KR20230105031A - Composition for improving female menopausal syndrome comprising mixed traditional herb extract as effective component - Google Patents
Composition for improving female menopausal syndrome comprising mixed traditional herb extract as effective component Download PDFInfo
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- KR20230105031A KR20230105031A KR1020220000109A KR20220000109A KR20230105031A KR 20230105031 A KR20230105031 A KR 20230105031A KR 1020220000109 A KR1020220000109 A KR 1020220000109A KR 20220000109 A KR20220000109 A KR 20220000109A KR 20230105031 A KR20230105031 A KR 20230105031A
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- dermis
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Abstract
본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 증상 개선용 조성물에 관한 것으로, 난소절제 동물모델에서 단삼, 진피 및 갈근 혼합 추출물의 투여는 체중의 증가를 감소시키고, 골다공증을 개선하며, 혈중 에스트라디올 함량을 증가시키는 효과가 있다. 또한, 골수 유래 대식세포에서 유도된 파골세포의 분화를 억제하고, 에스트로겐 수용체 α 및 β의 발현을 증가시겼으며, 활성 에스트로겐인 에스트라디올의 함량을 증가시켜 에스트로겐 활성을 나타내는 효과가 있으므로, 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 본 발명의 조성물은 여성 갱년기 증상 개선, 특히 골 다공증 개선용 건강기능식품 조성물 또는 치료제로 유용하게 사용될 수 있다. The present invention relates to a composition for improving female menopausal symptoms containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient. and has the effect of increasing the estradiol content in the blood. In addition, it inhibits the differentiation of osteoclasts derived from bone marrow-derived macrophages, increases the expression of estrogen receptors α and β, and increases the content of estradiol, an active estrogen, to show estrogenic activity. The composition of the present invention containing a mixture of dermis and root root extract as an active ingredient can be usefully used as a health functional food composition or therapeutic agent for improving women's menopausal symptoms, particularly osteoporosis.
Description
본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 증상 개선용 조성물에 관한 것이다. The present invention relates to a composition for improving women's menopausal symptoms containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient.
여성은 나이가 들면서 난소의 기능이 저하되고, 이로 인해 에스트로겐의 분비가 크게 감소한다. 이에 따라, 난포자극호르몬(FSH) 및 황체형성호르몬(LH)에 대한 반응이 감소되고, 프로게스테론의 생성도 감소된다. 특히, 50대 전후로 난소의 기능이 완전히 멈추는 폐경이 발생할 확률이 매우 높아지는데, 폐경기에 접어들기 시작하면서부터 완전한 폐경 이후의 시기에 호르몬 균형이 깨지면서 신체에 각종 증상이 야기되는 시기를 흔히 갱년기라 한다. 또한, 난소 제거술, 자궁 적출술 등의 이유로 조기 폐경을 맞게 되는 경우에도 갱년기 증상을 느낄 수 있다.As women age, ovarian function declines, resulting in a significant decrease in estrogen secretion. Accordingly, the response to follicle stimulating hormone (FSH) and luteinizing hormone (LH) is reduced, and production of progesterone is also reduced. In particular, there is a very high probability of menopause, in which ovarian function completely stops around the age of 50. . In addition, menopausal symptoms can be felt even in cases of early menopause due to ovarian removal or hysterectomy.
즉, 여성의 갱년기란 내분비 증후군의 일종으로 난소 기능의 노화로 인해 여성호르몬인 에스트로겐이 감소하게 되어 생리적 기능 및 성기능이 감소 내지 소실되는 시기를 의미한다. 이러한 갱년기의 증상으로는 안면홍조, 빈맥, 발한, 또는 두통과 같은 혈관성 변화에 의한 증상이 있고, 근육통, 관절통 및 요통과 같은 근골격계 변화에 의한 증상이 있으며, 빈뇨 또는 요실금과 같은 비뇨생식기 변화에 의한 증상과 함께 기억력 감퇴, 우울증, 집중력 감퇴 및 현기증과 같은 뇌신경계 변화에 의한 증상이 있다. 이 외에도 시력감퇴 및 피부와 모발이 변화되는 증상들이 발생되며, 호르몬 변화로 인한 골다공증이나 심혈관계 질환 등 여성의 건강에 치명적인 질환이 발생하기도 한다.That is, women's menopause is a kind of endocrine syndrome, and refers to a period in which physiological and sexual functions are reduced or lost due to a decrease in estrogen, a female hormone, due to aging of ovarian function. These menopausal symptoms include symptoms due to vascular changes such as hot flashes, tachycardia, sweating, or headaches, symptoms due to musculoskeletal changes such as muscle pain, arthralgia, and back pain, and symptoms caused by genitourinary changes such as frequent urination or urinary incontinence. Along with the symptoms, there are symptoms caused by changes in the brain nervous system, such as memory loss, depression, loss of concentration, and dizziness. In addition, symptoms such as loss of vision and changes in skin and hair occur, and diseases that are fatal to women's health, such as osteoporosis or cardiovascular disease due to hormonal changes, occur.
특히, 에스트로겐 결핍은 초기 폐경 여성에서 골교체의 증가 뿐만 아니라, 간접적으로 이차성 부갑상선 기능 항진증이 발생하여 후기 폐경 여성에서 골교체가 증가에 관여하게 되는데, 폐경 후 골다공증은 갱년기 후 10~15년 이내에 발생하는 것으로 원발성 골다공증의 약 80% 정도를 차지하며, 에스트로겐 결핍이 폐경 후 골다공증의 주 원인이다. In particular, estrogen deficiency not only increases bone turnover in early menopausal women, but also indirectly causes secondary hyperparathyroidism, which is involved in the increase in bone turnover in late menopausal women. Postmenopausal osteoporosis occurs within 10 to 15 years after menopause. It accounts for about 80% of primary osteoporosis, and estrogen deficiency is the main cause of postmenopausal osteoporosis.
골다공증이란 뼈가 약해져 골절이 되기 쉬운 상태에 있는 질환이며, 첫 골절이 일어나기 전까지 그 증상을 깨닫기 어렵다. 골다공증은 주로 엉덩이 및 고관절(hip), 척추(spine), 그리고 손목(wrist) 등의 부위에서 자주 발견되는 질환이며, 현재까지 완벽한 진단과 치료법이 정착되지 않아 기술적으로도 보다 잠재가능을 보이는 분야이다. Osteoporosis is a disease in which bones become weak and prone to fracture, and it is difficult to realize the symptoms until the first fracture occurs. Osteoporosis is a disease that is frequently found in areas such as the hip, hip, spine, and wrist, and it is a field that shows more potential technically because a perfect diagnosis and treatment have not been established until now. .
갱년기 증상의 가장 큰 원인이 에스트로겐 부족 때문이므로, 치료법으로는 에스트로겐 대체요법이 여성갱년기 증상 개선을 위해 주로 사용되어졌다. 그러나, 갱년기 증상을 갖는 여성의 35~40%만이 에스트로겐 대체요법을 사용하며, 이들조차도 대부분 이 방법을 지속적으로 사용하는 것을 기피한다. 에스트로겐 대체요법은 인위적인 호르몬 투여를 기반으로 하기 때문에 그 자체에 대한 거부반응과 부작용이 따르기 때문이다. 에스트로겐 대체요법의 부작용으로 자궁출혈, 뇌졸중, 심장발작, 유방암, 자궁암 발생 위험이 증가될 수 있음에 대한 많은 연구결과들이 보고되고 있다. 따라서 부작용이 없으면서도 갱년기의 증상, 특히 골다공증을 완화시키는 효과가 우수한 새로운 갱년기 증상 개선제의 개발이 요구되고 있다.Since the biggest cause of menopausal symptoms is estrogen deficiency, estrogen replacement therapy has been mainly used to improve female menopausal symptoms. However, only 35-40% of women with menopausal symptoms use estrogen replacement therapy, and even most of them avoid using this method continuously. This is because estrogen replacement therapy is based on artificial hormone administration, so it has its own rejection and side effects. As a side effect of estrogen replacement therapy, many studies have reported that the risk of uterine bleeding, stroke, heart attack, breast cancer, and uterine cancer may increase. Therefore, there is a demand for the development of a new menopausal symptom improving agent having excellent effects in relieving menopausal symptoms, especially osteoporosis, without side effects.
한편, 한국등록특허 제1165248호에 플라반계 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 갱년기 증후군 또는 골다공증의 예방 및 치료용 약학적 조성물이 개시되어 있고, 한국 등록특허 제1895972호에 번행초 추출물을 포함하는 갱년기 장애의 예방 또는 치료용 조성물이 개시되어 있지만, 본 발명의 한방 혼합 추출물, 구체적으로 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 증상 개선용 조성물에 관해 전혀 개시된 바 없다. Meanwhile, Korean Patent No. 1165248 discloses a pharmaceutical composition for preventing and treating menopausal syndrome or osteoporosis containing a flavan-based compound or a pharmaceutically acceptable salt thereof as an active ingredient, and Korean Patent No. 1895972 Although a composition for the prevention or treatment of menopausal disorders containing an extract of Haengbokcho is disclosed, there is no disclosure of a composition for improving female menopausal symptoms containing the herbal mixed extract of the present invention, specifically, the mixed extract of Danshen, dermis, and radish root as an active ingredient. no bar
본 발명은 상기와 같은 요구에 의해 도출된 것으로, 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 증상 개선용 조성물을 제공하고, 상기 유효성분이 혈중 에스트라디올 함량을 높이고, 체중을 감소시키며, 에스트로겐 분비 감소에 의해 유도된 골다공증 개선 효과가 있다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention has been derived from the above needs, and provides a composition for improving female menopausal symptoms containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient, and the active ingredient increases the content of estradiol in blood, reduces body weight, , By confirming that there is an effect of improving osteoporosis induced by a decrease in estrogen secretion, the present invention was completed.
상기 과제를 해결하기 위하여, 본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving women's menopausal disease, containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient.
또한, 본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 질환의 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of female menopausal diseases, containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient.
본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 증상 개선용 조성물에 관한 것으로, 난소 절제 동물모델에서 단삼, 진피 및 갈근 혼합 추출물의 투여는 체중의 증가를 감소시키고, 골다공증을 개선하며, 혈중 에스트라디올 함량을 증가시키는 효과가 있다. 또한, 골수 유래 대식세포에서 유도된 파골세포의 분화를 억제하고, 에스트로겐 수용체 α 및 β의 발현을 증가시겼으며, 활성 에스트로겐인 에스트라디올의 함량을 증가시켜 에스트로겐 활성을 나타내는 효과가 있다. The present invention relates to a composition for improving female menopausal symptoms containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient. and has the effect of increasing the estradiol content in the blood. In addition, it suppresses the differentiation of osteoclasts derived from bone marrow-derived macrophages, increases the expression of estrogen receptors α and β, and increases the content of estradiol, an active estrogen, to exhibit estrogenic activity.
도 1은 난소 절제 동물모델(OVX)에서 본 발명의 단삼, 진피 및 갈근 혼합 추출물(S1) 투여시 자궁 크기 변화(A) 및 무게 변화(B)를 확인한 결과이다. Sham 또는 Normal은 난소를 절제하지 않은 정상동물모델 대조군이다.
도 2는 난소 절제 동물모델(OVX)에서 본 발명의 단삼, 진피 및 갈근 혼합 추출물(S1) 투여시 혈중 에스트라디올 함량 변화를 확인한 결과이다. Sham은 난소를 절제하지 않은 정상동물모델 대조군이다. 도면 내 서로 다른 문자 a~c는 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.05이다.
도 3은 난소 절제 동물모델(OVX)에서 본 발명의 단삼, 진피 및 갈근 혼합 추출물(S1) 투여에 의한 혈중 ALP(A) 및 CTX(B) 함량 변화를 확인한 결과이다. 폐경기 여성의 경우 뼈 교체율이 증가하는 것을 ALP 수치 증가로 확인할 수 있고, CTX는 뼈 흡수 표지자이다. Sham은 난소를 절제하지 않은 정상동물모델 대조군이다. 도면 내 서로 다른 문자 a~c는 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.05이다.
도 4는 난소 절제 동물모델(OVX)에서 본 발명의 단삼, 진피 및 갈근 혼합 추출물(S1) 투여에 의한 혈중 OPG(A), RANKL(B) 및 TRAP(C) 함량 변화를 확인한 결과이다. OPG는 파골세포의 분화를 억제하는 역할을 하고, RANKL은 OPG 분비를 억제하는 역할을 한다. TRAP는 파골세포의 분화를 촉진하는 역할을 한다. Sham은 난소를 절제하지 않은 정상동물모델 대조군이다. 도면 내 서로 다른 문자 a~c는 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.05이다.
도 5는 난소 절제 동물모델(OVX)에서 본 발명의 단삼, 진피 및 갈근 혼합 추출물(S1)이 골다공증에 미치는 영향을 대퇴골 Micro-CT로 확인한 결과이다. BV/TV(%)는 뼈 부피/조직 부피, BS/BV는 뼈 표면/뼈 부피, Tb.Th는 섬유주의 두께(trabecular thickness), Tb.N는 섬유주의 수(trabecular number), Tb.Sp는 섬유주 분리(trabecular separation), BMD는 골밀도(bone mineral density)를 의미한다. Sham은 난소를 절제하지 않은 정상동물모델 대조군이다. 도면 내 서로 다른 문자 a~c는 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.05이다.
도 6은 용매별 본 발명의 단삼, 진피 및 갈근 혼합 추출물의 세포독성을 RAW 264.7 세포에서 확인한 결과이다.
도 7은 용매별 본 발명의 단삼, 진피 및 갈근 혼합 추출물이 파골세포 분화에 미치는 영향을 RAW 264.7 세포에서 확인한 결과이다. (A)는 TRAP(Tartrate Resistant Acid Phosphatase) 염색 결과이고, (B)는 TRAP 활성 분석 결과이다. RANKL(R)을 처리하여 파골세포의 분화를 유도하였다. ***은 Control 군에 비해 RANKL(R) 처리군의 TRAP 활성이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.001이다. ###은 RANKL(R) 처리군 대비 시료 처리군의 TRAP 활성이 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.001이다.
도 8은 용매별 본 발명의 단삼, 진피 및 갈근 혼합 추출물 처리에 의한 에스트로겐 수용체 α, β의 단백질 발현(A) 및 mRNA 발현(B 및 C)을 RAW 264.7 세포에서 확인한 결과이다. E2(17 β-estradiol, 0.01μM)는 양성대조군이다. RANKL(R)을 처리하여 파골세포의 분화를 유도하였다.
도 9는 용매별 본 발명의 단삼, 진피 및 갈근 혼합 추출물 처리에 의한 NFATC1 및 C-Fos의 단백질 발현(A), NFATC1의 mRNA 발현(B), NFATC1에 의해 조절되는 Acp5(C) 및 Ctsk(D) mRNA 발현을 확인한 결과이다. NFATC1 및 C-Fos는 파골세포 분화에 핵심 효소이다. E2(17 β-estradiol, 0.01μM)는 양성대조군이다. RANKL(R)을 처리하여 파골세포의 분화를 유도하였다. **, ***은 RANKL 단독처리군에 비해 통계적으로 유의미하게 유전자의 발현이 감소하였다는 것을 의미하며, **은 p<0.01, ***은 p<0.001이다.
도 10은 용매별 본 발명의 단삼, 진피 및 갈근 혼합 추출물 처리에 의한 에스트라디올 농도를 확인한 결과이다. E2(17 β-estradiol, 0.01μM)는 양성대조군이다. RANKL(R)을 처리하여 파골세포의 분화를 유도하였다. Figure 1 is a result confirming the size change (A) and weight change (B) of the uterus upon administration of the Danshen, dermis, and radish root mixed extract (S1) of the present invention in an ovariectomy animal model (OVX). Sham or Normal is a normal animal model control group in which ovaries are not resected.
Figure 2 is the result of confirming the change in the content of estradiol in blood when the Danshen, dermis, and radish root mixture extract (S1) of the present invention was administered in an ovariectomy animal model (OVX). Sham is a normal animal model control group in which ovaries are not resected. Different letters a to c in the figure mean that there is a statistically significant difference, p<0.05.
Figure 3 is a result confirming the changes in blood ALP (A) and CTX (B) content by administration of the Danshen, dermis, and radish root mixture extract (S1) of the present invention in an ovariectomy animal model (OVX). In the case of postmenopausal women, an increase in bone turnover can be confirmed by an increase in ALP level, and CTX is a marker for bone resorption. Sham is a normal animal model control group in which ovaries are not resected. Different letters a to c in the figure mean that there is a statistically significant difference, p<0.05.
Figure 4 is a result confirming the change in OPG (A), RANKL (B) and TRAP (C) content in the blood by administration of the Danshen, dermis, and radish root mixed extract (S1) of the present invention in an ovariectomy animal model (OVX). OPG serves to inhibit the differentiation of osteoclasts, and RANKL serves to inhibit OPG secretion. TRAP plays a role in promoting the differentiation of osteoclasts. Sham is a normal animal model control group in which ovaries are not resected. Different letters a to c in the figure mean that there is a statistically significant difference, p<0.05.
Figure 5 is the result of confirming the effect of the Danshen, dermis and radish root mixed extract (S1) of the present invention on osteoporosis in an ovariectomized animal model (OVX) with femur Micro-CT. BV/TV (%) is bone volume/tissue volume, BS/BV is bone surface/bone volume, Tb.Th is trabecular thickness, Tb.N is trabecular number, Tb.Sp denotes trabecular separation, and BMD denotes bone mineral density. Sham is a normal animal model control group in which ovaries are not resected. Different letters a to c in the figure mean that there is a statistically significant difference, p<0.05.
Figure 6 is a result of confirming the cytotoxicity of the mixed extract of Danshen, dermis, and radish root of the present invention for each solvent in RAW 264.7 cells.
Figure 7 is the result of confirming the effect of the solvent-specific Danshen, dermis and radish root mixed extract of the present invention on osteoclast differentiation in RAW 264.7 cells. (A) is a TRAP (tartrate resistant acid phosphatase) staining result, (B) is a TRAP activity assay result. Differentiation of osteoclasts was induced by treatment with RANKL(R). *** indicates that the TRAP activity of the RANKL(R) treated group increased statistically significantly compared to the control group, p<0.001. ### means that the TRAP activity of the sample treatment group was statistically significantly decreased compared to the RANKL (R) treatment group, p<0.001.
Figure 8 shows the results of confirming the protein expression (A) and mRNA expression (B and C) of estrogen receptors α and β in RAW 264.7 cells by treatment with the mixed extract of Danshen, dermis, and radish root of the present invention for each solvent. E2 (17 β-estradiol, 0.01 μM) is a positive control. Differentiation of osteoclasts was induced by treatment with RANKL(R).
Figure 9 is a protein expression of NFATC1 and C-Fos (A), mRNA expression of NFATC1 (B), Acp5 (C) and Ctsk ( D) This is the result of confirming mRNA expression. NFATC1 and C-Fos are key enzymes in osteoclast differentiation. E2 (17 β-estradiol, 0.01 μM) is a positive control. Differentiation of osteoclasts was induced by treatment with RANKL(R). **, *** means that the gene expression decreased statistically significantly compared to the RANKL alone treatment group, ** means p<0.01, *** means p<0.001.
Figure 10 is the result of confirming the concentration of estradiol by treatment with the mixed extract of Danshen, dermis, and dandelion root of the present invention for each solvent. E2 (17 β-estradiol, 0.01 μM) is a positive control. Differentiation of osteoclasts was induced by treatment with RANKL(R).
본 발명의 목적을 달성하기 위하여, 본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving women's menopausal disease, containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient.
상기 혼합 추출물의 추출 용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물일 수 있고, 바람직하게는 물 또는 에탄올인 것이지만, 이에 한정하는 것은 아니다. The extraction solvent of the mixed extract may be water, C 1 ~ C 4 lower alcohol or a mixture thereof, preferably water or ethanol, but is not limited thereto.
상기 단삼, 진피 및 갈근 혼합 추출물은 단삼 1 중량부에 대하여 진피 0.5~1.5 중량부 및 갈근 0.5~1.5 중량부를 혼합하여 추출한 것일 수 있고, 바람직하게는 단삼 1 중량부에 대하여 진피 1 중량부 및 갈근 1 중량부를 혼합하여 추출한 것이지만, 이에 한정하는 것은 아니다. The mixed extract of Danshen, dermis, and root root may be extracted by mixing 0.5 to 1.5 parts by weight of dermis and 0.5 to 1.5 parts by weight of root root with respect to 1 part by weight of Danshen root. It is extracted by mixing 1 part by weight, but is not limited thereto.
상기 갱년기 질환은 안면홍조, 발한, 피부건조, 질건조증, 질위축, 하부 요도 위축, 질염, 방광염, 배뇨통, 급뇨, 집중장애, 단기 기억장애, 불안, 신경과민, 기억력 감퇴, 근육통, 관절통 및 골다공증으로 이루어진 군에서 선택된 하나 이상인 것이고, 바람직하게는 난소기능 저하에 의한 골다공증인 것이지만, 이에 제한되는 것은 아니다. The menopausal diseases include hot flashes, sweating, dry skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, dysuria, urgency, concentration disorder, short-term memory disorder, anxiety, nervousness, memory loss, muscle pain, arthralgia, and osteoporosis. It is one or more selected from the group consisting of, preferably osteoporosis due to reduced ovarian function, but is not limited thereto.
상기 혼합 추출물은 혈중 에스트라디올(estradiol) 함량을 증진시키는 효과가 있다. The mixed extract has an effect of increasing the content of estradiol in blood.
상기 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조되는 것일 수 있으나, 이에 제한되지 않는다.The composition may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 총 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 하는 장기간의 섭취인 경우에, 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. Active ingredients can be appropriately used depending on their purpose of use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total amount of raw materials. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the type of health functional food. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in a conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 통상적으로 첨가되는 성분을 포함할 수 있다. 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.In addition, the health functional food composition of the present invention can be made into food, particularly functional food. The functional food of the present invention may contain ingredients that are commonly added. Examples include proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared as a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may further contain a carbonation agent used in beverages and the like. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
또한, 본 발명은 단삼, 진피 및 갈근 혼합 추출물을 유효성분으로 함유하는 여성 갱년기 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of female menopausal diseases, containing a mixed extract of Danshen, dermis, and dandelion root as an active ingredient.
상기 조성물은 캡슐제, 산제, 과립제, 정제, 현탁액, 에멀젼, 시럽 및 에어로졸 중에서 선택된 어느 하나의 제형으로 제조될 수 있다. The composition may be prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols.
본 발명의 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical composition of the present invention may further include suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions.
본 발명에 따른 조성물의 약학적 투여 형태는 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage form of the composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds as well as in suitable combinations.
본 발명에 따른 약학 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 있다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트 및 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 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, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and various compounds or mixtures including mineral oil and the like. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. is prepared In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 약학 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the drug type, the route and duration of administration, but can be appropriately selected by those skilled in the art.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 제조예 및 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using examples. These Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
실험 재료 및 방법Experimental Materials and Methods
1. 시험 물질의 제조1. Preparation of Test Substances
단삼, 진피 및 갈근을 1:1:1 중량비로 혼합한 후, 혼합물 1kg에 대하여 물 10L를 첨가한 다음 105℃에서 3시간 동안 추출한 후 여과하여 단삼, 진피 및 갈근 혼합 물 추출물을 제조하였다. After mixing Danshen, dermis, and Red root at a weight ratio of 1:1:1, 10 L of water was added to 1 kg of the mixture, followed by extraction at 105 ° C. for 3 hours, followed by filtration to prepare a mixed water extract of Danshen, dermis, and Red root.
또한, 단삼, 진피 및 갈근을 1:1:1 중량비로 혼합한 후, 혼합물 1kg에 대하여 에탄올 10L를 첨가한 다음 3시간 동안 추출한 후 여과하여 단삼, 진피 및 갈근 혼합 에탄올 추출물을 제조하였다.In addition, after mixing Danshen, dermis, and dandelion root at a weight ratio of 1:1:1, 10L of ethanol was added to 1 kg of the mixture, followed by extraction for 3 hours, followed by filtration to prepare a mixed ethanol extract of dandelion root, dermis, and dandelion root.
2. 동물 실험2. Animal Testing
2.1. 실험동물2.1. laboratory animal
실험동물은 샘타코(경기도, 한국)에서 SPF(Specific-pathogen free) 상태의 SD 랫트(rat) 12주령 암컷(female)을 분양받아 7일간 순화시켰다. 순화된 랫트에 대해 난소 절제술을 시행한 후 1주간의 회복기를 거쳐 실험에 사용하였다. 사육기간 중 식이는 일반 고형사료(Samtako, Gyunggi, Korea)를 공급하였으며, 음수는 필터링된 음용수를 매일 갈아주어 자유롭게 섭취하도록 하였다. 본 실험은 동물 실험윤리규정을 준수하여 수행하였다(승인번호 IV-RB-02-2107-21).As experimental animals, SPF (Specific-pathogen free) 12-week-old female SD rats were purchased from Samtaco (Gyeonggi-do, Korea) and acclimatized for 7 days. After ovariectomy was performed on the acclimatized rats, they were used in the experiment after a one-week recovery period. During the breeding period, regular solid feed (Samtako, Gyunggi, Korea) was supplied, and filtered drinking water was changed daily for drinking water so that they could freely consume it. This experiment was performed in compliance with the animal experimentation ethics regulations (approval number IV-RB-02-2107-21).
실험 환경 조건은 하기 표 1에 개시된 바와 같다. Experimental environmental conditions are as shown in Table 1 below.
2.2. 실험군 설정 및 사료 투여2.2. Experimental group setup and feed administration
순화기간 후 체중을 측정하고 난괴법에 따라 군간 평균값이 유사하도록 고르게 분리하였다. 실험군은 정상군(Normal group), 대조군(난소 절제 그룹, OVX), S1 (단삼, 진피, 갈근 혼합 물 추출물 7.2ml/kg)으로 나눠 군당 10두씩 실험에 사용하였고, 각 시료를 1주에 5번씩 총 12주간 경구투여 하였다.After the acclimatization period, the body weight was measured and divided evenly according to the egg mass method so that the mean values of the groups were similar. The experimental group was divided into a normal group, a control group (ovarian excision group, OVX), and S1 (7.2ml/kg of extract of Danshen, dermis, and radish root mixture), and 10 heads per group were used in the experiment, and each sample was 5 times a week. It was administered orally each time for a total of 12 weeks.
2.3. 주간 체중 및 바이오 마커 분석 및 부검2.3. Weekly weight and biomarker analysis and autopsy
주간 체중량은 주 1회 일정시간에 모두 측정하였다.Weekly body weight was measured once a week at a fixed time.
부검은 흡입마취 하에 진행하였고, 복대정맥에서 채혈하여 분리한 혈청은 바이오마커 분석에 사용하였고, 대퇴골(Femur)은 4%(v/v) 파라포름알데하이드(paraformaldehyde)에 고정하여 Micro-CT 측정하였으며, 자궁은 적출하여 무게를 측정하였다.The autopsy was performed under inhalational anesthesia, blood collected from the abdominal vena cava and separated serum was used for biomarker analysis, and the femur was fixed in 4% (v / v) paraformaldehyde and measured by Micro-CT. , Uterus was removed and weighed.
2.4. Micro-CT2.4. Micro-CT
부검 후 4%(v/v) 파라포름알데하이드(paraformaldehyde)에 고정한 대퇴골은 한국기초과학연구원 노화연구시설(전라남도 광주, 한국)에 의뢰하여 Micro-CT를 측정하였으며, 측정항목은 하기 표 2와 같다.After autopsy, the femur fixed in 4% (v/v) paraformaldehyde was measured by Micro-CT at the Korea Basic Science Institute Aging Research Facility (Gwangju, Jeollanam-do, Korea), and the measurement items are shown in Table 2 below. .
2.5. 혈액학적 분석2.5. hematological analysis
동물 부검시 복대정맥에서 채혈하고 실온에서 30분 동안 응고 과정을 거친 후 원심분리기로 10분 동안 3,000rpm으로 원심 분리하여 혈청을 회수하였다.At the time of animal necropsy, blood was collected from the abdominal vena cava, coagulated at room temperature for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes in a centrifuge to recover serum.
회수한 혈청은 ELISA kit를 이용하여 OPG(Osteoprotegertin, Abcam, USA), CTX-Ⅰ(C-telopeptide of type Ⅰ collagen, MBS, USA), TRAP(Tartrate Resistant Acid Phosphatase, MBS, USA), ALP(Alkaline Phosphatase, MBS, USA), RANKL(Receptor Activity Of Nuclear Factor Kappa B Ligand, MBS, USA)를 분석하였다.The collected serum was prepared using an ELISA kit for OPG (Osteoprotegertin, Abcam, USA), CTX-I (C-telopeptide of type I collagen, MBS, USA), TRAP (Tartrate Resistant Acid Phosphatase, MBS, USA), ALP (Alkaline Phosphatase, MBS, USA) and RANKL (Receptor Activity Of Nuclear Factor Kappa B Ligand, MBS, USA) were analyzed.
3. 세포 실험3. Cell experiments
3.1. 세포배양3.1. cell culture
Raw 264.7 세포는 한국 세포주 은행으로부터 구입하였다. Raw 264.7 세포는 10%(v/v) FBS(fetal bovine serum)와 100mg/L 페니실린-스트렙토마이신(penicillin-streptomycin)을 첨가한 DMEM 배지를 사용하여 CO2 배양기(37℃, 5% CO2)에서 배양하였다.Raw 264.7 cells were purchased from Korea Cell Line Bank. Raw 264.7 cells were grown in CO 2 incubator (37°C, 5% CO 2 ) using DMEM medium supplemented with 10% (v/v) FBS (fetal bovine serum) and 100 mg/L penicillin-streptomycin. cultured in.
3.2. 세포독성 평가3.2. Cytotoxicity evaluation
세포독성 평가는 CCK(Cell Counting Kit)-8 분석법으로 평가하였다. Cytotoxicity was evaluated by CCK (Cell Counting Kit)-8 assay.
CO2 배양기(37℃, 5% CO2)에서 배양한 세포를 24웰 플레이트에 1.5×105/웰로 분주하여 24시간 동안 배양하였다. 그 후, 다양한 농도(0.1, 0.2, 0.5, 1.0mg/mL)의 시료를 처리하고 24시간 동안 배양한 후 CCK-8 시약 50㎕를 분주한 후 2시간 동안 배양하였다. 2시간 후 마이크로 플레이트 리더기(micro plate reader)를 이용하여 450nm에서 흡광도를 측정하여 대조군과 비교하였다.Cells cultured in a CO 2 incubator (37° C., 5% CO 2 ) were dispensed into a 24-well plate at an amount of 1.5×10 5 /well and cultured for 24 hours. Thereafter, samples of various concentrations (0.1, 0.2, 0.5, 1.0 mg/mL) were treated and incubated for 24 hours, and then 50 μl of CCK-8 reagent was dispensed and cultured for 2 hours. After 2 hours, the absorbance was measured at 450 nm using a micro plate reader and compared with the control group.
3.3. 단백질 추출3.3. protein extraction
세포를 60mm 접시에 24시간 동안 배양한 후, 시료를 0.1, 0.2, 0.5mg/mL 처리하였다. 이 때 양성대조군으로 E2(17β-estradiol, 001μM)를 사용하였다. 시료 처리 후, 24시간 동안 배양하였고, 배양 후 스크랩퍼를 이용하여 세포를 걷어냈다. 걷어낸 세포 펠렛(pellet)에서 단백질을 추출하기 위해 단백질 분해효소 억제제(protease inhibitor)가 포함된 단백질 용해 버퍼(protein lysis buffer)를 첨가한 다음 현탁(voltexing)하였다. 그 후 얼음 위에서 20분 동안 정치시키고, 4℃, 12,000rpm으로 20분 동안 원심분리하였다. 그 후 상층액만 획득하여 -80℃에 보관했다. 추출한 단백질은 BCA 분석 키트를 이용해 정량하여 같은 양으로 실험에 사용하였다.After culturing the cells in a 60 mm dish for 24 hours, the samples were treated with 0.1, 0.2, or 0.5 mg/mL. At this time, E2 (17β-estradiol, 001 μM) was used as a positive control. After sample treatment, culture was performed for 24 hours, and after culture, the cells were removed using a scraper. To extract proteins from the skimmed cell pellet, a protein lysis buffer containing a protease inhibitor was added and then suspended (voltexing). After that, it was allowed to stand on ice for 20 minutes and centrifuged for 20 minutes at 4° C. and 12,000 rpm. After that, only the supernatant was obtained and stored at -80 ° C. The extracted protein was quantified using a BCA assay kit and used in the experiment in the same amount.
3.4. 웨스턴 블롯(Western Blot)3.4. Western Blot
SDS 10%-폴리아크릴아마이드 겔을 이용하여 전기영동을 통해 단백질을 분리했다. 분리한 단백질은 PDVF 멤브레인으로 이동시킨 후, TBS-T에 희석된 1%(w/v) BSA 또는 5%(w/v) skim milk를 이용하여 blocking시켰고, ERα, ERβ, NFATC1, c-Fos, c-Jun, β-actin 항체를 적절한 농도로 희석하여 넣고 4℃ 에서 16시간 동안 결합시켰다. 그 후, TBS-T로 10분간 3번 세척하였고, HRP(horseradish-peroxidase)가 붙어있는 2차 항체를 TBS-T에 적절한 농도로 희석한 후, 멤브레인과 실온에서 1시간 동안 반응시킨 후, ECL kit를 처리하여 LAS Image Gauge 프로그램을 이용하여 밴드의 밀도를 측정하였다. β-actin 항체 밴드를 대조군으로 사용하여 정량하였다.Proteins were separated through
3.5. RAN 추출 및 qRT-PCR(Quantitative reverse transcription-polymerase chain reaction) 분석3.5. RAN extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis
세포를 60mm 접시에 24시간 동안 배양한 후, 시료를 0.5mg/mL 처리하였다. 이 때 양성대조군으로 E2(17β-estradiol, 001μM)를 사용하였다. 시료 처리 후, 24시간 동안 배양하였고, 배양 후 스크랩퍼를 이용하여 세포를 걷어냈다. 걷어낸 세포 펠렛(pellet)에서 RNA 추출을 위해 trizol을 이용하여 현탁한 후, 클로로포름을 첨가하여 현탁한 다음 실온에서 10분 동안 정치하였다. 그 후, 4℃, 12,000rpm으로 20분 동안 원심분리하였다. 상층액을 제거한 다음 차가운 75%(v/v) 에탄올로 세척한 다음 건조한 후 DEPC 물로 RNA를 용해하였다. 추출한 RNA는 나노드랍 리더기를 이용하여 농도를 측정한 뒤 같은 양을 실험에 사용하였다.After culturing the cells in a 60 mm dish for 24 hours, the sample was treated with 0.5 mg/mL. At this time, E2 (17β-estradiol, 001 μM) was used as a positive control. After sample treatment, culture was performed for 24 hours, and after culture, the cells were removed using a scraper. After suspending with trizol for RNA extraction from the skimmed cell pellet, chloroform was added to suspend and then allowed to stand at room temperature for 10 minutes. Then, centrifugation was performed at 4°C and 12,000 rpm for 20 minutes. The supernatant was removed, washed with cold 75% (v/v) ethanol, dried, and RNA was dissolved in DEPC water. The extracted RNA was used in the experiment after measuring the concentration using a nanodrop reader.
추출한 25㎍의 RNA는 DiaStar™ RT kit를 이용하여 cDNA 합성에 사용하였다. qRT-PCR은 PowerSYBR® Green PCR Master Mix와 StepOnePlus™ Real-Time PCR System을 사용하여 수행되었다.25 μg of extracted RNA was used for cDNA synthesis using DiaStar™ RT kit. qRT-PCR was performed using the PowerSYBR® Green PCR Master Mix and the StepOnePlus™ Real-Time PCR System.
3.6. 파골세포 배양3.6. Osteoclast culture
파골세포로 분화시키기 위한 Raw 264.7 세포는 10%(v/v) FBS와 100mg/L 페니실린-스트렙토마이신(penicillin-streptomycin)을 첨가한 α-MEM 배지를 사용하여 CO2 배양기(37℃, 5% CO2)에서 배양하였다. Raw 264.7 세포에 100ng/mL 농도의 RANKL를 처리하여 파골세포로 분화시켜 사용하였다.Raw 264.7 cells for differentiation into osteoclasts were cultured in a CO 2 incubator (37°C, 5% CO 2 ). Raw 264.7 cells were treated with RANKL at a concentration of 100 ng/mL and differentiated into osteoclasts.
3.7. TRAP 염색 분석3.7. TRAP staining assay
세포를 96웰 플레이트에서 24시간 동안 배양한 후, 시료를 0.5mg/mL 처리하고, 동시에 RANKL을 100ng/mL 처리하였다. 5일 후, TRAP 염색을 진행하기 위해, TRAP 염색 키트를 사용하여 제조사의 프로토콜에 따라 염색하였다. After culturing the cells in a 96-well plate for 24 hours, the sample was treated with 0.5 mg/mL, and at the same time RANKL was treated with 100 ng/mL. After 5 days, in order to proceed with TRAP staining, staining was performed using a TRAP staining kit according to the manufacturer's protocol.
간략히, 배지 제거 후 PBS로 세척한 뒤, 10% 포르말린 중성 완충액(formalin neutral buffer solution)을 100㎕ 씩 분주한 후, 실온에서 30분 동안 방치하였다. 30분 후 증류수로 3회 세척한 뒤 타타르산염 용액(tartrate working solution)을 100㎕ 씩 분주한 후, 37℃에서 60분 동안 배양한 다음 용액을 제거하고, 증류수를 첨가하여 반응을 종료한 뒤 현미경으로 관찰하였다. Briefly, after removing the medium, washing with PBS, 100 μl of 10% formalin neutral buffer solution was dispensed, and then left at room temperature for 30 minutes. After 30 minutes, after washing three times with distilled water, 100 μl of tartrate working solution was dispensed, incubated at 37 ° C for 60 minutes, the solution was removed, distilled water was added to terminate the reaction, and the microscopic examination was performed. was observed.
3.8. TRAP 활성 분석3.8. TRAP activity assay
세포를 96웰 플레이트에 24시간 동안 배양한 후, 시료를 0.5mg/mL 처리하고, 동시에 RANKL을 100ng/mL 처리하였다. 5일 후 TRAP 활성도를 분석하기 위해 TRAP 활성 키트를 사용하여 제조사의 프로토콜에 따라 분석하였다. After culturing the cells in a 96-well plate for 24 hours, the sample was treated with 0.5 mg/mL, and at the same time RANKL was treated with 100 ng/mL. After 5 days, TRAP activity was analyzed using a TRAP activity kit according to the manufacturer's protocol.
간략히, 배지 제거 후 생리식염수로 세척한 뒤, 추출 용액(extraction solution)을 50㎕ 분주한 후 현탁시킨다음 기질용액 50㎕를 첨가하고 37℃에서 60분 동안 방치하였다. 그 후, 50㎕의 Stop 용액을 처리한 후, 마이크로 플레이트 리더기를 사용하여 405nm에서 흡광도를 측정하였다. Briefly, after removing the medium, washing with physiological saline, 50 μl of the extraction solution was dispensed and suspended, and then 50 μl of the substrate solution was added and allowed to stand at 37° C. for 60 minutes. Then, after processing 50 μl of the Stop solution, the absorbance was measured at 405 nm using a microplate reader.
3.9. 에스트라디올(estradiol)에 대한 정량적 평가3.9. Quantitative evaluation of estradiol
세포를 60mm 접시에 24시간 동안 배양한 후, 시료를 0.1, 0.2, 0.5mg/mL 처리하였다. 이 때 양성대조군으로 E2(17β-estradiol, 001μM)를 사용하였다. 세포 배양액 내 에스트라디올 농도는 ELISA 방법으로 제조사의 프로토콜에 따라 측정하였다. After culturing the cells in a 60 mm dish for 24 hours, the samples were treated with 0.1, 0.2, or 0.5 mg/mL. At this time, E2 (17β-estradiol, 001 μM) was used as a positive control. The concentration of estradiol in the cell culture medium was measured by ELISA according to the manufacturer's protocol.
4. 통계분석4. Statistical analysis
동물 실험 결과는 통계 프로그램(SPSS ver.12.0, SPSS Inc., Chicago, IL, USA)을 이용하여 평균±표준오차(Mean±S.E.)로 계산하였다. 각 실험군 간의 통계적 유의성 검정에 따른 통계분석은 ANOVA(one-way analysis of variance test)를 실시한 후 유의성이 있는 경우, p<0.05 미만일 때 Ducan's multiple range test로 사후 검정하였다.The animal test results were calculated as mean ± standard error (Mean ± S.E.) using a statistical program (SPSS ver.12.0, SPSS Inc., Chicago, IL, USA). Statistical analysis according to the statistical significance test between each experimental group was carried out after ANOVA (one-way analysis of variance test), and if there is significance, it was post-tested with Ducan's multiple range test when p <0.05 or less.
세포 실험 결과는 3회 반복실험에 대한 평균±표준오차(Mean±S.E.)로 나타내었다. 통계학적 유의성 검증은 GraphPad Prism 5 프로그램(ver. 5.01)을 이용하여 정상군과 대조군 혹은 대조군과 약물 처리군 간 ANOVA(one-way analysis of variance test)를 실시한 후, p<0.05 이하인 경우 유의성이 있는 것으로 판정하였다. Cell test results were expressed as mean±standard error (Mean±S.E.) for three replicates. Statistical significance verification was performed by ANOVA (one-way analysis of variance test) between the normal group and the control group or between the control group and the drug treatment group using the
실시예 1. 동물 실험Example 1. Animal testing
1. 체중 변화1. Weight change
폐경 후 난소 호르몬 생산의 감소는 지방 축적에 의한 체중의 증가를 야기한다. 따라서 본 발명의 단삼, 진피 및 갈근 혼합 물 추출물(S1)이 난소 호르몬 생산 감소에 의한 체중 증가에 미치는 영향을 난소 절제 동물모델(OVX)을 이용하여 확인하였다. 실험 시작 시점 각 실험군별 체중 측정 결과, 정상군(Sham)은 263.8±5.4g, 대조군(OVX)은 267.7±1.1g, S1 투여군은 264.4±4.0g으로 모든 군간 체중의 차이가 없었다. 난소 절제 실험 4주차 체중은 대조군(OVX)은 371.3±4.9g으로 S1 실험군의 체중 345.4±7.9g 보다 유의하게 증가되어 있었다. 실험 종료 시점인 12주차 각 군별 체중은 정상군(Sham)은 314.7±4.6g, 대조군(OVX)은 429.5±3.0g, S1 투여군은 393.5±8.4g으로, S1 투여군에서 대조군(OVX)에 비해 유의하게 낮은 체중량을 보였다. Decreased ovarian hormone production after menopause results in weight gain due to fat accumulation. Therefore, the effect of the extract (S1) of Danshen, dermis, and radish root mixture of the present invention on weight gain due to reduction in ovarian hormone production was confirmed using an ovariectomized animal model (OVX). At the start of the experiment, as a result of body weight measurement for each experimental group, the normal group (Sham) was 263.8±5.4g, the control group (OVX) 267.7±1.1g, and the S1-administered group 264.4±4.0g, showing no difference in body weight between all groups. At the 4th week of the ovariectomy experiment, the weight of the control group (OVX) was 371.3±4.9g, which was significantly higher than that of the S1 experimental group (345.4±7.9g). At the end of the experiment, at
2. 자궁 무게 변화2. Changes in cervical weight
난소 절제에 의한 에스트로겐의 부족은 자궁과 질 등 여성 생식기의 현저한 위축을 초래한다. 따라서 난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 자궁 변화에 미치는 영향을 확인하였다. Lack of estrogen due to ovariectomy results in marked atrophy of the female genital organs, such as the uterus and vagina. Therefore, the effect of Danshen, dermis, and radish root mixed extract (S1) on cervical changes was confirmed in an ovariectomy animal model (OVX).
그 결과, 도 1에 개시된 바와 같이 난소 절제에 의해 자궁의 크기가 현저히 감소하였고, S1을 투여한 경우 대조군(OVX)에 비해 자궁의 크기 및 무게가 증가되는 것을 확인할 수 있었다. As a result, as shown in FIG. 1, the size of the uterus was significantly reduced by ovariectomy, and when S1 was administered, it was confirmed that the size and weight of the uterus increased compared to the control group (OVX).
3. 혈액학적 분석(에스트라디올, ALP, CTX, OPG, RANKL, TRAP)3. Hematological analysis (estradiol, ALP, CTX, OPG, RANKL, TRAP)
3.1. 에스트라디올 함량 분석3.1. Estradiol content analysis
난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 혈중 에스트라디올에 미치는 영향을 확인하기 위해, 난소 절제 12주 후 부검하여 혈중 에스트라디올 함량을 분석하였다. 그 결과, 도 2에 개시된 바와 같이 대조군(OVX)에서 감소된 혈중 에스트라디올이 S1 투여시 증가되는 것을 확인하였다. In order to confirm the effect of the mixed extract (S1) of Danshen, dermis, and radish root on blood estradiol in an ovariectomy animal model (OVX), estradiol content in blood was analyzed by
3.2. ALP 함량 분석3.2. ALP content analysis
ALP의 수치는 뼈 교체율과 관계되며, 폐경기 여성의 경우 뼈 교체율이 증가하는 것을 ALP의 수치 증가로 확인할 수 있다. 따라서 난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 혈중 ALP 함량에 미치는 영향을 확인하였다. 그 결과, 도 3A에 개시된 바와 같이 대조군(OVX)에서 증가된 혈중 ALP 함량이 S1 투여시 감소되는 것을 확인하였다. The level of ALP is related to the bone turnover rate, and in the case of postmenopausal women, an increase in the bone turnover rate can be confirmed by an increase in the level of ALP. Therefore, in the ovariectomized animal model (OVX), the effect of the mixed extract (S1) of Danshen, dermis, and radish root on blood ALP content was confirmed. As a result, as shown in FIG. 3A , it was confirmed that the increased ALP content in blood in the control group (OVX) was reduced upon administration of S1.
3.3. CTX 함량 분석3.3. CTX content analysis
혈청 CTX는 골 용해 과정시 제1형 콜라겐의 분해 효소인 카텝신 K(cathepsin K)에 의해 매개되는 파골세포의 대사 산물로 뼈 흡수의 표지자로 알려져 있으며, 폐경 후 CTX 농도가 증가한다고 알려져 있다. 따라서 난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 혈중 CTX 함량에 미치는 영향을 확인하였다. 그 결과, 도 3B에 개시된 바와 같이 대조군(OVX)에서 증가된 혈중 CTX 함량이 S1 투여시 감소되는 것을 확인하였다. Serum CTX is a metabolite of osteoclasts mediated by cathepsin K, an enzyme that degrades
3.4. OPG, RANKL 및 TRAP 함량 분석3.4. Analysis of OPG, RANKL and TRAP content
에스트로겐의 감소는 파골세포의 분화를 억제하는 OPG를 감소시킴으로써 골 교체율 균형이 깨져 골다공증을 유발하고, 폐경기 골다공증 상태의 에스트로겐 결핍은 RANKL의 생성을 증가시키고 OPG 분비를 억제한다고 알려져 있다. 또한, 난소 절제는 E2 결핍을 유도하고 파골세포 형성을 증가시킬 뿐만 아니라 RANKL 및 TRAP 같은 파골세포를 분화시키는 단백질 수준을 증가시킨다. It is known that a decrease in estrogen decreases OPG, which inhibits the differentiation of osteoclasts, thereby breaking the balance of bone turnover and causing osteoporosis, and estrogen deficiency in postmenopausal osteoporosis increases RANKL production and inhibits OPG secretion. In addition, ovarian ablation induces E2 deficiency and increases osteoclastogenesis as well as increased levels of osteoclast-differentiating proteins such as RANKL and TRAP.
따라서 난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 혈중 OPG, RANKL 및 TRAP 함량에 미치는 영향을 확인하였다. 그 결과, 도 4A에 개시된 바와 같이 대조군(OVX)에서 감소된 혈중 OPG 함량이 S1 투여시 증가되는 것을 확인하였고, 도 4B 및 4C에 개시된 바와 같이 대조군(OVX)에서 증가된 혈중 RANKL 및 TRAP 함량이 S1 투여시 감소되는 것을 확인하였다. Therefore, in the ovariectomized animal model (OVX), the effects of the mixed extract (S1) of Danshen, dermis, and radish root on the contents of OPG, RANKL, and TRAP in blood were confirmed. As a result, as shown in FIG. 4A, it was confirmed that the reduced blood OPG content in the control group (OVX) was increased upon S1 administration, and the increased blood RANKL and TRAP contents in the control group (OVX), as shown in FIGS. 4B and 4C. It was confirmed that it decreased upon administration of S1.
4. Micro-CT4. Micro-CT
갱년기 여성에게 나타나는 대표적인 증상으로는 골 대사 이상에 따른 골다공증이 있다. 이는 폐경기 이후 조골세포 기능은 저하되고 파골세포 기능은 활발해져 골 흡수가 가속화되는 현상으로, 특히 여성의 골다공증은 폐경으로 인한 에스트로겐 감소가 가장 큰 원인인 것으로 알려져 있어 골밀도 등 뼈 건강은 갱년기 여성 건강의 주요 지표로 사용된다. 골 감소는 대부분 해면골에서 일어나므로 해면골 부피와 무기질 밀도 함량 측정은 골다공증 진단에 가장 유용한 지표로 사용되어 진다. 따라서 난소 절제 동물모델(OVX)에서 단삼, 진피 및 갈근 혼합 추출물(S1)이 골다공증에 미치는 영향을 Micro-CT를 통해 확인하였다. BV/TV(%)는 뼈 부피/조직 부피, BS/BV는 뼈 표면/뼈 부피, Tb.Th는 섬유주의 두께(trabecular thickness), Tb.N는 섬유주의 수(trabecular number), Tb.Sp는 섬유주 분리(trabecular separation), BMD는 골밀도(bone mineral density)를 의미한다. A representative symptom of menopausal women is osteoporosis due to abnormal bone metabolism. This is a phenomenon in which osteoblast function declines after menopause and osteoclast function becomes active, accelerating bone resorption. In particular, it is known that the decrease in estrogen due to menopause is the biggest cause of osteoporosis in women. used as an indicator Since most of the bone loss occurs in cancellous bone, measurements of cancellous bone volume and mineral density are used as the most useful indicators for diagnosing osteoporosis. Therefore, the effect of Danshen, dermis, and radish root mixed extract (S1) on osteoporosis in an ovariectomized animal model (OVX) was confirmed through Micro-CT. BV/TV (%) is bone volume/tissue volume, BS/BV is bone surface/bone volume, Tb.Th is trabecular thickness, Tb.N is trabecular number, Tb.Sp denotes trabecular separation, and BMD denotes bone mineral density.
그 결과, 도 5에 개시된 바와 같이 BV/TV(%), BS/BV 및 BDM은 대조군(OVX)과 비교시 S1을 투여한 경우 증가하는 경향을 보였다. As a result, as shown in FIG. 5, BV/TV (%), BS/BV, and BDM tended to increase when S1 was administered compared to the control group (OVX).
실시예 2. 세포 실험Example 2. Cell experiments
1. 세포독성1. Cytotoxicity
RAW 264.7 세포에서 본 발명의 단삼, 진피 및 갈근 혼합 물 추출물 및 단삼, 진피 및 갈근 혼합 에탄올 추출물의 독성을 확인한 결과, 도 6에 개시된 바와 같이 0.5mg/mL 농도 이하에서 세포 독성을 나타내지 않는 것을 확인하였고, 이후 실험에서 0.5mg/mL 이하의 농도까지 사용하였다. As a result of confirming the toxicity of the ethanol extract of Danshen, dermis, and Radix root mixed extract and Danshen, dermis, and Radix root mixture of the present invention in RAW 264.7 cells, as shown in FIG. In subsequent experiments, it was used up to a concentration of 0.5 mg/mL or less.
2. TRAP(Tartrate Resistant Acid Phosphatase) 분석2. TRAP (Tartrate Resistant Acid Phosphatase) Assay
2.1. TRAP 염색 분석2.1. TRAP staining assay
RANKL은 파골세포 분화에 중요한 인자이며, RANKL의 조절에 의해 성숙한 파골세포로 분화한다. 따라서 도 7A에 개시된 바와 같이 RANKL에 의해 분화가 유도된 파골세포는 자색으로 염색되는 것을 확인할 수 있었고, RANKL과 함께 본 발명의 혼합 추출물을 처리한 경우, 대조군인 RANKL 단독 처리군에 비해 염색의 강도 및 밀도가 감소하므로, 파골세포 분화가 억제된 것을 확인할 수 있었다. 특히 단삼, 진피 및 갈근 혼합 물 추출물 투여시 파골세포 분화가 현저히 억제되는 것을 확인하였다. RANKL is an important factor for osteoclast differentiation, and differentiates into mature osteoclasts under the control of RANKL. Therefore, as shown in Figure 7A, it was confirmed that the osteoclasts differentiated by RANKL were stained purple, and when the mixed extract of the present invention was treated with RANKL, the intensity of staining compared to the control group, RANKL alone treatment And since the density decreased, it was confirmed that osteoclast differentiation was inhibited. In particular, it was confirmed that osteoclast differentiation was significantly inhibited when the extract of Danshen, dermis, and root mixture was administered.
2.2. TRAP 활성 분석2.2. TRAP activity assay
도 7B에 개시된 바와 같이 RANKL 처리에 의해 증가된 TRAP 활성이 본 발명의 혼합 추출물 처리에 의해 현저히 감소되는 것을 확인하였다. As shown in Figure 7B, it was confirmed that the TRAP activity increased by RANKL treatment was significantly reduced by treatment with the mixed extract of the present invention.
3. 에스트로겐 수용체 단백질 및 mRNA 발현 확인3. Confirm estrogen receptor protein and mRNA expression
에스트로겐 수용체 ERα 및 ERβ에 대한 결합력을 평가하기 위해 ERα 및 ERβ의 단백질 및 mRNA 발현을 확인하였다. 그 결과, 도 8A에 개시된 바와 같이 본 발명의 단삼, 진피 및 갈근 혼합 에탄올 추출물은 ERα 및 ERβ의 단백질 발현을 대조군인 RANKL 단독 처리군에 비해 증가시키는 경향을 나타냈다. 또한, 도 8B에 개시된 바와 같이 본 발명의 단삼, 진피 및 갈근 혼합 에탄올 및 물 추출물은 ERα 및 ERβ의 mRNA 발현을 대조군인 RANKL 단독 처리군에 비해 증가시키는 것을 확인하였다. To evaluate the binding ability to the estrogen receptors ERα and ERβ, the protein and mRNA expression of ERα and ERβ was confirmed. As a result, as shown in FIG. 8A, the mixed ethanol extract of Danshen, dermis, and dandelion root of the present invention showed a tendency to increase the protein expression of ERα and ERβ compared to the control group, RANKL alone treatment. In addition, as shown in FIG. 8B, it was confirmed that the mixed ethanol and water extracts of Danshen, dermis, and dandelion root of the present invention increased the mRNA expression of ERα and ERβ compared to the control group, RANKL alone treatment group.
4. 파골세포 분화 억제 평가4. Assessment of inhibition of osteoclast differentiation
4.1. NFATC1, c-Fos 단백질 및 NFATC1 mRNA 발현 확인4.1. Confirmation of NFATC1, c-Fos protein and NFATC1 mRNA expression
NFATC1, c-Fos는 파골세포 분화에 핵심적인 효소이며, RANKL 처리에 의해 발현이 증가되는 것으로 알려져 있다. 본 발명의 혼합 추출물 처리에 의한 NFATC1, c-Fos 단백질 발현 변화를 확인한 결과, 도 9A에 개시된 바와 같이 RANKL 처리에 의해 증가된 NFATC1, c-Fos 단백질 발현이 단삼, 진피 및 갈근 혼합 에탄올 추출물 처리에 의해 감소되는 것을 확인하였고, 도 9B에 개시된 바와 같이 NFATC1의 mRNA 발현 역시 단삼, 진피 및 갈근 혼합 에탄올 및 물 추출물 처리에 의해 감소되는 것을 확인하였다. NFATC1 and c-Fos are key enzymes for osteoclast differentiation, and their expression is known to be increased by RANKL treatment. As a result of confirming the change in NFATC1 and c-Fos protein expression by the mixed extract treatment of the present invention, as shown in FIG. 9A, the increased NFATC1 and c-Fos protein expression by RANKL treatment was found in the treatment of Danshen, dermis, and red root mixed ethanol extracts. As shown in FIG. 9B, it was confirmed that the mRNA expression of NFATC1 was also reduced by treatment with ethanol and water extracts of Danshen, dermis, and dandelion root mixture.
4.2. Acp5, Ctsk mRNA 발현 확인4.2. Confirmation of Acp5, Ctsk mRNA expression
NFATC1에 의해 파골세포의 특이적인 유전자인 Acp5(tartrate-resistant acid phosphatase type 5) 및 Ctsk(cathepsin K)가 조절되고, RANKL에 의해 Acp5 및 Ctsk의 발현이 증가된다는 것이 알려져 있다. 본 발명의 혼합 추출물 처리에 의한 Acp5 및 Ctsk의 mRNA 발현 변화를 확인한 결과, 도 9C 및 9D에 개시된 바와 같이 본 발명의 단삼, 진피 및 갈근 혼합 에탄올 및 물 추출물 처리에 의해 Acp5 및 Ctsk의 발현이 감소하는 것을 확인하였다. It is known that the specific genes of osteoclasts, Acp5 (tartrate-resistant acid phosphatase type 5) and Ctsk (cathepsin K), are regulated by NFATC1, and the expression of Acp5 and Ctsk is increased by RANKL. As a result of confirming the mRNA expression changes of Acp5 and Ctsk by the mixed extract treatment of the present invention, as shown in Figures 9C and 9D, the expression of Acp5 and Ctsk was reduced by the treatment of the mixed ethanol and water extracts of Danshen, dermis, and radish root of the present invention. confirmed that.
5. 에스트라디올 정량 평가5. Quantitative evaluation of estradiol
세포에서 분비되는 에스트라디올의 함량을 ELISA 실험을 통해 확인한 결과, 도 10에 개시된 바와 같이 본 발명의 단삼, 진피 및 갈근 혼합 에탄올 및 물 추출물 처리에 의해 에스트라디올의 분비가 증가하는 것을 확인하였다. As a result of confirming the content of estradiol secreted from the cells through an ELISA experiment, as shown in FIG. 10 , it was confirmed that the secretion of estradiol increased by the treatment of the ethanol and water extracts of the mixed dandelion, dermis, and radish root of the present invention.
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