG50), (5) RANKL induction group + group administered the 100ug / ml SKG extract of Example 1 (SKG100).

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.
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.

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

상기 실시예 1의 SKG 추출물의 파골세포 분화 및 증식 관련 인자인 TRAP, MMP-9, Cathepsin K 등의 유전자 발현에 대한 영향을 알아보기 위해 하기와 같이 실험을 수행하였다.
In order to determine the effect on the gene expression of the osteoclast differentiation and proliferation-related factors, such as TRAP, MMP-9, Cathepsin K of the SKG extract 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 SKG 추출물은 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, AKT1은 RANKL 처리시 발현이 증가하였으며, 상기의 SGK 추출물은 0.1 ug/ml, 1 ug/ml, 농도에서 증가한 유전자 발현을 현저히 억제함을 확인하였다 (도 2, 도 3 및 도 4 참조). 따라서 SKG 추출물은 TRAP, JNK, AKT1 유전자 발현을 억제하는데 탁월한 효과가 있음을 알 수 있었다.
Treatment of receptor activator for nuclear factor B ligand (RANKL) in RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation, and SKG extract in this example was 0.1 ug / ml, 1 At ug / ml, concentrations were shown to inhibit the receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation (FIG. 1). The expression of tartarate resistance Acid Phosphatase (TRAP), JNK, and AKT1 increased in RANKL treatment. The SGK extract was found to significantly inhibit gene expression increased at 0.1 ug / ml, 1 ug / ml (see FIG. 2, FIG. 3 and FIG. 4). Therefore, SKG extract was found to have an excellent effect in inhibiting TRAP, JNK, AKT1 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. 건강 식품의 제조 1. Manufacture of healthy food

실시예의 SKG 1000 ㎎Example SKG 1000 mg

비타민 혼합물 적량Vitamin mixture proper amount

비타민 A 아세테이트 70 ㎍70 μg of Vitamin A Acetate

비타민 E 1.0 ㎎Vitamin E 1.0 mg

비타민 B1 0.13 ㎎Vitamin B1 0.13 mg

비타민 B2 0.15 ㎎Vitamin B2 0.15 mg

비타민 B6 0.5 ㎎Vitamin B6 0.5 mg

비타민 B12 0.2 ㎍0.2 μg of vitamin B12

비타민 C 10 ㎎Vitamin C 10 mg

비오틴 10 ㎍10 μg biotin

니코틴산아미드 1.7 ㎎Nicotinic Acid 1.7 mg

엽산 50 ㎍50 μg folic acid

판토텐산 칼슘 0.5 ㎎Calcium Pantothenate 0.5mg

무기질 혼합물 적량Mineral mixture

황산제1철 1.75 ㎎Ferrous Sulfate 1.75 mg

산화아연 0.82 ㎎Zinc Oxide 0.82 mg

탄산마그네슘 25.3 ㎎Magnesium carbonate 25.3 mg

제1인산칼륨 15 ㎎Potassium monophosphate 15 mg

제2인산칼슘 55 ㎎Dibasic calcium phosphate 55 mg

구연산칼륨 90 ㎎Potassium Citrate 90 mg

탄산칼슘 100 ㎎Calcium Carbonate 100 mg

염화마그네슘 24.8 ㎎Magnesium chloride 24.8 mg

상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

제제예Formulation example 2. 건강 음료의 제조 2. Manufacture of health drinks

실시예의 SKG 1000 ㎎Example SKG 1000 mg

구연산 1000 ㎎Citric acid 1000 mg

올리고당 100 g100 g of oligosaccharide

매실농축액 2 gPlum concentrate 2 g

타우린 1 gTaurine 1 g

정제수를 가하여 전체 900 ㎖Purified water was added to a total of 900 ml

통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2ℓ 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 수요계층, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.
Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (4)

파골세포 억제효능을 나타내는 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강으로 구성된 복합생약 추출물을 유효성분으로 함유하는 골대사성 질환 개선 및 예방용 식품첨가물.Improvement and prevention of bone metabolic diseases, which contain complex herbal extracts consisting of Sukjihwang, Eochochocho, Seongchocho, Dougong, Woojeol, Dongguk, Gojija, Baekbokyeong, Baekjak, Broiler, Sessin, Baekji, Wealthy, Licorice and Ginger, which show osteoclast inhibition Food additives. 제 1항에 있어서,
상기 복합생약은 숙지황, 어성초, 두충, 우슬, 당귀, 구기자, 백복령, 백작약, 육계, 세신, 백지, 부자, 감초 및 생강의 배합(w/w)비가 1∼10;1∼10:1∼10:1∼10:1∼10:1∼10:1∼10:1∼10:1∼10: 1∼10:1∼10:1∼10:1∼10: 1∼10의 배합비로 배합됨을 특징으로 하는 식품첨가물.The method of claim 1,
The compound herbal medicine is a mixture (w / w) ratio of Sukjihwang, Eoseongcho, Toesok, Wooseul, Angelica, Goji, Baekbokyeong, Baekjak, Broiler, Sessin, Baekji, Rich, Licorice and Ginger 1 to 10; 1 to 10: 1 to 10 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10: 1 to 10 Food additives. 제 1항에 있어서,
상기 추출물은 정제수를 포함한 물, 탄소수 1 내지 4의 저급 알코올 또는 이들의 혼합용매로부터 선택된 용매에 가용한 추출물임을 특징으로 하는 식품첨가물.The method of claim 1,
The extract is a food additive, characterized in that the extract is available in a solvent selected from water, including purified water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof. 제 1항에 있어서,
상기 골 대사성 질환은 골다공증(osteoprosis), 파제트병(paget disease), 치주질환(periodontal disease), 골전이암(metastatic cancer) 또는 류마티스 관절염(rheumatoid arthiritis)인 식품첨가물.
The method of claim 1,
The bone metabolic disease is osteoporosis (osteoprosis), paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (rheumatoid arthiritis) food additives (rheumatoid arthiritis).
KR1020110138596A 2011-12-20 2011-12-20 Food additives comprising extract of skg showing an anti-osteoporosis KR20130071216A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104587343A (en) * 2015-02-09 2015-05-06 王秋霞 Traditional Chinese medicine for replenishing qi and nourishing yin of hyperthyroidism patients and preparation method
WO2018174589A1 (en) * 2017-03-24 2018-09-27 주식회사 에이치엘사이언스 Method for alleviating gingivitis and periodontitis by antibiosis, antioxidation, anti-inflammation, suppression of periodontal bone loss, and regeneration of periodontal bone of complex of moringa o leifera leaf extract and eucommia ulmoides extract

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104587343A (en) * 2015-02-09 2015-05-06 王秋霞 Traditional Chinese medicine for replenishing qi and nourishing yin of hyperthyroidism patients and preparation method
WO2018174589A1 (en) * 2017-03-24 2018-09-27 주식회사 에이치엘사이언스 Method for alleviating gingivitis and periodontitis by antibiosis, antioxidation, anti-inflammation, suppression of periodontal bone loss, and regeneration of periodontal bone of complex of moringa o leifera leaf extract and eucommia ulmoides extract
CN110519997A (en) * 2017-03-24 2019-11-29 Hl科学株式会社 Pass through the antibiosis of moringa oleifera leaf extractive and the compound of eucommia ulmoides extracts, anti-oxidant, anti-inflammatory, inhibition periodontal bone lesion and the method for regenerating periodontal bone alleviation gingivitis and periodontitis
CN110519997B (en) * 2017-03-24 2023-06-02 Hl科学株式会社 Method for relieving gingivitis and periodontitis by antibiotic, antioxidant, antiinflammatory, periodontal bone loss inhibiting and regeneration of tooth Zhou Gu of Moringa oleifera leaf extract and eucommia ulmoides extract complex

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