KR100910622B1 - Functional food comprising extract of Nelumbo nucifera Gaertn for the prevention and amelioration of osteoporosis - Google Patents
Functional food comprising extract of Nelumbo nucifera Gaertn for the prevention and amelioration of osteoporosis Download PDFInfo
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- KR100910622B1 KR100910622B1 KR1020070059340A KR20070059340A KR100910622B1 KR 100910622 B1 KR100910622 B1 KR 100910622B1 KR 1020070059340 A KR1020070059340 A KR 1020070059340A KR 20070059340 A KR20070059340 A KR 20070059340A KR 100910622 B1 KR100910622 B1 KR 100910622B1
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- South Korea
- Prior art keywords
- lotus root
- extract
- root extract
- osteoporosis
- bone
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- A—HUMAN NECESSITIES
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- 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
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- 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
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- 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
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Abstract
본 발명은 연근 추출물을 포함하는 골다공증 예방 및 개선용 건강식품에 관한 것으로서, 구체적으로 조골세포 생성과 파골세포 억제능이 우수하여 골다공증 예방효과를 갖는 연근 추출물을 유효성분으로 포함하여 골다공증의 예방 및 개선을 위해 유용하게 사용될 수 있을 것이다.The present invention relates to a health food for preventing and improving osteoporosis, including lotus root extract, specifically, to prevent and improve osteoporosis by including lotus root extract having an osteoporosis prevention effect with excellent osteoblast production and osteoclast suppression. It may be useful for this purpose.
연근, 골다공증, 파골세포, 조골세포, 골밀도 Lotus root, osteoporosis, osteoclasts, osteoblasts, bone density
Description
도 1A는 본 발명에 사용한 연근을 추출 용매별로 추출하여 조골세포에 처리하였을 때 세포가 증식된 정도를 나타낸 그래프이고,Figure 1A is a graph showing the degree of proliferation of cells when treated with osteoblasts by extracting the lotus root used in the present invention for each extraction solvent,
도 1B는 본 발명에 사용된 연근을 추출 용매별로 추출하여 조골세포에 처리하였을 때 염기성 인산분해효소의 활성을 나타낸 그래프이고,1B is a graph showing the activity of basic phosphatase when the lotus root used in the present invention was extracted for each extraction solvent and treated on osteoblasts,
도 2A는 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 3일 배양하였을 때 조골세포의 증식에 미치는 영향을 MTT법으로 분석한 결과이고,Figure 2A is the result of analyzing the effect on the proliferation of osteoblasts when cultured for 3 days after the treatment of lotus root extract (30% ethanol extract) of the present invention to osteoblasts,
도 2B는 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 7일 배양하였을 때 조골세포의 증식에 미치는 영향을 MTT법으로 분석한 결과이고,Figure 2B is a result of analyzing the effect on the proliferation of osteoblasts when cultured for 7 days after the lotus root extract (30% ethanol extract) of the present invention in osteoblasts,
도 2C는 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 14일 배양하였을 때 조골세포의 증식에 미치는 영향을 MTT법으로 분석한 결과이고,Figure 2C is a result of analyzing the effect on the proliferation of osteoblasts when the lotus root extract of the present invention (30% ethanol extract) treated with osteoblasts, and cultured for 14 days,
도 3A은 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 3일 배양하였을 때 염기성 인산분해효소의 활성을 나타낸 그래프이고,3A is a graph showing the activity of basic phosphatase when cultured for 3 days after treatment of osteomyocyte extract (30% ethanol extract) of the present invention to osteoblasts,
도 3B은 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 7일 배양하였을 때 염기성 인산분해효소의 활성을 나타낸 그래프이고,Figure 3B is a graph showing the activity of basic phosphatase when the lotus root extract of the present invention (30% ethanol extract) treated with osteoblasts, and cultured for 7 days,
도 3C은 본 발명의 연근추출물(30%에탄올 추출물)을 조골세포에 처리 후, 14일 배양하였을 때 염기성 인산분해효소의 활성을 나타낸 그래프이고,Figure 3C is a graph showing the activity of basic phosphatase when the lotus root extract of the present invention (30% ethanol extract) treated with osteoblasts, and cultured for 14 days,
* 도 4는 본 발명의 연근추출물(30%에탄올 추출물)을 여러농도로 파골세포에 처리하였을 때 파골세포의 형성에 미치는 영향을 나타낸 그래프이고,* Figure 4 is a graph showing the effect on the formation of osteoclasts when the lotus root extract of the present invention (30% ethanol extract) treated with osteoclasts at various concentrations,
도 5는 본 발명의 연근추출물(30%에탄올 추출물)을 여러농도로 파골세포에 처리하였을 때 파골세포의 TRAP 활성에 미치는 영향을 영향을 나타낸 그래프이고 (TRAP 활성은 타르트레이트에 대해 저항성을 나타내는 산성 포스파타제 활성을 의미한다).5 is a graph showing the effect on the TRAP activity of osteoclasts when the lotus root extract of the present invention (30% ethanol extract) was treated to osteoclasts at various concentrations (TRAP activity is acidic resistance to tartrate) Phosphatase activity).
도 6은 본 발명의 연근 추출물(30%에탄올 추출물)에 의한 파골세포의 세포독성을 나타낸 그래프이다.Figure 6 is a graph showing the cytotoxicity of osteoclasts by lotus root extract (30% ethanol extract) of the present invention.
본 발명은 골다공증 예방 및 치료에 효과를 갖는 연근 추출물(lotus root)에 관한 것으로서, 보다 상세하게는 조골세포 생성과 파골세포 억제능이 우수하여 골다공증 예방 및 치료효과를 갖는 연근 추출물에 관한 것이다. The present invention relates to a lotus root extract (lotus root) having an effect on the prevention and treatment of osteoporosis, and more particularly, to a lotus root extract having an osteoporosis prevention and treatment effect with excellent osteoblast production and osteoclast inhibition ability.
골다공증(Osteoporosis)은 골 조직의 석회가 감소되어 뼈의 치밀질이 엷어지 고 그로 인해 골수강이 넓어지는 상태로, 증세가 진전됨에 따라 뼈가 약해지기 때문에 작은 충격에도 골절되기 쉽다. 골량은 유전적 요인, 영양섭취, 호르몬의 변화, 운동 및 생활습관의 차이 등 여러 요인들에 의해 영향을 받으며, 골다공증의 원인으로는 노령, 운동부족, 저체중, 흡연, 저칼슘 식이, 폐경, 난소 절제 등이 알려져 있다. 한편 개인차는 있지만 백인보다는 흑인이 골 재흡수 수준 (bone resorption level)이 낮아 골량이 더 높으며, 대개 골량은 14~18세에 가장 높고 노후에는 1년에 약 1%씩 감소한다. 특히 여성의 경우 30세 이후부터 골 감소가 지속적으로 진행되며, 폐경기에 이르면 호르몬 변화에 의해 골 감소가 급격히 진행된다. Osteoporosis is a condition in which bone mineralization is thinned due to the reduction of bone tissue and thus the bone marrow cavity is widened. As osteoporosis progresses, the bone becomes weaker as the symptoms progress. Bone mass is influenced by several factors, including genetic factors, nutritional intake, hormonal changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, and ovaries. Ablation and the like are known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, resulting in higher bone mass. Usually bone mass is highest at 14-18 years of age and decreases by about 1% per year in old age. Especially in women, bone reduction continues after 30 years of age, and when menopause reaches bone growth rapidly due to hormonal changes.
이와 같이 골다공증은 정도에 차이는 있으나 노년층, 특히 폐경기 이후의 여성에게 있어서는 피할 수 없는 증상으로, 선진국에서는 인구가 노령화됨에 따라 골다공증 및 그 치료제에 대한 관심이 점차 증가되고 있다. 또한 전세계적으로 골질환 치료와 관련되어 약 1300억 달러의 시장이 형성되어 있는 것으로 알려져 있으며 앞으로 더 증가할 것으로 예상되기 때문에, 세계적인 각 연구 기관과 제약회사에서는 골질환 치료제 개발에 많은 투자를 하고 있다. As described above, osteoporosis is unavoidable for elderly people, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases worldwide and is expected to increase further. Therefore, many research institutes and pharmaceutical companies around the world are investing heavily in the development of bone disease treatments. .
골다공증과 관련하여 과거에는 주로 골의 무기질, 즉 칼슘과 인의 대사이상을 중심으로 그 연구가 진행되어 왔으며, 이의 발병기전 규명에는 진전을 보지 못하였다 그러나, 최근 골형성에 중요한 무기질 뿐만 아니라 골기질 단백질의 대사에 관한 연구의 중요성이 새로이 부각되고 있다. 현재 이용되는 골다공증의 치료 및 예방법으로는 대개 칼슘이 함유된 식이요법, 폐경기의 여성들에게는 에스트로젠 (estrogen)을 투여하거나 비타민 D를 투여하여 골다공증의 진행을 완화시키는 방법이다. 골다공증 치료제로 널리 사용되는 칼슘 보강제재는 부갑상선 호르몬의 분비를 억제하며 골흡수에 의한 골량 감소를 방지하지만, 골량 유지의 개인 차이가 심하다고 알려져 있다 (Heaney, R.P., Princeples of Bone Biology, Academic press, 1007-1017, 1996). 에스트로젠이나 칼시토닌을 이용한 호르몬 요법의 경우 골밀도를 증가시키고, 직장암의 발생을 감소시킨다는 보고도 있으나, 유방암, 심근경색, 정맥 혈전증 등의 부작용이 보고된 바 있다 (Nelson, H.D. et al., JAMA, 288: 872-881, 2002; Lemay, A., J. Ostet. Bynaecol. Can., 24: 711-715). 비스포스포네이트는 파골세포를 억제하는 골흡수 억제제로써 새로운 대체 치료제로 주목받고 있으나, 올바르지 않은 경구 투여시 상기도에 병소가 관찰되기도 한다(Fleisch, H., Principles of Bone Biology, Academic press, 1007-1017, 1996; Cryer, R. and Bauer, D.C., Mayo. Clin. Proc., 77: 1031-1043, 2002). In the past, the research has been conducted mainly on bone minerals, namely, calcium and phosphorus metabolic abnormalities, and no progress has been made in the pathogenesis. However, recently, not only minerals important for bone formation but also bone matrix proteins The importance of research on metabolism is emerging. Currently, the treatment and prevention of osteoporosis is a calcium-containing diet, and estrogen or vitamin D is administered to postmenopausal women to alleviate the progression of osteoporosis. Calcium adjuvant, widely used as a treatment for osteoporosis, suppresses secretion of parathyroid hormone and prevents bone loss due to bone resorption, but it is known that individual differences in bone mass maintenance are severe (Heaney, RP, Princeples of Bone Biology, Academic press, 1007). -1017, 1996). Hormonal therapy with estrogen or calcitonin has been reported to increase bone density and reduce the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction and venous thrombosis have been reported (Nelson, HD et al., JAMA, 288). : 872-881, 2002; Lemay, A., J. Ostet. Bynaecol. Can., 24: 711-715). Bisphosphonates are attracting attention as a novel alternative therapeutic agent for inhibiting osteoclasts, but lesions can be observed in the upper respiratory tract with incorrect oral administration (Fleisch, H., Principles of Bone Biology, Academic press, 1007-1017, 1996 Cryer, R. and Bauer, DC, Mayo. Clin. Proc., 77: 1031-1043, 2002).
따라서, 새로운 작용 및 골격구조를 가지면서 독성 및 부작용이 적어 골다공증의 예방 및 치료에 효과적인 신물질의 개발이 절실히 요구되고 있다. 또한, 이러한 신물질은 민간요법으로 예로부터 적용되어 온 천연물에서 발견될 가능성이 매우 높기 때문에 천연물, 특히 생약으로부터 신약을 제조하려는 시도가 활발히 진행되고 있다. Therefore, there is an urgent need for the development of new substances effective in the prevention and treatment of osteoporosis due to the new action and skeletal structure and less toxicity and side effects. In addition, since these new substances are very likely to be found in natural products that have been applied to folk medicine since ancient times, attempts are being actively made to produce new drugs from natural products, particularly herbal medicines.
한편, 연근(lotus root)은 수련과에 속하는 다년생 초본인 연꽃(학명 Nelumbo nucifera Gaertn)의 뿌리줄기로, 재배는 표토(表土)가 깊고 유기질이 많은 양토(壤土)나 점질양토(粘質壤土)가 적당하며, 유기질 비료를 주로 사용한다. 주성 분은 당질로서 대부분이 녹말이다. 아미노산으로는 아스파라긴산, 이르기닌, 티로신이 함유되어 있으며 레시틴, 펙틴도 많다. 또한 일반 식물에는 적은 비타민B12가 함유되어 있는 것이 특색이다. On the other hand, lotus root is the root stem of the perennial herbaceous lotus (Nelumbo nucifera Gaertn), which belongs to the water lily family, and the cultivation is a loam or viscous loam with a deep topsoil and a lot of organic matter. Is suitable, and organic fertilizer is mainly used. The main component is sugar, most of which is starch. Amino acids contain aspartic acid, ylangine and tyrosine, as well as lecithin and pectin. It is also characterized by low levels of vitamin B12 in common plants.
효능으로는 정력강장, 피로회복, 정신안정에 도움을 주며, 폐병, 빈혈, 하혈, 각기, 출혈성 위궤양이나 위염에 좋은 것으로 알려져 있으며, 니코틴 (담배의 독) 등 각종 독성물질에 대한 중화, 해독작용 또한 알려져 있다. 그러나, 현재까지 상기 연근과 골다공증과의 상관 관계에 대한 보고는 없는 실정이다. Efficacy in helping to strengthen stamina, fatigue, mental stability, and is known to be good for lung disease, anemia, bleeding, each, hemorrhagic gastric ulcer or gastritis. Neutralizes and detoxifies various toxic substances such as nicotine Also known. However, there is no report on the correlation between the lotus root and osteoporosis.
이에, 본 발명자들은 연근 추출물이 골다공증 유발 억제작용이 뛰어남을 발견하여 연근 추출물을 골다공증 치료제 또는 예방제 및 건강식품으로 유용하게 사용할 수 있음을 밝힘으로써 본 발명을 완성하였다.Accordingly, the present inventors have found that the lotus root extract is excellent in inhibiting osteoporosis-induced action, thereby completing the present invention by revealing that the lotus root extract can be usefully used as a therapeutic agent or a preventive agent and a health food for osteoporosis.
본 발명의 목적은 골다공증 예방 및 치료효과를 갖는 연근 추출물, 이를 포함하는 약학적 조성물 및 건강식품을 제공하는 것이다.It is an object of the present invention to provide a lotus root extract, a pharmaceutical composition and health food comprising the same, having an osteoporosis prevention and treatment effect.
상기 목적을 달성하기 위해, 본 발명은 조골세포의 증식 활성과 파골세포의 증식 억제활성 및 골밀도 증가효능을 갖는 연근 추출물을 제공한다.In order to achieve the above object, the present invention provides a lotus root extract having proliferative activity of osteoblasts and proliferation inhibitory activity of osteoclasts and bone mineral density increasing effect.
또한, 본 발명은 연근 추출물을 유효성분으로 하는 골다공증 예방 및 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention and treatment of osteoporosis using lotus root extract as an active ingredient.
또한, 본 발명은 연근 추출물을 유효성분으로 하는 골다공증 예방 및 치료용 건강식품을 제공한다.In another aspect, the present invention provides a health food for preventing and treating osteoporosis using lotus root extract as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 조골세포의 증식 활성 및 파골세포의 증식 억제활성을 갖는 연근 추출물을 제공한다.The present invention provides a lotus root extract having proliferative activity of osteoblasts and inhibitory activity of osteoclasts.
추출을 위한 용매는 물, 알코올 또는 이의 혼합물이고 알코올은 C1 내지 C4의 저급 알코올이고 바람직하게는 5 내지 100%, 보다 바람직하게는 20 내지 80%, 보다 더 바람직하게는 20 내지 50%의 에틸알콜(ethyl alcohol) 또는 바람직하게는 5 내지 100%, 보다 바람직하게는 20 내지 80%, 보다 더 바람직하게는 20 내지 50%의 메틸알콜(methyl alcohol)이다. 물 추출은 열수추출일 수 있다. The solvent for extraction is water, alcohol or mixtures thereof and the alcohol is C 1 to C 4 lower alcohols and is preferably 5 to 100%, more preferably 20 to 80%, even more preferably 20 to 50%. Ethyl alcohol or preferably 5 to 100%, more preferably 20 to 80%, even more preferably 20 to 50% methyl alcohol. Water extraction may be hot water extraction.
본 발명자들은 연근에 각각 70% 에탄올, 30% 에탄올 및 증류수를 첨가하여 환류 추출 및 동결건조하여 연근 추출물을 수득한 후, 이를 조골세포에 첨가하여 조골세포 증식효과를 알아보았다(도 1A 참조). 특히, 30% 에탄올 연근 추출물은 대조군 보다 높은 증식효과가 나타남을 확인하였다(도 2 참조).The present inventors added 70% ethanol, 30% ethanol and distilled water to reflux extract and lyophilized to obtain lotus root extract, and then added it to osteoblasts to examine the osteoblast proliferation effect (see FIG. 1A). In particular, it was confirmed that the 30% ethanol lotus root extract showed a higher proliferative effect than the control (see Fig. 2).
연근 추출물 첨가 후 조골세포의 특징 중 하나인 ALP(Alkaline phosphatase) 활성이 어떻게 변화하는지 알아본 결과, 연근 추출물 첨가군이 대조군에 비해 ALP 활성이 증가하였다(도 1B 참조). 특히, 30% 에탄올 연근 추출물은 대조군에 비해 ALP 활성이 농도의존적으로 증가함을 확인하였다(도 3 참조). As a result of examining how the ALP (Alkaline phosphatase) activity, which is one of the characteristics of osteoblasts, changes after the addition of lotus root extract, the ALP activity of the lotus root extract added group was increased compared to the control group (see FIG. 1B). In particular, 30% ethanol lotus root extract was found to increase the concentration-dependent ALP activity compared to the control (see Figure 3).
파골세포는 골수내의 단핵구/대식세포 계통의 세포로부터 유래하고, 이 단핵전구세포는 혈액내로 순환되며 골내막층에서 전구세포들이 증식되어 다핵세포를 형성하기 위해 융합된다고 알려져 있으며 (Scheven, B.A.A. etal., Nature, 321:79-81, 1986), 파골세포에는 특징적으로 타르트레이트 (tartrate)에 대해 저항성을 나타내는 산성 포스파타제 (acid phosphatase)인 TRAP을 가지며 이는 다른 골조직 세포와 구별할 수 있는 파골세포의 세포화학적 표지효소로 이용된다고 알려져 있다(Minkin, C., Calcif. Tissue Int., 34: 285-290, 1982). 본 발명자들은 연근 추출물에 의한 파골세포의 생성 억제와 세포 활성도 감소 확인을 위하여, 하기와 같은 실험을 실시하였다.Osteoclasts are derived from cells of the monocyte / macrophage lineage in the bone marrow, which are circulated into the blood and are known to proliferate and proliferate in the endothelial layer to form multinucleated cells (Scheven, BAA et al. , Nature, 321: 79-81, 1986), osteoclasts have an acidic phosphatase, TRAP, which is characteristically resistant to tartrate, which is a cell of osteoclasts that can be distinguished from other bone tissue cells. It is known to be used as a chemical marker (Minkin, C., Calcif. Tissue Int., 34: 285-290, 1982). The inventors carried out the following experiment to confirm the inhibition of the production of osteoclasts and decrease in cell activity by the lotus root extract.
구체적으로, ICR 마우스의 대퇴골과 경골을 적출하고 골수를 모아 파골세포의 전구세포가 되는 미부착세포를 수득하였다. 이에, 실시예에서 제조한 연근 추출물을 처리한 뒤, 파골세포의 생성을 검사하기 위하여, TRAP(Tartrate-resistant acid phosphatase) 염색을 실시하였다. TRAP에 양성(+)이며 다핵(3개 이상의 핵)을 갖는 TRAP-양성 다핵세포들을 파골세포로 측정하고 다핵세포수의 변화를 측정하였다. 그 결과, 연근 추출물을 처리한 군은 대조군에 비해 TRAP(+) 다핵세포의 수가 현저히 감소하는 것을 확인하였다. TRAP 활성도도 현저히 감소하는 것을 확인하였다. 상기 결과로부터, 본 발명의 연근 추출물이 농도 의존적으로 파골세포의 생성 억제 작용을 한다는 것을 확인하였다(도 4 및 5 참조). Specifically, the femur and tibia of the ICR mouse were removed, and the bone marrow was collected to obtain unattached cells that become progenitor cells of osteoclasts. Thus, after treating the lotus root extract prepared in Example, to test the production of osteoclasts, TRAP (Tartrate-resistant acid phosphatase) staining was performed. TRAP-positive multinucleated cells positive (+) and multinuclear (more than 3 nuclei) in TRAP were measured as osteoclasts and the change in the number of multinucleated cells. As a result, the group treated with lotus root extract was found to significantly reduce the number of TRAP (+) multinucleated cells compared to the control group. It was also confirmed that the TRAP activity was also significantly reduced. From the above results, it was confirmed that the lotus root extract of the present invention has a concentration-dependent inhibition of the production of osteoclasts (see FIGS. 4 and 5).
또한, 본 발명은 연근 추출물을 유효성분으로 하는 골다공증 예방 및 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention and treatment of osteoporosis using lotus root extract as an active ingredient.
본 발명의 연근 추출물을 유효성분으로 하는 약학적 조성물은 투여시에 경구로 투여가 가능하며 일반적인 의약품제제의 형태로 사용될 수 있다. 본 발명의 조성물은 실제로 경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용 될 수 있다. 또한, 본 발명의 조성물은 생리식염수 또는 유기용매와 같이 약제로 허용된 여러 담체와 혼합하여 사용될 수 있고, 안정성이나 흡수성을 증가시키기 위하여 글루코스, 수크로스 또는 덱스트란과 같은 카보하이드레이트, 아스코르브 산(ascorbic acid) 또는 글루타치온과 같은 항산화제(antioxidants), 킬레이팅 물질(chelating agents), 저분자 단백질 또는 다른 안정화제(stabilizers)들이 약제로 사용될 수 있다.Pharmaceutical compositions comprising the lotus root extract of the present invention as an active ingredient can be administered orally at the time of administration and can be used in the form of general pharmaceutical preparations. The compositions of the present invention may be administered in various oral dosage forms, which are formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, the composition of the present invention can be used in admixture with various carriers acceptable as medicaments, such as physiological saline or organic solvents, carbohydrates such as glucose, sucrose or dextran, ascorbic acid to increase the stability or absorption acids) or antioxidants such as glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.
본 발명의 조성물 내 추출물의 유효용량은 10 내지 5000 ㎎/㎏이고, 바람직하기로는 30 내지 3000 ㎎/㎏ 이며, 하루 1회 내지 3회 투여될 수 있다. The effective dose of the extract in the composition of the present invention is 10 to 5000 mg / kg, preferably 30 to 3000 mg / kg, it can be administered once to three times a day.
또한, 본 발명은 연근 추출물을 유효성분으로 하는 골다공증 예방 및 치료용 건강식품을 제공한다. In another aspect, the present invention provides a health food for preventing and treating osteoporosis using lotus root extract as an active ingredient.
본 발명의 연근 추출물을 식품으로 사용할 경우, 상기 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 본 발명의 연근 추출물을 식품 또는 음료의 제조 시에 원료에 대하여 0.001 내지 90 중량%, 바람직하게는 0.1 내지 40 중량%의 양으로 첨가될 수 있다. When the lotus root extract of the present invention is used as a food, the extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, the lotus root extract of the present invention may be added in an amount of 0.001 to 90% by weight, preferably 0.1 to 40% by weight based on the raw materials in the manufacture of food or beverage.
본 발명의 연근 추출물의 유효용량은 상기 약학적 조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of lotus root extract of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or for health control. It is evident that the component can be used in an amount above the range because there is no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없다. 상기 연근 추출물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the kind of food. Examples of the food to which the lotus root extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, and drinks. Alcoholic beverages and vitamin complexes.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발 명을 예시하는 것일 뿐, 본 발명을 한정하지는 않는다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, but not to limit the present invention.
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실시예Example
1> 연근추출물의 제조 1 1> Preparation of
연근(lotus root, 구입처 : 경기도 부천 소재 대영제약) 10.4g을 플라스크에 넣고 300ml 70% 에탄올을 첨가하여 2시간동안 2회 환류추출하였다. 얻어진 추출액을 거름종이로 여과하여 진공감압하여 농축시킨 후, 동결건조하여 연근 추출물 50.9mg을 얻었다.10.4 g of lotus root (lotus root, Daeyeong Pharmaceutical Co., Ltd., Bucheon, Gyeonggi-do) was placed in a flask, and 300
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실시예Example
2> 연근 추출물의 제조 2 2> Preparation of
연근 10.3g을 플라스크에 넣고 300ml 30% 에탄올을 첨가하여 2시간동안 2회 환류추출하였다. 얻어진 추출액을 거름종이로 여과하여 진공감압하여 농축시킨 후, 동결건조하여 연근 추출물 128mg을 얻었다.10.3 g of lotus root was added to the flask, and 300 ml of 30% ethanol was added thereto, followed by two reflux extractions for 2 hours. The obtained extract was filtered through a filter paper, concentrated under reduced pressure and concentrated in vacuo, and then lyophilized to yield 128 mg of lotus root extract.
<< 실시예Example 3> 연근 추출물의 제조 3 3> Preparation of Lotus Root Extract 3
연근 10g을 플라스크에 넣고 300ml 증류수를 첨가하여 2시간동안 2회 환류추출하였다. 얻어진 추출액을 거름종이로 여과하여 진공감압하여 농축시킨 후, 동결건조하여 연근 추출물 299mg을 얻었다.10 g of lotus root was added to the flask, and 300 ml of distilled water was added thereto, and the mixture was refluxed twice for 2 hours. The obtained extract was filtered through a filter paper, concentrated under reduced pressure and concentrated in vacuo, and then lyophilized to obtain 299 mg of lotus root extract.
<< 실험예Experimental Example 1> 조골세포에서 연근 추출물의 골다공증 치료효과 확인 1> Confirmation of osteoporosis treatment effect of lotus root extract in osteoblasts
<1-1> 조골세포의 배양<1-1> Culture of Osteoblasts
Saos-2 세포는 한국세포주은행에서 구입하였으며, 10% FBS, 페니실린 100 Unit/ml, 스트렙토마이신 100ug/ml을 포함하는 RPMI 1640 배지(Gibco BRL, 미국)를 사용하여 습식조건, 37℃로 5% CO2 배양기에서 배양하였다. Saos-2 cells were purchased from Korea Cell Line Bank, and were wetted using RPMI 1640 medium (Gibco BRL, USA) containing 10% FBS, 100 units / ml of penicillin and 100 ug / ml of streptomycin, 5% at 37 ° C. Cultured in a CO 2 incubator.
<1-2> 연근 추출물 처리에 따른 조골세포의 <1-2> Osteoblasts Treated with Lotus Root Extract 증식정도Growth 측정 Measure
상기 세포주를 96 웰 플레이트에 1x104 세포주/웰로 접종하고 상기 실시예 1, 2, 3에서 수득한 연근 추출물을 DMSO(Dimethylsulfoxide)에 용해시켜(배지내 최종 DMSO 농도 1%) 20 ug/ml의 농도가 되도록 웰에 첨가하였다. 대조군으로는 연근 추출물을 첨가하지 않은 것을 사용하였다.The cell line was inoculated with 1 × 10 4 cell lines / well in a 96 well plate, and the lotus root extract obtained in Examples 1, 2, and 3 was dissolved in DMSO (Dimethylsulfoxide) (1% final DMSO concentration in medium) at a concentration of 20 ug / ml. Was added to the wells. As a control, one without addition of lotus root extract was used.
상기에서 준비된 것을 37℃ 배양기에서 7일간 배양하고 여기에 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)을 0.05mg/ml 농도로 가하여 같은 조건에서 3시간 더 배양하였다. 생성된 포르마잔(Formazan)결정을 DMSO로 용해시켜 엘리자 판독시(ELISA reader)로 570nm에서의 흡광도를 측정하여 세포증식률(%)을 알아보았다. 비교군으로는 현재 골다공증 치료제로 주로 사용되고 있는 NaF를 사용하였다.The prepared above was incubated for 7 days in an incubator at 37 ° C., and MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) was added thereto at a concentration of 0.05 mg / ml for 3 hours under the same conditions. It was further cultured. Formazan crystals were dissolved in DMSO and absorbance at 570 nm was measured using an ELISA reader to determine the cell growth rate (%). As a comparison group, NaF, which is mainly used as a treatment for osteoporosis, was used.
세포증식율(%)=추출물 첨가 웰의 흡광도/대조군 웰의 흡광도% Cell proliferation = absorbance of extract-added wells / absorbance of control wells
그 결과, 실시예 1의 70% 에탄올 추출물, 실시예 2의 30% 에탄올 추출물, 실시예 3의 열수 추출물을 20ug/ml 농도로 처리한 경우에 모두 증식효과가 나타고, 70% 에탄올 추출물 39.12 ± 3.21% 증가, 열수추출물 41.21 ± 2.14% 증가, 30% 에 탄올 추출물 52.84 ± 0.59% 증가로 30% 에탄올 추출물에서 세포 증식효과가 가장 뛰어남을 확인할 수 있었다(도 1A). As a result, when the 70% ethanol extract of Example 1, the 30% ethanol extract of Example 2, the hydrothermal extract of Example 3 was treated at a concentration of 20 ug / ml, all proliferative effects appeared, and 70% ethanol extract 39.12 ± 3.21% increase, hot water extract 41.21 ± 2.14% increase, 30% ethanol extract 52.84 ± 0.59% increase was found that the best cell proliferation effect in 30% ethanol extract (Fig. 1A).
<1-3> 연근 추출물 처리에 따른 ALP 활성 측정<1-3> ALP activity measurement by lotus root extract treatment
조골세포의 특징 중 하나는 ALP(Alkaline phosphatase) 활성을 나타낸다는 것이므로 연근 추출물이 조골세포인 Saos-2 세포에서 ALP 활성에 어떤 변화를 가져오는지 알아보았다.One of the characteristics of osteoblasts is that it exhibits ALP (Alkaline phosphatase) activity. Therefore, we investigated how the lotus root extract changes ALP activity in osteoblast Saos-2 cells.
실시예 <1-2>의 MTT 실험에서와 동일한 세포수의 Saos-2 세포주에 연근 추출물을 20ug/ml 농도로 처리하고 동일한 조건에서 7일동안 배양하였다. 한편 ALP는 p-니트로페닐포스페이트(p-nitrophenylphosphate)를 p-니트로페놀(p-nitrophenol)과 포스페이트(phosphate)로 분해시키는 것을 이용하여 405nm에서의 흡광도의 변화로 ALP 활성을 측정하였다. 비교군으로는 현재 골다공증 치료제로 주로 사용되고 있는 NaF(0.001ug/ml)을 첨가한 군을 사용하였다. Saos-2 cell line of the same cell number as in the MTT experiment of Example <1-2> was treated with 20ug / ml concentration of lotus root extract and incubated for 7 days under the same conditions. Meanwhile, ALP measured the ALP activity by changing the absorbance at 405 nm by decomposing p-nitrophenylphosphate into p-nitrophenol and phosphate. As a comparison group, a group containing NaF (0.001 ug / ml), which is currently used mainly for treating osteoporosis, was used.
그 결과, 실시예 1의 70% 에탄올 추출물, 실시예 2의 30% 에탄올 추출물, 실시예 3의 열수추출물을 20ug/ml농도로 처리한 경우에 모두 대조군에 비해 ALP의 활성이 증가함을 확인하였고 70% 에탄올 추출물 40.32 ± 1.33% 증가, 열수추출물 46.52 ± 3.14% 증가, 30% 에탄올 추출물 87.62 ± 1.2% 증가로 30% 에탄올 추출물에서의 활성이 약 두 배 정도 탁월함을 확인하였다(도 1B) As a result, when the 70% ethanol extract of Example 1, 30% ethanol extract of Example 2, the hot water extract of Example 3 was treated at a concentration of 20ug / ml, it was confirmed that the activity of ALP was increased compared to the control group. 70% ethanol extract 40.32 ± 1.33% increase, hot water extract 46.52 ± 3.14% increase, 30% ethanol extract 87.62 ± 1.2% increase was confirmed that the activity in 30% ethanol extract is about twice as excellent (Fig. 1B).
<1-4> 연근 추출물(30% 에탄올) 처리에 따른 조골세포의 <1-4> Osteoblasts Treated with Lotus Root Extract (30% Ethanol) 증식정도Growth 측정 Measure
상기 세포주를 96 웰 플레이트에 1X104 세포주/웰로 접종하고 실험예 <1-2>와 <1-3>의 실험을 통해 가장 뛰어난 활성을 나타낸 실시에 2의 30% 에탄올 추출물을 0.2,1,5,20 ug/ml의 농도가 되도록 각 농도별로 웰에 첨가하였다. 연근 추출물은 DMSO(Dimethylsulfoxide)에 용해시켜 사용하였으며 배양조건에서 DMSO의 최종농도는 1% 이하가 되도록 하였다. 한편 대조군으로는 연근 추출물을 첨가하지 않은 것을 사용하였고 비교군으로는 NaF를 사용하였다.The cell line was inoculated with 1 × 10 4 cell lines / well in a 96 well plate, and the 30% ethanol extract of Example 2 showed the highest activity through the experiments of Experimental Examples <1-2> and <1-3>, 0.2,1,5 Each well was added to the wells to a concentration of 20 ug / ml. Lotus root extract was used by dissolving in DMSO (Dimethylsulfoxide) and the final concentration of DMSO was below 1% under culture conditions. Meanwhile, as the control group, the lotus root extract was not added, and as the comparison group, NaF was used.
상기 세포주를 37℃ 배양기에서 3, 7, 14 일간 각각 배양하고 여기에 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)을 0.05mg/ml 농도로 가하여 같은 조건에서 3시간 더 배양하였다. 생성된 포르자마잔(Formazan)결정을 DMSO로 용해시켜 엘리자 판독시(ELISA reader)로 570nm에서의 흡광도를 측정하여 세포증식율(%)을 알아보았다(연근 추출물을 첨가하지 않은 대조군 사용)The cell lines were incubated for 3, 7, and 14 days in a 37 ° C. incubator, and MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) was added at a concentration of 0.05 mg / ml. Incubated for 3 more hours under conditions. Formazan crystals were dissolved in DMSO and the absorbance at 570 nm was measured using an ELISA reader to determine the cell growth rate (%) (using the control without addition of lotus root extract).
세포증식율(%)=추출물 첨가 웰의 흡광도/대조군 웰의 흡광도% Cell proliferation = absorbance of extract-added wells / absorbance of control wells
그 결과, 본 발명의 30% 에탄올 연근 추출물은 도 2에서와 같이, 대조군 보다 3, 7, 14 일 모든에서 높은 증식효과가 나타남을 알 수 있었다. As a result, it was found that the 30% ethanol lotus root extract of the present invention showed a higher proliferative effect in all 3, 7, 14 days than the control.
상기 실험으로부터 본 발명에 따른 30% 에탄올 연근 추출물의 조골세포 증식효과는 NaF처리군에 비해 고농도에서 나타나기는 하나, 비슷하거나 더 높은 활성을 나타내었으며, 농도의존적으로 조골세포의 증식을 촉진하는 효과가 있음을 확인하였다.Osteoblast proliferation effect of the 30% ethanol lotus root extract according to the present invention from the above experiments was shown at higher concentrations than NaF treatment group, but showed similar or higher activity, and concentration-dependent effect of promoting osteoblast growth. It was confirmed that there is.
<1-5> 연근 추출물(30% 에탄올) 처리에 따른 ALP 활성 측정<1-5> ALP Activity Measurement of Lotus Root Extract (30% Ethanol)
MTT 실험에서와 동일한 세포수의 Saos-2 세포주에 실험예 <1-3>과 같이 처리하고 동일한 조건에서 3, 7, 14일 동안 배양한 후 수확하여 ALP 활성을 측정한 결과, 30% 에탄올 연근 추출물은 도 3에서와 같이, 대조군에 비해 3,7,14 일 모든 측정에서 염기성 인산분해효소의 활성이 농도 의존적으로 증가함을 확인하였다. The ALP activity was obtained by treating the Saos-2 cell line with the same cell number as in Experimental Example <1-3> and incubating for 3, 7, 14 days under the same conditions as the MTT experiment, and then harvesting it. As shown in FIG. 3, it was confirmed that the activity of the basic phosphatase increased in a concentration-dependent manner at all measurements for 3,7,14 days compared to the control.
<< 실험예Experimental Example 2> 파골세포에서 연근 추출물의 골다공증 치료효과 확인 2> Confirmation of osteoporosis treatment effect of lotus root extract in osteoclasts
연근 추출물에 의한 파골세포의 생성 억제와 세포 활성도 감소 확인을 위하여, 다음과 같은 실험들을 시행하였다. In order to confirm the inhibition of the production of osteoclasts and the decrease of cell activity by the lotus root extract, the following experiments were conducted.
<2-1> 파골세포 배양<2-1> osteoclast culture
ICR 마우스(중앙실험동물)의 대퇴골(femur)과 경골(tibia)을 무균적으로 적출하고 주위의 근육조직을 깨끗이 제거하여 대퇴골과 경골을 분리하고 양끝을 절단한 후, 30G 주사침을 이용하여 골수가 완전히 빠질 때까지 α-MEM (α-minimum essential medium, Gibco BRL) 배지로 씻어내렸다. 골수를 모아 10% FBS가 포함된 α-MEM에 24시간 전 배양한 후 파골세포의 전구세포가 되는 미부착세포를 수득하여 웰 당 2× 105개의 세포가 되도록 분주하여 배양하였다. 배양하는 동안 30 ng/ml M-CSF(macrophage-colony stimulating factor)와 50 ng/ml RANKL이 포함된 α-MEM에 실시예 2에서 제조된 연근 추출물을 최종 농도가 5, 10, 20 ug/ml이 되도록 처 리하여 배양하였다. 대조군으로는 연근 추출물을 첨가하지 않은 것을 사용하였고, 비교군으로는 골다공증 예방에 효능이 있는 것으로 알려진 제니스테인(genistein) 30uM을 사용하였다(Ishimi Y. et al., Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen defficiency. Endocrinol., 140(4): 189301900, 1999). Aseptically remove the femur and tibia of the ICR mouse (Central experiment animal), clean the surrounding muscle tissue, separate the femur and tibia, cut both ends, and then use the 30G needle Washed with α-MEM (α-minimum essential medium, Gibco BRL) medium until complete dissipation. Bone marrow was collected and cultured in α-MEM containing 10% FBS for 24 hours, and thus, non-adherent cells, which became osteoclast progenitor cells, were obtained and divided and cultured to 2 × 10 5 cells per well. During incubation, the lotus root extract prepared in Example 2 was added to α-MEM containing 30 ng / ml macrophage-colony stimulating factor (M-CSF) and 50 ng / ml RANKL, and the final concentration was 5, 10, 20 ug / ml. Treatment was carried out to incubate. As a control group, no lotus root extract was added, and as a control group, genistein 30uM, which is known to be effective in preventing osteoporosis, was used (Ishimi Y. et al., Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen defficiency.Endocrinol., 140 (4): 189301900, 1999).
<2-2> 연근 추출물 처리에 따른 파골세포 생성 억제 확인<2-2> Confirmation of inhibition of osteoclast production by lotus root extract treatment
추출물을 첨가한 지 6일째에 배양을 완료한 후 파골세포의 생성을 검사하기 위하여, 세포를 고정하여 TRAP(Tartrate-resistant acid phosphatase) 염색을 실시하였다. 염색은 시판되는 키트(Acid phosphatase, Sigma)를 사용하였다. On the 6th day after the addition of the extract, the cells were fixed and stained with TRAP (Tartrate-resistant acid phosphatase) in order to examine the production of osteoclasts. Staining was done using a commercial kit (Acid phosphatase, Sigma).
구체적으로 부착세포를 PBS로 세척하고 시트레이트-아세테이트-포름알데히드(citrate-acetate-formaldehyde)로 5분간 고정시키고 나프톨(naphthol) AS-BI 포스페이트, 패스트가넷(fast Garnet) GBC 용액과 7mM 타트레이트(tartrate) 완충용액(pH 5.0)을 함유하는 37℃ 아세테이트 버퍼(pH 5.0)에 1시간 동안 배양하여 염색을 하였다.Specifically, adherent cells were washed with PBS, fixed with citrate-acetate-formaldehyde for 5 minutes, naphthol AS-BI phosphate, fast garnet GBC solution and 7 mM tartrate ( tartrate was stained by incubating for 1 hour in 37 ℃ acetate buffer (pH 5.0) containing buffer (pH 5.0).
TRAP에 양성(+)이며 다핵(3개 이상의 핵)을 갖는 TRAP-양성 다핵세포들을 파골세포로 측정하고 6일 동안 배양한 후 TRAP 양성 다핵세포수의 변화를 측정하였다. 그 결과, 연근 추출물을 처리한 군은 대조군 100%에 비해 TRAP(+) 다핵세포의 수가 5, 10, 20 ug/ml에서 각각 17.58%, 15.45%, 13.58%로 현저히 감소하는 것을 확인하였다. 상기 결과로부터, 본 발명의 연근 추출물이 농도 의존적으로 파골세 포의 생성 억제 작용을 한다는 것을 확인하였다(도 4). TRAP-positive multinucleated cells having positive (+) and multinucleus (more than 3 nuclei) in TRAP were measured as osteoclasts and cultured for 6 days, and then the change in the number of TRAP positive multinucleated cells was measured. As a result, in the group treated with lotus root extract, the number of TRAP (+) multinucleated cells was significantly reduced from 5, 10 and 20 ug / ml to 17.58%, 15.45% and 13.58%, respectively, compared to 100% of the control group. From the above results, it was confirmed that the lotus root extract of the present invention has a concentration-dependent inhibition of the production of osteoclasts (Fig. 4).
<2-3> 연근 추출물 처리에 따른 TRAP 활성 억제 확인<2-3> Confirmation of TRAP activity inhibition by lotus root extract treatment
추출물을 첨가한 지 4일째에 TRAP 활성을 측정하였다. 구체적으로, 부착세포의 배지를 제거하여 PBS로 세척하고 10% 포름알데히드로 고정한 세포에 기질 용액(1.35 mg/ml 4-nitrophenyl phosphate disodium salt, 10 mM tartarate, 50 mM citrate buffer)을 가하여 37℃에서 10분간 반응시켰다. 반응 후 0.1N NaOH로 반응을 중지시켜 405 nm에서 흡광도를 측정하였다. TRAP 활성은 시료의 흡광도를 대조군의 흡광도에 대한 백분율로 표시하였다. TRAP activity was measured 4 days after the addition of the extract. Specifically, the medium of the adherent cells was removed, washed with PBS, and the substrate solution (1.35 mg / ml 4-nitrophenyl phosphate disodium salt, 10 mM tartarate, 50 mM citrate buffer) was added to the cells fixed with 10% formaldehyde at 37 ° C. The reaction was carried out for 10 minutes. After the reaction, the reaction was stopped with 0.1N NaOH and absorbance was measured at 405 nm. TRAP activity expressed the absorbance of the sample as a percentage of the absorbance of the control.
그 결과, 연근 추출물을 처리한 군은 대조군에 비해 TRAP 활성도가 5, 10, 20 ug/ml에서 각각 13.84%, 12.39%, 11.84%로 현저히 감소하는 것을 확인하였다. 상기 결과로부터, 본 발명의 연근 30% 에탄올 추출물이 파골세포의 TRAP 활성 억제 작용을 한다는 것을 확인하였다(도 5). As a result, the group treated with lotus root extract was found to significantly reduce TRAP activity to 13.84%, 12.39% and 11.84% at 5, 10 and 20 ug / ml, respectively, compared to the control group. From the above results, it was confirmed that the
<2-4> 연근 추출물에 의한 세포독성 측정<2-4> Cytotoxicity Measurement by Lotus Root Extract
연근 추출물에 의한 세포독성을 측정하기 위해 파골세포의 전구세포를 연근 추출물과 함께 배양하고, MTT 실험을 실시하여 세포 증식을 측정하였다. 추출물을 가한지 4일째에 0.05% MTT(3-〔4,5-dimethylthiazol-2-yl〕-2,5-diphenyltetrazolium bromide; Triazolyl Blue)를 가하여 3시간 더 배양하여 생성된 포르마잔(formazan) 결정을 DMSO로 용해시켜 570 nm에서의 흡광도를 측정하였 다. In order to measure the cytotoxicity by the lotus root extract, the progenitor cells of osteoclasts were incubated with the lotus root extract, and MTT experiments were performed to measure cell proliferation. Formazan crystals formed by adding 0.05% MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; Triazolyl Blue) on the 4th day of the extract for 3 hours Was dissolved in DMSO and the absorbance at 570 nm was measured.
세포 증식율을 측정한 결과, 연근 추출물을 처리한 군은 대조군에 비해 5, 10, 20 ug/ml에서 각각 108%, 106.66%, 107.43%로 대조군과 크게 차이가 없는 것을 확인하였다. 상기 결과로부터, 본 발명의 연근 추출물이 파골 전구세포에 독성이 없는 것을 확인하였다(도 6).As a result of measuring the cell proliferation rate, the group treated with lotus root extract was 108, 106, 66.66% and 107.43% at 5, 10 and 20 ug / ml, respectively, compared with the control group, and it was confirmed that there was no significant difference from the control group. From the above results, it was confirmed that the lotus root extract of the present invention is not toxic to osteoclast progenitor cells (Fig. 6).
<< 실험예Experimental Example 3> 동물모델을 3> animal models 이용한 이용한Used 임상적 효능시험 Clinical efficacy test
250g 내외의 백서의 암컷을 대상으로 하여 본 발명의 연근 추출물이 골다공증의 치료 및 예방에 어떠한 영향을 미치는지 알아보기 위하여, 난소절제 백서에 추출물을 투여한 후, 골밀도 측정을 실시하였다. In order to examine the effect of the lotus root extract of the present invention on the treatment and prevention of osteoporosis in females of 250 g or more white rats, bone density measurement was performed after administration of the extract to the ovariectomized white paper.
<3-1> 난소 절제술<3-1> ovarian resection
100mg/Kg의 케타민(Ketamine, Ketara)과 2% 자이라진(Xylazine, Rompun0.15ml/Kg)으로 전신마취 후 통상의 방법에 따라 제모 및 술전 무균처리(10% povidone-iodine scrub followed by 70% alcohol wipe)를 수행하였다.100mg / Kg ketamine (Ketamine, Ketara) and 2% xyazine (Xylazine, Rompun 0.15ml / Kg) followed by general anesthesia followed by conventional hair removal and aseptic treatment (10% povidone-iodine scrub) followed by 70% alcohol wipe) was performed.
백서 복측 중앙에 1cm 가량의 절개를 시행한 뒤 횡격막이나 간 등 주용 장기에 손상이 가해지지 않도록 주의를 하며 자궁을 따라 난소를 확인하였다. 봉합용 실로 난소를 결찰한 뒤 난소 절제를 양측으로 시행하였다. 난소 절제 후 각 장기를 복강 내로 재위치 시킨 후 봉합용 실로 층별 봉합을 시행하였다. 수술 후 감염방지를 위해 항생제 세파졸린(cefazolin 50mg/Kg)을 주사하였다.A 1 cm incision was made in the center of the abdomen, and care was taken to prevent damage to main organs such as the diaphragm and liver. The ovary was ligated with sutures and ovarian resection was performed on both sides. After ovarian resection, each organ was relocated into the abdominal cavity and layered sutures were performed with suture thread. The antibiotic cefazoline (cefazolin 50mg / Kg) was injected to prevent infection after surgery.
<3-2> 골 밀도 측정기(Bone mineral densitometer ; BMD )를 이용한 골다공증 예방 및 치료효능 시험 <3-2> Osteoporosis prevention and treatment test using Bone mineral densitometer ( BMD )
250g 내외의 백서를 선택하여 실험군(군당 7마리)과 대조군(7마리)으로 나누어 난소 절개를 하고, 물에 녹인 약물을 8주간 마리당 400mg 약물을 경구 투여하였다. 난소절개 전 골밀도 측정을 시행한 후 2주, 4주, 8주 후에 반복하여 골밀도(BMD) 측정을 시행하였다. BMD 측정은 독일에서 제조한 XCT 540 Research SA를 이용하였다.The white paper of about 250g was selected and divided into the experimental group (7 per group) and the control group (7). The ovary was incision, and the drug dissolved in water was orally administered with 400mg drug per 8 weeks. BMD was measured at 2, 4, and 8 weeks after ovarian bone mineral density measurement. BMD measurement was performed using XCT 540 Research SA made in Germany.
케이지 안에 사육중인 백서를 Ketamin HCl(ketara 10mg/Kg)과 2% xylazine HCl(Roupun 0.15ml/Kg) 혼합액을 복강에 투여하여 전신 마취시킨 후 voxel size0.1mm× 0.1mm, BMD 산출을 위한 Threshould값은 280mg/cm2(해면골 측정), 500mg/cm2(치밀골 측정)으로 설정하였다. Scout scan(10mm/sec)를 통하여 경골(Proxima tibia)의 측정위치를 정하고 CT scan(7mm/sec)을 통해 정해진 위치에서 3개의 slice에서 골밀도(BMD)값을 측정하였으며 동일 위치에서 2회 이상 촬영하고 2, 4, 8주간 매회 반복하였다.White rats in cages were administered with a mixture of Ketamin HCl (ketara 10mg / Kg) and 2% xylazine HCl (Roupun 0.15ml / Kg) intraperitoneally for general anesthesia, and then voxel size0.1mm × 0.1mm, Threshould value for BMD Was set to 280 mg / cm 2 (spongy bone measurement) and 500 mg / cm 2 (dense bone measurement). The measurement position of the tibia (Proxima tibia) was determined through a scout scan (10mm / sec), and the bone density (BMD) value was measured at three slices at the specified position by CT scan (7mm / sec). And repeated every 2, 4, 8 weeks.
그 결과, 난소절제를 시행한 대조군에서는 시간이 경과함에 따라 골밀도가 평균 27%정도 감소하였으나 연근 추출물을 투여한 실험군에서는 난소절제 전과 비슷한 상태를 계속 유지하였으며 4주 후에는 난소절제 전보다 더 증가된 골밀도를 나타내었다. 따라서 본 발명의 연근 30%에탄올 추출물은 골밀도 변화, 즉 골 형성 및 감소에 영향을 주고 있음을 확인할 수 있고 골다공증 예방 및 치료에 효과가 있음을 알 수 있다(표 1).As a result, in the control group treated with ovarian resection, bone density decreased by 27% on average, but the experimental group administered with lotus root extract remained similar to that before ovarian resection, and after 4 weeks the bone density increased more than before ovarian resection. Indicated. Therefore, the
<< 제제예Formulation example 1> 연질캅셀제의 제조 1> Preparation of soft capsule
실시예 2에 따라 조제된 연근 추출물 100.0mg, 콩기름 175.0mg, 황납 45.0 mg, 야자경화유 127.5mg, 대두인지질 21.0mg, 젤라틴 212.0mg, 글리세린(비중 1.24) 50.0mg, 디-소르비톨 76.0mg, 파라옥시안식향산메칠 0.54mg, 파라옥시안식향산프로필 0.90mg, 메칠바닐린 0.56mg, 황색 203호 적량의 성분이 1 캅셀 중에 함유되도록 약전 제제총칙중 연질캅셀의 제법에 따라 제조하였다.Lotus root extract 100.0 mg, soybean oil 175.0 mg, lead 45.0 mg, coconut hardened oil 127.5 mg, soybean phospholipid 21.0 mg, gelatin 212.0 mg, glycerin (specific gravity 1.24) 50.0 mg, di-sorbitol 76.0 mg, paraoxy according to Example 2 Methyl benzoate 0.54 mg, paraoxybenzoic acid propyl 0.90 mg, methyl vanillin 0.56 mg, yellow No. 203 was prepared in accordance with the manufacturing method of the soft capsule in the formulation of the general formula to contain in an appropriate amount of the ingredients.
<< 제제예Formulation example 2> 음료의 제조 2> Manufacture of beverage
본 발명자들은 연근 추출물을 유효성분으로 함유하는 음료 조성물을 하기와 같이 제조하였다.The present inventors prepared a beverage composition containing lotus root extract as an active ingredient as follows.
실시예 2에 따라 제조된 연근 추출물 0.48 ∼ 1.28㎎, 꿀 522㎎, 치옥토산아미드 5㎎, 니코틴산아미드 10㎎, 염산리보플라빈나트륨 3㎎, 염산피리독신 2㎎, 이노시톨 30㎎, 오르트산 50㎎ 및 물 200㎖의 조성 및 함량으로 하여 통상적인 방법을 사용하여 음료를 제조하였다. Lotus root extract 0.48-1.28 mg, honey 522 mg,
본 발명의 연근 추출물은 조골세포의 증식 활성 및 파골세포의 증식 억제활성을 가지므로 본 발명의 연근 추출물을 포함하는 약학적 조성물 및 건강식품은 골다공증 등 골 관련 질환의 예방 및 치료에 유용하게 사용될 수 있을 것이다. Since the lotus root extract of the present invention has proliferative activity of osteoblasts and inhibits the growth of osteoclasts, the pharmaceutical composition and health food containing the lotus root extract of the present invention can be usefully used for the prevention and treatment of bone-related diseases such as osteoporosis. There will be.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20190042999A (en) | 2017-10-17 | 2019-04-25 | 김좌진 | Composition for Prophylaxis and Treatment of Osteoporosis Comprising Abeliophyllum Distichum Extract |
| KR20240051542A (en) | 2022-10-13 | 2024-04-22 | 대구가톨릭대학교산학협력단 | Composition for inhibitiong osteoclast differentiation comprising extract of Nelumbo nucifera Gaertn. or compound isolated therefrom as an active ingredient |
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| KR100891690B1 (en) * | 2007-09-27 | 2009-04-03 | 무안군 | Preparing Method of Chocolates Using Lotus |
| KR100891691B1 (en) * | 2007-09-27 | 2009-04-03 | 무안군 | Preparing Method of Health Functional Cookie Using Lotus |
| KR101606494B1 (en) * | 2014-04-02 | 2016-03-28 | 연세대학교 산학협력단 | Composition for treating and preventing of bone disease comprising bisbenzylisoquinoline alkaloid |
| KR101655274B1 (en) * | 2015-11-10 | 2016-09-07 | 연세대학교 산학협력단 | Composition for treating and preventing of bone disease comprising aporphine alkaloid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20190042999A (en) | 2017-10-17 | 2019-04-25 | 김좌진 | Composition for Prophylaxis and Treatment of Osteoporosis Comprising Abeliophyllum Distichum Extract |
| KR20240051542A (en) | 2022-10-13 | 2024-04-22 | 대구가톨릭대학교산학협력단 | Composition for inhibitiong osteoclast differentiation comprising extract of Nelumbo nucifera Gaertn. or compound isolated therefrom as an active ingredient |
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