KR102455690B1 - Anti-cancer composition comprising an extract of Elaeocarpus sylvestris or purified extract thereof as an active ingredient - Google Patents
Anti-cancer composition comprising an extract of Elaeocarpus sylvestris or purified extract thereof as an active ingredient Download PDFInfo
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- KR102455690B1 KR102455690B1 KR1020220043755A KR20220043755A KR102455690B1 KR 102455690 B1 KR102455690 B1 KR 102455690B1 KR 1020220043755 A KR1020220043755 A KR 1020220043755A KR 20220043755 A KR20220043755 A KR 20220043755A KR 102455690 B1 KR102455690 B1 KR 102455690B1
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- extract
- dampalsu
- cancer
- leaf
- methanol
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Abstract
Description
본 발명은 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 유효성분으로 함유하는 항암용 조성물에 관한 것이다.The present invention relates to an anticancer composition comprising an extract of Dampalsu ( Elaeocarpus sylvestris ) or a purified product thereof as an active ingredient.
암은 현재 전세계적으로 가장 많은 사망자를 내는 질병 중 하나로서, 암 발생 연령은 점차 낮아지고 있는 반면 평균 수명은 점차 연장되어가고 있어 암 발생률은 더욱 증가할 것으로 전망되고 있다.Cancer is currently one of the diseases that cause the highest number of deaths worldwide, and the cancer incidence rate is expected to further increase as the age of cancer onset is gradually decreasing while the average lifespan is gradually increasing.
대표적인 암 치료 방법으로는 항암화학요법이 있으나, 이는 암세포 뿐만 아니라 빠르게 분열증식하는 정상 세포에도 손상을 가할 수 있어 암세포에 대한 표적성이 낮고, 항암화학요법 후 빈혈, 백혈구 및 혈소판 수의 감소, 구토, 오심, 및 설사 등의 부작용이 발생할 수 있다. A typical cancer treatment method is chemotherapy, but it can damage not only cancer cells but also rapidly dividing and proliferating normal cells, so the target for cancer cells is low. , nausea, and diarrhea may occur.
한편, 대장암은 전세계적으로 성인 남녀의 암 발생률 및 사망률의 주요 원인이며, 식생활방식의 서구화로 인하여 최근 국내에서도 대장암 발병률이 급격히 증가하고 있다. 대장암은 주로 노령인구에서 발생이 증가하는 것으로 알려져 있으며 우리나라의 인구가 점차 고령화되고 음식문화가 서구화됨에 따라 사회적으로 매우 주목 받고 있는 질환으로서 이를 예방하고 치료하기 위한 방법을 찾는 연구에 대한 관심이 높아지고 있다. On the other hand, colorectal cancer is a major cause of cancer incidence and mortality in adult males and females worldwide, and the incidence of colorectal cancer is rapidly increasing in Korea due to the westernization of diet. Colorectal cancer is known to occur mainly in the elderly, and as the population of Korea gradually ages and food culture becomes westernized, there is a growing interest in research to find ways to prevent and treat it. have.
신장암(renal cell carcinoma)은 대부분 신장에서 소변을 만드는 세포들이 모여 있는 부분인 실질(수질과 피질로 구성)에서 발생하는 신장세포 암을 의미한다 신장암의 위험 인자로는 유전학적 요인이 있으나, 일반적으로는 흡연, 과도한 지방 섭취 등을 들 수 있다. 또한 장기간 투석을 받고 있는 환자에게서 종양의 발생률이 높다고 알려져 있다. 신장암은 종양의 크기가 작을 때는 증상이 거의 없으며, 종양이 어느 정도 커져서 장기를 밀어낼 정도가 되어야 비로소 증상이 나타난다. Renal cell carcinoma refers to renal cell cancer that occurs mostly in the parenchyma (composed of the medulla and cortex), which is the part of the kidney where urine-producing cells are gathered. Although there are genetic factors as risk factors for kidney cancer, Common examples include smoking and excessive fat intake. In addition, it is known that the incidence of tumors is high in patients undergoing dialysis for a long time. Kidney cancer has almost no symptoms when the size of the tumor is small, and symptoms appear only when the tumor grows to a certain extent and pushes the organs away.
신장암의 치료를 위해, 신장과 그 주위 정상 조직을 광범위하게 절제하는 수술을 하는 외과적 수술이 일반적이다. 종양이 크지 않은 경우에는 복강경을 이용하여 절제 수술을 한다. 그 외의 치료 방법으로는 면역요법, 호르몬요법, 항암 화학요법, 방사선요법 등이 있지만, 치료 효과는 크지 않은 것으로 알려져 있어, 아직까지는 외과적 수술 이외에는 확실한 효과가 있는 치료법이 없다.For the treatment of kidney cancer, a surgical operation in which the kidney and surrounding normal tissues are extensively removed is common. If the tumor is not large, laparoscopic resection is performed. Other treatment methods include immunotherapy, hormone therapy, chemotherapy, radiation therapy, etc., but the therapeutic effect is not known to be large, so there is no treatment with a definite effect other than surgical operation.
또한, 대장암과 신장암 등 여러 암종에 항암 효과를 가지는 치료제를 개발하기 위한 다수의 연구가 있었으나, 정상 세포에 독성을 가지는 등 부작용이 많았다. 이에 정상 세포에는 독성이 없으면서 암 세포에만 특이적으로 독성을 나타내는 보다 안전하면서 효과가 뛰어난 천연 항암제의 개발이 필요한 실정이다. In addition, there have been many studies to develop a therapeutic agent having an anticancer effect on various carcinomas such as colorectal cancer and kidney cancer, but there were many side effects such as toxicity to normal cells. Accordingly, there is a need to develop a safer and more effective natural anticancer agent that is non-toxic to normal cells and specifically toxic to cancer cells.
본 발명의 목적은 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 유효성분으로 함유하는 암 치료 또는 예방용 약학 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for treating or preventing cancer containing an extract or a purified product thereof as an active ingredient.
또한, 본 발명의 다른 목적은 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 유효성분으로 함유하는 암 예방 또는 개선용 식품 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a food composition for preventing or improving cancer containing an extract or a purified product thereof as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위하여, 하나의 양태로 본 발명은 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 유효성분으로 함유하는 암 치료 또는 예방용 약학 조성물을 제공한다.In order to achieve the object of the present invention as described above, in one aspect, the present invention provides a pharmaceutical composition for treating or preventing cancer containing an extract or a purified product thereof as an active ingredient.
본 발명의 용어 "담팔수(Elaeocarpus sylvestris)"는 제주도 남쪽 산기슭에서 자라는 상록교목으로 높이 20m 정도이며, 중국 및 일본의 남부를 포함한 아열대지역에서도 서식하고 있다. 효능이 거의 알려져 있지 않으므로, 연구개발의 희귀성이 매우 높은 소재라 할 수 있다. The term "dampalsu ( Elaeocarpus sylvestris )" of the present invention is an evergreen tree growing at the southern foot of Jeju Island, about 20 m in height, and also inhabits subtropical regions including southern China and Japan. Since its efficacy is hardly known, it can be said that it is a material with very high R&D rarity.
본 발명에서 상기 조성물은 담팔수를 건조 및 파쇄하여 분말화한 것을 추출에 적용할 수 있고, 담팔수의 잎, 줄기, 뿌리, 꽃 또는 이들 모두를 포함할 수 있으며, 구체적으로 담팔수의 잎 또는 줄기를 사용할 수 있다.In the present invention, the composition may be applied to extraction of dried and crushed dampalsu, and may include leaves, stems, roots, flowers, or both of dampalsu. Specifically, the leaves or stems of dampalsu may be used. can
본 발명에서 용어, "추출물"은 추출 처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 여과액, 또는 상기 추출액의 여과액의 농축액, 상기 농축액의 재여과액, 상기 추출액, 희석액, 여과액, 농축액, 또는 재여과액을 건조하여 얻어지는 건조물, 상기 추출액, 희석액, 여과액, 농축액, 재여과액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. As used herein, the term "extract" refers to an extract obtained by extraction treatment, a dilution or filtrate of the extract, or a concentrate of the filtrate of the extract, re-filtrate of the concentrate, the extract, dilution, filtrate, concentrate, Or the extract obtained by drying the re-filtrate, the extract, the diluent, the filtrate, the concentrate, the adjusted or purified product of the re-filtrate, or a mixture thereof, and the extract of all formulations that can be formed using the extract itself and the extract. include
본 발명에서 추출물의 건조는 채취한 식물로부터 유용한 성분들이 파괴되지 않는 범위에서 공지의 방법으로 진행될 수 있고, 예를 들어 음지에서 자연건조의 방법으로 진행될 수 있다. 또한, 파쇄 또는 분쇄는 이후 추출과정에서 식물의 유용한 성분들이 충분하게 추출될 수 있을 정도로 파쇄 또는 분쇄하여 분말화할 수 있다. 상기 건조와 파쇄 또는 분쇄 공정은 필요에 따라서 순서를 뒤바꿔서 진행하거나 반복하여 실시할 수 있다.Drying of the extract in the present invention may be carried out by a known method in the range in which useful components from the harvested plant are not destroyed, for example, it may be carried out by a method of natural drying in the shade. In addition, crushing or pulverization can be pulverized by crushing or pulverizing to an extent that useful components of plants can be sufficiently extracted in the subsequent extraction process. The drying, crushing, or pulverizing processes may be performed in reverse order or repeated if necessary.
본 발명의 추출에 있어서, 상기 추출하는 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다.In the extraction of the present invention, the extraction method is not particularly limited, and extraction may be performed according to a method commonly used in the art.
본 발명에서 담팔수(Elaeocarpus sylvestris) 추출물은 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합 용매로 추출하여 수득될 수 있으며, 구체적으로 담팔수 잎 또는 줄기를 물, C1-C4의 알코올 또는 이들의 혼합용매로 추출한 것일 수 있다. 보다 구체적으로 담팔수 잎 또는 줄기를 물 또는 메탄올로 추출한 것일 수 있다. 또한, 상기 저급 알코올은 에탄올 또는 메탄올 일 수 있으며, 상기 메탄올은 50~70% 메탄올일 수 있다. Dampalsu ( Elaeocarpus sylvestris ) extract in the present invention can be obtained by extraction with water, C 1 to C 4 lower alcohol or a mixed solvent thereof, specifically, Dampalsu leaves or stems with water, C 1 to C 4 alcohol or It may be extracted with these mixed solvents. More specifically, the leaves or stems of Dampalsu may be extracted with water or methanol. In addition, the lower alcohol may be ethanol or methanol, and the methanol may be 50 to 70% methanol.
본 발명에서 상기 "담팔수 추출물"은 담팔수의 추출액의 여과액을 농축하여 얻은 농축액일 수 있으며, 상기 "담팔수 정제물"은 상기 농축액을 여과하여 얻은 재여과액을 정제하여 수득한 것일 수 있다. In the present invention, the "dampalsu extract" may be a concentrate obtained by concentrating the filtrate of the extract of Dampalsu, and the "purified Dampalsu" may be obtained by purifying the re-filtrate obtained by filtering the concentrate.
본 발명에서 상기 담팔수 추출물의 정제물은 담팔수 줄기 추출물의 정제물일 수 있으며, 구체적으로, 담팔수 줄기의 메탄올 추출물의 정제물로 HPLC를 수행하는 과정에서 시료를 넣고 크로마토그램 피크가 나타날 때까지의 시료의 체류시간이 2~8분, 16~22분, 22~28분, 27~35분, 및 34~40분으로 이루어진 군에서 선택되는 하나 이상의 획분(fraction)일 수 있으며, 구체적으로 시료의 체류시간이 22~28분인 획분일 수 있다. In the present invention, the purified product of the Dampalsu extract may be a purified product of the Dampalsu stem extract, specifically, the purified water of the methanol extract of the Dampalsu stem. The retention time may be one or more fractions selected from the group consisting of 2-8 minutes, 16-22 minutes, 22-28 minutes, 27-35 minutes, and 34-40 minutes, specifically, the retention time of the sample This may be a fraction that is 22 to 28 minutes.
또한, 본 발명에서 상기 담팔수 추출물의 정제물은 담팔수 잎 추출물의 정제물일 수 있으며, 구체적으로, 담팔수 잎의 메탄올 추출물의 정제물로 HPLC를 수행하는 과정에서 시료를 넣고 크로마토그램 피크가 나타날 때까지의 시료의 체류시간이 2~5분, 18~26분, 26~33분, 및 33~40분으로 이루어진 군에서 선택되는 하나 이상의 획분(fraction)일 수 있으며, 구체적으로 시료의 체류시간이 26~33분인 획분일 수 있다. In addition, in the present invention, the purified product of the Dampalsu extract may be the purified product of the Dampalsu leaf extract, and specifically, it is a purified product of the methanol extract of Dampalsu leaf until a sample is added and a chromatogram peak appears in the process of performing HPLC. The retention time of the sample may be one or more fractions selected from the group consisting of 2-5 minutes, 18-26 minutes, 26-33 minutes, and 33-40 minutes, and specifically, the retention time of the sample is 26- It may be a fraction that is 33 minutes.
또한, 본 발명의 조성물은 담팔수 추출물 또는 이의 정제물 이외에 상기 담팔수 추출물의 분획물을 더 포함할 수 있다. In addition, the composition of the present invention may further include a fraction of the dampalsu extract in addition to the dampalsu extract or a purified product thereof.
본 발명에서 사용되는 용어, "분획물"은 여러 다양한 구성 성분들을 포함하는 혼합물로부터 특정 성분 또는 특정 성분 그룹을 분리하기 위하여 분획을 수행하여 얻어진 결과물을 의미한다.As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture including various components.
본 발명에서 상기 분획물을 얻는 분획 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 수행될 수 있다. 상기 분획 방법의 비제한적인 예로는, 담팔수(Elaeocarpus sylvestris) 추출물에 소정의 용매를 처리하여 상기 추출물로부터 분획물을 얻는 방법을 들 수 있다.The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a method of obtaining a fraction from the extract by treating the extract with a predetermined solvent ( Elaeocarpus sylvestris ).
본 발명에서 상기 분획물을 얻는 데에 사용되는 용매의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 분획 용매의 비제한적인 예로는 물, 탄소수 1 내지 4의 알코올, 헥산(Hexane), 에틸아세테이트(Ethyl acetate), 클로로포름(Chloroform), 부탄올 또는 이들의 혼합용매를 들 수 있다. The kind of solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, butanol, or a mixed solvent thereof.
또한 본 발명에서 "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다. Also, in the present invention, the term "active ingredient" means a component that can exhibit the desired activity alone or can exhibit activity together with a carrier that is not active by itself.
본 발명에서, 용어 "암"은 일반적으로 비조절된 세포 성장의 특징을 갖는 포유동물의 생리학적 상태를 나타내거나 설명한다. "암"이란 세포의 정상적인 분열, 분화 및 사멸의 조절 기능에 문제가 발생하여 비정상적으로 과다 증식하여 주위 조직 및 장기에 침윤하여 덩어리를 형성하고 기존의 구조를 파괴하거나 변형시키는 상태를 의미한다.As used herein, the term “cancer” refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. "Cancer" refers to a condition in which a problem occurs in the control function of normal division, differentiation and death of cells, so that it proliferates abnormally, invades surrounding tissues and organs, forms a mass, and destroys or deforms the existing structure.
상기 암은 본 발명에서 제공하는 담팔수 추출물 또는 이의 정제물에 의하여 증상이 완화, 경감, 개선 또는 치료될 수 있는 한 특별히 이에 제한되지 않으나, 구체적인 예로, 위암, 대장암, 소장암, 유방암, 간암, 및 신장암으로 이루어진 군에서 선택되는 하나 이상의 질환일 수 있으며, 더욱 구체적으로 신장암, 대장암 및 유방암으로 이루어진 군에서 선택될 수 있다.The cancer is not particularly limited thereto, as long as the symptoms can be alleviated, alleviated, improved or treated by the dampalsu extract or a purified product thereof provided in the present invention, but specifically, gastric cancer, colon cancer, small intestine cancer, breast cancer, liver cancer, And it may be one or more diseases selected from the group consisting of kidney cancer, more specifically, it may be selected from the group consisting of kidney cancer, colorectal cancer and breast cancer.
상기 조성물은 상기 암세포에 세포독성을 나타내어, 항암 효과를 가지나, 정상 세포에는 독성이 없거나 약하다. 특히 정상 장관계 세포, 구체적으로 인간 장(intestine) 상피 세포에는 독성이 없거나 약하다. 또한 인체 정상 피부세포, 구체적으로 인체피부섬유모세포에 독성이 없다. The composition exhibits cytotoxicity to the cancer cells and has an anticancer effect, but has no or weak toxicity to normal cells. In particular, there is little or no toxicity to normal intestinal system cells, specifically human intestinal epithelial cells. In addition, it is not toxic to normal human skin cells, specifically human dermal fibroblasts.
본 발명의 용어, "예방"이란, 상기 담팔수 추출물 또는 이의 정제물을 포함하는 조성물의 투여로 암을 억제 또는 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any act of inhibiting or delaying cancer by administering a composition comprising the dampalsu extract or a purified product thereof.
본 발명의 용어, "치료"란, 상기 담팔수 추출물 또는 이의 정제물을 조성물의 투여로 암의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any action in which the symptoms of cancer are improved or beneficially changed by administering the composition of the dampalsu extract or a purified product thereof.
본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어, "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 예를 들면, 상기 추출물 또는 분획물은 유효성분으로 1일 0.001 내지 500 mg/kg으로, 구체적으로 0.1 내지 100 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한 번 또는 수회 나누어 투여할 수도 있다. 또한, 본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 추출물 또는 분획물을 0.001 내지 50% 중량 백분율로 포함할 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. For example, the extract or fraction may be administered as an active ingredient at a dose of 0.001 to 500 mg/kg per day, specifically 0.1 to 100 mg/kg, and the administration may be administered once or several times a day. may be In addition, the pharmaceutical composition of the present invention may include the extract or fraction in an amount of 0.001 to 50% by weight based on the total weight of the composition.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.
본 발명의 암의 예방 또는 치료용 약학 조성물은 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition for preventing or treating cancer of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredients described above. The carrier, excipient and diluent include lactose, dextrose, sucrose, 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.
본 발명의 상기 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 구체적으로, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하지만, 이에 제한되는 것은 아니다. 이러한 고형제제는 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제 등도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have. Specifically, in the case of formulation, it can be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, and a surfactant commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.
본 발명의 상기 약학 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.
상기 담팔수(Elaeocarpus sylvestris) 추출물은 신장암 또는 대장암 세포를 사멸함으로써 항암 효과가 있다. 따라서, 본 발명의 담팔수 추출물 또는 이의 정제물은 암 치료 또는 예방용 약학 조성물에 매우 유용하게 이용할 수 있다.The Dampalsu ( Elaeocarpus sylvestris ) extract has an anticancer effect by killing kidney cancer or colon cancer cells. Therefore, the dampalsu extract or a purified product thereof of the present invention can be very usefully used in a pharmaceutical composition for treating or preventing cancer.
다른 하나의 양태로 본 발명은 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 유효성분으로 함유하는 암 예방 또는 개선용 식품 조성물을 제공한다.In another aspect, the present invention provides a food composition for preventing or improving cancer containing an extract or a purified product thereof as an active ingredient.
본 발명에서, 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물, 암, 예방에 대한 설명은 전술한 바와 같다. In the present invention, Dampalsu ( Elaeocarpus sylvestris ) extract or a purified product thereof, cancer, and prevention are the same as described above.
본 발명의 용어 "개선"이란 본 발명의 담팔수 추출물 또는 이의 정제물을 포함하는 조성물의 투여로 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "improvement" refers to any action that at least reduces the parameters related to the condition to be treated, for example, the severity of symptoms by administration of a composition comprising the dampalsu extract or a purified product thereof of the present invention.
본 발명의 상기 식품 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다.When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method.
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함할 수 있으며, 본 발명의 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물을 첨가할 수 있는 식품의 종류에는 별다른 제한이 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있다.The food composition of the present invention may include the form of pills, powders, granules , needles, tablets, capsules or liquids, etc. There are no restrictions. Examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, Alcoholic beverages and vitamin complexes are available.
상기 식품 조성물에는 담팔수(Elaeocarpus sylvestris) 추출물 또는 이의 정제물 이외에도 다른 성분을 추가할 수 있으며, 그 종류는 특별히 제한되지 않는다. 예를 들어, 통상의 식품과 같이 여러 가지 생약 추출물, 식품학적으로 허용되는 식품보조첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있으며, 이에 제한되지 않는다.Other ingredients may be added to the food composition in addition to the dampalsu ( Elaeocarpus sylvestris ) extract or a purified product thereof, and the type thereof is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.
본 발명에서 용어, "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to prepare food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc., but the above examples are not limited to the types of food supplement additives of the present invention.
상기 천연 탄수화물의 예는 포도당, 과당 등의 단당류; 말토스, 수크로스 등의 이당류; 및 덱스트린, 시클로덱스트린 등의 다당류와, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 있으며, 상기한 것 이외의 향미제로서 천연 향미제(타우마틴 등), 스테비아 추출물(레바우디오시드 Ａ, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에서 사용된 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, and it can be excellent in portability.
유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 유효성분은 원료 조성물 중 0.01 내지 50 중량%, 바람직하게는 0.1 내지 10 중량%의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food, the active ingredient of the present invention may be added in an amount of 0.01 to 50% by weight, preferably 0.1 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term ingestion for health and hygiene purposes or for health control, the above amount may be used below the above range.
본 발명의 담팔수 추출물 또는 이의 정제물은 암 세포의 사멸 효과를 가지므로, 항암용 조성물로 이용하다. Since the Dampalsu extract of the present invention or a purified product thereof has an apoptosis effect on cancer cells, it is used as an anticancer composition.
도 1은 실시예 1의 담팔수 줄기의 추출물 원액의 여과액을 Prep HPLC를 수행하여 얻어진 크로마토그램이다.
도 2는 실시예 3의 담팔수 잎 추출물 원액의 여과액을 Prep HPLC를 수행하여 얻어진 크로마토그램이다.
도 3은 실시예 1과 실시예 3의 담팔수 시료를 대상으로 신장암 세포주에 대한 세포 독성을 나타낸 것이다.
도 4는 실시예 2-3과 실시예 4-3의 담팔수 시료를 대상으로 신장암 세포주에 대한 세포 독성을 나타낸 것이다.
도 5는 실시예 1과 실시예 3의 담팔수 시료를 대상으로 인체피부섬유모세포 에 대한 세포 독성을 나타낸 것이다.
도 6은 실시예 5-2의 담팔수 시료를 대상으로 대장암 세포주와 정상 장관계 세포주에 대한 세포 독성을 나타낸 것이다.
도 7은 실시예 5-1과 5-2의 담팔수 시료를 대상으로 대장암 세포주에 대한 세포 독성을 나타낸 것이다.
도 8은 실시예 5-1과 5-2의 담팔수 시료를 대상으로 유방암 세포주에 대한 세포 독성을 나타낸 것이다. 1 is a chromatogram obtained by performing Prep HPLC on the filtrate of an extract stock solution of Dampalsu stem of Example 1.
2 is a chromatogram obtained by performing Prep HPLC on the filtrate of the undiluted dampalsu leaf extract of Example 3. FIG.
Figure 3 shows the cytotoxicity to the renal cancer cell line for the samples of Example 1 and Example 3 Dampalsu.
Figure 4 shows the cytotoxicity to the renal cancer cell line targeting the dampalsu samples of Examples 2-3 and 4-3.
5 shows the cytotoxicity to human dermal fibroblasts in the dampalsu samples of Examples 1 and 3;
6 shows the cytotoxicity of the dampalsu sample of Example 5-2 to colon cancer cell lines and normal intestinal system cell lines.
7 shows the cytotoxicity to colon cancer cell lines for the samples of Examples 5-1 and 5-2 in Dampalsu.
FIG. 8 shows cytotoxicity to breast cancer cell lines in Dampalsu samples of Examples 5-1 and 5-2.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 첨부한 도면을 참고로 하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.
[실시예 제조][Example Preparation]
실시예 1. 담팔수 줄기 추출물 제조Example 1. Preparation of Dampalsu Stem Extract
건조된 담팔수 줄기 1kg에 담팔수 줄기 중량의 10배(v/w)에 해당하는 60% 메탄올 10L를 투입하고 60℃에서 17시간 동안 추출하여 담팔수 줄기 추출액을 얻었다. 다음으로 상기 담팔수 줄기 추출액을 10μm 여과포를 이용하여 여과하였다. 이 후, 60℃, 0.1~0.3 MPa에서 진공감압으로 농축하여 담팔수 줄기의 메탄올 추출물 원액을 얻었다. To 1 kg of dried dampalsu stems, 10L of 60% methanol corresponding to 10 times (v/w) the weight of dampalsu stems was added and extracted at 60°C for 17 hours to obtain a dampalsu stem extract. Next, the dampalsu stem extract was filtered using a 10 μm filter cloth. Thereafter, it was concentrated under vacuum at 60° C. and 0.1 to 0.3 MPa to obtain a undiluted methanol extract of Dampalsu stem.
실시예 2. 담팔수 줄기 추출물의 정제물 제조Example 2. Preparation of purified product of Dampalsu stem extract
실시예 1에서 얻은 담팔수 줄기의 메탄올 추출물 원액을 PTFE(Polytetrafluoroethylene) Syringe Filter(0.45μm pore size, 25mm diameter)로 여과하였다. The methanol extract stock solution of Dampalsu stem obtained in Example 1 was filtered with a PTFE (Polytetrafluoroethylene) Syringe Filter (0.45 μm pore size, 25 mm diameter).
상기와 같이 여과하여 얻은 여과액 3ml을 Prep HPLC(고성능액체크로마토그래피)를 이용하여 정제하였다. 컬럼은 C18 컬럼(Medium Flash Column C18 20μm, 20mg)을 사용하였고, 이동상 용매로 물(용매 A)과 메탄올(용매 B)을 사용하였으며, 254 nm과 280nm의 파장과 유속 60mL/min (Agela Technologies, USA)의 조건에서 분석 및 검출을 실시하였다. 3 ml of the filtrate obtained by filtration as described above was purified using Prep HPLC (High Performance Liquid Chromatography). A C18 column (Medium
< Prep HPLC 조건>< Prep HPLC conditions >
컬럼: Medium Flash Column C18 20μm, 20mg Column: Medium
유속: 60 ml/minFlow rate: 60 ml/min
주입량: 3mL (담팔수 줄기 추출물 원액의 여과액)Injection amount: 3mL (filtrate of Dampalsu stem extract undiluted solution)
컬럼 온도: 상온Column temperature: room temperature
용매: 용매 A; DI Water, 용매 B; MethanolSolvent: Solvent A; DI Water, solvent B; Methanol
Detector 파장: Dual UV Detector 254nm, 280nmDetector Wavelength: Dual UV Detector 254nm, 280nm
용매 조건은 표 1과 같다. The solvent conditions are shown in Table 1.
도 1은 실시예 1의 담팔수 줄기의 추출물 원액의 여과액을 Prep HPLC를 수행하여 얻어진 크로마토그램이다. 적색(낮은) 피크는 254nm에서 분석한 결과이고, 파란색(높은) 피크는 280nm에서 분석한 결과이다. 1 is a chromatogram obtained by performing Prep HPLC on the filtrate of an extract stock solution of Dampalsu stem of Example 1. The red (low) peak is the result of analysis at 254 nm, and the blue (high) peak is the result of the analysis at 280 nm.
그 결과 도 1에서 확인되는 바와 같이 HPLC를 수행하기 위해 시료(담팔수 잎 추출물 원액의 여과액)를 넣을 때부터 해당 성분의 봉우리(chromatogram peak)가 나타나기까지의 체류 시간(retentime time) 별로, ①, ②, ③, ④, ⑤ 총 5개의 획분(fraction)에 해당하는 피크를 얻었으며, 이를 분취 (分取)하여 5개의 정제시료를 얻었다. 5개의 획분에 대한 체류 시간은 표 2와 같다.As a result, as confirmed in FIG. 1, from the time the sample (filtrate of the dampalsu leaf extract stock solution) is added to the chromatogram peak to perform HPLC, by retention time (retention time), ①, ②, ③, ④, ⑤ Peaks corresponding to a total of 5 fractions were obtained, and these were fractionated to obtain 5 purified samples. The retention times for the five fractions are shown in Table 2.
실시예 3. 담팔수 잎 추출물 제조Example 3. Preparation of Dampalsu Leaf Extract
건조된 담팔수 잎 1kg에 담팔수 잎 중량의 10배(v/w)에 해당하는 60% 메탄올 10L를 투입하고 60℃에서 17시간 동안 추출하여 담팔수 잎 추출액을 얻었다. 다음으로 상기 담팔수 잎 추출액을 10μm 여과포를 이용하여 여과하였다. 이 후, 60℃, 0.1~0.3 MPa에서 진공감압으로 농축하여 담팔수 잎의 메탄올 추출물 원액을 얻었다. To 1 kg of dried dampalsu leaves, 10L of 60% methanol corresponding to 10 times (v/w) of the weight of dampalsu leaves was added and extracted at 60° C. for 17 hours to obtain a dampalsu leaf extract. Next, the dampalsu leaf extract was filtered using a 10 μm filter cloth. Thereafter, the mixture was concentrated under vacuum at 60° C. and 0.1 to 0.3 MPa to obtain a undiluted methanol extract of Dampalsu leaf.
실시예 4. 담팔수 잎 추출물의 정제물 제조Example 4. Preparation of purified product of Dampalsu leaf extract
실시예 3에서 얻은 담팔수 잎의 메탄올 추출물 원액을 PTFE(Polytetrafluoroethylene) Syringe Filter(0.45μm pore size, 25mm diameter)로 여과하였다. The methanol extract stock solution of Dampalsu leaf obtained in Example 3 was filtered with a PTFE (Polytetrafluoroethylene) Syringe Filter (0.45 μm pore size, 25 mm diameter).
상기와 같이 여과하여 얻은 여과액 0.8ml을 Prep HPLC(High-performance liquid chromatography, 고성능액체크로마토그래피)를 이용하여 정제하였다. 컬럼은 C18 컬럼(Medium Flash Column C18 20μm, 20mg)을 사용하였고, 이동상 용매로 물(용매 A)과 메탄올(용매 B)을 사용하였으며, 254 nm과 280nm의 파장과 유속 60mL/min (Agela Technologies, USA)의 조건에서 분석 및 검출을 실시하였다. 0.8 ml of the filtrate obtained by filtration as described above was purified using Prep HPLC (High-performance liquid chromatography). A C18 column (Medium
< Prep HPLC 조건>< Prep HPLC conditions >
컬럼: Medium Flash Column C18 20μm, 20mg Column: Medium
유속: 60 ml/minFlow rate: 60 ml/min
주입량: 0.8mL (담팔수 잎 추출물 원액의 여과액)Injection amount: 0.8mL (filtrate of dampalsu leaf extract stock solution)
컬럼 온도: 상온Column temperature: room temperature
용매: 용매 A; DI Water, 용매 B; MethanolSolvent: Solvent A; DI Water, solvent B; Methanol
Detector 파장: Dual UV Detector 254nm, 280nmDetector Wavelength: Dual UV Detector 254nm, 280nm
용매 조건은 표 3과 같다. The solvent conditions are shown in Table 3.
도 2는 실시예 3의 담팔수 잎 추출물 원액의 여과액을 Prep HPLC를 수행하여 얻어진 크로마토그램이다. 적색(낮은) 피크는 254nm에서 분석한 결과이고, 파란색(높은) 피크는 280nm에서 분석한 결과이다. 2 is a chromatogram obtained by performing Prep HPLC on the filtrate of the undiluted dampalsu leaf extract of Example 3. FIG. The red (low) peak is the result of analysis at 254 nm, and the blue (high) peak is the result of the analysis at 280 nm.
그 결과 도 2에서 확인되는 바와 같이 HPLC를 수행하기 위해 시료(담팔수 잎 추출물 원액의 여과액)를 넣을 때부터 해당 성분의 봉우리(chromatogram peak)가 나타나기까지의 체류 시간(retentime time) 별로 ①, ②, ③, ④ 4개의 획분(fraction)에 해당하는 피크를 얻었으며, 이를 분취 (分取)하여 4개의 정제시료를 얻었다. 4개의 획분에 대한 체류 시간은 표 4와 같다.As a result, as shown in Figure 2, from the time the sample (the filtrate of the dampalsu leaf extract stock solution) is added to the chromatogram peak to perform HPLC, ①, ② by retention time , ③, ④ Peaks corresponding to four fractions were obtained, and these were fractionated to obtain four purified samples. The retention times for the four fractions are shown in Table 4.
실시예 5. 담팔수 열수 추출물 제조Example 5. Preparation of hot water extract from Dampalsu
5-1: 담팔수 줄기의 열수 추출물5-1: Hot water extract of Dampalsu stem
담팔수 줄기 2kg에 10배(v/w)의 증류수를 넣고 100℃에서 4시간 동안 교반하여 추출 및 여과하였다. 이 후, 대형회전증발농축기(Buchi, R-220)를 이용하여 55℃, 36~39 mmTorr에서 12시간 농축 후 건조하여 담팔수 줄기의 열수 추출물을 얻었다. 10 times (v/w) distilled water was added to 2 kg of Dampalsu stem, and the mixture was stirred at 100° C. for 4 hours, followed by extraction and filtration. Thereafter, using a large rotary evaporator (Buchi, R-220), the mixture was concentrated at 55° C., 36-39 mmTorr for 12 hours and dried to obtain a hot water extract of Dampalsu stem.
5-2: 담팔수 잎의 열수 추출물5-2: Hot water extract of Dampalsu leaf
담팔수 줄기 2kg에 10배(v/w)의 증류수를 넣고 100℃에서 4시간 동안 교반하여 추출 및 여과하였다. 이 후, 대형회전증발농축기(Buchi, R-220)를 이용하여 55℃, 36~39 mmTorr에서 12시간 농축 후 건조하여 담팔수 잎의 열수 추출물을 얻었다. 10 times (v/w) distilled water was added to 2 kg of Dampalsu stem, and stirred at 100° C. for 4 hours, followed by extraction and filtration. Thereafter, using a large rotary evaporator (Buchi, R-220), the mixture was concentrated at 55°C and 36-39 mmTorr for 12 hours and dried to obtain a hot water extract of Dampalsu leaf.
실험예 1. 신장암 세포주에 대한 세포독성 평가(항암 효과)Experimental Example 1. Evaluation of cytotoxicity against renal cancer cell lines (anticancer effect)
1-1. 세포주 및 세포독성 평가 방법1-1. Cell Lines and Cytotoxicity Assessment Methods
인간 신장암 세포주 A489과 인체피부섬유모세포 Detroit 551는 Korean Cell Line Bank (Seoul, Korea)에서 분양 받았다. 세포배양과 독성 평가를 위하여 A489, Detroit 551 세포는 MEM 배지에서 37℃, 5% CO2 조건을 유지하여 배양하였다. 각 배지에는 10% fetal bovine serum (FBS)과 항생제(100 U/mL penicillin, 0.1 mg/mL streptomycin)를 첨가하였으며, Detroit 551세포를 위한 MEM 배지에는 1% 비필수아미노산(non-essential amino acid)을 주입하였다. Human kidney cancer cell line A489 and human dermal fibroblast Detroit 551 were purchased from Korean Cell Line Bank (Seoul, Korea). For cell culture and toxicity evaluation, A489 and Detroit 551 cells were cultured in MEM medium at 37° C. and 5% CO 2 condition. 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) were added to each medium, and 1% non-essential amino acid was added to the MEM medium for Detroit 551 cells. was injected.
세포독성 평가를 위하여 96-well plate에 well당 세포수를 1.5×104이 되도록 분주하여 24~48시간 동안 배양한 후, 시료가 포함된 serum-free 배지로 교환하여 24시간 더 처리하였다. 이후 처리 배지를 제거하고, 0.5 mg/mL MTT 용액을 각 well당 100 μL 씩 첨가하여, 37℃에서 약 1~2시간 반응시켰다. 생성된 MTT formazan을 100 μL DMSO에 용해하여 microplate reader (SpectraMax i3x, Molecular Devices, CA, USA)로 550 nm에서 흡광도를 측정하여 세포 생존율을 평가하였다.For cytotoxicity evaluation, the number of cells per well was aliquoted in a 96-well plate to 1.5×10 4 and cultured for 24 to 48 hours. Thereafter, the treatment medium was removed, and 100 μL of 0.5 mg/mL MTT solution was added to each well, followed by reaction at 37° C. for about 1 to 2 hours. The resulting MTT formazan was dissolved in 100 μL DMSO and absorbance was measured at 550 nm with a microplate reader (SpectraMax i3x, Molecular Devices, CA, USA) to evaluate cell viability.
1-2. 신장암 세포주에 대한 세포독성 평가1-2. Cytotoxicity evaluation for renal cancer cell lines
상기 실시예 1의 담팔수 줄기의 메탄올 추출물 원액(stem extracts)과 실시예 3의 담팔수 잎의 메탄올 추출물 원액(Leaf extracts), 실시예 2-3의 담팔수 줄기 추출물의 정제물(stem 3), 실시예 4-3의 담팔수 잎 추출물의 정제물(leaf 3)을 신장암 세포주 A489에 처리하고 세포 독성을 평가하였다. The methanol extract of dampalsu stem extract of Example 1, the methanol extract of dampalsu leaf of Example 3, and the purified product of the dampalsu stem extract of Example 2-3 (stem 3), Example The purified product (leaf 3) of the dampalsu leaf extract of 4-3 was treated with the renal cancer cell line A489 and cytotoxicity was evaluated.
도 3은 실시예 1과 실시예 3의 담팔수 시료를 대상으로 신장암 세포주에 대한 세포 독성을 나타낸 것이다. Figure 3 shows the cytotoxicity to the renal cancer cell line for the samples of Example 1 and Example 3 Dampalsu.
그 결과, 실시예 3의 담팔수 잎 추출물 원액은 62.5 μg/mL에서 약 35%로 신장암 세포주의 생존율을 저해했으나, 실시예 1의 담팔수 줄기 추출물 원액은 62.5 μg/mL에서 세포주 성장 저해가 10%에 불과하였으며, 125 μg/mL부터 약 37%로 신장암 세포주의 생존율을 저해하였다. 따라서, 동일 농도에서 담팔수 잎 추출물 원액이 줄기 추출물 원액에 비해 신장암 세포주에 높은 세포 독성을 나타냈다. As a result, the dampalsu leaf extract stock solution of Example 3 inhibited the viability of the renal cancer cell line by about 35% at 62.5 μg/mL, but the stock solution of Dampalsu leaf extract of Example 1 showed 10% inhibition of cell line growth at 62.5 μg/mL. , and inhibited the survival rate of renal cancer cell lines from 125 μg/mL to about 37%. Therefore, at the same concentration, the undiluted dampalsu leaf extract showed higher cytotoxicity to kidney cancer cell lines than the undiluted stem extract.
도 4는 실시예 2-3과 실시예 4-3의 담팔수 시료를 대상으로 신장암 세포주에 대한 세포 독성을 나타낸 것이다. Figure 4 shows the cytotoxicity to the renal cancer cell line targeting the dampalsu samples of Examples 2-3 and 4-3.
또한, 실시예 2-3의 담팔수 줄기 추출물의 정제물의 경우, 신장암 세포에 대한 독성이 없었으며, 실시예 4-3의 담팔수 잎 추출물의 정제물의 경우 250μg/mL 에서 56.7%의 세포 생존율을 저해하였다. In addition, in the case of the purified dampalsu stem extract of Example 2-3, there was no toxicity to kidney cancer cells, and in the case of the purified dampalsu leaf extract of Example 4-3, 56.7% of cell viability was inhibited at 250 μg/mL. did.
따라서, 실시예 3의 담팔수 잎 추출물 원액과 실시예 4-3의 담팔수 잎 추출물의 정제물이 담팔수 줄기 추출물 원액과 담팔수 줄기 추출물의 정제물에 비해 신장암 세포주에 대한 높은 세포 독성을 나타내어 항암 효과가 높다는 것을 알 수 있었다. Therefore, the purified product of the dampalsu leaf extract stock solution of Example 3 and the dampalsu leaf extract of Example 4-3 showed high cytotoxicity against kidney cancer cell lines compared to the purified product of the dampalsu stem extract stock solution and the dampalsu stem extract, thereby increasing the anticancer effect. was found to be high.
1-3. 정상세포주로 인체피부섬유모세포에 대한 세포독성 평가1-3. Cytotoxicity evaluation of human skin fibroblasts with normal cell lines
상기 실시예 1의 담팔수 줄기의 메탄올 추출물 원액(stem extracts)과 실시예 3의 담팔수 잎의 메탄올 추출물 원액(Leaf extracts)을 인체피부섬유모세포(Detroit 551 cell)에 처리하고 세포 독성을 평가하였다. Detroit 551 cells were treated with the methanol extracts of the stem extracts of Dampalsu stem of Example 1 and the methanol extracts of the leaves of Dampalsu leaves of Example 3, and cytotoxicity was evaluated.
도 5는 실시예 1과 실시예 3의 담팔수 시료를 대상으로 인체피부섬유모세포 에 대한 세포 독성을 나타낸 것이다. 5 shows the cytotoxicity to human dermal fibroblasts in the dampalsu samples of Examples 1 and 3;
그 결과, 실시예 3의 담팔수 잎 추출물 원액은 인체피부섬유모세포에 대한 독성이 없었으며, 오히려 500 μg/mL에서 인체피부섬유모세포의 증식을 유도하였다. 반면, 실시예 1의 담팔수 줄기 추출물 원액은 저농도에서는 세포의 증식을 유도하였으며, 125 μg/mL에서 인체피부섬유모세포의 생존율을 저해하였다. As a result, the undiluted dampalsu leaf extract of Example 3 had no toxicity to human dermal fibroblasts, and rather induced the proliferation of human dermal fibroblasts at 500 μg/mL. On the other hand, the undiluted dampalsu stem extract of Example 1 induced cell proliferation at a low concentration, and inhibited the survival rate of human dermal fibroblasts at 125 μg/mL.
따라서, 실시예 1과 실시예 3의 담팔수 줄기와 잎 추출물 원액은 인체피부섬유모세포에 대한 독성이 거의 없는 것을 알 수 있었다.Therefore, it was found that the undiluted solution of the dampalsu stem and leaf extracts of Examples 1 and 3 had little to no toxicity to human skin fibroblasts.
실험예 2. 대장암 세포주에 대한 세포독성 평가(항암 효과)Experimental Example 2. Evaluation of cytotoxicity against colorectal cancer cell lines (anticancer effect)
2-1. 세포주 및 세포독성 평가 방법2-1. Cell Lines and Cytotoxicity Assessment Methods
인간 대장암 세포주 HT-29, 정상 장관계 세포주 INT-407는 American Type Culture Collection (Manassas, VA, USA)에서 분양 받았다. 세포배양과 독성 평가를 위하여 HT-29과 INT-407 세포 각각 DMEM 및 MEM 배지에서 37℃, 5% CO2 조건을 유지하여 배양하였다. 각 배지에는 10% fetal bovine serum (FBS)과 항생제(100 U/mL penicillin, 0.1 mg/mL streptomycin)를 첨가하였으며, INT-407 세포를 위한 MEM 배지에는 1% 비필수 아미노산(non-essential amino acid)을 주입하였다. Human colorectal cancer cell line HT-29 and normal intestinal system cell line INT-407 were purchased from the American Type Culture Collection (Manassas, VA, USA). For cell culture and toxicity evaluation, HT-29 and INT-407 cells were cultured in DMEM and MEM media, respectively, at 37°C and 5% CO 2 conditions. 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) were added to each medium, and 1% non-essential amino acid (non-essential amino acid) was added to the MEM medium for INT-407 cells. ) was injected.
세포독성 평가를 위하여 HT-29와 INT-407 세포를 96-well plate에 well당 1.5Х104이 되도록 분주하여 24~48시간 동안 배양한 후, 시료가 포함된 serum-free 배지로 교환하여 24시간 더 처리하였다. 이 후 처리 배지를 제거하고, 0.5 mg/mL MTT 용액을 각 well당 100 μL 씩 첨가하여, 37℃에서 약 1~2시간 반응시켰다. 생성된 MTT formazan을 100 μL DMSO에 용해하여 microplate reader로 550 nm에서 흡광도를 측정하여 세포 생존율을 평가하였다.For cytotoxicity evaluation, HT-29 and INT-407 cells were aliquoted in a 96-well plate at a volume of 1.5Х104 per well, incubated for 24 to 48 hours, and then exchanged with a serum-free medium containing the sample for 24 hours. further processed. After that, the treatment medium was removed, and 100 μL of 0.5 mg/mL MTT solution was added to each well, followed by reaction at 37° C. for about 1 to 2 hours. Cell viability was evaluated by dissolving the generated MTT formazan in 100 μL DMSO and measuring the absorbance at 550 nm with a microplate reader.
2-2. 대장암 세포주와 정상 장관계 세포주에 대한 세포독성 평가2-2. Cytotoxicity evaluation for colorectal cancer cell lines and normal intestinal system cell lines
상기 실시예 5-2의 담팔수 잎의 열수 추출물을 이용하여 대장암 세포주와 정상 장관계 세포주에 대한 세포독성을 평가하였다. Cytotoxicity to colon cancer cell lines and normal intestinal system cell lines was evaluated using the hot water extract of Dampalsu leaf of Example 5-2.
도 6은 실시예 5-2의 담팔수 잎의 열수 추출물 시료를 대상으로 대장암 세포주와 정상 장관계 세포주에 대한 세포 독성을 나타낸 것이다. 6 shows the cytotoxicity to colon cancer cell lines and normal intestinal system cell lines for the hot water extract sample of Dampalsu leaf of Example 5-2.
실시예 5-2의 담팔수 잎의 열수 추출물은 정상 장관계 세포주(INT-407)에 비해, 대장암 세포주(HT-29)의 세포 생존율을 저해하였다. 따라서, 실시예 5-2의 담팔수 잎의 열수 추출물은 정상 장관계 세포주(인간 장 상피 세포, INT-407)에 대한 독성은 거의 없고 대장암 세포주에 대한 세포 독성을 보여 항암 효과를 가지는 것을 알 수 있었다.The hot water extract of Dampalsu leaf of Example 5-2 inhibited the cell viability of the colon cancer cell line (HT-29) compared to the normal intestinal system cell line (INT-407). Therefore, it can be seen that the hot water extract of Dampalsu leaf of Example 5-2 has little toxicity to normal intestinal system cell lines (human intestinal epithelial cells, INT-407) and cytotoxicity to colorectal cancer cell lines, indicating that it has an anticancer effect. there was.
2-3. 대장암 세포주에 대한 세포독성 평가2-3. Cytotoxicity evaluation for colorectal cancer cell lines
상기 실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물을 이용하여 대장암 세포주에 대한 세포독성을 평가하였다. Cytotoxicity to colon cancer cell lines was evaluated using the hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2.
도 7은 실시예 5-1과 실시예 5-2의 담팔수 열수 추출물 시료를 대상으로 대장암 세포주에 대한 세포 독성을 나타낸 것이다. 7 is a graph showing the cytotoxicity to colorectal cancer cell lines of the hot water extract samples of Examples 5-1 and 5-2.
실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물은 3.125~12.5 μg/Ml의 낮은 농도에서도 대장암 세포주(HT-29)의 세포 생존율을 저해하였다. 따라서, 실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물 모두 대장암 세포주에 대한 세포 독성을 보여 항암 효과를 가지는 것을 알 수 있었다.The hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2 inhibited the cell viability of the colon cancer cell line (HT-29) even at a low concentration of 3.125 to 12.5 μg/Ml. Therefore, both the hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2 showed cytotoxicity to colon cancer cell lines, thereby confirming that they had anticancer effects.
실험예 3. 유방암 세포주에 대한 세포독성 평가(항암 효과)Experimental Example 3. Cytotoxicity evaluation for breast cancer cell lines (anticancer effect)
3-1. 세포주 및 세포독성 평가 방법3-1. Cell Lines and Cytotoxicity Assessment Methods
인간 유방암 세포주 MCF-7은 American Type Culture Collection (Manassas, VA, USA)에서 분양받았다. 세포배양과 독성 평가를 위하여 MCF-7 세포는 DMEM 배지에서 37℃, 5% CO2 조건을 유지하여 배양하였다. 각 배지에는 10% fetal bovine serum (FBS)과 항생제(100 U/mL penicillin, 0.1 mg/mL streptomycin)를 첨가하였다.The human breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (Manassas, VA, USA). For cell culture and toxicity evaluation, MCF-7 cells were cultured in DMEM medium at 37° C. and 5% CO 2 condition. 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) were added to each medium.
세포독성 평가를 위하여 96-well plate에 well당 세포수를 1.5×104이 되도록 분주하여 24~48시간 동안 배양한 후, 시료가 포함된 serum-free 배지로 교환하여 24시간 더 처리하였다. 이후 처리 배지를 제거하고, 0.5 mg/mL MTT 용액을 각 well당 100 μL 씩 첨가하여, 37℃에서 약 1~2시간 반응시켰다. 생성된 MTT formazan을 100 μL DMSO에 용해하여 microplate reader (SpectraMax i3x, Molecular Devices, CA, USA)로 550 nm에서 흡광도를 측정하여 세포 생존율을 평가하였다.For cytotoxicity evaluation, the number of cells per well was aliquoted in a 96-well plate to 1.5×10 4 and cultured for 24 to 48 hours. Thereafter, the treatment medium was removed, and 100 μL of 0.5 mg/mL MTT solution was added to each well, followed by reaction at 37° C. for about 1 to 2 hours. The resulting MTT formazan was dissolved in 100 μL DMSO and absorbance was measured at 550 nm with a microplate reader (SpectraMax i3x, Molecular Devices, CA, USA) to evaluate cell viability.
3-2. 유방암 세포주에 대한 세포독성 평가3-2. Cytotoxicity evaluation for breast cancer cell lines
상기 실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물을 이용하여 유방암 세포주에 대한 세포독성을 평가하였다. Cytotoxicity to breast cancer cell lines was evaluated using the hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2.
도 8은 실시예 5-1과 실시예 5-2의 담팔수 열수 추출물 시료를 대상으로 유방암 세포주에 대한 세포 독성을 나타낸 것이다. FIG. 8 shows cytotoxicity to breast cancer cell lines in Dampalsu hot water extract samples of Examples 5-1 and 5-2.
실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물은 10~25 μg/mL의 농도에서 유방암 세포주(MCF-7)의 세포 생존율을 저해하였다. 따라서, 실시예 5-1의 담팔수 줄기의 열수 추출물과 실시예 5-2의 담팔수 잎의 열수 추출물 모두 유방암 세포주에 대한 세포 독성을 보여 항암 효과를 가지는 것을 알 수 있었다.The hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2 inhibited the cell viability of the breast cancer cell line (MCF-7) at a concentration of 10 to 25 μg/mL. Therefore, both the hot water extract of Dampalsu stem of Example 5-1 and the hot water extract of Dampalsu leaf of Example 5-2 showed cytotoxicity against breast cancer cell lines, thereby confirming that they had an anticancer effect.
결론적으로, 담팔수의 잎 또는 줄기로부터 유래한 추출물 또는 이의 정제물은 정상 세포에는 세포독성이 거의 없거나 오히려 증식을 유도하면서, 신장암, 유방암 그리고 대장암 특이적으로 사멸을 유도하는 것을 알 수 있었다. In conclusion, it was found that the extract or the purified product derived from the leaf or stem of Dampalsu had little or no cytotoxicity to normal cells, or rather induces proliferation, and specifically induces apoptosis in kidney cancer, breast cancer, and colorectal cancer.
이상에서 본 발명의 바람직한 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다.Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the
Claims (9)
상기 정제물은 담팔수 잎의 메탄올 추출물을 대상으로 C18 컬럼(Medium Flash Column C18) 및 이동상 용매로 물과 메탄올을 사용하여 Prep HPLC를 실시하여 시료의 체류시간이 26~33분에서 얻은 획분인 것인,
신장암 치료 또는 예방용 약학 조성물.Contains the purified water of Dampalsu ( Elaeocarpus sylvestris ) extract as an active ingredient,
The purified product is a fraction obtained by performing Prep HPLC on a methanol extract of Dampalsu leaf using a C18 column (Medium Flash Column C18) and water and methanol as a mobile phase solvent, and the retention time of the sample is 26 to 33 minutes. ,
A pharmaceutical composition for treating or preventing kidney cancer.
상기 정제물은 담팔수 잎의 메탄올 추출물을 대상으로 C18 컬럼(Medium Flash Column C18) 및 이동상 용매로 물과 메탄올을 사용하여 Prep HPLC를 실시하여 시료의 체류시간이 26~33분에서 얻은 획분인 것인,
신장암 예방 또는 개선용 식품 조성물.Contains the purified water of Dampalsu ( Elaeocarpus sylvestris ) extract as an active ingredient,
The purified product is a fraction obtained by performing Prep HPLC on a methanol extract of Dampalsu leaf using a C18 column (Medium Flash Column C18) and water and methanol as a mobile phase solvent, and the retention time of the sample is 26 to 33 minutes. ,
A food composition for preventing or improving kidney cancer.
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| KR20100001756A (en) | 2008-06-27 | 2010-01-06 | 아주대학교산학협력단 | Anti-cancer composition comprising aquatic microbial extract |
| KR101834872B1 (en) * | 2016-09-09 | 2018-04-13 | 주식회사 제넨셀 | Pharmaceutical composition for prevention or treatment of alpha-type herpes virus infection comprising the extract of Elaeocarpus sylvestris or fraction thereof as an active ingredient |
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| KR20100001756A (en) | 2008-06-27 | 2010-01-06 | 아주대학교산학협력단 | Anti-cancer composition comprising aquatic microbial extract |
| KR101834872B1 (en) * | 2016-09-09 | 2018-04-13 | 주식회사 제넨셀 | Pharmaceutical composition for prevention or treatment of alpha-type herpes virus infection comprising the extract of Elaeocarpus sylvestris or fraction thereof as an active ingredient |
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