KR100703180B1 - A pharmaceutical composition comprising the extract of herb mixture for treating or preventing osteoporosis disease - Google Patents
A pharmaceutical composition comprising the extract of herb mixture for treating or preventing osteoporosis disease Download PDFInfo
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- KR100703180B1 KR100703180B1 KR1020050073236A KR20050073236A KR100703180B1 KR 100703180 B1 KR100703180 B1 KR 100703180B1 KR 1020050073236 A KR1020050073236 A KR 1020050073236A KR 20050073236 A KR20050073236 A KR 20050073236A KR 100703180 B1 KR100703180 B1 KR 100703180B1
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- extract
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- osteoporosis
- pharmaceutical composition
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Abstract
본 발명은 혼합 생약제 추출물을 포함하는 골다공증 질환의 예방 및 치료를 위한 약학조성물에 관한 것으로, 더욱 상세하게는 서목태(Rhynchosia volubilis; 쥐눈이콩)와 황금 추출물을 유효 성분으로 포함하고, 상기 추출물에 더하여 멸치, 흑임자, 미역, 다시마, 홍화씨, 들깨, 로열 젤리로 구성된 군에서 선택된 하나 또는 그 이상을 추출물의 형태로 더 포함하는 골다공증 예방 및 치료를 위한 약학조성물로서, 골다공증, 퇴행성 골질환 및 류마티스 관절염과 같은 골질환 등의 예방 및 치료에 유용하게 이용될 수 있다.The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis disease, including a mixed herbal extract, more specifically, Rhynchosia volubilis (Rice Bean) and golden extract as an active ingredient, in addition to the extract anchovy As a pharmaceutical composition for the prevention and treatment of osteoporosis further comprising one or more selected from the group consisting of black sesame, seaweed, kelp, safflower seed, perilla, royal jelly in the form of extracts, such as osteoporosis, degenerative bone disease and rheumatoid arthritis It can be usefully used for the prevention and treatment of bone diseases.
골다공증, 황금 추출물, 서목태(쥐눈이콩), 약학조성물, 건강기능식품 Osteoporosis, Golden Extract, Seomoktae (Rice Eye Bean), Pharmaceutical Composition, Health Functional Food
Description
도 1은 본 발명의 동물 실험을 행하는데 있어서의 실험 스케줄을 도식화한 것이다.1 is a diagram illustrating an experiment schedule for carrying out an animal experiment of the present invention.
도 2a 내지 도 2b는 본 발명의 F식을 공급한 후 체중변화를 나타낸 것이며, 도 2c는 난소적출과 저 칼슘 식이의 공급으로 인한 골다공증 유발 후 각 군의 장기 무게를 측정하여 나타낸 결과이다.Figure 2a to Figure 2b shows the weight change after feeding the F formula of the present invention, Figure 2c is a result of measuring the organ weight of each group after induction of osteoporosis due to ovarian extraction and low calcium diet.
도 3는 본 발명에 따른 각 군의 경골과 대퇴골 무게를 측정한 결과를 나타낸 것이다.Figure 3 shows the results of measuring the tibia and femur weight of each group according to the present invention.
도 4는 본 발명에 따른 혈청 중 대사 산물의 농도를 측정한 결과로서, 도 4a는 ALP 활성도이며, 도 4b는 무기인의 농도, 도 4c는 칼슘의 농도를 나타낸 것이다.Figure 4 is a result of measuring the concentration of metabolites in the serum according to the present invention, Figure 4a is ALP activity, Figure 4b is the concentration of inorganic phosphorus, Figure 4c shows the concentration of calcium.
도 5는 본 발명에 따른 뼈지주(trabecular)의 조직학적 밀도 변화를 나타낸 것이다.Figure 5 shows the histological density change of the trabecular (trabecular) according to the present invention.
도 6은 본 발명에 따른 뼈지주(trabecular)의 조직학적 밀도 변화를 나타낸 사진으로, 도 6a는 골다공증 유발 후의 골밀도이며, 도 6b는 본 발명의 F식을 4주 간 투여한 후의 것이고, 도 6c는 8주 투여 후의 사진이다. Figure 6 is a photograph showing a histological density change of the trabecular bone (trabecular) according to the invention, Figure 6a is a bone density after osteoporosis induced, Figure 6b is after administration of the formula F of the present invention for 4 weeks, Figure 6c Is a photograph after 8 weeks of administration.
본 발명은 혼합 생약제 추출물을 포함하는 골다공증 예방 및 치료용 약학조성물에 관한 것으로, 더욱 상세하게는 서목태(Rhynchosia volubilis; 쥐눈이콩)와황금 추출물을 유효 성분으로 포함하고, 상기 추출물에 더하여 멸치, 흑임자, 미역, 다시마, 홍화씨, 들깨, 로열 젤리로 구성된 군에서 선택된 하나 또는 그 이상을 추출물의 형태로 더 포함하는 골다공증 예방 및 치료를 위한 약학조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis comprising a mixed herbal extract, more specifically, Rhynchosia volubilis (Rice Bean) and golden extract as an active ingredient, in addition to the extract anchovy, black sesame, It relates to a pharmaceutical composition for the prevention and treatment of osteoporosis further comprising one or more selected from the group consisting of seaweed, kelp, safflower seed, perilla, royal jelly in the form of extract.
골다공증은 동일 연령과 성별의 정상인에 비하여 골밀도가 현저하게 감소한 상태를 말하는데, 그 발명 원인으로는 내분비학적, 영양학적, 유전학적 인자들이 관여하고 있는 것으로 알려져 있다(Gauley et al., Am. J. Epidemiol, 124, p752, 1986). 특히, 여성의 경우 폐경과 함께 골밀도의 감소 현상이 더욱 심하여 골다공증에 대한 발병률이 남성보다 현저히 높은 것으로 나타나 이에 대한 연구가 활발히 진행되고 있다.Osteoporosis is a condition in which bone density is markedly reduced compared to normal people of the same age and sex, and the cause of the invention is known to be involved in endocrine, nutritional and genetic factors (Gauley et al., Am. J. Epidemiol , 124 , p752, 1986). In particular, the decrease in bone density with menopause is more severe in women, and the incidence of osteoporosis is significantly higher than in men, and research on this is being actively conducted.
폐경기 골다공증의 치료법으로는 호르몬 대체요법인 에스트로겐 보충이 시도되어 왔으며, 골량 유지에 효과적인 것으로 보고되고 있다. 그러나, 이 치료방법은 60세 이상의 고령층에서는 골량 감소 억제효과가 폐경 초기에 비하여 낮으며, 최소 5년 이상의 장기치료를 요하고, 불규칙한 자궁출혈, 유방암, 자궁내막암 및 고혈압 발생 빈도 증가 등의 위험이 있는 것으로 지적되고 있다.As a treatment for postmenopausal osteoporosis, estrogen supplementation, a hormone replacement therapy, has been attempted and is reported to be effective in maintaining bone mass. However, this method of treatment is less effective than the early menopause in elderly people over 60 years of age, requires at least five years of long-term treatment, risk of irregular uterine bleeding, breast cancer, endometrial cancer, and high blood pressure It is pointed out that there is.
한편, 칼슘과 비타민 D, 칼시토닌, 비스포스페이트(bisphosphate), 안드로겐(androgen) 및 후라보노이드(flavonoid) 등을 이용한 골다공증 치료제에 대한 연구가 진행되고 있으나, 아직까지 어떠한 약물도 감소된 골량을 완전히 회복시킬 수 없으며, 그 치료 방법도 제한되어 있는 실정이다. 따라서, 골다공증은 발병 후의 치료보다 예방의 중요성이 강조되고 있다.Meanwhile, studies on the treatment of osteoporosis using calcium and vitamin D, calcitonin, bisphosphate, androgen, and flavonoids have been conducted, but none of the drugs can fully restore the reduced bone mass. No, the treatment is also limited. Therefore, osteoporosis is emphasized the importance of prevention rather than treatment after onset.
최근에는 에스트로겐 투여에 의한 위험성을 보완하기 위하여 식품 등 천연물을 활성 성분으로 이용하는 대체요법에 대한 연구가 활발히 진행되고 있다. 폐경기 골다공증의 예방 및 치료를 위한 대표적인 대체 요법으로는 식물성 에스트로겐(phyroestrogen)의 경구 투여 또는 이를 다량 함유하고 있는 식품의 섭취가 시도되고 있다. 식물성 에스트로겐은 알파파(alfafa), 완두콩, 오트밀, 팥, 쌀 및 대두 등에 다량 함유되어 있으며, 특히 이소플라본(isoflavone)계 식물성 에스트로겐은 콩류에 많이 함유되어 있다. 이소플라본은 아글루콘(aglucon)인 제니스테인(genistein), 다이제인(daidzein), 글리시틴(glicitin)과 이들에 당이 결합된 배당체 등 지금까지 12 종류가 밝혀져 있고, 이들 중 다이제인과 제니스테인은 에스트로겐과 유사한 구조로 되어 있어 세포 내에서 에스트로겐 수용체와 결합하여 에스크로겐 효과뿐만 아니라 항에스트로겐 효과를 동시에 나타내는 것으로 밝혀졌다. 그 결과, 에스트로겐 투여에 의한 여러 가지 부작용을 유발하지 않는다는 장점 때문에 골다공증 예방을 위한 에스트로겐 대체 물질로서 각광을 받고 있다.Recently, researches on alternative therapies using natural products such as foods as active ingredients have been actively conducted to compensate for the dangers caused by the administration of estrogen. As a representative alternative therapy for the prevention and treatment of postmenopausal osteoporosis, oral administration of phyroestrogen or ingestion of a food containing a large amount thereof has been attempted. Phytoestrogens are contained in alfafa, peas, oatmeal, red beans, rice, and soybeans. In particular, isoflavone-based phytoestrogens are found in soybeans. Isoflavones have been identified to date, such as aglucon genistein, daidzein, glycidin, and glycosides in which sugars are bound, and among these, dizein and genistein Has a structure similar to that of estrogen, and it is found to bind to the estrogen receptor in the cell and simultaneously exhibit not only an estrogen effect but also an antiestrogenic effect. As a result, it has been in the spotlight as an estrogen replacement material for the prevention of osteoporosis due to the advantage that it does not cause various side effects by the administration of estrogen.
이와 같이, 서양에서는 이소플라본을 보충식으로 급여해야 한다는 주장이 현실화되고 있는 가운데, 골다공증 환자의 혈액 및 뇨에 함유된 이소플라본의 농도를 정량하여 골다공증과의 연관성 연구도 진행되고 있다. As described above, claims to supplement isoflavones in the West have been realized, and studies on the relationship between osteoporosis and quantitative concentrations of isoflavones in the blood and urine of osteoporosis patients are also underway.
상술한 바와 같이, 골다공증의 예방 및 치료를 위해 서양에서는 대두가 널리 이용되고 있는 반면, 한의학이나 민간요법에서는 쥐눈이콩(서목태), 멸치, 참깨, 미역, 홍화 등이 사용되어 왔다. 그러나, 골다공증과 연관된 약제들의 과학적 연구는 콩의 추출물 급여가 난소를 절제한 흰쥐에서 골생성 증진을 유도하거나 조골세포의 증식이 증가시킨다는 보고만이 있을 뿐 이소플라본계 식물성 에스트로겐을 다량 함유하고 있는 쥐눈이콩에 관한 연구는 미비한 상태이다. 또한, 골다공증에 유효한 기능을 가지고 있는 주요 기능성 성분을 배합한 식이의 효과에 대한 연구는 이루어지지 않는 상황이다.As described above, soybeans are widely used in the West for the prevention and treatment of osteoporosis, while rat or soybean (seomoktae), anchovy, sesame, seaweed, safflower and the like have been used in oriental medicine and folk medicine. However, scientific studies of drugs associated with osteoporosis have only reported that soy extracts induce ovarian growth or increase osteoblast proliferation in ovarian-controlled rats, and rats containing large amounts of isoflavone-based phytoestrogens. The research on is poor. In addition, there is no study on the effect of diet containing the main functional ingredients that have an effective function in osteoporosis.
한편, 황금(Scutellaria baicalensis)은 꿀풀과에 속하는 다년생 초본인 속썩은풀(Scutellaria baicalensis GEORGI)의 주피를 벗긴 뿌리를 건조한 것으로, 독성이 적고 주성분은 후라보노이드계 화합물 30여종이 분리되어 있다. 이러한 황금의 약리작용으로는 해열작용, 이뇨작용, 항바이러스작용, 항균작용, 진정작용, 혈압강하작용, 혈당상승작용, 이담작용(利膽作用), 장관(腸管) 운동억제작용, 아나필라시스(anaphylaxis) 작용, 소염작용, 항아러지작용, 항산화작용 및 세포면역 촉진작용 등이 있어서, 한방에서는 이들 작용을 기초로 하여 각종 한약의 상용 처방약으로 70여종의 황금탕 등에 사용하고 있다. Meanwhile, golden ( Scutellaria baicalensis ) is a dried root of the pericarp herbaceous perennial herb ( Scutellaria baicalensis GEORGI) belonging to the Lamiaceae, is less toxic and the main component is separated from 30 species of flavonoid compounds. These pharmacological actions of gold include antipyretic, diuretic, antiviral, antibacterial, sedative, blood pressure lowering, blood sugar elevation, edema (利 膽 作用), intestinal (腸管) exercise, anaphylaxis Anaphylaxis, anti-inflammatory, anti-allergic, anti-oxidant and cell-immune-promoting functions are used in herbal medicines, which are used in over 70 kinds of golden baths as commercial prescription drugs.
황금의 뿌리 성분인 3가지 후라보노이드(baicalin, baicalein, wogonin) 중 baicalein은 다른 후라보노이드 성분과 비교하여 염증성 동물 모델에서 염증 증상 을 가장 많이 완화시켰고, baicalein(20 ㎎/㎏)의 항염증 효과는 설파살라진(sulfasalazone) 50 ㎎/㎏의 효능과 유사하다고 보고된 바가 있다.Of the three flavonoids (baicalin, baicalein and wogonin), the roots of gold, baicalein was most relieved of inflammatory symptoms in inflammatory animal models compared to other flavonoids. sulfasalazone) has been reported to be similar to the efficacy of 50 mg / kg.
반면, baicalin의 경우, 카라기난 유도 랫트 발바닥 부종(carrageenan-induced rat paw eduma)에 대한 억제 효과와 방광암 세포(bladder cancer cell)을 이용한 항종양 효과 실험 결과 암세포의 증식 억제효과가 baicalein 및 wogonin에 비해 높게 나타났음이 보고되었다. baicalin의 화학구조는 baicalein의 하이드록시(hydroxy) 그룹이 글루크로니록시(glycuronyloxy) 그룹으로 대체되어 있어, 이로 인하여 두 성분 간에 서로 다른 약리 작용을 초래하는 것으로 여겨진다. On the other hand, in the case of baicalin, the inhibitory effect on carrageenan-induced rat paw eduma and antitumor effect using bladder cancer cells showed that the inhibition of the proliferation of cancer cells was higher than that of baicalein and wogonin. It was reported. The chemical structure of baicalin is that the hydroxy group of baicalein is replaced by the glucuronyloxy group, which is thought to cause different pharmacological action between the two components.
wogonin은 마우스의 포식세포계(macrophage cell line)인 RAW 264.7 세포에서 COX-2의 발현을 선택적으로 저해하고, iNOS(inducible macrophage-type nitric oxide synthase) 작용을 저해하여 각각 PGE2 생성을 감소시키고, NO 생성을 억제하는 효과가 보고되어 있다. 또한, TAP(12-O-tetradecanoylphoral-13-acetate)으로 염증을 유도한 마우스의 피부에서 50~200 ㎍/site/treatment 농도로 3일간 5번 발라 주었을 때, 선택적으로 COX-2의 발현을 억제함과 동시에 PGE2 생성을 저해하여 염증예방 및 치료에 효과가 있는 것으로 보고되어 있다.wogonin selectively inhibits the expression of COX-2 in RAW 264.7 cells, a macrophage cell line in mice, inhibits the action of inducible macrophage-type nitric oxide synthase (iNOS) and reduces PGE 2 production, respectively. The effect of inhibiting production has been reported. In addition, the expression of COX-2 was selectively inhibited when applied 5 times at 50 to 200 ㎍ / site / treatment concentration in the skin of mice induced with inflammation by TAP (12-O-tetradecanoylphoral-13-acetate). At the same time, it has been reported to be effective in preventing and treating inflammation by inhibiting PGE 2 production.
상기와 같이 황금의 단일 성분에 대한 연구는 부분적으로 이루어져 있으나, 황금 자체에 대한 연구는 어떠한 개시나 교시된 바가 없다.As described above, the study of a single component of gold is partially made, but the study of gold itself has not been disclosed or taught.
이에 본 발명자들은 골다공증을 치료할 수 있는 물질을 비교적 안전성이 보장되어 있는 전통 한방약재로부터 확보하고자 예의 노력한 결과, 본 발명의 황금 추출물이 한의학적 약리이론 이외에 골다공증으로 발생하는 염증의 치료 효과를 확인함으로써 본 발명을 완성하게 되었다.Therefore, the present inventors have made diligent efforts to secure a substance capable of treating osteoporosis from traditional herbal medicines that are relatively safe. As a result, the golden extract of the present invention confirms the therapeutic effect of inflammation caused by osteoporosis in addition to the herbal pharmacological theory. To complete.
결국 본 발명의 주된 목적은 골다공증 질환의 치료 및 예방 효과를 갖는 혼합 생약제 추출물을 제공하는데 있다.After all, the main object of the present invention is to provide a mixed herbal extract having a therapeutic and prophylactic effect of osteoporosis disease.
또한, 본 발명의 다른 목적은 상기 혼합 생약제 추출물을 유효성분으로 포함하는 골다공증 질환의 예방 및 치료를 위한 약학조성물을 제공하는데 있다.In addition, another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of osteoporosis disease comprising the mixed herbal extract as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 이소플라본계 식물성 에스트로겐을 다량 함유하고 있는 쥐눈이콩(서목태)과 항산화작용 및 항염증작용의 효과를 나타내는 황금 추출물을 포함하는 혼합 생약제 추출물을 제공한다.In order to achieve the above object, the present invention provides a mixed herbal extract comprising a rat eye bean (seomoktae) containing a large amount of isoflavone-based phytoestrogens and a golden extract showing the effects of antioxidant and anti-inflammatory action.
상기 혼합 생약제는 상기 쥐눈이콩 및 황금 추출물에 더하여 멸치, 흑임자, 미역, 다시마, 홍화씨, 들깨, 로열 젤리로 구성된 군에서 선택된 하나 또는 그 이상을 추출물의 형태로 더 포함하는 것을 특징으로 한다.The mixed herbal medicine is characterized in that it further comprises one or more selected from the group consisting of anchovy, black sesame, seaweed, kelp, safflower seed, perilla, royal jelly in addition to the rat bean and golden extract in the form of extract.
본 발명의 혼합 생약제 추출물은 물 및 메탄올, 에탄올, 부탄올 등과 같은 C1 내지 C4의 저급 알코올 및 이들의 혼합용매, 바람직하게는 메탄올에 가용한 추출물을 포함한다.The mixed herbal extract of the present invention includes water and C 1 to C 4 lower alcohols such as methanol, ethanol, butanol and the like, and a mixture thereof, preferably an extract available in methanol.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 혼합 생약제 추출물은 다음과 같이 수득될 수 있다.The mixed herbal extract according to the present invention can be obtained as follows.
본 발명의 생약제 시료 중량의 약 2배 내지 30배, 바람직하게는 3배 내지 20배에 달하는 부피의 메탄올, 에탄올, 부탄올 등과 같은 C1 내지 C4의 저급 알코올의 극성용매 또는 이들의 약 1:0.1 내지 1:10의 혼합비를 갖는 혼합용매로 열수추출, 냉침추출, 환류냉각추출 또는 초음파추출 등의 추출방법을 사용하여 진공여과에 의해 상층액을 회수한 다음, 상기의 과정을 수차례 반복 수행하여 상층액을 모으고, 감압 농축하여 생약제 추출물을 수득할 수 있다.Polar solvents of C 1 to C 4 lower alcohols, such as methanol, ethanol, butanol, and the like, about 1 to 30 times, preferably 3 to 20 times the weight of the herbal drug sample of the present invention or about 1: The supernatant was recovered by vacuum filtration using an extraction method such as hot water extraction, cold immersion extraction, reflux cooling extraction, or ultrasonic extraction with a mixed solvent having a mixing ratio of 0.1 to 1:10, and then the above procedure was repeated several times. The supernatant can be collected and concentrated under reduced pressure to obtain the herbal extract.
본 발명은 상기의 제법으로 얻어진 생약제 추출물을 유효성분으로 함유하는 골다공증 질환의 예방 및 치료용 약학조성물을 제공한다. 본 발명의 생약제는 오랫동안 생약으로 사용되어 오던 약재로서 이로부터 추출된 본 발명의 추출물들 역시 독성 및 부작용 등의 문제가 없다.The present invention provides a pharmaceutical composition for the prevention and treatment of osteoporosis disease containing the herbal extract obtained by the above-mentioned method as an active ingredient. The herbal medicine of the present invention has been used as a herbal medicine for a long time, the extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.
본 발명에 따른 골다공증 질환의 예방 및 치료용 약학조성물은, 조성물 총 중량에 대하여 상기 추출물을 0.1 내지 50 중량%로 포함한다.The pharmaceutical composition for preventing and treating osteoporosis disease according to the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.
본 발명의 추출물을 포함하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담채, 부형제 및 희석제를 더 포함할 수 있다.The pharmaceutical composition comprising the extract of the present invention may further comprise suitable tints, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명의 추출물의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
본 발명에 따른 추출물을 포함하는 약학조성물은, 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. 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에이스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐리롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘카보네이트(calcium carbonate), 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁액, 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, 좌제가 포함된다. 비수용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 웨텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, aceitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate and sucrose in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 추출물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서 본 발명의 추출물은 1일 0.0001 내지 100 ㎎/㎏, 바람직하게는 0.001 내지 100 ㎎/㎏으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
본 발명은 상기 추출물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 골다공증 질환의 예방 및 개선을 위한 건강기능식품을 제공한다.The present invention provides a health functional food for the prevention and improvement of osteoporosis disease, including the extract and a food supplement acceptable food supplement.
본 발명의 혼합 생약제 추출물을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다.Examples of the food to which the mixed herbal medicine extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
또한, 골다공증 질환의 예방 효과를 목적으로 식품 또는 음료에 첨가될 수 있다. 이때, 식품 또는 음료 중의 상기 추출물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다.It may also be added to foods or beverages for the purpose of preventing the prevention of osteoporosis diseases. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에 다른 성분에는 특별한 제한이 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등의 추가 성분을 함유할 수 있다. 상술한 천연 탄수화물의 예로는 모노사카라이드, 예를 들어 포도당, 과당 등; 디사카라이드, 예를 들어 말토오스, 수크로오스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 솔비톨, 에리트리톨 등의 당 알콜이다. 상술한 것 이외의 향미제로서 천연 향미제인 타우마틴, 스테비아 추출물, 예를 들어 레바우디오시드 A, 글리시르히진 등; 및 합성 향미제, 예를 들어 사카린, 아스파르탐 등을 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited to other ingredients in addition to containing the extract as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent other than the above-mentioned, a natural flavoring agent, taumartin, a stevia extract, for example, rebaudioside A, glycyrrhizin, etc .; And synthetic flavoring agents such as saccharin, aspartame and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 및 천연 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 추출물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이때, 첨가제의 비율은 그다지 중요하지는 않지만 본 발명의 추출물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract of the present invention has various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties Colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. At this time, the ratio of the additive is not very important, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.
이하, 본 발명을 하기 실시예 및 실험예에 의거하여 보다 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail based on the following Examples and Experimental Examples.
단, 하기의 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 이들에 한정되는 것은 아니다.However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.
실시예 1. 쥐눈이콩 추출물의 제조Example 1 Preparation of Rat Eye Extract
쥐눈이콩(서목태) 1 ㎏을 증류수로 세척하여 마쇄기로 분말화 한 후, 5 ℓ의 에탄올을 가하여 상온에서 1주간 추출한 다음, 진공 여과하여 상층액을 회수하였다. 상기 과정을 3회 반복하여 상층액을 모은 후, 감압농축한 다음 72시간 동결결조하여 쥐눈이콩 추출물을 수득하였다.1 kg of rat eye bean (Seomok Tae) was washed with distilled water and powdered with a crusher, and 5 L of ethanol was added thereto, extracted for 1 week at room temperature, and the supernatant was recovered by vacuum filtration. The process was repeated three times to collect the supernatant, and then concentrated under reduced pressure and then freeze-dried for 72 hours to obtain rat eye extract.
실시예 2. 황금 추출물의 제조Example 2. Preparation of Golden Extract
황금은 전라남도 여천에 위치한 한약방에서 전라남도 여수산 황금을 구입하여 사용하였다. 황금 뿌리 100 g에 물 2 ℓ를 가한 후 120 ℃에서 2시간 동안 진탕기로 열수 추출 과정을 2회 반복하고, 감압농축한 후 농축액을 동결건조하여 황금 추출물을 수득하였다.Gold was purchased and used in Yeosan, Jeollanam-do, Yeocheon. 2 g of water was added to 100 g of golden roots, followed by repeated hot water extraction with a shaker at 120 ° C. for 2 hours, concentrated under reduced pressure, and freeze-dried the concentrate to obtain a golden extract.
실시예 3. K 추출물의 제조Example 3. Preparation of K Extract
멸치, 흑임자, 미역, 다시마, 홍화씨, 들깨 각각 10 g에 증류수를 가하여 6시간 동안 열수 추출한 후, 실온으로 냉각하고, 하루 정치시켜 침전물을 제거하여 K 추출물을 제조하였다.10 g of anchovy, black sesame, seaweed, kelp, safflower seed, perilla were added to distilled water and extracted with hot water for 6 hours, then cooled to room temperature and allowed to stand for one day to remove precipitates.
실시예 4. F식이의 제조Example 4 Preparation of F Diet
상기 실시예 1 내지 실시예 3에서 제조한 쥐눈이콩 추출물, 황금추출물 및 K 추출물을 동일비율(중량비)로 혼합하고, 상기 혼합물 1 ㎏에 대하여 첨가물로서 CPPII 20 ㎎, 콘드로이틴 황산(condroitin sulfate) 50 ㎎, 글루코사민(glucosamin) 50 ㎎, 콜라겐(collagen) 50 ㎎, 상어연골 50 ㎎, 비타민 C 50 ㎎, 해조칼슘 70 ㎎, L-Lysine HCl 10 ㎎ 및 이소플라본 10 ㎎을 배합하여 F식을 제조하였다.The rat eye extract, golden extract and K extract prepared in Examples 1 to 3 were mixed at the same ratio (weight ratio), and with respect to 1 kg of the mixture, 20 mg of CPPII and 50 mg of condroitin sulfate as an additive. Formula 50 was prepared by combining 50 mg of glucosamine, 50 mg of collagen, 50 mg of shark cartilage, 50 mg of vitamin C, 70 mg of algae calcium, 10 mg of L-Lysine HCl, and 10 mg of isoflavones.
실험예 1. 실험준비Experimental Example 1. Experiment Preparation
1-1. 실험 동물1-1. Experimental animals
스프라그-도울리(Sprague-Dawely)계 자성(雌性) 흰쥐(랫트)를 샘타코 바이오코리아(오산, 한국)로부터 구입하여 랫트용 고형 압축 사료와 물을 충분히 공급하면서 약 3주간 실험실 환경에 적응시킨 후, 체중이 200~300 g이 되었을 때 난소적출을 시행하고, 각각 대조군(controll), sham군(비교군), OVX군 및 F-식이군(실험군)으로 분류하였다.Sprague-Dawely female rats were purchased from Samtaco Bio Korea (Osan, Korea) and adapted to the laboratory environment for approximately three weeks with sufficient supply of solid compressed feed and water for rats. After the weight, the ovary extraction was carried out when the body weight was 200-300 g, and classified into control group, sham group (comparative group), OVX group and F-diet group (experimental group), respectively.
난소적출은 케타라(염산 케타민, 유한양행)와 럼푼 주사액(바이엘 코리아)을 각각 2 ㎖/㎏ BW 및 0.4 ㎖/㎏ BW의 비율로 혼합하여 복강 내에 주입하여 전신마취한 다음, 등쪽의 털을 삭모하고 피부 및 근육까지 절개하여 난소를 노출시켜 난관을 실로 결찰한 후 난소를 절제하고 남은 실을 사용하여 근육을 봉합한 다음 피부를 동물용 스테이플러(stapler)로 봉합하였다. 반대측에 대해서도 동일한 방법으로 난소를 적출하였다. 이때, 동일한 방법으로 수술을 하되 난소는 적출하지 않은 채로 봉합하는 가장 수술(sham operator)을 시행하여 이를 비교군으로 사용하였다.Ovarian extraction was performed by mixing ketara (ketamine hydrochloride, Yuhan Corporation) and lumpoon injection solution (Bay Korea) at a rate of 2 ml / kg BW and 0.4 ml / kg BW, respectively, and injecting into the abdominal cavity for general anesthesia, and then removing the dorsal hair. After excision, skin and muscles were excised to expose the ovary, ligation of the fallopian tubes with thread, the ovaries were excised, the muscles were sutured with the remaining thread, and the skin was sutured with an animal stapler. The ovary was also extracted on the other side in the same way. In this case, surgery was performed in the same manner, but the ovary was performed without the ovary and a sham operator was used as a comparison group.
1-2. 실험 식이의 조제 및 최종 식이식의 투여1-2. Preparation of Experimental Diet and Administration of Final Diet
난소를 적출한 후에는 하기의 표 1에 제시한 바와 같이, 실험 식이를 조제하 고, 난소를 적출하고 약물을 투여하지 아니하는 대조군에게는 대조식을 섭식시켰으며, 비교군(sham군)과 난소를 적출한 OVX군 및 F-식이군(실험군)에게는 저칼슘 식이를 제공하여 확실한 골다공증을 유발하였다. 상기 식이의 공급은 난소 적출 후 4주간 실시하였다.After extraction of the ovary, as shown in Table 1 below, the experimental diet was prepared, and the control group was fed to the control group without the ovary and drug administration, the control group (sham group) and ovary OVX group and F-diet (experimental group), which had been extracted, were given a low calcium diet to induce osteoporosis. The diet was carried out for 4 weeks after ovarian extraction.
[표 1]TABLE 1
최종 식이식은 매일 1회 8주간 체중 100 g당 2 ㎎의 상기 실시예 4에서 제조한 본 발명의 F식을 경구투여하였으며(F-식이군), sham군과 OVX군은 매일 체중 100 g당 1 ㎖의 증류수를 경구투여하였다.The final diet was administered orally to the F diet of the present invention prepared in Example 4 of 2 mg per 100 g body weight once every 8 weeks (F- diet group), sham group and OVX group 1 per 100 g body weight daily ML of distilled water was orally administered.
실험예 2. 체중측정Experimental Example 2. Weight Measurement
난소적출 직전과 난소적출 후 실험종료 시까지 1주일에 1회 전자저울을 사용하여 체중을 측정하였다.Body weight was measured using an electronic scale once a week just before ovarian extraction and after ovarian extraction.
실험예 3. 식이 효율(Food efficiency ratio:FER) 산출Experimental Example 3. Calculation of food efficiency ratio (FER)
실험 식이를 공급한 후, 1주일에 2회 사료 섭취량을 측정하였으며, 사육 기간 동안의 체중 증가량을 동일 기간 동안 섭취한 식이량으로 나누어 식이 효율을 산출하였다.After feeding the experimental diet, feed intake was measured twice a week, and the dietary efficiency was calculated by dividing the weight gain during the breeding period by the diet intake during the same period.
실험예 4. 장기, 경골 및 대퇴골 무게 측정 및 골밀도 측정Experimental Example 4. Measurement of organ, tibia and femur weight and bone density
사육 기간이 끝난 실험 동물을 에틸에테르(ethyl ether)로 마취시킨 후 개복하여 간 문맥을 통해 혈액을 채취하고, 각종 장기와 경골 및 대퇴골을 채취하였다. After completion of the breeding period, the animals were anesthetized with ethyl ether, and then opened, and blood was collected through the liver portal vein. Various organs, tibia, and femur were collected.
채취한 혈액은 실온에서 30분 정도 방치한 다음 3000 rpm에서 15분간 원심분리하여 혈청을 얻은 후 -70 ℃에서 냉동보관하였으며, 혈액채취 직후 간, 신장, 비장을 분리하여 생리 식염수로 세척하고, 여과지로 물기를 제거한 뒤 무게를 측정하여 냉동보관하였다.The collected blood was left at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes to obtain serum, and frozen and stored at -70 ° C. Immediately after blood collection, liver, kidney and spleen were separated, washed with physiological saline, and filtered After removing the water with a weighing was stored frozen.
또한, 경골과 대퇴골을 채취하여 무게를 재고 골밀도를 측정하였다.In addition, the tibia and femur were collected and weighed to measure bone density.
도 2a는 본 발명의 F식을 공급한 4주 후의 체중변화를 나타낸 것이며, 도 2b는 8주 후 체중변화를 나타낸 것이다. 또한, 도 2c는 난소를 적출하고 저 칼슘 식이의 공급에 의한 골다공증 유발한 후 및 본 발명의 F식을 공급한 후 각 군의 장기 무게를 측정하여 나타낸 결과이다.Figure 2a shows the weight change after 4 weeks of feeding the F formula of the present invention, Figure 2b shows the weight change after 8 weeks. In addition, Figure 2c is a result of measuring the organ weight of each group after ovarian extraction and osteoporosis-induced by the supply of low calcium diet and after feeding the F formula of the present invention.
도 2a 및 도 2b에서 보이는 바와 같이, sham군, OVX군 및 F-식이군 모두 체중 증가를 보인 가운데, sham군 28.67±22.62 g, OVX군 76.67±28.29 g 및 F-식이군 106.33±42.77 g으로 특히, sham군에 비해 다른 군에서의 유의적인 체중 증가가 확인되었다.As shown in Figures 2a and 2b, the sham group, OVX group and F-die group all showed weight gain, with sham group 28.67 ± 22.62 g, OVX group 76.67 ± 28.29 g and F- diet group 106.33 ± 42.77 g In particular, significant weight gain was observed in the other groups compared to the sham group.
골다공증 유발 후 장기 무게 측정 결과, 간 무게는 OVX군>sham군>대조군의 순서였으며, 신장은 OVX군>대조군>sham군, 비장의 경우에는 sham군>대조군>OVX군의 순이었다. As a result of long-term weight measurement after osteoporosis induction, liver weight was in the order of OVX group> sham group> control group, and height was OVX group> control group> sham group, and sham group> control group> OVX group in spleen.
그러나, 본 발명의 F식을 4주간 급여 후 장기 무게의 변화는 간이 F-식이군>sham군>OVX군, 신장의 경우 sham군>OVX군>F-식이군 및 비장의 무게는 F-식이군>sham군>OVX군의 순서로 나타났다. 한편, 본 발명의 F식을 8주간 급여한 후 장기 무게를 측정한 결과는 간 무게가 F-식이군>sham군>OVX군, 신장의 무게는 F-식이군>sham군>OVX군, 비장의 경우 F-식이군>OVX군>sham군으로 나타났다(도 2c 참조). However, after 4 weeks of feeding the F diet of the present invention, the change in the weight of the organs was simple F-diet group> sham group> OVX group, and in case of kidney, sham group> OVX group> F-diet group and spleen weight of F-diet Group> sham group> OVX group. On the other hand, after feeding the F formula of the present invention for 8 weeks, the weight of the organ was measured. The liver weight was F-diet group> sham group> OVX group, and the weight of kidney was F-diet group> sham group> OVX group, spleen In the case of F-diet> OVX group> sham group (see Figure 2c).
각 군의 경골과 대퇴골 무게를 측정한 결과는 도 3에 정리하였다. 도 3에서 확인할 수 있듯이, 골다공증 유발 후 각 군의 경골의 무게는 대조군>sham군>OVX군의 순서였으며, 대퇴골은 sham군>OVX군>대조군의 순이었다. 한편, 4주간 본 발명의 F식을 투여한 후에는 경골의 무게가 sham군>OVX군>F-식이군의 순서였으며, 대퇴골은 F-식이군>OVX군>sham군의 순으로 나타났다. 그러나, 8주 후에는 경골과 대퇴골의 무게가 모두 F-식이군>OVX군>sham군의 순서로 확인되었다.The results of measuring the tibia and femur weight of each group are summarized in FIG. 3. As can be seen in Figure 3, the weight of the tibia of each group after osteoporosis induction was in the order of the control> sham group> OVX group, and the femur was in the order of sham group> OVX group> control. On the other hand, after administering the F diet of the present invention for 4 weeks, the tibial weight was in the order of sham group> OVX group> F-diet group, and the femur was in the order of F-diet group> OVX group> sham group. However, after 8 weeks, the tibia and femur weights were confirmed in the order of F-diet> OVX> sham group.
즉, 본 발명의 F식을 4주간 섭취시킨 군의 대퇴골의 무게가 다른 군들과 비교하여 많이 증가하였으며, 특히 8주 투여 후에는 경골과 대퇴골에서 모두 유의적인 증가를 나타내었음을 확인하였다. That is, the weight of the femur of the group fed the F diet of the present invention for 4 weeks was significantly increased compared to the other groups, especially after 8 weeks it was confirmed that the tibia and femur showed a significant increase.
실험예 5. 혈청 중 대사 산물 측정Experimental Example 5. Measurement of Metabolites in Serum
혈청 중의 대사 산물 측정을 위한 시약은 Bio clinical system corporation사의 자동분석기용 시약을 사용하였으며, BECKMEN DU530를 사용하여 흡광도를 측정하였다.Reagents for measuring metabolites in serum were measured using an automatic analyzer of Bio clinical system corporation, and absorbance was measured using BECKMEN DU530.
5-1. 알칼리성 인산분해효소(alkaline phosphatase; ALP) 측정5-1. Alkaline phosphatase (ALP) measurement
혈청 중의 알칼리성 인산분해효소의 측정을 위하여, Kind-king 변법으로 기질 완충액 2 ㎖을 37 ℃ 항온 수조에서 5분간 가온시킨 후 시료 0.05 ㎖를 잘 혼합하고 항온 수조에서 정확히 15분간 가온하여 실온에서 10분간 방치한 다음 60분 이내에 맹검을 대조로 500 ㎚에서 흡광도를 측정하였다.For the determination of alkaline phosphatase in serum, 2 ml of substrate buffer was warmed for 5 minutes in a 37 ° C constant temperature water bath using a Kind-king method, and 0.05 ml of the sample was mixed well, followed by warming for 15 minutes in a constant temperature water bath for 10 minutes at room temperature. Absorption was measured at 500 nm as a control within 60 minutes after standing.
5-2. 무기인 측정5-2. Inorganic Measurement
혈청 중의 무기인 측정은 인산 키트(phosphorus kit; Bio clinical system corporation)를 사용한 몰리브덴 색소법을 이용하였다. Measurement of inorganic phosphorus in serum was performed using the molybdenum pigment method using a phosphorus kit (Bio clinical system corporation).
정색 시약 8 ㎖에 시료 0.02 ㎖를 잘 혼합하여 실온에서 30분간 방치한 후 파장 650 ㎚에서 시약 맹검을 대조로 하여 흡광도를 측정하였다.0.02 ml of the sample was well mixed with 8 ml of the color reagent, and the mixture was left at room temperature for 30 minutes, and then absorbance was measured using the reagent blind as a control at a wavelength of 650 nm.
5-3. 칼슘 측정5-3. Calcium measurement
혈액 중의 칼슘 측정은 OCPC법을 사용하되, 측정시약은 자동분석기용 칼슘 키트(calcium kit; Bio clinical system corporation)를 사용하여 완충액 5 ㎖에 정색 시약 0.5 ㎖를 혼합하고, 검체 0.05 ㎖를 충분히 혼합한 후 실온에서 5 분간 방치한 다음 90분 이내에 맹검을 대조로 하여 파장 575 ㎚ 흡광도를 측정하였다.Calcium in blood is measured by OCPC method.Measurement reagent is mixed with 5 ml of buffer with 0.5 ml of color reagent using a calcium kit (bio clinical system corporation). After standing at room temperature for 5 minutes, the absorbance was measured by blinding within 90 minutes as a control.
각 측정 결과는 도 4a 내지 도 4c에 정리하였다. Each measurement result was put together in FIGS. 4A-4C.
ALP 활성도를 보면, 골다공증 유발 후에는 OVX군이 sham군에 비해 ALP 활성도가 증가하였다. 그러나, F식을 투여한 4주 후에는 F-식이군이 OVX군보다 감소하였고, 8주 투여 후에도 sham군>OVX군>F-식이군의 순서로 나타났다(도 4a 참조).In terms of ALP activity, OVX group showed higher ALP activity than sham group after osteoporosis induction. However, four weeks after the F diet, the F-diet group decreased compared to the OVX group, and even after eight weeks, the F-diet group showed the order of sham group> OVX group> F-diet group (see FIG. 4A).
혈중 무기인의 농도는 골다공증 유발 후에는 OVX군에서 가장 높았고, 다음으로 sham군, 대조군의 순서였으나, 본 발명의 F식을 섭취한 4주 후에는 F-식이군>sham군>OVX군이었으며, 8주 후에는 OVX군>F-식이군>sham군의 순으로 나타났는데 군간의 유의차는 없었다(도 4b 참조).The concentration of inorganic phosphorus in blood was highest in OVX group after osteoporosis induction, followed by sham group and control group, but 4 weeks after F diet of the present invention, F-diet> sham group> OVX group. After 8 weeks, OVX group> F-diet group> sham group appeared in order, and there was no significant difference between groups (see FIG. 4B).
한편, 혈중 칼슘의 농도는 골다공증 유발 후에는 sham군>OVX군>대조군의 순이었으며, 본 발명의 F식 투여 4주 후에는 F-식이군>sham군>OVX군, 8주 후에는 sham군>OVX군>F-식이군의 순서로 나타났으나 역시 군간의 유의차는 없었다(도 4c 참조).On the other hand, the concentration of calcium in the blood was sham group> OVX group> control group after osteoporosis induction, F- diet group> sham group> OVX group, 8 weeks after sham group> F diet administration of the present invention> OVX group> F-diet group appeared in order, but also there was no significant difference between groups (see Figure 4c).
실험예 6. 골광밀도(bone mineral density; BMD) 측정Experimental Example 6. Measurement of bone mineral density (BMD)
각 군의 랫트로부터 경골을 채취하여 Bouin's solution(또는 10% 포르말린 완충용액)으로 고정하고 5% 질산용액으로 5~7일간 탈회한 다음 12시간 흐르는 물로 씻어서 통상적인 조직 표본 제작과정을 거쳐 파라핀 포매(embedding) 실시 후, 마이크로톱을 이용하여 5 ㎛ 두께의 조직 표본을 만들었으며, Gomori 염색(thrichrom stain)을 하였다.Tibia was collected from rats of each group, fixed with Bouin's solution (or 10% formalin buffer solution), dehydrated with 5% nitric acid solution for 5-7 days, washed with running water for 12 hours, and then paraffin embedded through normal tissue specimen preparation. After embedding, a 5 μm thick tissue specimen was prepared using a microtop, followed by Gomori staining.
XCT research SA(STRATEC, 독일) 기기의 70% 알코올이 잠긴 튜브에 경골을 넣고 홀드에 고정하여 SV 스캔을 통해 경골의 전체적인 모양을 확인하고, 원하는 위치를 선정, 선정된 위치에서 CT로 촬영 분석하였다. Tibia was placed in a tube in which 70% alcohol of XCT research SA (STRATEC, Germany) was locked and fixed to the hold to check the overall shape of the tibia through SV scan, and the desired position was selected and analyzed by CT at the selected position. .
도 5는 난소 적출로 골다공증이 유발된 래트의 결골 부위 골단부 2 ㎜ 하방에서 뼈지주(trabecular bone)의 밀도를 나타낸 것으로, 대조군과 sham군에 비하여 OVX군에서 확실하게 골다공증이 유발되었음을 확인할 수 있었다. FIG. 5 shows the density of trabecular bones at 2 mm below the end of the bone site of the osteoporosis-induced osteoporosis of rats, and it was confirmed that osteoporosis was clearly induced in the OVX group compared to the control and sham groups. .
본 발명의 F식을 섭취한 4주 후에는 전체 밀도(total density)와 뼈지주 밀도(trabecular density)는 유의한 차이는 나타나지 않았다. 그러나, 8주 후에는 대조군과 비교하여 유의하게 향상되는 경향을 나타내었다.Four weeks after the F meal of the present invention, there was no significant difference between the total density and the trabecular density. However, after 8 weeks there was a tendency to be significantly improved compared to the control.
상기의 결과는 본 발명의 F식 투여에 따른 뼈지주의 조직학적 밀도 변화를 나타낸 도 6a 내지 도 6c에서도 확인 가능한 것으로서, 본 발명의 F식이 골다공증 질환의 예방 및 치료에 효과적임을 설명해 준다. The above results can be confirmed also in Figures 6a to 6c showing the histological density change of the bone graft according to the F-form administration of the present invention, illustrating that the F-form of the present invention is effective in the prevention and treatment of osteoporosis disease.
실험예 7. 조골세포와 조골세포의 증식 측정Experimental Example 7. Measurement of proliferation of osteoblasts and osteoblasts
MG-63 인간 조골세포(human osteoblast)를 폴리스틸렌 세포 배양접시에 부착시키고, 페니실린 및 스트렙토마이신이 함유된 1% antibacterial-antifungal 용액(Gibco, 미국)과 10% FBS(Gibco, 미국)를 첨가한 DMEM(Gibco, 미국)을 사용하여 배양하되, 습도는 95%, 온도 37 ℃를 유지하면서 5% CO2를 계속 공급하였다.DMG with MG-63 human osteoblast attached to a polystyrene cell culture dish and added 1% antibacterial-antifungal solution containing penicillin and streptomycin (Gibco, USA) and 10% FBS (Gibco, USA) (Gibco, USA) was incubated with 5% CO 2 while maintaining a humidity of 95%, temperature 37 ℃.
조골세포의 증식 측정은 단기간에 대량 검색이 가능한 MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyohenyl)-2-(4-sulfophenyl)-2H-tetra-zolim)] colourimetric assay 방법(Cell Titer 96 Aqueous One Solution Cell Proliferation Assay: Promega)을 사용하여 측정하였다. 96 well plate에 0.5ㅧ104 cell/well의 농도로 분주하고, 24시간 후에 쥐눈이콩 및 대두 메탄올 추출물, E2, 다이아제인, 제니스테인을 농도별로 투여하여 5일간 배양하였다.The proliferation of osteoblasts can be measured in a short period of time with the mass search of MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxy-methoxyohenyl) -2- (4-sulfophenyl) -2H-tetra- zolim)] was measured using a colorimetric assay method (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay: Promega). After dispensing at a concentration of 0.5 ㅧ 104 cell / well in a 96 well plate, and after 24 hours, rats and soybean methanol extract, E2, diazein, and genistein were incubated for 5 days.
5일째 되는 날 MTS(30 ㎕/well) 시약을 첨가하고 2시간 30분 동안 37 ℃에서 배양한 후 MTS가 포르마잔(formazan)으로 분해되는 양을 ELISA reader를 이용하여 490 ㎚에서 흡광도를 측정하여 결정하였다.On the fifth day, add MTS (30 μl / well) reagent, incubate at 37 ° C. for 2 hours 30 minutes, and measure the absorbance at 490 nm using an ELISA reader to determine the amount of MTS that is decomposed into formazan. Decided.
각각의 처리군을 12 well씩 분비하고, 3회 반복실험을 하였으며, 각 약제에 대한 세포증식 효과는 3번 반복실험의 평균값을 취한 후 DMSO만을 처리한 대조군에 대한 백분율로 표시하였다. Each treatment group secreted 12 wells, repeated three times, and the effect of cell proliferation on each drug was expressed as a percentage of the control group treated with DMSO only after taking the average of three replicates.
그 결과를 나타낸 표 2에서 볼 수 있는 바와 같이, 본 발명의 F식이 조골 세포 증식에 효과적임을 확인하였다.As can be seen in Table 2 showing the results, it was confirmed that the F formula of the present invention is effective for osteoblast proliferation.
[표 2]TABLE 2
실험예 8. 통계처리Experimental Example 8. Statistical Processing
통계방법-평균(mean)±SD로 표시하였고, 두 군간의 통계처리는 스튜던트 t-테스트로 하였으며, p값이 0.05 이하인 경우를 유의한 차이로 간주하였다.Statistical method-mean ± SD was used. Statistical processing between the two groups was performed by Student's t-test, and the p-value less than 0.05 was considered as a significant difference.
본 발명의 추출물을 포함하는 약학조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.
제제예 1. 산제의 제조Formulation Example 1 Preparation of Powder
혼합 생약제 추출물 분말 20 ㎎Mixed Herbal Extract Powder 20mg
유당 100 ㎎Lactose 100 mg
탈크 10 ㎎Talc 10 mg
상기의 성분들을 혼합하고, 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제제예 2. 정제의 제조Formulation Example 2 Preparation of Tablet
혼합 생약제 추출물 분말 10 ㎎Mixed Herbal Medicine Extract Powder 10mg
옥수수 전분 100 ㎎100 mg corn starch
유당 100 ㎎ Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
제제예 3. 캅셀제의 제조Formulation Example 3 Preparation of Capsule
혼합 생약제 추출물 분말 10 ㎎Mixed Herbal Medicine Extract Powder 10mg
결정성 셀룰로오스 3 ㎎3 mg of crystalline cellulose
락토오스 14.8 ㎎Lactose 14.8 mg
마그네슘 스테아레이트 2 ㎎Magnesium Stearate 2mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
제제예 4. 주사제의 제조Formulation Example 4 Preparation of Injection
혼합 생약제 추출물 분말 10 ㎎Mixed Herbal Medicine Extract Powder 10mg
만니톨 180 ㎎Mannitol 180 mg
주사용 멸균 증류수 2794 ㎎Sterile distilled water for injection 2794 mg
Na2HPO4·12H2O 26 ㎎ Na 2 HPO 4 · 12H 2 O 26 ㎎
통상의 주사제의 제조방법에 따라 1 앰플(2 ㎖)당 상기의 성분 함량으로 제조한다.According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).
제제예 5. 액제의 제조Formulation Example 5 Preparation of Liquid
혼합 생약제 추출물 분말 10 ㎎Mixed Herbal Medicine Extract Powder 10mg
이성화당 10 g10 g of isomerized sugar
만니톨 5 g 5 g of mannitol
정제수 적당량Purified water
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고, 레몬향을 적당량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 100 ㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100 ml, and then sterilized by filling in a brown bottle. To prepare.
제제예 6. 건강 음료의 제조Formulation Example 6 Preparation of Healthy Drink
혼합 생약제 추출물 분말 10 ㎎Mixed Herbal Medicine Extract Powder 10mg
비타민 C 15 g15 g of vitamin C
비타민 E(분말) 100 g100 g of vitamin E (powder)
젖산철 19.75 gIron lactate 19.75 g
산화아연 3.5 g3.5 g of zinc oxide
니코틴산아미드 3.5 gNicotinamide 3.5 g
비타민 A 0.2 g0.2 g of vitamin A
비타민 B1 0.25 g0.25 g of vitamin B1
비타민 B2 0.3 g 0.3 g of vitamin B2
물 적당량Water
통상의 건강 음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85 ℃에서 교반 가열 후, 만들어진 용액을 여과하여 멸균된 2 ℓ의 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관하여 본 발명에 따른 건강 음료 조성물 제조에 사용한다.After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and stored in the present invention It is used to prepare a healthy beverage composition according to.
상기 조성비는 비교적 기호 음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 수요계층이나 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the composition ratio is mixed with a component suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.
이상에서 설명한 바와 같이, 본 발명의 혼합 생약제 추출물은 체중 증가를 억제하고, 경골과 대퇴골의 뼈지주의 유의적인 증가 및 조골 세포의 증식 효과를 나타내므로, 골다공증, 퇴행성 골질환 및 류마티스 관절염과 같은 골질환의 예방 및 치료를 위한 약학조성물로 이용될 수 있다.As described above, the mixed herbal extract of the present invention suppresses weight gain, exhibits a significant increase in osteoporosis of the tibia and the femur, and proliferative effects of osteoblasts. It can be used as a pharmaceutical composition for the prevention and treatment of diseases.
또한, 본 발명의 혼합 생약제는 독성과 부작용이 없는 천연물질을 사용함으로써 건강기능식품으로도 유용하게 사용될 수 있다.In addition, the mixed herbal medicine of the present invention can be usefully used as a health functional food by using natural substances without toxicity and side effects.
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KR20190109308A (en) | 2018-03-15 | 2019-09-25 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising fermented extracts from aronia and black rice |
KR20200079203A (en) | 2018-12-24 | 2020-07-02 | 건국대학교 글로컬산학협력단 | Composition for preventing or treating bon disease comprising extract of blueberry |
KR102231424B1 (en) | 2019-11-28 | 2021-03-23 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising Aronia melanocarpa 'Viking' extract and Petasites japonicus extract |
KR20220057673A (en) | 2020-10-29 | 2022-05-09 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising bacteria fermented black rice extract containing aronia and bluberry extract |
KR20230011713A (en) | 2021-07-14 | 2023-01-25 | 경북대학교 산학협력단 | Composition for preventing or treating bone disease comprising yam derived extracellular vesicles |
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KR20040107713A (en) * | 2003-06-10 | 2004-12-23 | 대한민국 (부경대학교총장) | Pharmaceutical composition comprising the extract of Scutellaria baicalensis or Baicalin, Baicalein, Wogonin or 5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone isolated therefrom for prevention and inhibition of aging |
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KR20030031418A (en) * | 2001-10-10 | 2003-04-21 | 한국 한의학 연구원 | Extract of herbal mixture and pharmaceutical compositions for prevention or treatment of osteoporosis |
KR20040107713A (en) * | 2003-06-10 | 2004-12-23 | 대한민국 (부경대학교총장) | Pharmaceutical composition comprising the extract of Scutellaria baicalensis or Baicalin, Baicalein, Wogonin or 5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone isolated therefrom for prevention and inhibition of aging |
Cited By (6)
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KR101502392B1 (en) | 2013-06-12 | 2015-03-16 | 한국원자력연구원 | Pharmaceutical Formulation for Preventing or Treating Inflammatory Diseases Comprising Isoegomaketone |
KR20190109308A (en) | 2018-03-15 | 2019-09-25 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising fermented extracts from aronia and black rice |
KR20200079203A (en) | 2018-12-24 | 2020-07-02 | 건국대학교 글로컬산학협력단 | Composition for preventing or treating bon disease comprising extract of blueberry |
KR102231424B1 (en) | 2019-11-28 | 2021-03-23 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising Aronia melanocarpa 'Viking' extract and Petasites japonicus extract |
KR20220057673A (en) | 2020-10-29 | 2022-05-09 | 건국대학교 글로컬산학협력단 | Composition for preventing and treating of bone diseases comprising bacteria fermented black rice extract containing aronia and bluberry extract |
KR20230011713A (en) | 2021-07-14 | 2023-01-25 | 경북대학교 산학협력단 | Composition for preventing or treating bone disease comprising yam derived extracellular vesicles |
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