KR101745504B1 - Method for Isolating of Vomifoliol and Stigmasterol Having Anti-cancer Activity from Silkworm Feces - Google Patents
Method for Isolating of Vomifoliol and Stigmasterol Having Anti-cancer Activity from Silkworm Feces Download PDFInfo
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- KR101745504B1 KR101745504B1 KR1020140137441A KR20140137441A KR101745504B1 KR 101745504 B1 KR101745504 B1 KR 101745504B1 KR 1020140137441 A KR1020140137441 A KR 1020140137441A KR 20140137441 A KR20140137441 A KR 20140137441A KR 101745504 B1 KR101745504 B1 KR 101745504B1
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Abstract
본 발명은 항암 활성을 갖는 잠분 추출물을 함유하는 항암 조성물에 관한 것으로, 더욱 상세하게는 잠분 추출물, 잠분 추출물을 분획하여 얻은 잠분 분획물, 잠분 분획물로부터 분리되어 수득되는 화합물을 포함하는 항암 조성물에 관한 것이다.
본 발명에 따른 잠분 추출물, 이를 분획하여 얻은 잠분 분획물, 또는 이로부터 분리되어 수득되는 화합물인 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)은 우수한 암 세포 증식 억제능을 나타내므로, 천연물 유래의 신규 항암제로 이용이 가능하다.The present invention relates to an anticancer composition containing a subcutaneous extract having anticancer activity, more particularly to a subcutaneous extract, a subcutaneous fraction obtained by fractionation of a subcutaneous extract, and a compound obtained by separation from a subcutaneous fraction .
The vaginal extract according to the present invention, the subcutaneous fraction obtained by fractionation thereof, or the compounds obtained therefrom, vomifoliol and stigmasterol exhibit excellent cancer cell proliferation inhibitory ability, and thus can be used as a novel anticancer agent derived from natural products It is available.
Description
본 발명은 항암 활성을 갖는 잠분 추출물을 함유하는 항암 조성물에 관한 것으로, 더욱 상세하게는 잠분 추출물, 잠분 추출물을 분획하여 얻은 잠분 분획물, 잠분 분획물로부터 분리되어 수득되는 화합물을 포함하는 항암 조성물에 관한 것이다.
The present invention relates to an anticancer composition containing a subcutaneous extract having anticancer activity, more particularly to a subcutaneous extract, a subcutaneous fraction obtained by fractionation of a subcutaneous extract, and a compound obtained by separation from a subcutaneous fraction .
암은 음식물, 화학적 오염물 등의 환경요인에 의하여 유전자에 돌연변이가 일어났을 때 발생된다. 이들 돌연변이는 조절 불능의 세포증식을 초래하고, 종양 (tumor) 이라는 세포 덩어리를 형성시키며, 그들 중 일부가 다른 조직으로 침투하여 악성종양 (malignant tumor)을 만드는 전이 (metastasis)를 일으켜 주위의 정상조직까지 파괴시켜 개체의 생명을 위협할 수 있는 질환 군을 총칭한다.Cancer occurs when mutations occur in genes due to environmental factors such as food, chemical contaminants, and the like. These mutations lead to uncontrolled cell proliferation, the formation of cell masses called tumors, and some of them penetrate into other tissues to cause metastasis, which makes malignant tumors, , Which is a group of diseases that can destroy the life of an individual.
최근 세계보건기구의 통계자료에 의하면 전 세계적으로 암환자가 연간 1,000만 명 이상이 발생하며, 미국의 경우 1,300,000명의 새로운 환자가 발생하고 약 550,000명이 암으로 사망하는 것으로 추정된다고 한다. 우리나라의 경우에도 매년 암환자 수가 꾸준하게 늘고 있으며 질병으로 인한 사망 원인 중 가장 높은 비율을 차지한다. 또한 각종 암의 발생률이나 그 양상 또한 선진국형으로 점차 바뀌고 있다. According to the latest statistics from the World Health Organization, more than 10 million cancer cases occur annually worldwide, and about 1,300,000 new cases occur in the United States and about 550,000 people die from cancer. Even in Korea, the number of cancer patients is steadily increasing every year, and it accounts for the highest percentage of deaths caused by diseases. In addition, the incidence and patterns of various cancers are gradually changing to advanced countries.
특히, 폐암은 일부 정상세포들이 비정상적으로 성장해서 통제가 불가할 때 발생되는데, 폐암 세포들은 폐에서 분리된 후 혈관을 통해 다른 부위들로 침입하게 된다. 폐암은 가장 흔히 진단되는 암일 뿐 만 아니라 남성들의 암 사망률이 가장 높은 암 중 하나이며, 여성들의 경우 4번째로 흔하게 진단되는 질병으로 전 세계적으로 사망률이 2번째로 높은 질병이다(Jemal, A., Bray, et al., Global cancer statics. CA Cancer J. Clin., 61: 69~90, 2011). In particular, lung cancer occurs when some normal cells grow abnormally and can not be controlled. Lung cancer cells are separated from the lungs and then invade other parts through the blood vessels. Lung cancer is not only the most commonly diagnosed cancer, it is also one of the highest cancer deaths among men, and the fourth most commonly diagnosed disease in women worldwide (Jemal, A., Bray, et al., Global cancer statics. CA Cancer J. Clin., 61: 69-90, 2011).
한국의 경우, 지난 수세기 동안 대다수 서부 국가들 보다 폐암 발병율 및 사망률이 훨씬 더 낮았으나, 흡연인구의 증가, 간접흡연, 실내 공기 오염 및 미세먼지와 같은 다양한 위해요인들로 인해 폐암 발병률 및 사망률이 증가하고 있는 추세이다. In Korea, the incidence and mortality rates of lung cancer are much lower than those of most western countries in the past centuries, but the incidence and mortality rates of lung cancer have increased due to various risk factors such as increased smoking, secondhand smoke, indoor air pollution and fine dust. .
폐암 치료는 기본적으로 방사선 치료, 화학요법 및 외과적 수술 등으로 이뤄지고 있다. 화학요법은 가장 효과적인 방법이지만, 약제학적 내성이 생기거나 기타 부작용을 일으키기도 한다. 따라서 이러한 기존 화학제들을 대체할만한 치료제의 개발이 필요하다.
Treatment of lung cancer is fundamentally based on radiotherapy, chemotherapy, and surgical surgery. Chemotherapy is the most effective method, but it may cause pharmacological resistance or other side effects. Therefore, it is necessary to develop therapeutic agents that replace these existing chemicals.
한편, 천연물은 다양한 생리활성 물질들을 함유하고 있고, 상대적으로 안전하며, 그 작용점이 복합적이고 신규해서 내성 발생을 지연시키거나 감소시키는 기능을 보이므로 현재 사용되고 있는 합성 항암제들의 문제를 극복할 수 있는 가장 이상적인 대안 방안으로 대두되고 있다(Raskin, I., et al., Trends Biotechnol., 20: 522~531, 2002; Jassim, S.A. and Naji, M.A. J. Appl. Microbiol., 95: 412~427, 2003). On the other hand, natural products contain a variety of physiologically active substances, are relatively safe, and their action points are complex and novel. Thus, they exhibit a function of delaying or reducing the generation of tolerance. Therefore, natural products It has emerged as an ideal alternative methods (Raskin, I., et al, Trends Biotechnol, 20:.... 522 ~ 531, 2002; Jassim, SA and Naji, MA J. Appl Microbiol, 95: 412 ~ 427, 2003 ).
누에는 대개 소비한 뽕 잎 중 60% 가량을 소화시키지 못한 채 배출하므로 누에 배설물인 잠분에는 뽕 잎 성분을 비롯해 누에 장내에서 효소나 미생물들로 인해 변형된 다양한 물질들도 함유하고 있다. Since silkworms generally discharge 60% of the consumed mulberry leaves without digesting it, the silkworm, which is silkworm excrement, contains various materials modified by enzymes and microorganisms in the silkworm, including mulberry leaves.
동의보감에서 잠분은 무독하여 내장기관을 강화시키고, 당뇨 치료용으로 이용될 수 있고, 신농본초경에서 잠분은 숙취 및 마비증상을 치료하는데 사용된다고 기재되어있다. It is said that the immune system is used to strengthen the internal organs, to be used for the treatment of diabetes, and to prevent the hangover and paralysis symptoms in the HbMcN.
또한, 최근에는 누에나방[Bombyx mori (L.) (Lepidoptera: Bombycidae)]의 배설물이, 아토피 억제, 항염증, 항산화 등의 활성이 있다고 알려졌다(Park, J.H. et al., J. Korean Soc. Appl. Biol. Chem., 53: 734~739, 2010; Costa-Neto, E.M., J. Ethnobiol., 25: 93~114, 2005). Recently, feces of Bombyx mori (L.) (Lepidoptera: Bombycidae) have been reported to have activities such as atopy suppression, anti-inflammation and antioxidant (Park, JH et al., J. Korean Soc. Appl Biol. Chem., 53: 734-739 , 2010; Costa-Neto, EM, J. Ethnobiol., 25: 93-114 , 2005).
잠분에서 분리된 성분들로는 β-carotene, 1-deoxynojirimycin, fagomine, 3-epifagomine 등을 비롯한 지질류(corchorifatty acid E methyl ester, (8E)-10-hydroxydec-8-enoic acid methyl ester) 등이 알려졌으며, 잠분의 80% 메탄올 추출물들에서 유래한 리그난들이 항염증 활성을 갖는다는 것이 보고되었다(Park, J.H. et al., J. Korean Soc. Appl. Biol. Chem., 53: 734~739, 2010).(8E) -10-hydroxydec-8-enoic acid methyl ester), including β- carotene, 1-deoxynojirimycin, fagomine and 3-epifagomine (Park, JH et al., J. Korean Soc. Appl. Biol. Chem., 53: 734-739, 2010), and that lignans derived from 80% .
그러나, 누에나방 배설물인 잠분으로부터 항암 활성을 갖는 화합물을 분리하고, 그 작용기전이 어떻게 되는지에 대한 연구는 아직 이루어지지 않았다.
However, no study has yet been made on how to isolate compounds having anticancer activity from the silkworm moth, which is a silkworm moth, and how its mechanism is affected.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 예의 노력한 결과, 잠분 추출물 및 이로부터 분리된 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)이 우수한 항암활성을 갖는다는 것을 확인하고, 본 발명을 완성하게 되었다.
As a result, the inventors of the present invention have made efforts to solve the above problems, and as a result, it has been confirmed that vigorous extracts, vomifoliol and stigmasterol isolated therefrom have excellent anticancer activities, and thus completed the present invention .
본 발명의 목적은 천연물인 누에나방 배설물(잠분)로부터 분리된 항암 활성이 우수한 추출물 및 화합물을 포함하는 항암 조성물을 제공하는데 있다.
It is an object of the present invention to provide an anticancer composition comprising an extract and a compound having excellent anticancer activity isolated from a natural product, silkworm mite (excipient).
상기 목적을 달성하기 위하여, 본 발명은 항암 활성을 갖는 잠분 추출물을 함유하는 항암 조성물을 제공한다.In order to accomplish the above object, the present invention provides an anticancer composition containing a subcutaneous extract having anticancer activity.
본 발명에 있어서, 상기 잠분 추출물은 마쇄된 잠분을 유기용매로 추출하여 얻어진 추출물인 것을 특징으로 한다.In the present invention, the secretion extract is an extract obtained by extracting the crushed secretion with an organic solvent.
본 발명에 있어서, 상기 잠분 추출물은 에탄올에 마쇄된 잠분을 침지시켜 추출하고 여과한 후, 여과된 여액을 감압 농축하여 얻어진 에탄올 추출물인 것을 특징으로 한다.In the present invention, the drowning extract is an ethanol extract obtained by dipping undissolved impurities in ethanol, filtering and then filtering the filtrate under reduced pressure.
본 발명은 또한, 상기 잠분 추출물로부터 분획하여 얻은 잠분 분획물을 유효성분으로 함유하는 것을 특징으로 하는 항암 조성물을 제공한다.The present invention also provides an anticancer composition, which comprises, as an active ingredient, a subcutaneous fraction obtained by fractionation from said subcutaneous extract.
본 발명에 있어서, 상기 잠분 분획물은 잠분 추출물에 순차적으로 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 분획시켜 얻은 것을 특징으로 한다.In the present invention, the subcutaneous fraction is characterized by being obtained by fractionating the subcutaneous extract sequentially with hexane, chloroform, ethyl acetate, butanol and water.
본 발명은 또한, 상기 잠분 분획물로부터 분리되어 수득되는 화합물을 유효성분으로 함유하는 항암 조성물을 제공한다.The present invention also provides an anticancer composition comprising, as an active ingredient, a compound obtained by separation from the above-mentioned subcutaneous fraction.
본 발명에 있어서, 상기 화합물은 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)로 구성된 군으로부터 선택되는 1 이상인 것을 특징으로 한다.In the present invention, the compound is characterized by being at least one selected from the group consisting of bomipolol and stigmasterol.
본 발명에 있어서, 상기 보미폴리올(vomifoliol)은 잠분 클로로포름 분획물을 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 실리카겔 컬럼 크로마토그래피로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 이를 다시 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 중압용 크로마토그래피(MPLC)로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 메탄올과 물의 혼합액을 이동상으로 하여 분취용 고성능액체크로마토그래피(HPLC)로 분리하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 화합물을 분리 및 정제하여 획득한 것을 특징으로 한다.In the present invention, the vomifoliol fraction is fractionated by silica gel column chromatography while sequentially adding methanol to the chloroform mobile phase. The fraction having the highest activity is isolated by confirming the inhibition of cancer cell growth, The fraction with the highest activity was isolated by confirming the inhibition of cancer cell growth, and then the mixture of methanol and water was used as a mobile phase, and the resulting mixture was subjected to preparative high performance liquid chromatography (HPLC), followed by isolation and purification of the compound having the highest activity through confirmation of inhibition of cancer cell growth.
본 발명에 있어서, 상기 스티그마스테롤(stigmasterol)은 잠분 클로로포름 분획물을 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 실리카겔 컬럼 크로마토그래피로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 이를 다시 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 중압용 크로마토그래피(MPLC)로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 메탄올과 물의 혼합액을 이동상으로 하여 역상 중압용 크로마토그래피(Reverse MPLC)로 분리하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 화합물을 분리 및 정제하여 획득한 것을 특징으로 한다.In the present invention, the stigmasterol fraction is fractionated by silica gel column chromatography while sequentially adding methanol to the chloroform mobile phase. The fraction having the highest activity is isolated by confirming the inhibition of cancer cell growth, Then, methanol was sequentially added to the mobile phase of chloroform, and the fractions were fractionated by MPLC, and the fraction having the highest activity was isolated by confirming the inhibition of growth of cancer cells. Then, the mobile phase was subjected to reverse phase medium pressure chromatography (Reverse MPLC), and the compound having the highest activity was isolated and purified by confirming inhibition of cancer cell growth.
본 발명에 있어서, 상기 중압용 크로마토그래피(MPLC)를 통한 분획 및 암세포 성장 억제 확인을 통한 활성이 가장 높은 분획물을 분리하는 단계는 1~6회 수행하는 것을 특징으로 한다.In the present invention, the step of isolating the fraction having the highest activity through fractionation through MPLC and inhibition of cancer cell growth is performed 1 to 6 times.
본 발명에 있어서, 상기 암은 폐암, 위암, 전립선암, 자궁경부암, 대장암, 유방암, 간암, 식도암 및 난소암으로 구성된 군으로부터 선택되는 것을 특징으로 한다.In the present invention, the cancer is characterized in that the cancer is selected from the group consisting of lung cancer, stomach cancer, prostate cancer, cervical cancer, colon cancer, breast cancer, liver cancer, esophageal cancer and ovarian cancer.
본 발명에 있어서, 상기 항암 조성물은 안티 에이팝토시스 Akt1 및 Bcl-2 유전자의 발현을 억제시켜, 항암 활성을 갖는 것을 특징으로 한다.
In the present invention, the anticancer composition inhibits the expression of anti -apoptotic Akt1 and Bcl-2 genes and is characterized by having anticancer activity.
본 발명에 따른 잠분 추출물, 이를 분획하여 얻은 잠분 분획물, 또는 이로부터 분리되어 수득되는 화합물인 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)은 우수한 암 세포 증식 억제능을 나타내므로, 천연물 유래의 신규 항암제로 이용이 가능하다.
The vaginal extract according to the present invention, the subcutaneous fraction obtained by fractionation thereof, or the compounds obtained therefrom, vomifoliol and stigmasterol exhibit excellent cancer cell proliferation inhibitory ability, and thus can be used as a novel anticancer agent derived from natural products It is available.
도 1은 본 발명에 따른 잠분 유래 용매 분획물의 분리 공정도이다.
도 2는 본 발명에 따른 잠분 분획물로부터 항암 활성 화합물을 분리하는 과정을 나타낸 도면이다.
도 3 및 4는 본 발명의 일 실시예에 따라 분리된 항암 활성 화합물의 HPLC 크로마토그램이다.
도 5는 본 발명의 일 실시예에 따라 분리된 활성본체 1의 질량 스펙트럼이다(EI-MS).
도 6은 본 발명의 일 실시예에 따라 분리된 활성본체 1의 DEPT 스펙트럼이다.
도 7은 본 발명의 일 실시예에 따라 분리된 활성본체 2의 질량 스펙트럼이다(EI-MS).
도 8은 본 발명의 일 실시예에 따라 분리된 활성본체 2의 DEPT 스펙트럼이다.
도 9는 본 발명의 일 실시예에 따라 분리된 활성본체의 A549 세포내 Akt1 mRNA 발현 억제능을 확인한 그래프이다.
도 10은 본 발명의 일 실시예에 따라 분리된 활성본체의 A549 세포내 Bcl-2 mRNA 발현 억제능을 확인한 그래프이다.Fig. 1 is a process diagram showing the separation of the fraction derived from the solvent according to the present invention.
FIG. 2 is a view showing a process of separating an anticancer active compound from a subcutaneous fraction according to the present invention.
Figures 3 and 4 are HPLC chromatograms of isolated anti-cancer active compounds according to one embodiment of the present invention.
5 is a mass spectrum (EI-MS) of
Figure 6 is a DEPT spectrum of
Figure 7 is a mass spectrum of the
Figure 8 is a DEPT spectrum of
FIG. 9 is a graph showing the inhibitory activity of Akt1 mRNA expression in A549 cells of an isolated active body according to an embodiment of the present invention.
FIG. 10 is a graph showing the inhibitory activity of Bcl-2 mRNA expression in A549 cells of an active body isolated according to an embodiment of the present invention.
본 발명에서는 부작용 위험이 낮으면서, 효과가 우수한 천연물 유래의 항암 활성물질을 개발하고자 하였다. The present invention aims to develop an anticancer active material derived from a natural material having a low risk of side effects and having excellent effects.
본 발명에서는, 누에나방(Bombyx mori)이 뽕 잎을 먹고 배출한 배설물인 잠분 추출물이 항암 활성을 가지고 있다는 것을 확인하였다.In the present invention, it was confirmed that the subcutaneous extract of Bombyx mori , which is a fecal matter produced by eating the mulberry leaves, has anticancer activity.
즉, 본 발명의 일 실시예에서는 유기용매로 잠분을 추출하였고, 추출된 잠분 추출물이 폐암 세포주(A549 및 NCI-H727), 위암 세포주(AGS), 전립선암 세포주(PC-3 및 SNU-213), 자궁경부 암 세포주(Hela), 대장암 세포주(HT-29), 유방암 세포주(MCF-7), 간암 세포주(SK-Hep-1), 식도암 세포주(Hep-2) 및 난소암 세포주(SK-OV-3)에서 강한 세포증식 억제활성을 갖는 것을 확인할 수 있었다.(A549 and NCI-H727), a gastric cancer cell line (AGS), a prostate cancer cell line (PC-3 and SNU-213), and the like. SK-Hep-1, an oesophageal cancer cell line (Hep-2) and an ovarian cancer cell line (SK-Hep-1) OV-3), indicating that it has strong cell proliferation inhibitory activity.
따라서, 본 발명은 일 관점에서, 항암 활성을 갖는 잠분 추출물을 함유하는 항암 조성물에 관한 것이다.Accordingly, the present invention relates, in one aspect, to an anticancer composition containing a subcutaneous extract having anticancer activity.
상기 잠분은 누에 배설물로서, 통상의 누에나방과에 속하는 누에나방의 배설물이라면 특별한 제한없이 이용할 수 있다.The excrement may be used as silkworm excrement without any particular limitation as long as it is an excrement of the silkworm moth belonging to the common silkworm moth.
본 발명에 있어서, 상기 잠분 추출물은 잠분을 마쇄시킨 후, 유기용매 또는 물로 추출하여 얻을 수 있다. 상기 유기 용매로는 에탄올, 메탄올, 부탄올, 프로판올 등의 극성 용매; 헥산, 에테르, 벤젠, 톨루엔 등의 무극성 용매; 클로로포름, 에틸아세테이트, 아세톤, 메틸렌클로라이드 등의 중간극성 용매를 예시할 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the immune extract may be obtained by pulverizing the immune complex and then extracting it with an organic solvent or water. Examples of the organic solvent include polar solvents such as ethanol, methanol, butanol, and propanol; Non-polar solvents such as hexane, ether, benzene, and toluene; Chloroform, ethyl acetate, acetone, methylene chloride and the like, but are not limited thereto.
상기 잠분 추출물은 에탄올 등의 유기용매 또는 물에 마쇄된 잠분을 침지, 중탕, 증류시켜 추출하거나 초임계 추출 또는 마이크로파 추출법으로 추출할 수 있으며, 여과 및 농축과정을 통하여 획득할 수 있다.
The secretion extract may be extracted by immersion, bathing, distillation, extraction with an organic solvent such as ethanol or water, or extraction with supercritical extraction or microwave extraction, and may be obtained through filtration and concentration.
한편, 항암 활성을 가진 것으로 확인된 잠분 추출물을 분획할 경우, 항암 활성 성분을 함유하는 분획물을 분리할 수 있을 것으로 예측하였다.On the other hand, it was predicted that fractions containing the anticancer active ingredient could be separated when the subcutaneous extract was confirmed to have anticancer activity.
본 발명의 일 실시예에서는 잠분 추출물을 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 순차 분획하여 각각의 분획물을 획득하고, 획득한 분획물을 가지고 항암 활성 실험을 수행하였다. 그 결과, 클로로포름 분획물, 헥산 분획물, 에틸아세테이트 분획물이 우수한 항암 활성을 갖는다는 것을 확인할 수 있었다.In one embodiment of the present invention, each fraction was sequentially fractionated with hexane, chloroform, ethyl acetate, butanol and water, and the obtained fractions were subjected to an anticancer activity test. As a result, it was confirmed that the chloroform fraction, the hexane fraction and the ethyl acetate fraction had excellent anticancer activity.
따라서, 본 발명은 다른 관점에서 잠분 추출물로부터 분획하여 얻은 잠분 분획물을 유효성분으로 함유하는 것을 특징으로 하는 항암 조성물에 관한 것이다.Accordingly, the present invention relates to an anticancer composition characterized by containing, as an active ingredient, a subcutaneous fraction obtained by fractionation from a subcutaneous extract in another aspect.
상기 잠분 분획물은 유기용매의 극성에 따라 용해되는 화합물들을 분리하는 통상의 방법으로 획득할 수 있으며, 본 발명에서는 먼저 추출한 잠분 추출물에 순차적으로 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 분획하여 헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 및 물 분획물을 획득하였다. The scum extract may be obtained by a conventional method of isolating compounds that are dissolved according to the polarity of the organic solvent. In the present invention, the scum extract first extracted is sequentially fractionated with hexane, chloroform, ethyl acetate, butanol and water to obtain hexane fraction, Chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction were obtained.
본 발명에서는 또한, 항암 활성이 가장 우수한 것으로 확인된 클로로포름 분획물을 대상으로 크로마토그래피를 수행하면 항암 활성 본체를 분리 및 동정할 수 있을 것으로 예측하였다.In the present invention, it was also predicted that the chromatographic fraction of chloroform fractions having the highest anticancer activity would be able to isolate and identify the anticancer activity body.
본 발명의 일 실시예에서는 중압용 크로마토그래피(MPLC), 박막크로마토그래피(TLC) 및 고성능액체크로마토그래피(HPLC)를 수행하여 항암 활성 본체를 분리하고, 전자충격 질량분석(EI-MS), 핵자기공명(NMR) 및 DEPT(Distortionless Enhancement by Polarization Transfer)을 수행하여 항암 활성 본체가 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)이라는 것을 확인할 수 있었다.In one embodiment of the present invention, the body of the anticancer activity is separated by performing MPLC, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), and analyzed by electron impact mass spectrometry (EI-MS) It was confirmed that the main body of anticancer activity was vomifoliol and stigmasterol by performing magnetic resonance (NMR) and DEPT (Distortionless Enhancement by Polarization Transfer).
따라서, 본 발명은 또 다른 관점에서 잠분 분획물로부터 분리되어 수득되는 화합물을 유효성분으로 함유하는 항암 조성물에 관한 것이다.Accordingly, the present invention relates to an anticancer composition containing, as an active ingredient, a compound obtained separately from a subcutaneous fraction from a different viewpoint.
본 발명에 있어서, 상기 항암 활성을 화합물은 보미폴리올(vomifoliol) 및 스티그마스테롤(stigmasterol)로 구성된 군으로부터 선택되는 1 이상인 것을 특징으로 한다.In the present invention, the anticancer activity compound is characterized by being at least one selected from the group consisting of vomifoliol and stigmasterol.
본 발명에 있어서, 상기 보미폴리올(vomifoliol)은 항암 활성이 우수한 것으로 확인된 잠분 클로로포름 분획물을 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 실리카겔 컬럼 크로마토그래피로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 이를 다시 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 중압용 크로마토그래피(MPLC)로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 메탄올과 물의 혼합액을 이동상으로 하여 분취용 고성능액체크로마토그래피(HPLC)로 분리하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 화합물을 분리 및 정제함으로서 획득할 수 있다.In the present invention, the isolated chloroform fraction, which has been confirmed to have excellent anticancer activity, is fractionated by silica gel column chromatography while sequentially adding methanol to the mobile phase of chloroform, and the activity of vomifoliol The fractions were separated and further fractionated by medium pressure chromatography (MPLC) while methanol was sequentially added to the mobile phase of chloroform. The fraction having the highest activity was isolated by confirming the inhibition of growth of cancer cells. Then, a mixture solution of methanol and water , Followed by separation by preparative high performance liquid chromatography (HPLC), and by isolating and purifying the compound having the highest activity through confirmation of inhibition of cancer cell growth, it can be obtained.
또한, 상기 스티그마스테롤(stigmasterol)은 잠분 클로로포름 분획물을 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 실리카겔 컬럼 크로마토그래피로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 이를 다시 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 중압용 크로마토그래피(MPLC)로 분획하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 분획물을 분리한 다음, 메탄올과 물의 혼합액을 이동상으로 하여 역상 중압용 크로마토그래피(Reverse MPLC)로 분리하고, 암세포 성장 억제 확인을 통하여 활성이 가장 높은 화합물을 분리 및 정제함으로서 획득할 수 있다.In addition, the stigmasterol fraction was fractionated by silica gel column chromatography while sequentially adding methanol to the chloroform mobile phase, and the fraction having the highest activity was isolated by confirming inhibition of cancer cell growth. Then, the fractions were separated by chloroform mobile phase (MPLC), and the fraction having the highest activity was isolated by confirming inhibition of growth of cancer cells. Then, the fraction having the highest activity was separated, and then, using a mixture of methanol and water as a mobile phase, reverse-phase medium pressure chromatography (Reverse MPLC ) And isolating and purifying the compound having the highest activity through confirmation of inhibition of cancer cell growth.
상기 실리카겔 컬럼 크로마토그래피 및 중압용 크로마토그래피(MPLC) 수행시 이동상은 클로로포름 및 메탄올을 이용하는 것이 바람직하며, 클로로포름 : 메탄올의 비율이 100:0에서 순차적으로 0:100이 되도록 하는 것이 더욱 바람직하다.In carrying out the silica gel column chromatography and MPLC, it is preferable to use chloroform and methanol as the mobile phase, and it is more preferable that the ratio of chloroform: methanol is sequentially 0: 100 at 100: 0.
또한, 상기 역상 중압용 크로마토그래피(Reverse MPLC) 수행시 이동상은 메탄올 및 물을 이용하는 것이 바람직하며, 메탄올 : 물의 비율이 100:0에서 순차적으로 0:100이 되도록 하는 것이 더욱 바람직하다.In the reverse MPLC, the mobile phase is preferably methanol and water, more preferably 0: 100 at a ratio of methanol: water of 100: 0.
항암 활성물질을 분리하기 위하여 중압용 크로마토그래피(MPLC)로 얻어진 분리물들은 박막 크로마토그래피(TLC)를 이용하여 비슷한 전개 값(Rf)을 갖는 분리물들을 합친 다음, 항암 활성 실험을 진행하고, 항암 활성이 가장 우수한 것으로 확인된 분리물을 대상으로 다시 중압용 크로마토그래피(MPLC)를 수행한 다음, 박막 크로마토그래피(TLC)를 수행하는 과정을 1~6회 반복 수행할 수 있다.
To isolate the anticancer active substances, isolates obtained by medium pressure chromatography (MPLC) were subjected to thin-layer chromatography (TLC) to obtain isolates having a similar expansion value (Rf) (MPLC) is carried out again on the separated material which has been confirmed to be the most excellent, and then thin film chromatography (TLC) is carried out one to six times.
본 발명에 따른 항암 조성물은 암세포에 대해 세포독성 효과를 가지며, 폐암, 위암, 전립선암, 자궁경부암, 대장암, 유방암, 간암, 식도암, 난소암 등의 예방 및 치료에 사용될 수 있다.The anticancer composition according to the present invention has a cytotoxic effect on cancer cells and can be used for prevention and treatment of lung cancer, stomach cancer, prostate cancer, cervical cancer, colon cancer, breast cancer, liver cancer, oesophageal cancer and ovarian cancer.
본 발명에 따른 항암 조성물은 잠분 추출물, 잠분 분획물, 보미폴리올(vomifoliol) 또는 스티그마스테롤(stigmasterol) 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제, 향미제 등을 사용할 수 있다.The anticancer composition according to the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredient, the subcutaneous fraction, the vomifoliol or the stigmasterol, A disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant, and a flavoring agent.
상기 항암 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.The anticancer composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient described above for administration.
상기 약제학적 조성물의 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효 성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.
The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1: 잠분 추출물 및 잠분 분획물 분리Example 1: Separate Extracts and Separate Fractions
잠분은 전남 순천시 소재 약재시장에서 구입하여 사용하였고, 발명에 사용한 시료(SF-01)는 서울대학교 농생명과학연구원에 기탁하였다. The sample (SF-01) used in the invention was deposited with the Institute of Agronomy Science, Seoul National University.
먼저, 건조된 잠분 30.84kg을 곱게 마쇄한 다음 에탄올 155L로 실온에서 3일 동안 3회 추출 후, 여과하여 그 여과액을 회전진공농축기로 농축시켜 662g의 잠분 에탄올 추출물(수율: 약 2.2%)을 획득하였다.First, 30.84 kg of the dried suspension was crushed finely, and then extracted with 155 L of ethanol three times at room temperature for 3 days after filtration. The filtrate was concentrated by using a rotary vacuum concentrator to obtain 662 g of distilled ethanol extract (yield: about 2.2%) .
획득한 잠분 에탄올 추출물의 암세포 성장저해활성을 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]법을 이용하여 확인하고, 그 결과를 표 1에 나타내었다.The inhibitory activity of the acquired ethanol extract on cancer cells was confirmed by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] method, and the results are shown in Table 1 .
암세포 성장저해활성 평가를 위한 NCI-H727 (human lung carcinoma cell line), MRC-5 (human lung normal cell line), L-132 (human lung normal cell line), PC-3 (human prostate adenocarcinoma cell line), HT-29 (human colon adenocarcinoma cell line), AGS (human stomach adenocarcinoma cell line), MCF-7 (human breast adenocarcinoma cell line), SK-OV-3 (human ovary adenocarcinoma cell line), SNU-213 (human pancreas cancer), HEP-2 (human larynx adenocarcinoma cell line) 및 SK-HEP (human liver adenocarcinoma cell line)은 한국세포주은행(Korean Cell Line Bank, Seoul, South Korea)에서 구입하였고, HeLa (human cervix adenocarcinoma cell line) 및 A549 (human lung carcinoma cell line)는 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 구입하였다.NCI-H727 (human lung carcinoma cell line), MRC-5 (human lung normal cell line), L-132 (human lung normal cell line), PC-3 (human prostate adenocarcinoma cell line) , Human colon adenocarcinoma cell line (HT-29), human stomach adenocarcinoma cell line (MCF-7), human ovarian adenocarcinoma cell line SK-OV-3, SNU-213 pancreas cancer), HEP-2 (human larynx adenocarcinoma cell line) and SK-HEP (human liver adenocarcinoma cell line) were purchased from Korean Cell Line Bank, Seoul, South Korea and HeLa (human cervix adenocarcinoma cell line) and A549 (human lung carcinoma cell line) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
NCI-H727, PC-3, HT-29, AGS, MCF-7, SNU-213 및 SK-OV-3 세포주들은 37℃, 5% CO2와 95% 공기 조건의 10% FBS와 1% antibiotic-antimycotic 용액을 함유한 RPMI 1640(Life Technologies, USA)에서 배양하였다. Hela, A549, SK-HEP 및 MRC-5 세포주들은 37℃, 5% CO2와 95% 공기 조건의 10% FBS(Life Technologies, USA)와 1% antibiotic-antimycotic 용액 및 1% 글루타민(glutamine)을 함유한 MEM(Life Technologies, USA)에서 배양하였다. HEP-2 및 L-132 세포주는 37℃, 5% CO2와 95% 공기 조건의 10% FBS와 1% antibiotic-antimycotic 용액을 함유한 DMEM(Life Technologies, USA)에서 배양하였다. 모든 세포들은 페트리디쉬(2*104cm-Corning Costar, NY, USA)에서 배양하였다.NCI-H727, and PC-3, HT-29, AGS, MCF-7, SNU-213 and SK-OV-3 cell line are 37 ℃, 5% CO 2 Were cultured in RPMI 1640 (Life Technologies, USA) containing 10% FBS and 1% antibiotic-antimycotic solution in 95% air condition. Hela, A549, SK-HEP and MRC-5 cell lines were incubated at 37 ° C in 5% CO 2 (Life Technologies, USA) containing 95% air in 10% FBS (Life Technologies, USA) and 1% antibiotic-antimycotic solution and 1% glutamine. And HEP-2 and L-132 cell line is 37 ℃, 5% CO 2 Were cultured in DMEM (Life Technologies, USA) containing 10% FBS and 1% antibiotic-antimycotic solution in 95% air. All cells were cultured in Petri dishes (2 * 10 4 cm-Corning Costar, NY, USA).
MTT(Sigma-Aldrich) 5mg/mL 기질을 PBS (pH 7.4)에 녹인 용액을 멸균여과하여 실험에 사용할 때까지 -20℃에 냉동 보관하였다. 세포주들은 각기 다른 농도로 준비된 물질들과 100μL의 complete culture medium을 함유한 96-웰 플레이트 각 웰에 5*10-3개로 준비하였다. 시험 시료 각각은 최종 농도가 0.1% 또는 그 이하의 농도가 되도록 DMSO Hybri-Max에 녹여 사용하였다. 배양 플레이트들은 가습한 5% 이산화탄소 대기에서 37℃ 조건으로 2일 동안 배양하였고, 100μL PBS로 1회 세척하였다. 0.05% MTT를 함유한 100μL 배지를 각 웰에 분주한 후 동일 조건에서 4시간 동안 배양하였다. MTT 용액은 배양 4시간 후 제거하였고 200μL DMSO를 각 웰에 첨가하고, 마지막으로 플레이트에 형성된 보라색 포마잔(formazan) 결정들을 녹이기 위해 10분 동안 흔들어 주었다.A solution of MTT (Sigma-Aldrich) 5 mg / mL substrate in PBS (pH 7.4) was sterile filtered and stored frozen at -20 ° C until used. Cell lines were prepared at 5 * 10 -3 cells per well in 96-well plates containing 100 μL complete culture medium and materials prepared at different concentrations. Each test sample was dissolved in DMSO Hybri-Max to a final concentration of 0.1% or less. The culture plates were incubated for 2 days at 37 ° C in a humidified 5
항암제로 널리 이용되는 시스플라틴(Sigma-Aldrich, USA)을 포지티브 대조구로 이용하였고, DMSO 용액을 네거티브 대조구로 이용하였다. OD(optical density) 값은 560nm와 670nm에서 Molecular Devices VersaMax microplate reader (Sunnyvale, CA, USA)를 이용해서 측정하였고, 대조구 값들은 실측치들에서 추출하여 활용하였다. 모든 검정은 3반복으로 하였다. Cisplatin (Sigma-Aldrich, USA), widely used as an anticancer drug, was used as a positive control and DMSO solution was used as a negative control. Optical density (OD) values were measured at 560 nm and 670 nm using a Molecular Devices VersaMax microplate reader (Sunnyvale, CA, USA), and the control values were extracted from the measured values. All tests were performed in 3 replicates.
암세포 성장저해활성은 대조구 웰들과 비교하여 50%로 세포들의 활력을 감소시키는 물질의 50% 억제농도(IC50)로 제시하였다. 시험물질들의 IC50 값들은 Prism 5 software program (GraphPad Software, La Jolla, CA, USA)을 이용하여 계산하였다. 퍼센트 성장 저해력은 다음 식으로 산출하였다.The cancer cell growth inhibitory activity was shown as a 50% inhibitory concentration (IC 50 ) of the substance which reduces cell viability by 50% as compared to the control well. The IC 50 values of the test materials were calculated using the
[식] % growth inhibition = A/B × 100, A; 물질 처리한 웰들 세포들의 OD값, B; 대조구 웰들 세포들의 OD값.[Expression]% growth inhibition = A /
통계분석은 SAS 9.13 program (SAS Institute, Cary, NC)을 이용하였고, 평균간 비교는 ANOVA(P=0.05)로 하였다.
Statistical analysis was performed using the SAS 9.13 program (SAS Institute, Cary, NC) and the mean comparison was made by ANOVA (P = 0.05).
a 세포주들에 대한 50% 세포성장억제농도. a cell growth inhibitory concentration of 50%.
b 포지티브 대조구. b Positive control.
c 상대독성(RT), 잠분 에탄올 추출물의 IC50값/시스플라틴의 IC50값.
c Relative toxicity (RT), jambun IC 50 value / IC 50 value of the ethanol extract of cisplatin.
표 1에 나타난 바와 같이, 잠분 에탄올 추출물은 모든 암세포주들에 대하여 항암 활성(IC50, 9.25∼58.78g/mL)을 보였다. 잠분 에탄올 추출물의 IC50은 모든 암세포주들에 대하여 시스플라틴 IC50을 기준으로 약 1.69~37.06배의 농도에서 항암 활성을 나타내었다.As shown in Table 1, the inhibitory ethanol extract showed an anticancer activity (IC 50 , 9.25-58.78 g / mL) in all cancer cell lines. The IC 50 of the ethanol extract showed an anticancer activity at a concentration of about 1.69 to 37.06 times based on the cisplatin IC 50 for all cancer cell lines.
그러나, 정상 인간 폐세포주들(L-132, MRC-5)에 대해서는 별다른 독성을 나타내지 않았다(IC50, >100 g/mL).
However, it did not show any toxicity to normal human lung cell lines (L-132, MRC-5) (IC 50 ,> 100 g / mL).
도 1과 같이, 암세포 성장저해 활성이 확인된 잠분 에탄올 추출물로부터 활성 본체를 분리하기 위하여, 잠분 에탄올 추출물 660g을 순차적으로 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 분획하여 각 분획물 418.44g, 51.48g, 8.58g, 60.39g 및 121.11g을 확보하고, A549 폐암 세포주에 대하여 동일한 방법으로 각 분획물들의 암세포 성장저해활성을 평가하고, 그 결과를 표 2에 나타내었다.As shown in FIG. 1, in order to isolate the active substance from the distant ethanol extract having inhibited cancer cell growth inhibition activity, 660 g of the distilled ethanol extract was sequentially fractionated with hexane, chloroform, ethyl acetate, butanol and water to obtain 418.44 g, 51.48 g, 8.58 g, 60.39 g, and 121.11 g were obtained, and the inhibitory activity against cancer cell growth of each fraction was evaluated for the A549 lung cancer cell line by the same method. The results are shown in Table 2.
a 세포주들에 대한 50% 세포성장억제농도. a cell growth inhibitory concentration of 50%.
b CL은 신뢰한계를 의미. b CL means confidence limit.
c 적합도 검정을 위한 피어슨 카이자승 값(χ2).
c Pearson's square value (χ 2 ) for fitness tests.
표 2에 나타난 바와 같이, IC50 값을 기준으로 클로로포름 분획물이 가장 강한 암세포 성장저해활성을 보였고, 헥산 분획물과 에틸아세테이트 분획물이 중간 정도의 암세포 성장저해활성을 보였다. 반면 부탄올 분획물과 물 분획물은 전혀 활성을 나타내지 않았다.
As shown in Table 2, the chloroform fraction showed the strongest inhibitory activity against cancer cell growth on the basis of the IC 50 value, and the hexane fraction and the ethyl acetate fraction showed moderate cancer cell growth inhibitory activity. On the other hand, the butanol fraction and the water fraction did not show any activity at all.
실시예 2: 항암 활성물질 분리 및 정제Example 2: Separation and purification of anticancer active substances
2-1: 활성본체 1 분리 및 정제2-1: Isolation and purification of
도 2에 도시된 바와 같이, 실시예 1에서 가장 암세포 성장저해활성이 우수한 것으로 확인된 클로로포름 분획물을 컬럼 크로마토그래피(5.5 cm i. d. × 70 cm, 실리카겔 600g 충진)에 로딩한 후, 전개용매 클로로포름과 메탄올을 순차적으로 공급[100:0 (1 L), 97:3 (1 L), 95:5 (2 L), 90:10 (1 L), 80:20 (1 L), 70:30 (1 L), 0:100 (1 L), v/v]하여 37개(각 180 mL) 분획물을 수득하였다. As shown in Fig. 2, the chloroform fraction, which was confirmed to have the best inhibitory activity against cancer cell growth in Example 1, was loaded on a column chromatography (5.5 cm id x 70 cm, filled with 600 g silica gel), followed by elution with chloroform and methanol (1 L), 95: 5 (2 L), 90:10 (1 L), 80:20 (1 L), 70:30 (1 L) L), 0: 100 (1 L), v / v] to obtain 37 fractions (180 mL each).
수득한 분획물들은 TLC로 분석(chloroform:methanol = 95:5, v/v)하여 동일한 전개값을 갖는 것들은 동일한 분획물로 간주하고 혼합하였다. 분획물 23~36(C4, 14.86g)을 중압용 크로마토그래피(MPLC)(Isolera one Biotage® medium-pressure liquid chromatograph, Uppsala, Sweden)로 더욱 분획하였다. 분획은 클로로포름 이동상에 메탄올을 순차적으로 추가시켰다[100:0 (847 mL), 98:2 (657 mL), 97:3 (382 mL), 95:5 (2740 mL), 92:8 (536 mL), 90:10 (281 mL), 86:14 (127 mL), 85:15 (369 mL), 80:20 (335 mL), 0:100 (800 mL), v/v]. 분당 이동상의 유량은 25mL/min로 하여 총 190개 분획층(각각 약 22 mL)을 확보하였고, 이들 각 칼럼 분획물들은 TLC로 분석(chloroform:methanol = 95:5, v/v)하였다. TLC 상에서 비슷한 전개 값(Rf)을 가진 분획물들은 5% 황산용액을 뿌리고 열을 가해 확인 후 하나로 합쳤다.The obtained fractions were analyzed by TLC (chloroform: methanol = 95: 5, v / v), and those having the same expansion value were regarded as the same fraction and mixed. Of fractions 23 ~ 36 (C4, 14.86g) a chromatographic medium pressure for Photography (MPLC) (Isolera one Biotage ® medium-pressure liquid chromatograph, Uppsala, Sweden) was further fractionated. The fractions were added to the chloroform mobile phase sequentially with methanol (100: 0 (847 mL), 98: 2 (657 mL), 97: 3 (382 mL), 95: ), 90:10 (281 mL), 86:14 (127 mL), 85:15 (369 mL), 80:20 (335 mL), 0: 100 (800 mL), v / v]. A total of 190 fractions (about 22 mL each) were obtained at a flow rate of 25 mL / min per minute. The column fractions were analyzed by TLC (chloroform: methanol = 95: 5, v / v). The fractions with similar expansion values (Rf) on TLC were sprayed with 5% sulfuric acid solution, and after heating, they were combined into one.
수득된 82~165 분획물(C43, 5.40g)을 중압용 크로마토그래피(MPLC)로 더욱 분획하였다. 분획은 클로로포름 이동상에 메탄올을 순차적으로 추가시켰다[100:0 (924 mL), 99:1 (99 mL), 98:2 (46.2 mL), 95:5 (396 mL), 93:7 (567.6 mL), 90:10 (1379.4 mL), 85:15 (508.2 ml), 83:17 (481.8 ml), 80:20 (323.4 ml), 0:100 (800 mL), v/v]. 분당 이동상의 유량은 25mL/min로 하여 총 175개 분획층(각 25ml)을 확보하였고, 이들 각 칼럼 분획물들은 TLC로 분석(chloroform:methanol = 90:10, v/v)하였다. The resulting fractions 82 to 165 (C43, 5.40 g) were further fractionated by medium pressure chromatography (MPLC). The fractions were added to the chloroform mobile phase sequentially with methanol [99: 1 (992 mL), 98: 2 (46.2 mL), 95: 5 (396 mL), 93: ), 90:10 (1379.4 mL), 85:15 (508.2 mL), 83:17 (481.8 mL), 80:20 (323.4 mL), 0: 100 (800 mL), v / v]. A total of 175 fractions (25 ml each) were obtained at a flow rate of 25 mL / min per minute. The column fractions were analyzed by TLC (chloroform: methanol = 90:10, v / v).
수득된 17~40 분획물(C432, 2.13g)을 중압용 크로마토그래피(MPLC)로 더욱 분획하였다. 분획은 클로로포름 이동상에 메탄올을 순차적으로 추가시켰다[100:0 (699.6 mL), 97:3 (59.4 mL), 95:5 (1023 mL), 90:10 (910.8 mL), 85:15 (40.0 mL), 80:20 (699.6 ml), 70:30 (330 ml), 0:100 (800 mL), v/v] 분당 이동상의 유량은 25mL/min로 하여 총 235개 분획층(각 22ml)을 확보하였고, 이들 각 칼럼 분획물들은 TLC로 분석(chloroform:methanol = 90:10, v/v)하였다. The obtained 17-40 fractions (C432, 2.13 g) were further fractionated by medium pressure chromatography (MPLC). The fractions were successively added to the chloroform mobile phase with methanol sequentially (100: 0 (699.6 mL), 97: 3 (59.4 mL), 95: 5 (1023 mL), 90:10 (910.8 mL), 85:15 ), 80:20 (699.6 ml), 70:30 (330 ml), 0: 100 (800 ml), v / v] The flow rate of the mobile phase per minute was 25 ml / min. The column fractions were analyzed by TLC (chloroform: methanol = 90:10, v / v).
수득된 13~50 분획물(C4322, 0.84g)을 중압용 크로마토그래피(MPLC)로 더욱 분획하였다. 분획은 클로로포름 이동상에 메탄올을 순차적으로 추가시켰다[100:0 (580.8 mL), 99:1 (165 mL), 98:2 (132 mL), 97:3 (270.6 ml), 96:4 (983.4 ml), 95:5 (534.6 ml), 94:6 (316.8 ml), 93:7 (415.8 ml), 92:8 (330 ml), 91:9 (297 ml), 90:10 (343.2 ml), and 0:100 (800 mL)]. 분당 이동상의 유량은 25mL/min로 하여 총 165개 분획층(각 22ml)을 확보하였고, 이들 각 칼럼 분획물들은 TLC로 분석(chloroform:methanol = 90:10, v/v)하였다. The resulting 13-50 fractions (C4322, 0.84 g) were further fractionated by medium pressure chromatography (MPLC). The fractions were sequentially added to the chloroform mobile phase with methanol sequentially (100: 0 (580.8 mL), 99: 1 (165 mL), 98: 2 (132 mL), 97: ), 95: 5 (534.6 ml), 94: 6 (316.8 ml), 93: 7 (415.8 ml), 92: 8 (330 ml), 91: 9 (297 ml) and 0: 100 (800 mL)]. A total of 165 fractions (22 ml each) were obtained at a flow rate of 25 mL / min per minute. The column fractions were analyzed by TLC (chloroform: methanol = 90:10, v / v).
수득된 1~20 분획물(C43221, 6.0mg)을 분취용 HPLC로 더욱 분획하여 활성층 활성본체 1인 C432212(머무름 시간 28.83분)를 수득하였다(도 3). 분취용 HPLC에 사용한 칼럼은 μBondapak C18 (7.8 mm i.d.*300 mm; Waters, Milford, MA, USA)이었고 이동상은 메탄올과 물을 50:50(v/v)으로 고정하여 분당 1.0 ml로 흘려주면서 분취하였다. UV 검출기는 244nm로 고정하였다.
The resulting
2-2: 활성본체 2 분리 및 정제2-2: Isolation and purification of
2-1과 동일한 방법으로 분획물(C4322, 0.84g)을 중압용 크로마토그래피(MPLC)로 더욱 분획하여 수득된 35~82 분획물(C43224, 0.76g)을 중압용 크로마토그래피(MPLC)로 더욱 분획하였다. 분획은 클로로포름 이동상에 메탄올을 순차적으로 추가시켰다[100:0 (448.8 mL), 99:1 (59.4 mL), 98:2 (52.8 mL), 95:5 (19.8 ml), 92:8 (66 ml), 90:10 (257.4 ml), 89:11 (85.8 ml), 88:12 (158.4 ml), 85:15 (237.6 ml), 83:17 (237.6 ml), 80:20 (198 ml), 0:100 (800 mL), v/v]. 분당 이동상의 유량은 25mL/min로 하여 총 65개 분획층(각 22ml)을 확보하였고, 이들 각 칼럼 분획물들은 TLC로 분석(chloroform:methanol = 90:10, v/v)하였다.The fraction (C43224, 0.76 g) obtained by further fractionation of the fraction (C4322, 0.84 g) by medium pressure chromatography (MPLC) in the same manner as in 2-1 was further fractionated by medium pressure chromatography (MPLC) . The fractions were successively added to the chloroform mobile phase with methanol sequentially [100: 0 (448.8 mL), 99: 1 (59.4 mL), 98: 2 (52.8 mL), 95: ), 90:10 (257.4 mL), 89:11 (85.8 mL), 88:12 (158.4 mL), 85:15 (237.6 mL), 83:17 (237.6 mL), 80:20 (198 mL) 0: 100 (800 mL), v / v]. A total of 65 fractions (22 ml each) were obtained at a flow rate of 25 mL / min per minute. The column fractions were analyzed by TLC (chloroform: methanol = 90:10, v / v).
수득된 45~50 분획물(C432242, 9.5mg)을 역상 중압용 크로마토그래피(Reverse MPLC)로 더욱 분획하여 활성층 활성본체 2인 C4322426(머무름 시간 44.84분)를 수득하였다(도 4). 이동상은 분당 1.0 mL로 메탄올과 물을 80:20 (v/v) 흘려주었고, UV 검출기는 214 nm로 고정하였다.The resulting 45-50 fractions (C432242, 9.5 mg) were further fractionated by reverse phase medium pressure chromatography (Reverse MPLC) to obtain C4322426 (retention time 44.84 min) as the
중압용 크로마토그래피(MPLC) 및 박막 크로마토그래피(TLC)로 분획된 분획층들 및 최종적으로 HPLC로 분리된 분획층들에 대해서는 A549 폐암 세포주에 대하여 실시예 1과 동일한 방법으로 암세포 성장저해활성을 평가하고, 그 결과를 표 3에 나타내었다.As to the fraction layers separated by the medium pressure chromatography (MPLC) and the thin layer chromatography (TLC) and finally the fraction layers separated by HPLC, the A549 lung cancer cell line was evaluated for inhibiting cancer cell growth by the same method as in Example 1 And the results are shown in Table 3.
a 세포주들에 대한 50% 세포성장억제농도. a cell growth inhibitory concentration of 50%.
b CL은 신뢰한계를 의미. b CL means confidence limit.
c 적합도 검정을 위한 피어슨 카이자승 값(χ2).
c Pearson's square value (χ 2 ) for fitness tests.
실시예 3: 항암 활성물질 동정Example 3: Identification of antitumor active substance
실시예 2에서 분리된 무색 분말의 C432212 분획물 및 C4322426 분획물을 확인하기 위하여 EI-MS, 1H-NMR(600 MHz, Rheinstetten, Baden-Wurttemberg, Germany), 13C-NMR(150 MHz, Rheinstetten, Baden-Wurttemberg, Germany) 및 DEPT 분석을 서울대학교 농생명과학공동기기원(NICEM)에 의뢰하였다. 1 H-NMR (600 MHz, Rheinstetten, Baden-Württemberg, Germany), 13 C-NMR (150 MHz, Rheinstetten, Baden, Germany) to identify the C432212 fraction and the C4322426 fraction of the colorless powder isolated in Example 2 -Wurttemberg, Germany) and DEPT analysis were commissioned to NICEM (National Institute of Agronomy Science, Seoul National University).
3-1: 활성본체 1(C432212) 동정3-1: Identification of active body 1 (C432212)
도 5에 나타난 바와 같이, EI-MS 결과, C432212의 질량 스펙트럼은 124m/z에서 가장 강한 피크[M]+를 보였다 (EI-MS (70 eV), m/z (relative intensity): 124 [M]+ (100), 55 (10), 59 (10), 95 (10), 111 (15), 124 (100), 135 (11), 150 (11), 168 (13)).5, the mass spectrum of C432212 showed the strongest peak [M] + at 124 m / z (EI-MS (70 eV), relative intensity: 124 [M] ] + (100), 55 (10), 59 (10), 95 (10), 111 (15), 124 (100), 135 (11), 150 (11), 168 (13).
또한 C432212의 1H-NMR (MeOD, 600MHz) 및 13C-NMR (MeOD, 150MHz) 데이터는 표 4에 나타내었다. 1 H-NMR (MeOD, 600 MHz) and 13 C-NMR (MeOD, 150 MHz) data of C432212 are shown in Table 4.
structure Partial
structure
Xiaoxi et al. (1999) [delta] H (ppm)
Xiaoxi et al. (1999)
(J = 18.0 Hz) 2.42, d
( J = 18.0 Hz)
(J = 17.0 Hz) 2.45, d
( J = 17.0 Hz)
(J = 16.2 Hz) 5.88, d
( J = 16.2 Hz)
(J = 15.5 Hz) 5.79, d
( J = 15.5 Hz)
(J = 16.2, 6.2 Hz) 5.88, dd
( J = 16.2, 6.2 Hz)
(J = 15.5 , 6.0 Hz) 5.86, dd
( J = 15.5, 6.0 Hz)
(J = 1.8 Hz) 1.27, d
( J = 1.8 Hz)
(J = 1.5 Hz) 1.30, d
( J = 1.5 Hz)
표 4에 나타난 바와 같이, C432212는 기본적으로 13개 탄소로 구성된 물질임을 NMR 스펙트럼을 통해 얻었고, 도 6의 DEPT 데이터로부터 4개의 메틸 그룹, 2개의 hydroxyl 그룹, 하나의 케톤 그룹을 갖고 있음을 확인하였다.As shown in Table 4, it was confirmed that C432212 was basically a material composed of 13 carbons through NMR spectrum, and it was confirmed from the DEPT data of FIG. 6 that it had four methyl groups, two hydroxyl groups, and one ketone group .
이상의 결과와 기존 문헌(Liua X., Tiana F., Zhanga H.B., Pilarinoub E., and McLaughlina J.L., Biologically Active Blumenol A from the Leaves of Annona Glabra. Natural Product Letters, 14(1):77-81, 1999)에 근간해서 C432212는 C13H20O3 분자식을 가지며, 화학식 1로 표시되는 보미폴리올(vomifoliol) (blumenol A)이라는 것을 확인할 수 있었다.The above results are compared with those of the conventional literature (Liua X., Tiana F., Zhanga HB, Pilarinoub E., and McLaughlina JL, Biologically Active Blumenol from the Leaves of Annona Glabra. Natural Product Letters, 14 (1): 77-81, 1999 ), It was confirmed that C432212 had a C 13 H 20 O 3 molecular formula and was a vomifoliol (blumenol A) represented by the general formula (1).
3-2: 활성본체 2(C4322426) 동정3-2: Identification of active body 2 (C4322426)
도 7에 나타난 바와 같이, EI-MS 결과, C4322426의 질량 스펙트럼은 69m/z에서 가장 강한 피크[M]+를 보였다 (EI-MS (70 eV), m/z (%relative intensity): 69 [M]+ (100), 55 (71), 69 (100), 251 (65), 269 (40), 379 (73), 383 (50), 395 (67), 412 (88)).7, the mass spectrum of C4322426 showed the strongest peak [M] + at 69 m / z (EI-MS (70 eV), m / z (% relative intensity): 69 [ M] + (100), 55 (71), 69 (100), 251 (65), 269 (40), 379 (73), 383 (50), 395 (67), 412 (88).
또한 C4322426의 1H-NMR (MeOD, 600MHz) 및 13C-NMR (MeOD, 150MHz) 데이터는 표 5에 나타내었다. 1 H-NMR (MeOD, 600 MHz) and 13 C-NMR (MeOD, 150 MHz) data of C4322426 are shown in Table 5.
structure Partial
structure
Peter and Katalin (2004) [delta] H (ppm)
Peter and Katalin (2004)
(J = 15.8 Hz) 5.48, dd
( J = 15.8 Hz)
(J = 15.2 Hz) 5.18, dd
( J = 15.2 Hz)
(J = 9.2 Hz) 5.48, dd
( J = 9.2 Hz)
(J = 8.6 Hz) 5.04, dd
( J = 8.6 Hz)
표 5에 나타난 바와 같이, C4322426는 기본적으로 수소 20개 및 탄소 29개로 구성된 물질임을 NMR 스펙트럼을 통해 얻었고, 도 8의 DEPT 데이터로부터 스테로이드 골격을 갖고 C-17 위치에 부착된 10개의 탄소 원자들을 함유한 측쇄의 phytosterol기를 갖고 있음을 확인하였다.As shown in Table 5, the C4322426 was basically a substance consisting of 20 hydrogen atoms and 29 carbon atoms, and it was found from the DEPT data of FIG. 8 that the 10 carbon atoms having a steroid skeleton attached at the C-17 position It was confirmed that the phytosterol group of one side chain was present.
이상의 결과와 기존 문헌(Forgo P. & Kover K.E., Gradient enhanced selective experiments in the 1H-NMR chemical shift assignment of the skeleton and side-chain resonances of stigmasterol, a phytosterol derivative. Steroids, 69(1):43∼50, 2004)에 근간해서 C4322426는 C29H48O 분자식을 가지며, 화학식 2로 표시되는 스티그마스테롤(stigmasterol)이라는 것을 확인할 수 있었다.The above results are compared with those of the existing literature (Forgo P. & Kover KE, Gradient enhanced selective experiments in the 1 H-NMR chemical shift assignment of the skeleton and side-chain resonances of stigmasterol, a phytosterol derivative. Steroids, 69 , 2004), it was confirmed that C4322426 had a molecular formula of C 29 H 48 O and was a stigmasterol represented by the formula (2).
실험예 1: 보미폴리올 및 스티그마스테롤의 항암세포 성장억제활성 확인Experimental Example 1: Confirmation of anticancer cell growth inhibitory activity of bamipolol and stigmasterol
1-1: A549 폐암 세포주1-1: A549 lung cancer cell line
실시예 2에서 분리되고, 실시예 3에서 확인된 보미폴리올, 스티그마스테롤 및 항암제 시스플라틴(Sigma-Aldrich, USA)의 A549 폐암 세포주에 대한 세포성장 억제활성을 실시예 1에 기재한 MTT법으로 평가하고, 그 결과를 표 6에 나타내었다.The cell growth inhibitory activity of bupivir polyol, stigmasterol and anticancer agent cisplatin (Sigma-Aldrich, USA) isolated in Example 2 and identified in Example 3 on A549 lung cancer cell line was evaluated by the MTT method described in Example 1 , And the results are shown in Table 6.
a 세포주들에 대한 50% 세포성장억제농도 a 50% cell growth inhibitory concentration
b CL은 신뢰한계를 의미 b CL means trust limit
c 적합도 검정을 위한 피어슨 카이자승 값(χ2) c Pearson's square value (χ 2 ) for fitness tests
d 시스플라틴 IC50 값에 대한 상대적인 보미폴리올 또는 스티그마스테롤의 IC50 값
d IC 50 value of biphasic polyol or stigmasterol relative to cisplatin IC 50 value
표 6에 나타난 바와 같이, 잠분에서 분리된 천연 보미폴리올 및 스티그마스테롤은 A549 폐암 세포주에 대한 IC50 값이 각각 51.66 및 48.41로써, 시스플라틴 보다는 세포증식억제력이 다소 낮았지만, 항암제로 이용이 가능함을 확인할 수 있었다.
As shown in Table 6, the natural Bomi polyol and stigmasterol isolated from jambun the A549 as an IC 50 value of each of 51.66 and 48.41 for the lung cancer cell line, a cell proliferation inhibitory effect rather natatjiman than cisplatin, to determine the possible use as an anticancer agent there was.
1-2: NCI-H727 폐암 세포주1-2: NCI-H727 lung cancer cell line
실시예 2에서 분리되고, 실시예 3에서 확인된 보미폴리올, 스티그마스테롤 및 항암제 시스플라틴(Sigma-Aldrich, USA)의 NCI-H727 폐암 세포주에 대한 세포성장 억제활성을 실시예 1에 기재한 MTT법으로 평가하고, 그 결과를 표 7에 나타내었다.The cell growth inhibitory activity of the bumipolol, stigmasterol and anticancer agent cisplatin (Sigma-Aldrich, USA) isolated in Example 2 and identified in Example 3 on the NCI-H727 lung cancer cell line was measured by the MTT method described in Example 1 The results are shown in Table 7.
a 세포주들에 대한 50% 세포성장억제농도 a 50% cell growth inhibitory concentration
b CL은 신뢰한계를 의미 b CL means trust limit
c 적합도 검정을 위한 피어슨 카이자승 값(χ2) c Pearson's square value (χ 2 ) for fitness tests
d 시스플라틴 IC50 값에 대한 상대적인 보미폴리올 또는 스티그마스테롤의 IC50 값
d IC 50 value of biphasic polyol or stigmasterol relative to cisplatin IC 50 value
표 7에 나타난 바와 같이, 잠분에서 분리된 천연 보미폴리올 및 스티그마스테롤은 NCI-H727 폐암 세포주에 대한 IC50 값이 각각 51.84 및 46.21로써, 시스플라틴 보다는 세포증식억제력이 다소 낮았지만, 항암제로 이용이 가능함을 확인할 수 있었다.
As shown in Table 7, this is possible using a natural Bomi polyol and stigmasterol are slightly NCI-H727 as an IC 50 value of each of 51.84 and 46.21 for the lung cancer cell lines, cell growth inhibitory effect than cisplatin natatjiman, anticancer agents isolated from jambun I could confirm.
실험예 2: 보미폴리올 및 스티그마스테롤의 Experimental Example 2: Preparation of Bream Polyol and Stigmasterol Akt1Akt1 및 And Bcl-2Bcl-2 유전자 발현에 미치는 영향 평가 Evaluation of effect on gene expression
실시예 2에서 분리되고, 실시예 3에서 확인된 보미폴리올 및 스티그마스테롤의 Akt1 및 Bcl -2 유전자 발현 억제능을 real-time qRT-PCR(real-time quantitative reverse transcription-polymerase chain reaction)로 평가하였다. Time-resolved quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to evaluate the inhibition of Akt1 and Bcl- 2 gene expression of bamipolol and stigmasterol isolated in Example 2 and identified in Example 3.
유전자 발현 수준을 평가하기 위해 SYBR Green dye를 활용하여 real-time qRT-PCR을 수행하였다. 세포배양플라스크(25cm2, Corning Costar, NY, USA)에서 배양한 A549 세포주에 각 시험 화합물(보미포리올, 스티그마스테롤) 12.5, 25 및 50μg/mL를 처리하였다. Real-time qRT-PCR was performed using SYBR Green dye to assess gene expression levels. A549 cell lines cultured in a cell culture flask (25 cm 2 , Corning Costar, NY, USA) were treated with 12.5, 25 and 50 μg / mL of each test compound (bamipholol, stigmasterol).
처리 2일 후, total RNA를 RNeasy kit (Qiagen, Hilden, Germany)로 추출한 후, 오염된 genomic DNA를 RQ1 RNeas-free DNAase (Promega, Madison, WI, USA)로 제거한 다음, SuperScript First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA)을 이용하여 cDNA를 합성하였다. After 2 days of treatment, total RNA was extracted with RNeasy kit (Qiagen, Hilden, Germany), and the contaminated genomic DNA was removed with RQ1 RNeas-free DNAase (Promega, Madison, WI, USA) (Invitrogen, Carlsbad, Calif.).
PCR 효율성(100ng ~ 10pg per reaction)을 확인하기 위해 각 RNA에 대한 cDNA를 log10-fold로 희석(5개 구간)한 후, qRT-PCR(Applied Biosystem StepOne-plus real-time PCR system, Applied Biosystems, Foster, CA USA)을 수행하였다.To verify PCR efficiency (100 ng to 10 pg per reaction), cDNA for each RNA was diluted (5 sections) with log10-fold and then subjected to qRT-PCR (Applied Biosystem StepOne-plus real-time PCR system, Applied Biosystems, Foster, CA USA).
먼저, 10μL Maxima SYBR Green/ROX qPCR Master Mix (2×), 2μL forward 및 reverse 프라이머들(각 5 pmol)(표 8 참조), 1 μL cDNA (8ng) 그리고 7μL diethylpyrocarbonate-treated water의 각 반응 혼합물(전체 20μL)을 준비한 다음 50℃에서 2분, 95℃에서 10분, 95℃에서 15초 동안 50회 실시한 후, 60℃에서 30초 동안 GAPDH, Akt1 및 Bcl-2의 qRT-PCR을 수행하였다.First, each reaction mixture of 10 μL Maxima SYBR Green / ROX qPCR Master Mix (2x), 2 μL forward and reverse primers (5 pmol each) (see Table 8), 1 μL cDNA (8 ng) and 7 μL diethylpyrocarbonate- 20 μL total) was prepared and then subjected to 50 cycles of 50 minutes at 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, and 95 ° C. for 15 seconds, followed by qRT-PCR of GAPDH, Akt1 and Bcl-2 at 60 ° C. for 30 seconds.
목적 유전자의 mRNA 발현수준을 housekeeping gene GAPDH의 mRNA 수준에 맞춘 후, StepOne Software v2.1/DataAssist Software (Applied Biosystems)를 이용하여 분석하고, 그 결과를 도 9 및 도 10에 나타내었다. 각 그래프는 3반복 처리구의 평균과 표준오차(mean±SE)로 제시했고, 평균간 비교는 본페로니를 이용하였다(P = 0.05, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns = no significant).
The mRNA expression level of the target gene was adjusted to the level of the housekeeping gene GAPDH mRNA and analyzed using StepOne Software v2.1 / DataAssist Software (Applied Biosystems). The results are shown in Figs. 9 and 10. Each graph was presented with mean and standard error (mean ± SE) of 3 replicates, and the mean liver comparisons were using the ferroni (P = 0.05, ***: p <0.001, **: p <0.01, *: p < 0.05, ns = no significant).
and Reverse primer sequenceForward primer
and Reverse primer sequence
R: GGATGCAGGGATGATGTTCTG F: TGCACCACCAACTGCTTAG
R: GGATGCAGGGATGATGTTCTG
R: GGATCACCTTGCCGAAAGTG F: CAAGCCCAAGCACCGC
R: GGATCACCTTGCCGAAAGTG
R: TGCATTCTTGGACGAGGG F: ATTGGGAAGTTTCAAATCAGC
R: TGCATTCTTGGACGAGGG
도 9에 도시된 바와 같이, A549 세포내 Akt1 유전자의 발현량은 보미폴리올 또는 스티그마스테롤 처리 후 유의하게 감소되었다. 특히, 보미폴리올을 12.5μg/mL 이상 처리할 경우 2.85배 이상 감소시켰고, 스티그마스테롤은 약 1.22배 이상 감소시키는 것을 확인하였다.As shown in Fig. 9, the expression level of Akt1 gene in A549 cells was significantly decreased after treatment with bamipolol or stigmasterol. In particular, it was confirmed that the treatment with bomipolol decreased more than 2.85 times when treated with 12.5 μg / mL or more, and decreased about 1.22 times or more with stigmasterol.
또한, 도 10에 도시된 바와 같이, A549 세포내 bcl-2 유전자의 발현량은 보미폴리올 또는 스티그마스테롤 처리 후 유의하게 감소되었다. 특히, 보미폴리올을 12.5μg/mL 이상 처리할 경우 1.27배 이상 감소시켰고, 스티그마스테롤은 약 14.3배 이상 감소시키는 것을 확인하였다.In addition, as shown in Fig. 10, the expression amount of bcl-2 gene in A549 cells was significantly decreased after treatment with bamipolol or stigmasterol. In particular, it was confirmed that the treatment with bomipolol decreased more than 1.27 times when treated with 12.5 μg / mL or more, and decreased more than 14.3 times with stigmasterol.
즉, 잠분에서 추출된 보미폴리올 및 스티그마스테롤은 안티 에팝토시스(anti-apoptosis) 유전자인 Akt1 및 bcl - 2 의 발현을 억제함으로써, 암세포의 세포자살을 촉진시켜, 암세포를 사멸시킬 수 있다.
That is, the bamipolol and stigmasterol extracted from the immune cells inhibit the expression of anti-apoptosis genes Akt1 and bcl - 2 , thereby promoting apoptosis of cancer cells and killing cancer cells.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (12)
The isolated chloroform fraction obtained by adding chloroform to the remaining ethanol extract fraction was fractionated by silica gel column chromatography while sequentially adding methanol to the mobile phase of chloroform. The fraction having the highest activity was isolated by confirming inhibition of cancer cell growth, After fractionation by MPLC, the highest activity fraction was isolated by confirmation of the inhibition of cancer cell growth, followed by separation by preparative high performance liquid chromatography (HPLC) A method for separating a vomifoliol having anticancer activity from an immobilizing agent, which comprises separating and purifying a high compound.
[8] The method according to claim 7, wherein the step of separating the fraction having the highest activity through fractionation through MPLC and inhibiting the growth of cancer cells comprises the steps of (i) sequentially adding methanol to the chloroform mobile phase, Separating the fractions; (Ii) separating the fraction having the highest activity by sequentially adding methanol to the chloroform mobile phase in the fraction separated in the step (i); (Iii) separating the fraction having the highest activity by sequentially adding methanol to the chloroform mobile phase in the fraction separated in the step (ii); (Iv) sequentially adding methanol to the chloroform mobile phase to separate the fractions separated in step (iii), thereby separating the fraction having the highest activity, wherein the mobile phase of the preparative high performance liquid chromatography (HPLC) Water (50: 50 (v / v)). The method of separating the vomifoliol having anticancer activity from the immobilization.
The isolated chloroform fraction obtained by adding chloroform to the remaining ethanol extract fraction was fractionated by silica gel column chromatography while sequentially adding methanol to the mobile phase of chloroform. The fraction having the highest activity was isolated by confirming inhibition of cancer cell growth, The fractions with the highest activity were separated by reverse transcription chromatography (MPLC) to confirm the inhibition of cancer cell growth. Then, the fractions were separated by reversed-phase medium pressure chromatography (reverse MPLC) A method for separating stigmasterol having anticancer activity from a suspension comprising separating and purifying a high compound.
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