KR20240003562A - A composition for improving, preventing and treating of breast cancer comprising Cirsium setidens extract - Google Patents
A composition for improving, preventing and treating of breast cancer comprising Cirsium setidens extract Download PDFInfo
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- KR20240003562A KR20240003562A KR1020220081298A KR20220081298A KR20240003562A KR 20240003562 A KR20240003562 A KR 20240003562A KR 1020220081298 A KR1020220081298 A KR 1020220081298A KR 20220081298 A KR20220081298 A KR 20220081298A KR 20240003562 A KR20240003562 A KR 20240003562A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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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
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
Abstract
본 발명은 유방암의 개선, 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 엉겅퀴 추출물을 유효성분으로 포함함으로써, 유방암을 개선, 예방 또는 치료할 수 있으므로, 식품 조성물, 나아가 건강기능식품 또는 약학 조성물로 활용될 수 있다.The present invention relates to a composition for improving, preventing or treating breast cancer, and more specifically, by containing milk thistle extract as an active ingredient, breast cancer can be improved, prevented or treated, and thus it can be used as a food composition, furthermore, as a health functional food or pharmaceutical composition. It can be utilized.
Description
본 발명은 엉겅퀴 추출물을 유효성분으로 포함하는 유방암의 개선, 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for improving, preventing or treating breast cancer containing thistle extract as an active ingredient.
유방암(breast cancer)은 유방에 발생하는 모든 악성 종양을 통틀어 말하는 것으로, 유방에 비정상적인 세포조직이 계속 자라거나 다른 장기로 퍼지는 치명적인 질병이다. 한국 여성들에게 있어서 유방암 발병률은 갑상선암에 이어서 두 번째이며, 유방암 발병률은 20대 및 30대에서 증가하다가 40대에서 최고에 이른다. 우리나라의 경우, 2000년 이후 유방암 발병 증가율이 연 6.8%로 세계에서 가장 빠른 증가 속도를 보이고 있다(2010년 WHO 보고). Breast cancer refers to all malignant tumors that occur in the breast. It is a fatal disease in which abnormal cell tissue in the breast continues to grow or spread to other organs. Among Korean women, the incidence of breast cancer is second only to thyroid cancer, and the incidence of breast cancer increases in people in their 20s and 30s and reaches a peak in their 40s. In Korea, the rate of increase in breast cancer incidence has been 6.8% per year since 2000, the fastest rate of increase in the world (WHO report in 2010).
실제 유방암은 2012, 2013년 한국보건산업진흥원 기관의 통계에 의하면, 총 3,232,417명의 검진 환자 중 약 885,735명이 유방암 판정을 받았고, 전국의 암환자 중 두 번째로 많은 진료비를 내고 있다. 우리나라의 유방암 발병 증가율을 고려할 때 유방암의 치료는 대단히 중요하며 의약 조성물의 개발이 요구된다.In fact, according to statistics from the Korea Health Industry Development Institute in 2012 and 2013, about 885,735 out of a total of 3,232,417 patients were diagnosed with breast cancer, and it pays the second highest medical expenses among cancer patients nationwide. Considering the increasing rate of breast cancer in Korea, treatment of breast cancer is very important and development of pharmaceutical compositions is required.
유방암의 치료는 조기 진단시 외과적 절제술로 치료가 가능하나 많은 환자에서 폐, 뼈, 간 등 원격적인 전이 형태로 재발된다. 암 세포의 전이(metastasis)는 암 치료를 어렵게 하는데, 이때 세포를 둘러싸고 있는 조직으로 침투해 들어가는 세포 침윤(invasion) 과정이 필수적이며 이는 악성 암세포의 특징 중 하나이다(Aznavoorian et al., Cancer 71,1368-1383, 1993). Breast cancer can be treated with surgical resection when diagnosed early, but in many patients, it recurs in the form of distant metastases such as the lungs, bones, and liver. Metastasis of cancer cells makes cancer treatment difficult, and at this time, the process of cell invasion into the tissue surrounding the cells is essential, which is one of the characteristics of malignant cancer cells (Aznavoorian et al., Cancer 71,1368 -1383, 1993).
암세포의 침윤성에는 세포외 기질(extracellular matrix, ECM)과 기저막(basement membrane)을 분해하는 단백질 분해과정과 분해된 매트릭스(matrix)를 통하여 이동하는 세포 이동성(migration)이 관여한다.The invasiveness of cancer cells involves a proteolytic process that decomposes the extracellular matrix (ECM) and basement membrane, and cell migration through the decomposed matrix.
상기 외과적 절제술 외에 방사선조사 및 화학요법의 치료법이 있는데, 화학요법은 세포 복제 또는 세포 대사를 붕괴시키며, 흔히 유방, 폐 및 정소의 암을 치료하는데 많이 사용된다. 종양 질병을 치료하는데 사용되는 전신성 화학요법의 부작용은 암 치료를 받는 환자들에게 가장 문제가 된다. 이러한 부작용 중에서 멀미와 구토는 가장 일반적이며 심각한 부작용이다. 화학요법에 의한 부작용은 환자의 생명에 큰 영향을 미치며 치료에 대한 환자의 순응성을 급격하게 변화시킬 수 있다. 또한, 화학치료제와 관련된 부작용으로는 일반적으로 이러한 약물의 투여시 주의해야 하는 용량 제한 독성(DLT)이 있다. 예를 들어, 점막염은 여러 항암제, 예컨대 항대사물질 세포 독소제인 5-플루오로우라실(5-Fluorouracil), 메토트렉세이트(metotrexate) 및 항종양 항생제(예, 독소루비신) 등에 대한 용량 제한 독성이다. In addition to the above surgical resection, there are treatments including radiation and chemotherapy. Chemotherapy disrupts cell replication or cell metabolism and is commonly used to treat breast, lung, and testis cancer. The side effects of systemic chemotherapy used to treat neoplastic disease are most problematic for patients receiving cancer treatment. Among these side effects, motion sickness and vomiting are the most common and serious side effects. Side effects from chemotherapy can have a significant impact on a patient's life and can drastically change a patient's compliance with treatment. Additionally, side effects associated with chemotherapy drugs generally include dose-limiting toxicities (DLTs), which require caution when administering these drugs. For example, mucositis is a dose-limiting toxicity for several anticancer drugs, such as the antimetabolite cytotoxins 5-Fluorouracil, methotrexate, and antitumor antibiotics (e.g., doxorubicin).
이러한 화학요법 유래의 부작용 중 대부분은 심한 경우 입원을 요하거나 통증을 치료하기 위해 진통제를 필요로 하기도 한다. 이와 같이 화학치료제 및 방사선 치료에 의한 부작용들은 암 환자의 임상적 처치 시 주요 문제가 되고 있다. 또한, 종래의 항암제들은 정상세포까지 죽이는 경우가 많았기 때문에, 최근에는 정상세포에는 작용하지 않고 암세포에만 작용할 수 있는 새로운 기전의 항암제에 대한 연구가 진행되고 있다.Many of these chemotherapy-derived side effects can be severe, requiring hospitalization or requiring painkillers to treat the pain. Likewise, side effects caused by chemotherapy and radiation therapy have become a major problem in the clinical treatment of cancer patients. In addition, because conventional anticancer drugs often kill normal cells, research has recently been conducted on anticancer drugs with a new mechanism that can only act on cancer cells and not normal cells.
따라서, 유방암에 대하여 부작용이 적고 치료 효능이 높은 표적 약물의 발굴은 유방암 치료를 위한 약물 개발 시 충족되지 않은 요구사항이며, 그 필요성이 절실하게 대두되고 있다.Therefore, the discovery of target drugs with low side effects and high therapeutic efficacy for breast cancer is an unmet requirement in drug development for breast cancer treatment, and the need is urgently emerging.
본 발명의 목적은 엉겅퀴 추출물을 유효성분으로 포함하는 유방암의 예방 또는 치료용 약학 조성물을 제공하는데 있다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating breast cancer containing thistle extract as an active ingredient.
또한, 본 발명의 다른 목적은 엉겅퀴 추출물을 유효성분으로 포함하는 유방암의 예방 또는 개선용 식품 조성물을 제공하는데 있다.In addition, another object of the present invention is to provide a food composition for preventing or improving breast cancer containing thistle extract as an active ingredient.
상기한 목적을 달성하기 위한 본 발명의 유방암을 예방 또는 치료하기 위한 약학 조성물은 엉겅퀴 추출물을 유효성분으로 포함할 수 있다.The pharmaceutical composition for preventing or treating breast cancer of the present invention to achieve the above object may include milk thistle extract as an active ingredient.
상기 엉겅퀴 추출물은 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로 추출된 것일 수 있다.The thistle extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
상기 혼합용매는 20 내지 100 부피%의 메탄올, 에탄올, 부탄올 또는 프로판올 수용액일 수 있다.The mixed solvent may be a 20 to 100% by volume aqueous solution of methanol, ethanol, butanol, or propanol.
상기 엉겅퀴 추출물은 20 내지 50 kHz의 진동수 및 50 내지 700 W의 파워의 초음파기로 5 내지 60 분 동안 40 내지 90 ℃ 하에서 처리된 것일 수 있다.The thistle extract may be treated at 40 to 90° C. for 5 to 60 minutes with an ultrasonicator with a frequency of 20 to 50 kHz and a power of 50 to 700 W.
상기 엉겅퀴 추출물은 엉겅퀴 줄기와 잎이 1 : 0.3-1의 중량비로 혼합된 혼합물의 추출물일 수 있다.The thistle extract may be an extract of a mixture of thistle stems and leaves at a weight ratio of 1:0.3-1.
상기 엉겅퀴 추출물의 지표물질은 클로로겐산(chlorogenic acid)일 수 있다.The indicator substance of the thistle extract may be chlorogenic acid.
또한, 상기한 다른 목적을 달성하기 위한 본 발명의 유방암을 예방 또는 개선하기 위한 식품 조성물은 엉겅퀴 추출물을 유효성분으로 포함할 수 있다. In addition, the food composition for preventing or improving breast cancer of the present invention to achieve the above-described other purposes may include milk thistle extract as an active ingredient.
본 발명의 고려엉겅퀴 추출물을 유효성분으로 포함하는 조성물은 유방암세포의 세포증식을 억제시키고, 유방암세포의 이동을 억제시킬 뿐만 아니라 암세포에서 세포사멸의 핵심유전자인 AMPK-α, p53 및 Bax의 발현을 증가시키고 Bcl-2의 발현을 감소시키므로 유방암 특이적 항암활성이 있다.The composition containing the Korean thistle extract of the present invention as an active ingredient not only inhibits the cell proliferation of breast cancer cells and the migration of breast cancer cells, but also inhibits the expression of AMPK-α, p53 , and Bax , which are key genes for apoptosis in cancer cells. It has breast cancer-specific anticancer activity because it increases and decreases the expression of Bcl-2 .
도 1a는 클로로겐산(chlorogenic acid)을 측정한 HPLC 그래프이며, 도 1b는 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물을 HPLC로 측정한 후 주요 지표물질을 확인한 HPLC 그래프이다.
도 2a는 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물을 농도별로 처리 시 p53, AMPK-α, Bax 및 Bcl-2의 발현을 나타낸 PCR이며; 도 2b 내지 도 2e는 상기 도 2a에서 측정한 p53, AMPK-α, Bax 및 Bcl-2의 발현량을 각각 정량화한 그래프이다.Figure 1a is an HPLC graph measuring chlorogenic acid, and Figure 1b is an HPLC graph confirming major indicators after measuring the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention by HPLC.
Figure 2a is a PCR showing the expression of p53, AMPK-α, Bax, and Bcl-2 when treating Korean thistle ultrasonic extract prepared according to Example 1 of the present invention at different concentrations; Figures 2b to 2e are graphs quantifying the expression levels of p53, AMPK-α, Bax, and Bcl-2 measured in Figure 2a.
본 발명은 엉겅퀴 추출물을 유효성분으로 포함하는 유방암의 개선, 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for improving, preventing or treating breast cancer containing thistle extract as an active ingredient.
구체적으로, 본 발명에서 사용되는 엉겅퀴는 고려엉겅퀴이며, 초음파로 추출한 추출물을 사용하는 것이 바람직하다.Specifically, the thistle used in the present invention is Korean thistle, and it is preferable to use an extract extracted by ultrasonic waves.
이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 유방암을 개선, 예방 또는 치료할 수 있는 조성물은 엉겅퀴 추출물을 유효성분으로 포함한다.The composition of the present invention that can improve, prevent or treat breast cancer includes milk thistle extract as an active ingredient.
상기 엉겅퀴는 고려엉겅퀴로서, 고려엉겅퀴(Cirsium setidens)는 쌍떡잎식물 초롱꽃목 국화과의 여러해살이풀이다. 상기 고려엉겅퀴는 산과 들에서 높이 약 1 m까지 자라고, 뿌리가 곧으며 가지가 사방으로 퍼진다. 상기 고려엉겅퀴의 뿌리에 달린 잎과 밑부분의 잎은 꽃이 필 때 시든다. The thistle is Korean thistle, and Korean thistle ( Cirsium setidens ) is a perennial herb of the dicotyledonous plant Campanula order Asteraceae. The Korean thistle grows to a height of about 1 m in mountains and fields, has straight roots and branches spread in all directions. The leaves on the roots and lower leaves of the Korean thistle wither when the flowers bloom.
상기 고려엉겅퀴의 줄기에 달린 잎은 타원 모양 바소꼴 또는 달걀 모양으로서 밑쪽 잎은 잎자루가 길고 위쪽 잎은 잎자루가 짧다. 상기 고려엉겅퀴 잎의 앞면은 녹색에 털이 약간 나며 뒷면은 흰색에 털이 없고 가장자리가 밋밋하거나 가시 같은 톱니가 있다. 상기 고려엉겅퀴는 한국 특산종으로 전국에 분포한다. The leaves on the stem of the Korean thistle are oval-shaped or egg-shaped, and the lower leaves have long petioles and the upper leaves have short petioles. The front side of the Korean thistle leaf is green and slightly hairy, and the back side is white and hairless and has a smooth edge or thorn-like teeth. The Korean thistle is a Korean endemic species and is distributed throughout the country.
본 발명에서는 고려엉겅퀴의 줄기와 잎을 혼합하여 사용하며, 바람직하게는 줄기와 잎을 1 : 0.3-1의 중량비, 더욱 바람직하게는 1 : 0.5-0.8의 중량비로 혼합하여 사용한다. 줄기를 기준으로 잎의 함량이 상기 범위를 벗어나는 경우에는 유방암의 개선, 예방 또는 치료 효과가 낮을 수 있다.In the present invention, the stems and leaves of Korean thistle are mixed and used, preferably at a weight ratio of 1:0.3-1, more preferably at a weight ratio of 1:0.5-0.8. If the content of leaves relative to the stem is outside the above range, the effect of improving, preventing or treating breast cancer may be low.
또한, 고려엉겅퀴의 줄기와 잎의 혼합물 대신 꽃 및/또는 뿌리를 사용하는 경우에는 유방암의 개선, 예방 또는 치료 효과가 현저히 낮을 수 있다.Additionally, if flowers and/or roots of Korean thistle are used instead of a mixture of stems and leaves, the effectiveness of improving, preventing, or treating breast cancer may be significantly lower.
상기 고려엉겅퀴는 추출용매 하에서 초음파처리를 통해 추출되는 것으로서, 구체적으로 고려엉겅퀴와 추출용매가 1 : 5 내지 25의 중량비, 바람직하게는 1 : 8 내지 15의 중량비로 혼합되어 20 내지 50 kHz, 바람직하게는 30 내지 40 kHz의 진동수 및 50 내지 700 W, 바람직하게는 150 내지 400 W 파워의 초음파기로 40 내지 90 ℃, 바람직하게는 50 내지 85 ℃ 하에서 5 내지 60 분, 바람직하게는 15 내지 40 분 동안 처리된다. The Korean thistle is extracted through ultrasonic treatment under an extraction solvent. Specifically, the Korean thistle and the extraction solvent are mixed at a weight ratio of 1:5 to 25, preferably 1:8 to 15, and extracted at 20 to 50 kHz, preferably. Preferably, the ultrasonicator has a frequency of 30 to 40 kHz and a power of 50 to 700 W, preferably 150 to 400 W, at 40 to 90° C., preferably 50 to 85° C., for 5 to 60 minutes, preferably 15 to 40 minutes. processed while
고려엉겅퀴를 추출 시 초음파 추출이 아니라 열수 추출, 용매 추출 또는 침지 추출인 경우에는 유효성분이 소량 추출될 뿐만 아니라 유방암의 개선, 예방 또는 치료 효과가 낮을 수 있다.If Korean thistle is extracted using hot water extraction, solvent extraction, or immersion extraction rather than ultrasonic extraction, not only a small amount of the active ingredient is extracted, but the effectiveness of improving, preventing, or treating breast cancer may be low.
상기 고려엉겅퀴와 추출용매의 중량비가 상기 범위를 벗어나는 경우에는 추출물에 고려엉겅퀴의 유효성분이 적은 양으로 추출될 수 있다. If the weight ratio of the Korean thistle and the extraction solvent is outside the above range, the active ingredient of the Korean thistle may be extracted in a small amount in the extract.
또한, 초음파기의 진동수 및 파워가 상기 하한치 미만인 경우에는 고려엉겅퀴의 유효성분이 적은 양으로 추출될 수 있으며, 상기 상한치 초과인 경우에는 유효물질의 열분해, 중합 또는 유효성분 외에 다른 물질도 다량으로 추출되어 효과가 저하될 수 있다. In addition, if the frequency and power of the ultrasonicator are less than the above lower limit, the active ingredients of Korean thistle can be extracted in a small amount, and if it is greater than the above upper limit, the active substances may be thermally decomposed, polymerized, or other substances in addition to the effective ingredients may be extracted in large quantities, resulting in an effect. may deteriorate.
또한, 추출온도 및 추출시간이 상기 하한치 미만인 경우에는 고려엉겅퀴의 유효성분이 적은 양으로 추출될 수 있으며, 상기 상한치 초과인 경우에는 독성물질이 발생할 수 있다.In addition, if the extraction temperature and extraction time are less than the above lower limit, the active ingredients of Korean thistle may be extracted in a small amount, and if the extraction temperature and extraction time exceed the above upper limit, toxic substances may be generated.
상기 추출물을 추출하는 추출용매는 물, 탄소수 1 내지 4의 저급알코올, 에틸렌글리콜, 에틸에테르 또는 이들의 혼합용매이다. 상기 저급알코올로는 20 내지 99 부피%의 메탄올, 에탄올, 부탄올 또는 프로판올 수용액을 들 수 있으며, 바람직하게는 우수한 유방암의 개선, 예방 또는 치료 효과를 위하여 40 내지 99 부피%의 에탄올 수용액을 들 수 있다.The extraction solvent for extracting the extract is water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The lower alcohol may include a 20 to 99 volume % aqueous solution of methanol, ethanol, butanol or propanol, and preferably a 40 to 99 volume % ethanol aqueous solution for excellent improvement, prevention or treatment of breast cancer. .
본 발명의 고려엉겅퀴 추출물은 약학 조성물 외에 식품 조성물에도 사용될 수 있다.The Korean thistle extract of the present invention can be used in food compositions in addition to pharmaceutical compositions.
본 발명에서 사용되는 용어 ‘추출물’은 상기 용매를 이용하여 고려엉겅퀴에 포함된 성분을 추출한 추출물, 이들로부터 분획한 분획물, 이들 추출물 또는 분획물을 추가적으로 농축한 농축물, 이를 정제 또는 분리한 정제물도 포함하고, 상기 추출물, 분획물, 농축물 또는 정제물을 건조한 건조물 또는 그를 분쇄한 분말을 포함하는 의미로 사용된다. The term 'extract' used in the present invention also includes extracts obtained by extracting components contained in Korean thistle using the above solvent, fractions obtained from them, concentrates obtained by additionally concentrating these extracts or fractions, and purified products obtained by purifying or separating them. And, it is used to include the dried product of the extract, fraction, concentrate or purified product or the powder obtained by pulverizing the same.
상기 정제물의 제조를 위해 분자량 컷-오프 값을 갖는 한외 여과막을 통과시키거나, 또는 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 부가할 수 있다.For the preparation of the purified product, various additional methods are used, such as passage through an ultrafiltration membrane with a molecular weight cut-off value, or separation by various chromatographs (designed for separation according to size, charge, hydrophobicity or affinity). Purification methods can be added.
본 발명은 고려엉겅퀴 추출물을 유효성분으로 포함하는 유방암의 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for preventing or improving breast cancer containing extract of Korean thistle as an active ingredient.
상기 ‘식품 조성물’은 유효성분으로 고려엉겅퀴 추출물 이외에, 식품 제조에 통상적으로 사용되는 식품의 기준 및 규격(‘식품공전’)에 기재된 식품으로 사용가능한 식품 원료, 식품첨가물 공전에 기재된 식품첨가물을 포함한다.The 'food composition' includes, in addition to Korean thistle extract as an active ingredient, food raw materials that can be used as foods as described in the standards and specifications for foods commonly used in food production ('Food Code'), and food additives listed in the Food Additive Code. do.
특별히 한정할 필요는 없으나 예를 들어 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상기 탄수화물은 단당류, 예를 들어, 포도당, 과당 등; 이당류, 예를 들어 말토스, 설탕, 유당 등; 올리고당 또는 폴리사카라이드, 예를 들어 덱스트린, 물엿, 사이클로덱스트린 등; 당알코올, 예를 들어 자일리톨, 소르비톨, 에리트리톨 등을 사용할 수 있다. 상기 향미제는 천연 향미제[타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)] 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다.There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).
상기 고려엉겅퀴 추출물을 유효성분으로 식품 조성물을 제조하는 경우에 고려엉겅퀴 추출물은 유방암을 예방 또는 치료할 수 있는 함량이면 특별히 한정할 필요는 없으나, 예를 들어 0.1 내지 99 중량%, 0.5 내지 95 중량%, 1 내지 90 중량%, 2 내지 80 중량%, 3 내지 70 중량%, 4 내지 60 중량%, 5 내지 50 중량%로 포함될 수 있다.When preparing a food composition using the Korean thistle extract as an active ingredient, there is no need to specifically limit the content of the Korean thistle extract as long as it can prevent or treat breast cancer, for example, 0.1 to 99% by weight, 0.5 to 95% by weight, It may be included in 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, and 5 to 50% by weight.
상기 식품 조성물에서 유효성분인 고려엉겅퀴 추출물은 섭취자의 상태, 체중, 질병의 유무나 정도 및 기간에 따라 다르지만, 통상의 기술자에 의해 적절하게 선택될 수 있다. 예들 들어 1일 투여량을 기준으로 1 내지 5,000 mg, 바람직하게는 5 내지 2,000 mg, 더욱 바람직하게는 10 내지 1,000 mg, 더더욱 바람직하게는 20 내지 800 mg, 가장 바람직하게는 50 내지 500 mg일 수 있고, 투여 횟수는 특별히 한정할 필요는 없으나 1일 3회 내지 1주일에 1회의 범위 내에서 통상의 기술자가 조절할 수 있다. 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있다.The Korean thistle extract, which is an active ingredient in the above food composition, varies depending on the condition, body weight, and presence, degree, and duration of the disease of the consumer, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.
상기 식품 조성물은 특별히 한정할 필요는 없으나 예를 들어 산제, 과립제, 정제, 캡슐제, 환제, 엑스제, 젤리 제형, 티백 제형 또는 음료 제형일 수 있다.The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.
또한 일반 식품에 유방암의 예방 또는 개선의 기능성을 부여하기 위하여 상기 고려엉겅퀴 추출물을 첨가할 수 있으며, 첨가가 가능한 식품은, 특별히 한정할 필요는 없으나 예를 들어 식품위생법 제7조에 따른 식품의 기준 및 규격(‘식품공전’)에 예시된 과자류, 빵 또는 떡류, 코코아가공품류 또는 초콜릿류, 식육 또는 알가공품, 어육가공품, 두부류 또는 묵류, 면류, 다류, 커피, 음료류, 특수용도식품, 장류, 조미식품, 드레싱류, 김치류, 젓갈류, 절임식품, 조림식품, 주류, 건포류, 기타 식품류 등에 첨가될 수 있다. 또한 축산물위생관리법 제4조에 따른 축산물의 가공기준 및 성분규격(‘축산물공전’)에 예시된 유가공품, 식육가공품 및 포장육, 알가공품에 첨가될 수 있다.In addition, the above-mentioned Korean thistle extract can be added to general foods to provide functionality for preventing or improving breast cancer. Foods that can be added do not need to be specifically limited, but for example, the food standards and regulations under Article 7 of the Food Sanitation Act Confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish products, tofu or jelly, noodles, tea, coffee, beverages, special-purpose foods, sauces, and seasonings as exemplified in the standards ('Food Code of Conduct'). It can be added to foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, dried raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.
한편, 상기 고려엉겅퀴 추출물을 유효성분으로 하는 식품 조성물은 단독으로 “유방암의 예방 또는 개선용 건강기능식품”으로 이용될 수 있다. Meanwhile, the food composition containing the Korean thistle extract as an active ingredient can be used alone as a “health functional food for preventing or improving breast cancer.”
상기 ‘건강기능식품’은 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 법적 기준에 따라 제조(가공을 포함)한 식품(건강기능식품에 관한 법률 제3조 제1호)을 말한다. 상기 ‘건강기능식품’은 국가마다 용어나 범위에 차이가 있을 수 있으나, 미국의 ‘식이 보충제(Dietary Supplement)’, 유럽의 ‘식품 보충제(Food Supplements)’, 일본의 ‘보건기능식품’ 또는 ‘특정보건용 식품(Food for Special Health Uses, FOSHU)’, 중국의 ‘보건식품’ 등에 해당할 수 있다.The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplements' in Europe, and 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Uses (FOSHU)’ or ‘health food’ in China.
상기 식품 조성물 또는 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, 식품첨가물로서의 적합여부는 다른 규정이 없는 한 ‘식품첨가물공전’의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 따른다.The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.
또한 본 발명은 인간, 또는 인간을 제외한 동물에게 상기 조성물을 투여하는 유방암의 치료방법을 제공한다.Additionally, the present invention provides a method of treating breast cancer by administering the composition to humans or non-human animals.
또한 본 발명은 유방암의 예방 또는 치료용 의약, 또는 동물용 의약 제조를 위한 고려엉겅퀴 추출물의 신규 용도를 제공한다.In addition, the present invention provides a new use of Korean thistle extract for the manufacture of medicine for the prevention or treatment of breast cancer, or medicine for animals.
상기 ‘약학 조성물’또는 ‘의약’은 유효성분으로 고려엉겅퀴 추출물 이외에, 약학 조성물 등의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The ‘pharmaceutical composition’ or ‘medicine’ may further include appropriate carriers, excipients, and diluents commonly used in the production of pharmaceutical compositions, etc., in addition to Korean thistle extract as an active ingredient.
상기 ‘담체’는 세포 또는 조직 내로의 화합물의 부가를 용이하게 하는 화합물이다. 상기 ‘희석제’는 대상 화합물의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 화합물을 용해시키게 되는 물에서 희석되는 화합물이다. The ‘carrier’ is a compound that facilitates the addition of the compound into cells or tissues. The ‘diluent’ is a compound diluted in water that not only stabilizes the biologically active form of the target compound, but also dissolves the compound.
상기 담체, 부형제 및 희석제로는 특별히 한정할 필요는 없으나 예를 들어, 유당, 포도당, 설탕, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다.There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약의 사용량은 환자 또는 치료대상 동물의 나이, 성별, 체중에 따라 달라질 수 있으며, 무엇보다도, 치료대상 개체의 상태, 치료 대상 질환의 특정한 카테고리 또는 종류, 투여 경로, 사용되는 치료제의 속성에 의존적일 것이다.The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, gender, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 체내에서 활성성분의 흡수도, 배설속도, 환자 또는 치료대상 동물의 연령 및 체중, 성별 및 상태, 치료할 질병의 중증정도 등에 따라 적절히 선택되나, 일반적으로 1일 0.1 내지 1,000 mg/kg, 바람직하게는 1 내지 500 mg/kg, 더욱 바람직하게는 5 내지 250 mg/kg, 가장 바람직하게는 10 내지 100 mg/kg으로 투여하는 것이 바람직하다. 이렇게 제형화된 단위 투여형 제제는 필요에 따라 일정시간 간격으로 수회 투여할 수 있다.The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , it is generally preferred to administer 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.
상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 개별적으로 예방제 또는 치료제로서 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다.The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
상기 약학조성물, 의약, 동물용 약학 조성물 또는 동물용 의약은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 트로키제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구 제형으로 제형화하여 사용될 수 있다. 제형화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical compositions, medicines, pharmaceutical compositions for animals, or medicines for animals can be used by formulating them into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. there is. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 카보네이트, 설탕 또는 유당, 젤라틴 등을 섞어 조제될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .
상기 유방암의 치료방법은 인간, 또는 인간을 제외한 동물, 특히 포유동물에게 상기 조성물을 투여 하는 것으로, 예를 들어 유방암인 치료대상 개체에게 상기 조성물을 투여하는 것이다.The method of treating breast cancer involves administering the composition to humans or non-human animals, especially mammals, for example, administering the composition to an individual to be treated who has breast cancer.
상기 치료를 위한 투여량, 투여 방법 및 투여 횟수는 상기 약학 조성물, 의약, 동물용 약학 조성물 또는 동물용 의약의 투여량, 투여 방법 및 투여 횟수를 참고할 수 있다.The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.
실시예 1. 초음파 에탄올 추출물_줄기:잎=1:0.7의 중량비Example 1. Ultrasonic ethanol extract_stem:leaf = weight ratio of 1:0.7
고려엉겅퀴 혼합물(줄기:잎=1:0.7의 중량비)과 50 부피%의 에탄올 수용액을 1 : 10의 중량비로 혼합하여 초음파기(SD-250H, Mujigae Co., Seoul, Korea)에 투입 후 60 ℃ 하에서 초음파(40 kHz, 250 W)를 이용하여 30 분간 추출한 다음 추출물을 5,000 rpm에서 10 분간 원심분리하여 상등액을 회수함으로써 고려엉겅퀴 초음파 에탄올 추출물을 수득하였다.Mix Korean thistle mixture (stem:leaf = 1:0.7 weight ratio) and 50% by volume ethanol aqueous solution at a weight ratio of 1:10, put into an ultrasonicator (SD-250H, Mujigae Co., Seoul, Korea), and place at 60°C. After extraction for 30 minutes using ultrasound (40 kHz, 250 W), the extract was centrifuged at 5,000 rpm for 10 minutes to recover the supernatant, thereby obtaining an ultrasonic ethanol extract of Korean thistle.
비교예 1. 고려엉겅퀴 에탄올 추출물_초음파 생략Comparative Example 1. Korean thistle ethanol extract_ultrasound omitted
고려엉겅퀴 혼합물(줄기:잎=1:0.7의 중량비)과 50 부피%의 에탄올 수용액을 1 : 10의 중량비로 혼합하여 80 ℃ 하에서 50 분 동안 추출한 다음 추출물을 1,000 rpm에서 5 분간 원심분리하여 상등액을 회수함으로써 고려엉겅퀴 에탄올 추출물을 수득하였다.Korean thistle mixture (stem:leaf = 1:0.7 weight ratio) and 50% by volume ethanol aqueous solution were mixed at a weight ratio of 1:10 and extracted at 80°C for 50 minutes, then the extract was centrifuged at 1,000 rpm for 5 minutes and the supernatant was Through recovery, an ethanol extract of Korean thistle was obtained.
비교예 2. 줄기:잎=1:2의 중량비Comparative Example 2. Weight ratio of stem:leaf=1:2
상기 실시예 1과 동일하게 실시하되, 고려엉겅퀴의 줄기와 잎을 1 : 2의 중량비로 혼합하여 고려엉겅퀴 초음파 에탄올 추출물을 수득하였다.The same procedure as in Example 1 was performed, but the stems and leaves of Korean thistle were mixed at a weight ratio of 1:2 to obtain an ultrasonic ethanol extract of Korean thistle.
비교예 3. 줄기:뿌리=1:0.7의 중량비 Comparative Example 3. Weight ratio of stem: root = 1:0.7
상기 실시예 1과 동일하게 실시하되, 고려엉겅퀴의 줄기와 뿌리를 1 : 0.7의 중량비로 혼합하여 고려엉겅퀴 초음파 에탄올 추출물을 수득하였다.The same procedure as in Example 1 was performed, but the stems and roots of Korean thistle were mixed at a weight ratio of 1:0.7 to obtain an ultrasonic ethanol extract of Korean thistle.
비교예 4. 줄기 단독Comparative Example 4. Stem alone
상기 실시예 1과 동일하게 실시하되, 고려엉겅퀴의 줄기만을 사용하여 고려엉겅퀴 줄기 초음파 에탄올 추출물을 수득하였다.An ultrasonic ethanol extract of Korean thistle stems was obtained in the same manner as in Example 1, except that only the stems of Korean thistle were used.
비교예 5. 잎 단독Comparative Example 5. Leaves alone
상기 실시예 1과 동일하게 실시하되, 고려엉겅퀴의 잎만을 사용하여 고려엉겅퀴 잎 초음파 에탄올 추출물을 수득하였다.An ultrasonic ethanol extract of Korean thistle leaves was obtained in the same manner as in Example 1, except that only the leaves of Korean thistle were used.
비교예 6. 뿌리 단독Comparative Example 6. Root alone
상기 실시예 1과 동일하게 실시하되, 고려엉겅퀴의 뿌리만을 사용하여 고려엉겅퀴 뿌리 초음파 에탄올 추출물을 수득하였다.An ultrasonic ethanol extract of Korean thistle roots was obtained in the same manner as in Example 1, except that only the roots of Korean thistle were used.
<시험예> <Test example>
시험예 1. HPLC 측정Test Example 1. HPLC measurement
HPLC 측정: 고려엉겅퀴 초음파 추출물의 지표물질의 정량 및 정성 분석을 위해 고려엉겅퀴 초음파 추출물을 0.45 μm syringe filter (Hyundai Micro Co., Seoul, Korea)로 여과한 후 Poroshell 120 EC-C18 column (4.6 X 150 mm, Agilent technology Inc., California, USA)과 diode array detector (DAD)가 장착된 HPLC (Agilent 1260 series, Agilent technology Inc., California, USA)를 이용하여 분석하였다. 피크 분리를 위해 이동상 A (99.9% acetonitrile)와 이동상 B (1.0% acetic acid)의 혼합비율을 시간에 따라 변경하며 분석을 수행하였다. 분석을 위한 시료 주입량은 10 μL로 0.5 mL/min 유속에서 64 분간 분석을 진행하여 시료의 검출시간(retention time, RT)을 확인하였고 DAD를 이용하여 190 ~ 640 nm에서 흡수 스펙트럼을 확보하고 표준물질의 검출시간과 흡수 스펙트럼을 비교하여 정성평가를 수행하였으며 검출 물질을 표준물질의 피크 면적을 기준으로 검량선과 비교하여 정량분석하였다.HPLC measurement: For quantitative and qualitative analysis of indicators of Korean thistle ultrasonic extract, the Korean thistle ultrasonic extract was filtered through a 0.45 μm syringe filter (Hyundai Micro Co., Seoul, Korea) and then filtered using Poroshell 120 EC-C 18 columns (4.6 Analysis was performed using HPLC (Agilent 1260 series, Agilent technology Inc., California, USA) equipped with a 150 mm, Agilent technology Inc., California, USA) and diode array detector (DAD). For peak separation, analysis was performed by changing the mixing ratio of mobile phase A (99.9% acetonitrile) and mobile phase B (1.0% acetic acid) over time. The sample injection volume for analysis was 10 μL, and analysis was performed for 64 minutes at a flow rate of 0.5 mL/min to confirm the retention time (RT) of the sample. An absorption spectrum was obtained from 190 to 640 nm using DAD and a standard substance was obtained. Qualitative evaluation was performed by comparing the detection time and absorption spectrum, and the detected substances were quantitatively analyzed by comparing them with the calibration curve based on the peak area of the standard substance.
도 1a는 클로로겐산(chlorogenic acid)을 측정한 HPLC 그래프이며; 도 1b는 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물을 HPLC로 측정한 후 주요 지표물질을 확인한 HPLC 그래프이다.Figure 1a is an HPLC graph measuring chlorogenic acid; Figure 1b is an HPLC graph confirming the main indicator substances after measuring the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention by HPLC.
도 1a 및 도 1b에 도시된 바와 같이, 고려엉겅퀴 초음파 추출물의 주요 피크는 11.3 분에서 확인되었으며, 이를 폴리페놀 표준물질 중의 하나인 클로로겐산(chlorogenic acid)과 비교 시 RT가 일치하였고, 스펙트럼 분석에 있어 325 nm에서 최대 흡수파장을 나타내며 표준물질과 스펙트럼의 양상이 동일한 것을 확인되어 고려엉겅퀴 초음파 추출물의 주요 지표 물질이 클로로겐산(chlorogenic acid)인 것을 확인하였다.As shown in Figures 1a and 1b, the main peak of the Korean thistle ultrasonic extract was confirmed at 11.3 minutes, and when compared with chlorogenic acid, one of the polyphenol standards, the RT was consistent, and in the spectrum analysis It was confirmed that the maximum absorption wavelength was at 325 nm and that the spectrum was identical to that of the standard material, confirming that the main indicator substance of the Korean thistle ultrasonic extract was chlorogenic acid.
또한, 표준물질의 피크 면적을 기준으로 클로로겐산(chlorogenic acid)의 농도를 산출한 결과, 0.02 mg/g DM으로 확인되었다.In addition, as a result of calculating the concentration of chlorogenic acid based on the peak area of the standard material, it was confirmed to be 0.02 mg/g DM.
시험예 2. 유방암세포의 세포독성 및 세포 증식 억제 효과 측정Test Example 2. Measurement of cytotoxicity and cell proliferation inhibition effect of breast cancer cells
세포배양cell culture
시료의 유방암 증식 및 전이 억제 효과를 통한 항암 효과 평가를 위해 유방암 세포인 MDA-MB-231을 사용하였으며 정상 세포에 대한 무독성을 확인하기 위해 인간 신장세포인 HEK-293을 사용하여 두 세포간의 생존율을 비교하였다. 세포배양 시 10% FBS와 1% penicillin-streptomycin이 첨가된 DMEM 배지를 사용하였으며 세포 배양기(Panasonic, Osaka, Japan)에서 37 ℃, 5.0% CO2를 유지하며 배양하였다. 상기 배양된 세포는 72 시간 뒤 PBS를 이용하여 세척한 후 0.05% trypsin-EDTA로 부착된 세포를 분리 및 회수하여 4.0 ℃에서 2,000 rpm으로 2 분간 원심분리하고 상등액을 제거한 다음 부착된 세포에 DMEM 배지를 첨가하여 균일하게 분산 후 5.0 X 104 cells/mL로 분주하여 계대 배양을 수행하였다.MDA-MB-231, a breast cancer cell, was used to evaluate the anti-cancer effect of the sample by inhibiting breast cancer growth and metastasis. To confirm non-toxicity to normal cells, HEK-293, a human kidney cell, was used to measure the survival rate between the two cells. compared. When culturing cells, DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin was used, and the cells were cultured in a cell incubator (Panasonic, Osaka, Japan) at 37°C and 5.0% CO 2 . The cultured cells were washed with PBS after 72 hours, and then the attached cells were separated and recovered with 0.05% trypsin-EDTA, centrifuged at 2,000 rpm for 2 minutes at 4.0°C, the supernatant was removed, and the attached cells were added to DMEM medium. After adding and dispersing evenly, subculture was performed by dispensing at 5.0
세포독성 및 세포 증식 억제 측정Measurement of cytotoxicity and cell proliferation inhibition
시료의 세포독성과 암세포 증식억제 효과 확인을 위해 MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5 diphenyl tetrazolium bromide) 분석법을 측정하였으며 유방암 세포와 정상 세포를 각 5.0 X 103 cells/mL로 96 well cell culture plate에 180 μL씩 분주하여 세포 배양기에서 24 시간 배양하였다. 시료를 DMEM 배지에 희석하여 농도별로 20 μL씩 분주하였고 대조군에는 추출물 대신 DMEM을 동량 첨가하여 48 시간 추가 배양하였다. 세포독성 실험을 위해 배지를 제거하고 0.25 mg/mL MTT 시약을 각 100 μL씩 처리하여 세포 배양기에서 3 시간 배양 후 마이크로 플레이트 리더(Infiniteⓡ200, Tecan group Co. Ltd., Zurich, Switzerland)를 이용하여 540 nm에서 흡광도를 측정하고 시료의 처리 농도에 따른 세포 생존율을 백분율로 산출하였다.To confirm the cytotoxicity of the sample and its effect on inhibiting cancer cell proliferation, the MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5 diphenyl tetrazolium bromide) assay was measured, and 5.0 At cells/mL, 180 μL was dispensed into 96 well cell culture plates and cultured in a cell incubator for 24 hours. The sample was diluted in DMEM medium and dispensed at 20 μL for each concentration, and the same amount of DMEM was added instead of the extract to the control group and cultured for an additional 48 hours. For the cytotoxicity test, remove the medium, treat with 100 μL each of 0.25 mg/mL MTT reagent, culture for 3 hours in a cell incubator, and then use a microplate reader (Infinite ⓡ 200, Tecan group Co. Ltd., Zurich, Switzerland). Absorbance was measured at 540 nm, and cell viability was calculated as a percentage according to the treatment concentration of the sample.
[수학식 1][Equation 1]
세포 생존율(cell viability, %) = [1-시료 OD/대조군 OD]*100Cell viability (%) = [1-sample OD/control OD]*100
위 표 2에 나타낸 바와 같이, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 비교예 1 내지 6의 고려엉겅퀴 추출물에 비하여 유방암세포의 세포 생존율을 현저히 감소시키는 것을 확인하였다.As shown in Table 2 above, it was confirmed that the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention significantly reduced the cell survival rate of breast cancer cells compared to the Korean thistle extracts of Comparative Examples 1 to 6.
즉, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 2 mg/ml이하, 특히 0.5 mg/mL이하에서 정상세포에서 성장 저해없이 유방암세포의 성장을 억제시킬 수 있음을 확인하였다.That is, it was confirmed that the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention can inhibit the growth of breast cancer cells without inhibiting the growth of normal cells at a concentration of 2 mg/ml or less, especially 0.5 mg/mL or less.
반면, 비교예 1, 4 내지 6의 고려엉겅퀴 추출물은 모든 농도에서 유방암세포의 세포 생존율이 높으며, 비교예 2 및 3의 고려엉겅퀴 추출물은 2 mg/mL의 농도에서 유방암세포의 세포 생존율이 낮은 편이지만 정상세포의 세포 생존율 역시 낮으므로 유방암의 개선, 예방 또는 치료용 조성물로 적합하지 않다.On the other hand, the Korean thistle extracts of Comparative Examples 1, 4 to 6 had a high cell survival rate of breast cancer cells at all concentrations, and the Korean thistle extracts of Comparative Examples 2 and 3 had a low cell survival rate of breast cancer cells at a concentration of 2 mg/mL. However, the cell survival rate of normal cells is also low, so it is not suitable as a composition for improving, preventing, or treating breast cancer.
시험예 3. 유방암세포의 이동성 평가Test Example 3. Evaluation of mobility of breast cancer cells
시료의 유방암 세포에 대한 전이 억제 효과를 확인하기 위해 추출물이 세포 이동성에 미치는 영향을 평가하였다. 유방암 세포인 MDA-MB-231를 2.5 X 105 cells/mL로 24 well cell culture plate에 분주하고 세포 배양기에서 24 시간 배양하였으며 세포가 부착됨을 확인하고 배지를 제거한 후 0.35 mL PBS를 넣고 멸균된 피펫 팁으로 부착면을 횡단하여 0.9 mm가량의 절개부를 생성하고 PBS를 제거하였다. 이후 시료를 0.0, 0.25, 0.50과 1.0 mg/mL로 배지에 혼합하여 처리하고 도립현미경(Leitz Fluovert FS, Leica, Wetzlar, Germany)을 이용해 절개면에서의 세포이동을 12 시간 간격으로 관찰하였다. 추출물 처리에 의한 MDA-MB-231의 이동능은 36시간 후 관찰된 절개 면적을 이미지 분석 프로그램인 Image J (US National Institutes of Health, Bethesda, MD, USA)로 측정하여 아래의 식에 따라 백분율로 산출하였다.To confirm the metastasis inhibitory effect on breast cancer cells in the sample, the effect of the extract on cell mobility was evaluated. Breast cancer cells MDA-MB-231 were dispensed at 2.5 An incision of approximately 0.9 mm was created by crossing the attachment surface with the tip, and the PBS was removed. Afterwards, the samples were mixed and treated with medium at concentrations of 0.0, 0.25, 0.50, and 1.0 mg/mL, and cell migration on the cut surface was observed at 12-hour intervals using an inverted microscope (Leitz Fluovert FS, Leica, Wetzlar, Germany). The migration ability of MDA-MB-231 by extract treatment was measured by measuring the incision area observed after 36 hours with Image J (US National Institutes of Health, Bethesda, MD, USA), an image analysis program, and expressed as a percentage according to the formula below. Calculated.
[수학식 2][Equation 2]
세포 이동성(%) = [1-시료 OD/대조군 OD]*100Cell mobility (%) = [1-sample OD/control OD]*100
위 표 3에 나타낸 바와 같이, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 농도가 증가함에 따라 유방암세포의 이동성이 유의하게 감소하는 것을 확인하였다.As shown in Table 3 above, it was confirmed that the mobility of breast cancer cells significantly decreased as the concentration of the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention increased.
더욱이, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 비교예 1 내지 6의 고려엉겅퀴 추출물에 비하여 유방암세포의 이동성이 현저히 감소되는 것을 확인하였다.Moreover, it was confirmed that the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention significantly reduced the mobility of breast cancer cells compared to the Korean thistle extracts of Comparative Examples 1 to 6.
또한, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 정상세포에 독성이 없다고 확인된 농도 중에서 0.5 mg/mL 처리 시 유방암세포의 이동성이 대조군에 비하여 약 1.4배 이상 저해된 것을 확인하였다.In addition, it was confirmed that the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention inhibited the mobility of breast cancer cells by about 1.4 times or more compared to the control group when treated at 0.5 mg/mL among the concentrations confirmed to be non-toxic to normal cells. .
시험예 4. 암세포 표적 mRNA 발현 Test Example 4. Cancer cell target mRNA expression
세포사멸 유도에 관련된 유전자 발현을 분석하기 위해 MDA-MB-231을 24 well plate에 1 X 106 cells/mL로 분주한 후 시료를 0.0 ~ 1.0 mg/mL가 되도록 배지와 혼합하여 처리하고 24 시간 배양하였다. 배양 후 세포에 trypsin-EDTA를 처리하고 1,800 rpm으로 5 분간 원심분리하여 세포를 회수한 후 AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea)를 이용하여 추출된 전체 RNA 농도를 NanoDrop™ 2000 spectrophotometer (ThermoFisher Scientific, Wilmington, DE, USA)로 측정하였다. 역전사는 AmfiRivert cDNA synthesis platinum master mix (GenDEPOT, Barker, TX, USA)를 이용하여 [표 4]에 따라 p53, AMPK-α, Bax 및 Bcl-2의 cDNA를 주형으로 증폭하였다. 유전자 발현량은 Gel red® nucleic acid gel stain (Komabiotech, Seoul, Korea)을 첨가한 1.5% 아가로스 겔에서 전기영동한 후 Gel Doc™ XR+ System과 Quantity One software (Bio-Rad, Hercules, CA, USA)로 측정하였다.To analyze gene expression related to apoptosis induction, MDA-MB-231 was dispensed at 1 Cultured. After incubation, the cells were treated with trypsin-EDTA and centrifuged at 1,800 rpm for 5 minutes to recover the cells. The concentration of total RNA extracted using the AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea) was measured using a NanoDrop™ 2000 spectrophotometer ( ThermoFisher Scientific, Wilmington, DE, USA). For reverse transcription, cDNA of p53, AMPK-α, Bax, and Bcl-2 was amplified as a template according to [Table 4] using AmfiRivert cDNA synthesis platinum master mix (GenDEPOT, Barker, TX, USA). Gene expression level was measured by electrophoresis on a 1.5% agarose gel supplemented with Gel red® nucleic acid gel stain (Komabiotech, Seoul, Korea) using the Gel Doc™ XR+ System and Quantity One software (Bio-Rad, Hercules, CA, USA). ) was measured.
도 2a는 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물을 농도별로 처리 시 p53, AMPK-α, Bax 및 Bcl-2의 발현을 나타낸 PCR이며; 도 2b 내지 도 2e는 상기 도 2a에서 측정한 p53, AMPK-α, Bax 및 Bcl-2의 발현량을 각각 정량화한 그래프이다.Figure 2a is a PCR showing the expression of p53, AMPK-α, Bax, and Bcl-2 when treating Korean thistle ultrasonic extract prepared according to Example 1 of the present invention at different concentrations; Figures 2b to 2e are graphs quantifying the expression levels of p53, AMPK-α, Bax, and Bcl-2 measured in Figure 2a.
도 2a 내지 도 2e에 도시된 바와 같이, 본 발명의 실시예 1에 따라 제조된 고려엉겅퀴 초음파 추출물은 농도 의존적으로 유전자의 발현량이 변화하는 것을 확인하였으며(p < 0.05), 0.5 mg/mL를 기준으로 AMPK-α, p53 및 Bax의 발현이 대조군 대비 각각 1.45배, 1.43배 및 1.68배로 증가한 반면 Bcl-2의 발현은 0.14배 감소한 것을 확인하였다.As shown in Figures 2A to 2E, the Korean thistle ultrasonic extract prepared according to Example 1 of the present invention was confirmed to change the gene expression level in a concentration-dependent manner ( p < 0.05), based on 0.5 mg/mL. It was confirmed that the expression of AMPK-α, p53, and Bax increased by 1.45-fold, 1.43-fold, and 1.68-fold, respectively, compared to the control group, while the expression of Bcl-2 decreased by 0.14-fold.
세포 내 대사과정에서 DNA 손상에 의해 p53이 p21을 활성화하여 세포주기를 정지시키거나 활성화된 AMPK-α와 세포질 내에서 결합해 암 억제 인자로 작용하며 암세포 성장에 관여하는 Bcl-2 및 Bax 등과 같은 신호분자를 억제하여 세포사멸을 유도한다고 알려져 있어 본 발명의 고려엉겅퀴 초음파 추출물이 유방암세포에서 인산화된 AMPK-α가 증가함에 따라 축적된 p53이 미토콘드리아에서 Bcl-2 활성을 억제시키고 Bax를 증가시켜 세포사멸을 유도시킨 것으로 판단된다.During intracellular metabolism, DNA damage causes p53 to activate p21 to arrest the cell cycle or bind to activated AMPK-α in the cytoplasm to act as a cancer suppressor, such as Bcl-2 and Bax , which are involved in cancer cell growth. It is known to induce cell death by suppressing signaling molecules. As the Korean thistle ultrasonic extract of the present invention increases phosphorylated AMPK-α in breast cancer cells, accumulated p53 inhibits Bcl-2 activity in mitochondria and increases Bax , thereby inhibiting cell death. It is believed that death was induced.
즉, 본 발명의 고려엉겅퀴 초음파 추출물이 p53, AMPK-α, Bax 및 Bcl-2의 상호작용에 따른 유방암 특이적 항암활성이 있는 것으로 확인된다.In other words, it was confirmed that the Korean thistle ultrasonic extract of the present invention has breast cancer-specific anticancer activity due to the interaction of p53, AMPK-α, Bax, and Bcl-2 .
아래에 본 발명의 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Below, a formulation example of a composition containing the extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.
제제예 1: 산제의 제조Formulation Example 1: Preparation of powder
실시예 1의 추출물 분말 20 mg20 mg of extract powder of Example 1
유당 100 mg100 mg lactose
탈크 10 mgTalc 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled into an airtight bubble to prepare a powder.
제제예 2: 정제의 제조Formulation Example 2: Preparation of tablets
실시예 1의 추출물 분말 10 mg10 mg of extract powder of Example 1
옥수수전분 100 mgCorn starch 100 mg
유당 100 mg100 mg lactose
스테아린산 마그네슘 2 mgMagnesium stearate 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.
제제예 3: 캡슐제의 제조Formulation Example 3: Preparation of capsules
실시예 1의 추출물 분말 10 mg10 mg of extract powder of Example 1
결정성 셀룰로오스 3 mg3 mg crystalline cellulose
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.
제제예 4: 과립제의 제조Formulation Example 4: Preparation of granules
실시예 1의 추출물 분말 1,000 mg1,000 mg of extract powder of Example 1
비타민 혼합물 적량Vitamin mixture dosage
비타민 A 아세테이트 70 ㎍Vitamin A acetate 70 μg
비타민 E 1.0 mgVitamin E 1.0 mg
비타민 B1 0.13 mgVitamin B1 0.13 mg
비타민 B2 0.15 mgVitamin B2 0.15 mg
비타민 B6 0.5 mgVitamin B6 0.5 mg
비타민 B12 0.2 ㎍Vitamin B12 0.2 ㎍
비타민 C 10 mgVitamin C 10 mg
비오틴 10 ㎍Biotin 10 μg
니코틴산아미드 1.7 mgNicotinamide 1.7 mg
엽산 50 ㎍Folic acid 50 μg
판토텐산 칼슘 0.5 mgCalcium pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture appropriate amount
황산제1철 1.75 mgFerrous sulfate 1.75 mg
산화아연 0.82 mgZinc oxide 0.82 mg
탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg
제1인산칼륨 15 mgMonopotassium phosphate 15 mg
제2인산칼슘 55 mgDibasic calcium phosphate 55 mg
구연산칼륨 90 mgPotassium citrate 90 mg
탄산칼슘 100 mgCalcium carbonate 100 mg
염화마그네슘 24.8 mgMagnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 건강기능식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강기능식품 조성물 제조에 사용할 수 있다.The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a normal health functional food manufacturing method. Next, granules can be prepared and used to manufacture a health functional food composition according to a conventional method.
제제예 5: 음료 제형의 제조Formulation Example 5: Preparation of beverage formulation
실시예 1의 추출물 분말 1,000 mg1,000 mg of extract powder of Example 1
구연산 1,000 mg1,000 mg citric acid
올리고당 100 g100 g oligosaccharides
매실농축액 2 g2 g plum concentrate
타우린 1 g1 g taurine
정제수를 가하여 전체 900 mLAdd purified water to make a total of 900 mL.
통상의 음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1 시간 동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 L 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 기능성 음료 조성물 제조에 사용한다.After mixing the above ingredients according to a typical beverage production method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, and stored in the refrigerator. Then, the solution according to the present invention was mixed. It is used in the production of functional beverage compositions.
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| KR102360622B1 (en) | 2019-01-14 | 2022-02-09 | 동국대학교 경주캠퍼스 산학협력단 | Composition comprising extracts of Inonotus obliquus for the Prevention, Treatment or Improving of Radioresistant Cancer |
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| KR102360622B1 (en) | 2019-01-14 | 2022-02-09 | 동국대학교 경주캠퍼스 산학협력단 | Composition comprising extracts of Inonotus obliquus for the Prevention, Treatment or Improving of Radioresistant Cancer |
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