KR20120090118A - Anti-cancer and anti-cancer adjuvent composition comprising a stem bark extracts of albizzia julibrissin - Google Patents
Anti-cancer and anti-cancer adjuvent composition comprising a stem bark extracts of albizzia julibrissin Download PDFInfo
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- KR20120090118A KR20120090118A KR1020100139493A KR20100139493A KR20120090118A KR 20120090118 A KR20120090118 A KR 20120090118A KR 1020100139493 A KR1020100139493 A KR 1020100139493A KR 20100139493 A KR20100139493 A KR 20100139493A KR 20120090118 A KR20120090118 A KR 20120090118A
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- anticancer
- cancer
- bark
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- extract
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Abstract
Description
본 발명은 자귀나무 추출물을 포함하는 항암 또는 항암 보조용 조성물에 관한 것으로, 보다 상세하게는 자귀나무 추출물과 기존의 항암제를 포함하는 항암 또는 항암 보조용 조성물에 관한 것이다. The present invention relates to an anticancer or anticancer adjuvant composition comprising a silk tree extract, and more particularly, to an anticancer or anticancer adjuvant composition comprising a silk tree extract and an existing anticancer agent.
암이란 일반적으로 인체조직을 이루고 있는 세포의 세포주기에 이상이 생겨 정상적으로 분화하지 않고, 성장을 조절할 수 없이 커진 악성 종양을 말한다. 암은 개시(initiation), 촉진(promotion)및 진행 (progression)의 세 단계를 거쳐 발생하며, 충분한 양의 발암 물질만으로 발생될 수 있으나 실제적으로는 환경이나 음식물 속에 포함된 소량의 발암개시물질(initiator)에 의해 세포 돌연변이(개시세포)가 일어나고 이렇게 세포들의 수가 기하급수적으로 늘어나면서 발암 촉진물질(tumor promotor)의 자극에 의해 비정상적인 세포분열이 일어났을 때 비로소 암 조직이 형성된다고 알려져 있다. 환경의 악화와 노년 인구의 증가로 세계 암 발생률이 매년 5%이상 증가하고 있으며, 1997년 암을 원인으로 한 사망자가 600만 명으로 질병 사망률의 12%에 달했다. 국내에서도 매년 10만 명의 암 환자가 새롭게 발생하고 5만 여명이 매년 사망하고 있으며, 암환자 증가율도 10%에 이르고 있다. 1999년 국립암센터의 통계에 따르면 환자는 12 만명으로서 남자는 위암(24%), 간암(16%), 폐암(16%), 대장암(10%)의 순서이며, 여자는 위암(16%), 자궁경부암(12%), 유방암(15%), 대장암(10%)의 순서로 암 환자가 발생했다. Cancer generally refers to a malignant tumor that has grown abnormally in the cell cycle of the cells constituting the human tissue and does not differentiate normally and cannot control growth. Cancer occurs through three stages: initiation, promotion, and progression, and can occur with a sufficient amount of carcinogens, but in reality a small amount of carcinogens in the environment or food It is known that cancer tissues are formed only when abnormal cell division occurs due to stimulation of a tumor promotor as cell mutations (initiation cells) occur and the number of cells increases exponentially. Due to deterioration of the environment and the increase of the elderly population, the global cancer incidence is increasing by more than 5% every year. In 1997, 6 million people died of cancer, accounting for 12% of the mortality rate. In Korea, 100,000 new cases of cancer occur every year, about 50,000 people die every year, and the increase rate of cancer patients reaches 10%. According to the National Cancer Center's statistics in 1999, there were 120,000 patients, men in stomach cancer (24%), liver cancer (16%), lung cancer (16%), colon cancer (10%), and women in stomach cancer (16%). ), Cervical cancer (12%), breast cancer (15%) and colorectal cancer (10%).
이러한 암의 치료를 위하여 외과적 치료, 화학요법, 생물요법, 방사선요법 등이 이용되고 있다. 그 중에서도 항암제가 대표적으로 사용되고 있는데, 현재 사용중인 대부분의 항암제들은 심각한 독성 및 부작용으로 인해 사용이 제한적인데도 불구하고, 치료효과의 증강을 위하여 투여용량을 증가시키고 있어서 인체에 치명적이라는 문제점이 있다. 이러한 항암제로서, 5-플루러유러실(5-FU), 독소루비신(Doxorubicin)=아드리아마이신(Adriamycin), 미토마이신(Mitomycin), 시스플라틴(Cisplatin) 등이 많이 사용되고 있으며, 최근에 새로이 개발된 항암제로는 파클리탁셀(Paclitaxel), 도세탁셀(Docetaxel), 이리노테칸(Irinotecan), 젤로다(Xeloda), 옥살로플라틴(Oxalopatin) 등이 있다. 이 중에서도 특히, 독소루비신 (Doxorubicin)은 폐암, 소화 기관 암 등에 널리 사용되는 항암제이지만 심근병증 등의 부작용을 일으킬 수 있다는 문제점이 있다. 또한, 5-플루오로우라실(5-fluorouracil; 5-Fu)은 피리미딘 유도체로서 헥산대사를 억제하여 주로 유방암, 대장암, 위암, 췌장암등에 항암효과를 나타내지만 골수억제, 구강, 소화기 궤양, 간 및 신장손상등의 부작용이 있으며 (Katzung, B.G. Katzung's 임상약리학, 제8판, 대한의학서적, 서울, p977-1015, 2001), 사이클로포스파미드(cyclophosphamide; CY)는 알킬레이트화제로서 DNA 복제 및 RNA 전사를 억제하여 항암작용을 나타내며 급/만성 백혈병 및 기타 고형암에 널리 사용되는 항암제이지만 심각한 면역억제(면역독성) 작용을 나타낸다는 점으로 인하여 지속적인 사용이 어려운 실정이다. 이러한 기존에 사용되고 있는 항암제들의 독성 및 부작용을 경감시키기 위한 여러 약물 등이 현재 연구되고 있다. 예를 들어, 항암제와 인삼(Panax ginseng C.A. Meyer)으로부터 분획된 산성다당체를 병용투여한 예가 보고된 바 있다(Kim, Y.S. et.al., Arch. Pharm. Res. 13:330, 1990). 그런데 상기 종래기술은 항암제와 사이클로포스파미드와 인삼 산성다당체를 병용투여함으로써 면역독성 억제효과와 암세포(Sarcoma-180) 이식 마우스의 암발생 억제 및 망내계의 탐식활성 증대효과를 목적으로 하는 것이나, 항암제와 인삼 산성다당체의 병용투여에 의해서도 동등한 항암효과를 얻기 위해 필요로 하는 항암제의 투여용량을 현저히 저감시키지 못하여 항암제의 독성 및 부작용 문제를 근본적으로 해결하지는 못하였다. Surgical treatment, chemotherapy, biotherapy, radiotherapy and the like are used for the treatment of such cancer. Among them, anticancer drugs are typically used. Although most of the anticancer drugs currently in use are limited due to serious toxicity and side effects, there is a problem that they are fatal to the human body because of increasing the dosage to enhance the therapeutic effect. As such anti-cancer agents, 5-fluurerylsil (5-FU), doxorubicin (Axoamycin) = Adriamycin, mitomycin (Mitomycin), cisplatin (Cisplatin), etc. are widely used, and recently developed as an anticancer agent Paclitaxel, Docetaxel, Irinotecan, Xeloda, Oxaloplatin and the like. In particular, doxorubicin (Doxorubicin) is an anticancer agent widely used in lung cancer, gastrointestinal cancer, etc., but has a problem of causing side effects such as cardiomyopathy. In addition, 5-fluorouracil (5-Fu) is a pyrimidine derivative that inhibits hexane metabolism and mainly exhibits anticancer effects on breast cancer, colon cancer, gastric cancer, pancreatic cancer, etc., but myelosuppression, oral cavity, peptic ulcer and liver. And kidney damage (Katzung, BG Katzung's Clinical Pharmacology, 8th Edition, The Korean Medical Book, Seoul, p977-1015, 2001). Cyclophosphamide (CY) is an alkylating agent, It inhibits RNA transcription to show anticancer activity and is widely used in acute / chronic leukemia and other solid cancers, but it is difficult to use continuously due to its serious immunosuppressive (immunotoxic) action. Various drugs for reducing the toxicity and side effects of the existing anticancer drugs are currently being studied. For example, a combination of an anticancer agent and an acidic polysaccharide fractionated from Panax ginseng C.A.Meyer has been reported (Kim, Y.S. et.al., Arch. Pharm. Res. 13: 330, 1990). However, the above-mentioned conventional techniques are aimed at inhibiting immunotoxicity and inhibiting cancer development of cancer cells (Sarcoma-180) transplanted mice and enhancing phagocytic activity of the intranet system by administering a combination of an anticancer agent, cyclophosphamide, and ginseng acid polysaccharide. Combined administration of anticancer drugs and ginseng acid polysaccharides did not significantly reduce the dose of anticancer drugs required to achieve an equivalent anticancer effect, and did not fundamentally solve the toxicity and side effects of anticancer drugs.
본 발명은 상술한 바와 같은 문제점을 해결하기 위해 안출된 것으로서, 자귀나무 추출물과 항암제를 함유하는 항암 또는 항암 보조용 조성물을 제공하는 것을 목적으로 한다.The present invention has been made to solve the problems described above, and an object of the present invention is to provide an anticancer or anticancer composition comprising a silk tree extract and an anticancer agent.
또한 본 발명은 자귀나무 추출물과 항암제의 병용투여에 의해서 항암제의 적은 투여용량에서도 약물의 상승효과를 나타내어 항암 활성을 극대화시킬 수 있는 항암 또는 항암 보조용 조성물을 제공하는 것을 목적으로 한다. In addition, an object of the present invention is to provide an anticancer or anticancer adjuvant composition capable of maximizing anticancer activity by exhibiting synergistic effect of the drug even at a small dose of the anticancer agent by co-administration of the extract of the Albizia julibrissin and an anticancer agent.
아울러 본 발명은 적은 투여용량의 항암제를 사용함으로써 항암제 투여에 따른 독성 및 부작용을 줄일 수 있는 항암 또는 항암 보조용 약제학적 조성물을 제공하는 것을 목적으로 한다. In addition, an object of the present invention is to provide an anticancer or anticancer pharmaceutical composition that can reduce the toxicity and side effects of the anticancer drug administration by using a small dose of the anticancer agent.
상기 목적을 달성하기 위해, 본 발명은 자귀나무 껍질 추출물과 항암제를 포함하는 항암 또는 항암 보조용 조성물을 제공한다. 특히, 본 발명은 자귀나무 껍질 추출물과 독소루비신을 포함하는 것을 특징으로 하는 항암 또는 항암 보조용 조성물을 제공한다. In order to achieve the above object, the present invention provides an anticancer or anticancer composition comprising a bark extract and anticancer agent. In particular, the present invention provides an anticancer or anticancer composition comprising a bark extract and doxorubicin.
본 발명에 따른 자귀나무 껍질 추출물을 항암제 또는 항암 보조제로 사용할 경우, 기존의 항암제를 적은 투여량으로 사용하여도 약물의 상승효과가 나타나 항암 활성이 극대화될 수 있으며, 다량의 항암제 투여에 따른 독성 및 부작용도 줄일 수 있어 유용하다. When the bark extract according to the present invention is used as an anticancer agent or anticancer adjuvant, the synergistic effect of the drug may be maximized even when the existing anticancer agent is used in a small dose, and the anticancer activity may be maximized. It is also useful because it can reduce side effects.
도 1은 본 발명에 따른 자귀나무 추출물의 정제 공정 및 그의 항암 효과의 상승 작용을 조사를 위한 과정을 간단하게 도식화하여 나타낸 것이다.
도 2은 HPLC에 의한 자귀나무 추출물(HaBC18)의 분획물의 정제 공정을 도식화한 것이다.
도 3는 시료 HaBC18을 용매계 (MeOH/MeCN=80/20) 내에서 용해하여 GS320 컬럼으로 HPLC를 수행한 것이다 (각 분획물은 P1~P6을 나타냈다. 분획물은 UV(254nm) 와 IR detector로 검출하였다).
도 4은 HPLC에서의 HaBC18인 P4 분획물로부터 정제한 물질의 패턴을 나타낸 것으로, 용매계 (MeOH/MeCN=80/20) 내에서 GS320 컬럼으로 HPLC를 수행하였다. 분획물은 UV(254nm) 와 IR detector로 검출하였다.
도 5는 HPLC를 수행하여 HaBC18에서 얻은 정제 물질의 패턴과 TLC에서의 HaBC18 분획물로부터 정제된 세포 독성 물질 (Ha75F1)을 나타낸 것이다. 용매계 (MeOH/MeCN=80/20) 내에서 GS320 컬럼으로 HPLC를 수행하였다. ((A): 분획물은 UV(254nm) 와 IR detector로 검출, (B): 용매계 (MeOH/MeCN=80/20)는 RP-C18 TLC에서 developed 하였음. Anisadehyde 시약을 발색 시약으로 사용).
도 6는 Ha75F1의 질량 스펙트럼과 Julibroside III의 화학 구조를 나타낸 것이다. (A): Ha75F1의 질량 스펙트럼. EI-MS m/z (rel. int, %) 2219[M+] (B): 12C103 13CH164NO48의 분자식.1 is a schematic diagram illustrating a process for investigating the purification process and the synergistic effect of the anti-cancer effect of the extract of the silk tree according to the present invention.
Figure 2 is a schematic of the purification process of fractions of Albizia julibrissin extract (HaBC18) by HPLC.
FIG. 3 shows that the sample HaBC18 was dissolved in a solvent system (MeOH / MeCN = 80/20) and HPLC was performed on a GS320 column (each fraction showed P1 to P6. The fractions were detected by UV (254 nm) and an IR detector. ).
Figure 4 shows the pattern of the material purified from the P4 fraction, HaBC18 in HPLC, HPLC was performed on a GS320 column in a solvent system (MeOH / MeCN = 80/20). Fractions were detected by UV (254 nm) and IR detector.
FIG. 5 shows the pattern of purified material obtained on HaBC18 by HPLC and the cytotoxic material (Ha75F1) purified from the HaBC18 fraction in TLC. HPLC was performed on a GS320 column in a solvent system (MeOH / MeCN = 80/20). ((A): Fraction was detected by UV (254 nm) and IR detector, (B): Solvent system (MeOH / MeCN = 80/20) was developed in RP-C18 TLC using Anisadehyde reagent as color development reagent.
Figure 6 shows the mass spectrum of Ha75F1 and the chemical structure of Julibroside III. (A): Mass spectrum of Ha75F1. EI-MS m / z (rel. Int,%) 2219 [M + ] (B): Molecular formula of 12 C 103 13 CH 164 NO 48 .
자귀나무(A. julibrissin)는 우리나라에서 자생하고 있는 콩과식물로, 전통적으로 불면증, 이뇨, 강장, 구충 등에 동양의학의 약제로서 사용되어 왔는데12), 특히 자귀나무 껍질에는 자궁수축13), 설사 억제14), 수면15),16)등에 약리효과가 있다고 알려져 있다. 그 중에서도, 자귀나무 껍질의 트리테르페노이드 사포닌 (triterpenoid saponin)은 KB 암세포에 세포 독성을 나타낸다는 보고3)가 있었다. 그러나, 사포닌의 세포독성에 대한 체계적인 메카니즘 연구는 거의 이루어진바 없으며, 자귀나무의 껍질에 있는 구조가 다양한 사포닌1-6) 중 인체 급성백혈구세포인 Jurkat T 세포에 가장 강한 세포독성을 나타내는 사포닌 (Julibroside)의 구조와 특성은 아직 알려져 있지 않은 실정이다. Silk tree (A. julibrissin) is a legume that grows in the country, picked been traditionally used as a medicine for insomnia, oriental medicine, diuretic, tonic, anthelmintic, etc. 12), in particular, the hatchet 13 contractions bark), diarrhea It is known to have a pharmacological effect on inhibition 14) , sleep 15), 16) and the like. Among them, triterpenoid saponin of bark has been reported to be cytotoxic to KB cancer cells 3) . However, few systematic mechanisms of cytotoxicity of saponins have been studied, and saponins (Julibroside), which have the strongest cytotoxicity to Jurkat T cells, the human acute leukocytes of the various structures of saponins 1-6) in the bark of the silk tree The structure and characteristics of) are not known yet.
이러한 실정에서, 본 발명자들은 인체에 부작용을 일으키지 않으면서, 적은 용량의 항암제를 사용하더라도 상승 효과를 갖는 천연 재료를 찾고 연구하던 중, 자귀나무 껍질에서 부분 정제된 트리테르페노이드 사포닌을 사용하여 분취용 HPLC 상에서 세포 독성이 가장 강한 사포닌을 순수분리/정제하였는데, 분리된 세포 독성물질인“Ha75F1”은 질량 스펙트럼, 1H-NMR, 13C-NMR, HMQC 및 HMBC 스펙트럼을 통하여 구조를 확인한 결과, 분자량이 2219인 Julibroside III와 일치한다는 것을 발견하였다. 또한, 지금까지는 암세포에 대한 Julibroside III의 세포 독성이 알려진 바 없다. 특히, 현재 항암제로 사용되는 있는 독소루비신과 Julibroside III를 함께 사용한 경우, 약제의 상승 작용이 있는지에 대한 보고는 전혀 없었는데, 본 발명에서는 자귀나무 껍질 추출물에서 얻은 Julibroside III를 항암제로 사용되고 있는 독소루비신과 병용 투여시 약물의 상승작용에 의한 항암 활성의 상승 효과를 나타냄을 확인하였다 (실험예 3 참조). 따라서, 본 발명은 자귀나무 껍질 추출물을 포함하는 항암 또는 항암 보조용 조성물을 제공한다. In this situation, the inventors of the present invention, while searching for and studying a natural material having a synergistic effect even with a small dose of anticancer drugs without causing side effects on the human body, using the triterpenoid saponin partially purified from the bark of the bark Saponins with the highest cytotoxicity were purified / purified on preparative HPLC. The isolated cytotoxic substance, “Ha75F1”, was identified by mass spectra, 1 H-NMR, 13 C-NMR, HMQC and HMBC spectra. It was found to be consistent with Julibroside III having a molecular weight of 2219. In addition, until now, the cytotoxicity of Julibroside III against cancer cells is unknown. In particular, when doxorubicin and Julibroside III, which are currently used as an anticancer agent, are not reported, there is no report on whether there is a synergistic effect of the drug. It was confirmed that the synergistic effect of the anticancer activity due to the synergism of the drug (see Experimental Example 3). Therefore, the present invention provides an anticancer or anticancer composition comprising a bark extract.
이하, 본 발명을 더욱 상세하게 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명의‘자귀나무 껍질 추출물’은, 자귀나무 껍질에 추출 용매를 처리하여 얻은 것으로서, 추출 용매로는 물, 에탄올, 메탄올과 같은 저급알콜 또는 이들의 혼합용매로부터 선택된 용매를 사용할 수 있다. 자귀나무 껍질의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있으며, 추출 방법은 예를 들어 냉침 추출, 초음파 추출, 환류 냉각 추출, 열수추출, 초임계추출 등의 추출방법을 사용하여 추출할 수 있다. 상기 추출물에는, 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물도 포함된다. 본 발명의 구체적인 예에 의하면, 자귀나무 껍질은 통상 시판되는 것을 구입하여 사용할 수 있다. 구입한 자귀나무 껍질은 건조하여 분말로 만든 후 추출하여 본 발명에 따른 자귀나무 껍질 추출물을 제조할 수 있다. 더 구체적으로, 분말화한 자귀나무 껍질 시료에 메탄올을 가하여 감압 농축시킨 후, 분말화한 메탄올 자귀나무 껍질 추출물은 증류수에 용해하여 CH2Cl2, EtOAc 및 BuOH 등으로 분획하고, 다시 수회 감압 농축하여 얻은 분획물을 본 발명에 의한 자귀나무 껍질 추출물로 사용할 수 있다 (제조예 1, 도 2 참조). 더욱 구체적으로, 본 발명에 의하여 자귀나무 껍질 추출물로부터 추출하여 부분 정제한 정제 물질에 대한 세포 독성 시험을 수행하여, 세포 독성이 가장 강한 분획물을 얻었는데, 상기 얻은 분획물을 정제한 시료는 구조 분석 및 분자량을 측정 결과, Julibroside III (saponine)임을 확인할 수 있었다 (도 6B, 실험예 2). 따라서, 본 발명은 자귀나무 껍질 추출물을 이용한 Julibroside III의 순수 분리 방법도 제공한다. The 'Prune bark extract' of the present invention is obtained by treating the bark bark with an extraction solvent. As the extraction solvent, a solvent selected from lower alcohols such as water, ethanol and methanol, or a mixed solvent thereof can be used. The active ingredient of the bark tree bark can be extracted at room temperature or warmed under the conditions that are not destroyed or minimized, and the extraction method is, for example, cold extraction, ultrasonic extraction, reflux cooling extraction, hot water extraction, supercritical extraction, and the like. Can be extracted using. The extract also includes an extract obtained by an extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or these modifiers or purified products. According to a specific example of the present invention, the bark of the silk tree can be purchased commercially available. The purchased silk bark may be dried and then made into powder to extract the silk bark extract according to the present invention. More specifically, after adding methanol to the powdered powdered bark bark and concentrating under reduced pressure, the powdered methanol bark extract was dissolved in distilled water and fractionated with CH 2 Cl 2 , EtOAc and BuOH, and concentrated under reduced pressure several times. The fraction obtained by the above can be used as a silk tree bark extract according to the present invention (see Preparation Example 1, Figure 2). More specifically, according to the present invention by performing a cytotoxicity test on the purified material extracted and extracted from the bark bark extract, the fraction having the strongest cytotoxicity was obtained, the sample purified the obtained fractions were structural analysis and As a result of measuring the molecular weight, it could be confirmed that it was Julibroside III (saponine) (FIG. 6B, Experimental Example 2). Therefore, the present invention also provides a pure separation method of Julibroside III using the bark extract.
본 발명의 ‘항암 조성물’은 암 세포의 형성을 억제, 예방, 치료하는 효과를 갖는 조성물을 의미하는 것으로서, 구체적으로는 이미 형성된 암 세포의 크기를 억제하거나 세포 분열이 비정상적으로 왕성한 조직에 대하여 세포 증식을 억제하는 것, 인체 급성 혈액암 세포인 Jurkat T 세포에 대한 독성을 나타내는 것 모두를 포함한다. 본 발명에서는 항암 효과의 정도를 Jurkat T 세포에 대한 세포 독성 정도를 측정하여 평가하였다. "Anti-cancer composition" of the present invention refers to a composition having the effect of inhibiting, preventing and treating the formation of cancer cells, specifically, cells for tissues that suppress the size of cancer cells that have been formed or abnormally active cell division. It includes both inhibiting proliferation and showing toxicity against Jurkat T cells, which are human acute hematologic cancer cells. In the present invention, the degree of anticancer effect was evaluated by measuring the degree of cytotoxicity against Jurkat T cells.
앞서 설명한 바와 같이, 기존에 사용되고 있는 독소루비신 등과 같은 항암제는 오랜 시간 또는 다량으로 투여하면 심근병증 등의 부작용을 일으킬 수 있다는 문제점이 있었다. 상기 자귀나무 껍질 추출물은 적은 용량의 항암제와 병용하여도 목적하는 수준의 항암 효과를 나타낼 수 있도록 상승 작용을 가지므로 기존의 항암제와 함께 사용할 수 있다. 보다 구체적으로, 본 발명자들은 자귀나무 껍질 추출물의 BuOH 분획물인 HaBC18 (triterpenoid saponin)을 HPLC (GS320 컬럼)을 이용하여 순수 분리한 세포 독성이 강한 시료 (Ha75F1)를 얻었는데, 분리된 Ha75F1은 질량 스펙트럼, 1H-NMR, 13C-NMR, HMBC 및 HMQC 스펙트럼을 통하여 구조 분석 및 분자량 측정 결과 상기 시료가 Juribroside III임을 확인하였다. 따라서, 본 발명은 Juribroside III과 독소루비신을 포함하는 항암제 및 항암 보조용 조성물도 제공한다. As described above, an anticancer agent, such as doxorubicin, which has been used in the past, has a problem that it may cause side effects such as cardiomyopathy when administered for a long time or in a large amount. The silk tree bark extract has a synergistic effect to exhibit a desired level of anticancer effect even when used in combination with a small dose of anticancer agents, and thus can be used with conventional anticancer agents. More specifically, the present inventors obtained a cytotoxic sample (Ha75F1) from which HaBC18 (triterpenoid saponin), a BuOH fraction of Albizia bark extract, was purified by HPLC (GS320 column), and the isolated Ha75F1 mass spectrum , 1 H-NMR, 13 C-NMR, HMBC and HMQC spectra through the structural analysis and molecular weight measurement results confirmed that the sample was Juribroside III. Accordingly, the present invention also provides an anticancer agent and anticancer adjuvant composition comprising Juribroside III and doxorubicin.
자귀나무 껍질 추출물이 상기와 같은 항암효과를 나타내는지 탐색하기 위하여, 본 발명자들은 Jurkat T 세포에 대한 자귀나무 껍질 추출물의 독성 시험을 수행하였다. 자귀나무 껍질을 추출하여 얻은 분획물과 독소루비신의 병용 효과를 알아보기 위하여, 독소루비신과 자귀나무 껍질 분획물이 함유된 96-웰 플레이트에 Human Jurkat T 세포를 접종하여 배양한 후, 세포의 IC50을 측정하여 세포 독성 측정을 수행하였다 (실험예 3 참조). 상기 실험 결과, 본 발명에 의한 자귀나무 껍질 추출물과 독소루비신을 혼합하여 사용하는 경우, 독소루비신을 단독으로 사용하였을 때보다 Jurkat T 세포에 대한 세포 독성이 강하게 증가한다는 것을 발견하였다 (표 3 참조). 따라서, 본 발명에 따른 자귀나무 껍질 추출물과 독소루비신을 함유하는 항암 또는 항암 보조용 조성물은 다른 항암제 또는 기타 부형제와 함께 항암제로 사용될 수 있으며, 본 발명에 따른 자귀나무 껍질 추출물은 항암 보조제로서 기타의 항암제와 함께 항암 조성물로서 사용될 수 있다. 또한, 적은 양의 항암제를 사용하더라도 본 발명에 따른 자귀나무 껍질 추출물을 병용 투여하는 경우, 동일한 항암효능을 기대할 수 있으므로 항암제의 다량 사용에 의한 부작용도 피할 수 있다는 장점이 있다. In order to investigate whether the silkworm bark extract exhibits the anticancer effect as described above, the inventors conducted a toxicity test of the silkworm bark extract against Jurkat T cells. In order to examine the combined effect of the fraction obtained by extracting the bark and the doxorubicin, human Jurkat T cells were inoculated and cultured in 96-well plates containing the doxorubicin and the bark fraction, and the IC 50 of the cells was measured. Cytotoxicity measurements were performed (see Experimental Example 3). As a result of the above experiment, it was found that when mixed with the bark extract and doxorubicin according to the present invention, cytotoxicity against Jurkat T cells was increased more strongly than when doxorubicin alone was used (see Table 3). Therefore, the anti-cancer or anticancer composition containing the bark extract according to the present invention and doxorubicin may be used as an anticancer agent along with other anticancer agents or other excipients, and the bark extract according to the present invention is an anticancer agent as another anticancer agent. Together with the anticancer composition. In addition, even when a small amount of anticancer agent is used in combination administration of the bark extract according to the present invention, since the same anticancer effect can be expected, there is an advantage that side effects by using a large amount of anticancer agent can be avoided.
한편, 마우스의 육종 (sarcoma)에 대한 생체 실험에서 자귀나무 껍질 추출물인 Juribroside를 다량 주입하는 실험을 수행하였는데, 상기 실험 결과 마우스의 간이나 신장 등에 독성 징후가 없음을 확인하였다 (실험예 1 참조). 이로써, 본 발명에 의한 자귀나무 껍질 추출물은 신체에 유해하지 않으면서 독소루비신 등의 항암제와 함께 사용하는 경우, 독소루비신에 의한 부작용을 최소한으로 줄일 수 있음을 알 수 있다.
On the other hand, in vivo experiments on the sarcoma of the mouse was carried out experiments injecting a large amount of the bark extract Juribroside, it was confirmed that there are no signs of toxicity, such as liver or kidney of the mouse (see Experimental Example 1) . Thus, the bark extract according to the present invention can be seen that when used in combination with anticancer agents such as doxorubicin without harmful to the body, side effects caused by doxorubicin can be reduced to a minimum.
본 발명에 의한 자귀나무 껍질 추출물과 독소루비신은 생리학적으로 허용되는 담체와 배합하여 사용될 수 있다. 상기 생리학적으로 허용되는 담체로는 경구투여용 담체, 주사제용 담체 또는 국소투여용 담체를 들 수 있다. 보다 구체적인 경구투여용 담체의 예로는 결합제, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁제, 색소 또는 향료등을 들 수 있고, 주사제용 담체의 예로는 보존제, 무통화제, 가용화제 또는 안정화제등을 들 수 있으며, 그리고 국소투여용 담체의 예로는 기제, 부형제, 윤활제 또는 보존제등을 들 수 있다. 나아가 본 발명의 항암용 약제학적 조성물은 항암제 분야에서 당업자에게 첨가물로서 공지되어있는 다른 통상적인 성분들을 함유할 수 있다. 상기 첨가물로는 예를 들어, 경구투여시 약제가 위산에 의해 분해되는 것을 방지하기 위한 제산제등이 있다.The bark extract and doxorubicin according to the present invention can be used in combination with a physiologically acceptable carrier. Examples of the physiologically acceptable carrier include oral administration carriers, carriers for injection, or carriers for topical administration. More specific examples of carriers for oral administration include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, or flavoring agents. Examples of carriers for injection include preservatives, analgesics, Solubilizers or stabilizers, and examples of the carrier for topical administration include bases, excipients, lubricants, and preservatives. Furthermore, the anticancer pharmaceutical composition of the present invention may contain other conventional ingredients known as additives to those skilled in the art of anticancer drugs. The additives include, for example, an antacid to prevent the drug from being degraded by gastric acid upon oral administration.
또한, 본 발명에 의한 항암 또는 항암 보조용 조성물은 정제, 경질/연질 캅셀, 환제, 산제등의 고형제제 및 내복액제, 시럽, 현탁제등의 액제, 그리고 주사제등을 포함함으로써 경구적으로 투여되거나, 정맥내, 피하, 복강내 투여와 같이 비경구적으로 투여되거나, 또는 국소부위에 적용될 수 있다. 또한, 정제 등의 경구투여용 고형제제를 장용피로 피복된 제제로 제형화하여 투여할 수도 있다. 이 경우, 적정 투여량은 암의 종류와 중증정도, 연령, 환자의 상태등의 다양한 요인에 의해 변형될 수 있으며, 구체적인 투여량은 약제학적 분야에서 통상의 지식을 가진 자라면 후술하는 실시예 및 실험예를 토대로 하여 용이하게 추정할 수 있다.
In addition, the anticancer or anticancer composition according to the present invention may be administered orally by containing a solid preparation such as tablets, hard / soft capsules, pills, powders, and liquid preparations such as oral solution, syrup, suspension, and injections. It may be administered parenterally, such as intravenous, subcutaneous, intraperitoneal, or applied topically. In addition, oral administration solid preparations such as tablets may be formulated and administered in a preparation coated with enteric skin. In this case, the proper dosage may be modified by various factors such as the type and severity of cancer, age, and the condition of the patient, and the specific dosage is described below by those skilled in the art of pharmacy and It can be estimated easily based on an experimental example.
이하, 본 발명을 하기의 실시예 및 실험예에 의해 상세히 설명한다. 단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples. However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.
<< 제조예Manufacturing example 1> 자귀나무 껍질 추출물의 제조 1> Preparation of Alder Bark Extract
자귀나무 (Albizzia julibrissin.)의 껍질은 현대약업사(부산시 전포동)에서 구입(2007년 8월)한 것을 사용하였다. 자귀나무의 시료추출은 Won7 ) 등의 문헌에 기재된 방법에 따라 수행하였다. 즉, 자귀나무의 껍질분말(3.0㎏)에 메탄올 (MeOH) 10ℓ를 가하여 추출하였다. 추출된 MeOH 용액은 감압농축시키고, 분말화된 MeOH 추출물은 증류수에 용해하여, CH2Cl2, EtOAc 및 BuOH로 순차적으로 분획하고, BuOH분획물은 다시 감압농축하여 증류수에 녹인 후 Diaion HP20 레진 컬럼 크로마토그래피를 행하였다. 컬럼에 흡착된 물질은 증류수와 60% MeOH 용액을 순차적으로 충분히 씻어서 제거시킨 후 65%, 70% 및 80% MeOH 용액으로 용출되는 분획을 순차적으로 모았다. 이 분획물들 중에서 70% MeOH로 용출된 분획을 다시 감압 농축하고, MeOH에 용해하여 다시 Sephadex LH-20 gel filtration(Ф3x50 cm)을 행한 후 HPLC C18 컬럼 크로마토그래피를 행하였다. 여기서 얻은 각 분획들은 다시 감압 농축하여 TLC (n-BuOH:AcOH:H2O=4:1:5)를 행한 후 UV조사와 Anisaldehyde-H2SO4-HOAc로 발색하여 Rf값이 동일한 spot을 가진 분획들을 모아서 Jurkat T 세포에 강한 세포독성을 확인하였다. 이와 같이 얻어진 자귀나무 껍질 추출 분획물은 본 실험에 시료 “HaBC18”로 사용하였다. 하기 실험에서는 분획물을 더욱 정제하여 세포 독성이 강한 분획물을 얻고, 얻은 분획물의 구조 분석 및 상승적 항암 효과를 알아보았다.
Alder tree ( Albizzia The shell of julibrissin . was used by Hyundai Pharmaceutical Company (Jeonpo-dong, Busan) (August 2007). Sampling of the silk tree was performed according to the method described in Won 7 ) et al. That is, 10 liters of methanol (MeOH) was added to the bark powder (3.0 kg) of the silk tree and extracted. The extracted MeOH solution was concentrated under reduced pressure, and the powdered MeOH extract was dissolved in distilled water, fractionated sequentially into CH 2 Cl 2 , EtOAc and BuOH, and the BuOH fraction was concentrated under reduced pressure and dissolved in distilled water, followed by Diaion HP20 resin column chromatography. Photography was performed. The material adsorbed on the column was washed with distilled water and 60% MeOH solution sequentially and then removed, and the fractions eluted with 65%, 70% and 80% MeOH solution were collected sequentially. Among the fractions, the fractions eluted with 70% MeOH were concentrated under reduced pressure again, dissolved in MeOH, and again subjected to Sephadex LH-20 gel filtration (Ф3 × 50 cm), followed by HPLC C18 column chromatography. The fractions obtained here were concentrated under reduced pressure again, TLC (n-BuOH: AcOH: H2O = 4: 1: 5), followed by UV irradiation and Anisaldehyde-H2SO4-HOAc to form fractions with spots with the same Rf value. Strong cytotoxicity was confirmed on T cells. The silk bark extract fraction thus obtained was used as sample “HaBC18” in this experiment. In the following experiment, the fractions were further purified to obtain cytotoxic fractions, and the structural analysis and synergistic anticancer effect of the obtained fractions were examined.
<제조예 2> 시료 물질의 정제 Preparation Example 2 Purification of Sample Material
시료 HaBC18(3.96g)는 용매계(MeOH/MeCN=80/20)에 용해하여 recycling preparative HPLC(GS320 column)를 행한 결과 여러 물질이 섞여 있는 상태를 확인하였다 (도 3). 이에, 자귀나무의 껍질에서 추출하여 부분 정제된 “HaBC18”을 더욱 정제하기 위해 recycle용 HPLC(GS320 column)을 사용하였다. 즉, 시료 HaBC18을 용매계(MeOH/MeCN=8/2)에 녹인 다음 0.45?m filter로 여과한 후 HPLC(GS320 column)에 주입시킨 후 동일한 용매계로 용출하였다. 그 결과 각 단계별로 분리된 분획(P1-P5)들은 감압 농축한 다음 각각 인체 Jurkat T cell에 대한 세포 독성 시험을 수행하여, 이 중 가장 강한 세포독성을 나타내는 분획 P4 (Fr. no. 35-41)를 얻었다 (표 1 참조). P4 분획물은 다시 동일한 용매계에 용해시켜 HPLC로 4회 recycle을 행하여, 두 개의 분획물 P1 (Ha75F1), P2 (Ha75F2)를 얻었다 (도 4). 이때 분리된 각 분획물은 농축한 뒤 Jurkat T cells에 대해 독성시험을 행하였고, 이들 중 가장 세포독성이 강한 분획(Fr. no. 26?40)을 모아 감압 농축하였다. 상기 분획물의 Jurkat T 세포에 대한 IC50을 측정한 결과 P1 (Ha75F1)에서는 1.75ug/ml이였고 P2 (Ha75F2)에서는 5.15ug/ml으로 나타났다. 이에, P1 분획물은 2가지의 용매계를 사용하여 여러 번의 recycling을 수행하였고 최종적으로 단 하나의 peak를 나타내는 분획 (도 5A)을 확보하였으며, 이 분획물은 감압 농축하여 새로 조제된 용매계 (MeOH/MeCN/D.W = 80/20/42)에 녹인 다음 HPLC (동일한 GS320 컬럼)를 행하고 동일한 용매계로 6회 recycle하여 각 시료를 분획하였다. 이들 분획된 각 시료는 감압 농축하여 다시 상기와 같이 Jurkat T 세포에 대해 세포독성시험을 행하여 세포독성이 강한 분획(Fr. no. 30?37)을 얻었다. 이 분획은 동일한 용매계로 HPLC(10회 recycle)를 행하여 모든 불순물을 제거하고 Jurkat T 세포에 대해 세포독성을 가장 강하게 나타내는 분획 (Fr. no. 19?24)을 감압 농축하여 정제된 시료를 얻었다 (도 2). 상기 세포 독성을 가장 강하게 나타내는 분획 (Fr. no. 19~24)의 분획물을 TLC(RP-C18)행한 결과 하나의 spot를 확인하였다 (도 5B). 따라서 조정제 HaBC18 (3.96g)을 HPLC 후 Jurkat T cell에 대해 세포독성을 나타내는 정제된 물질(0.04g)을 얻었으며 이를 “Ha75F1”이라 명명하였고 수율은 1.01%였다.Sample HaBC18 (3.96 g) was dissolved in a solvent system (MeOH / MeCN = 80/20) and subjected to recycling preparative HPLC (GS320 column) to confirm the mixed state of several substances (FIG. 3). Accordingly, in order to further purify partially purified “HaBC18” extracted from the bark of the silk tree, recycled HPLC (GS320 column) was used. That is, sample HaBC18 was dissolved in a solvent system (MeOH / MeCN = 8/2), filtered through a 0.45 μm filter, injected into an HPLC (GS320 column), and eluted with the same solvent system. As a result, the fractions (P1-P5) separated in each step were concentrated under reduced pressure and then subjected to cytotoxicity tests on human Jurkat T cells, respectively. Among them, fraction P4 (Fr. no. 35-41) showed the strongest cytotoxicity. ) (See Table 1). The P4 fraction was again dissolved in the same solvent system and recycled four times by HPLC to obtain two fractions P1 (Ha75F1) and P2 (Ha75F2) (FIG. 4). At this time, the separated fractions were concentrated and subjected to toxicity test on Jurkat T cells, and the most cytotoxic fractions among them (Fr. no. 26-40) were concentrated under reduced pressure. IC 50 of the fraction of Jurkat T cells was measured to be 1.75 ug / ml in P1 (Ha75F1) and 5.15 ug / ml in P2 (Ha75F2). Thus, the P1 fraction was recycled several times using two solvent systems, and finally, a fraction showing only one peak was obtained (FIG. 5A). The fraction was concentrated under reduced pressure to prepare a newly prepared solvent system (MeOH / MeCN / DW = 80/20/42), followed by HPLC (same GS320 column) and recycled six times with the same solvent system to fractionate each sample. Each of these fractions was concentrated under reduced pressure and subjected to cytotoxicity test on Jurkat T cells as described above to obtain a cytotoxic fraction (Fr. no. 30 to 37). This fraction was subjected to HPLC (10 recycles) using the same solvent system to remove all impurities and concentrated under reduced pressure to obtain a purified sample (Fr. no. 19-24) which showed the strongest cytotoxicity against Jurkat T cells. 2). TLC (RP-C18) of the fraction of the fraction (Fr. no. 19-24) that shows the cytotoxicity was confirmed as one spot (Fig. 5B). Thus, the purified HaBC18 (3.96g) after purification to obtain a purified material (0.04g) showing cytotoxicity against Jurkat T cells, which was named "Ha75F1" and the yield was 1.01%.
<< 실험예Experimental Example 1> 1> JuribrosideJuribroside 의 안정성Stability
Juribroside III를 먹였을때 안전한지 여부를 조사하기 위하여, 마우스의 육종 (sarcoma)에 대한 생체 실험에서 Juribroside III를 100mg/kg의 양까지 주입하였다. 그러나, 마우스의 간이나 신장에 조직학적으로 독성을 나타내는 징후가 전혀 없음이 관찰되었다. 이로부터, 신체 내에 Juribroside III을 투여하여도 유해한 영향이 없을 것임을 알 수 있다.
To determine whether Juribroside III was safe for feeding, in vivo experiments on sarcoma of mice were injected with Juribroside III up to an amount of 100 mg / kg. However, no signs of histological toxicity were observed in the liver or kidney of mice. From this, it can be seen that the administration of Juribroside III in the body will not have a deleterious effect.
<실험예 2> 정제 물질의 순도 측정 및 구조 분석 Experimental Example 2 Purity Measurement and Structure Analysis of Purified Material
제조예 2에서 얻은 자귀나무 껍질 추출물의 정제된 시료 (Ha75F1)의 순도는 TLC에 의해 측정하였다. 즉, TLC는 전개 용매계(MeOH/MeCN/H2O=80/20/42)에서 RP-C18 TLC를 수행하였다. 또한 정제된 시료 Ha75F1의 구조분석을 위해 1H-NMR와 13C-NMR 스펙트럼은 JEOL JNM-ECR 스펙트럼을 사용하였고, HMQC와 HMBC 스펙트럼은 기울기 범위에서 파장을 사용하여 JEOL LNM-EPC으로 기록하였다. 또한, 정제된 Ha75F1의 구조를 알아보기 위해 1H-NMR, 13C-NMR, 및 HMQC 스펙트럼을 행하였다. 1H-NMR과 13C-NMR의 측정결, 그리고 HMQC (heteronuclear multiple quantum correlation)와 HMBC (heteronuclear multiple bond correlation)의 분석결과를 종합하여 표 2에 나타냈다. 분자량 측정을 위한 질량 스펙트럼은 Normal Ion [MF-Linear] spectrum type을 사용하였다. 그 결과, 분자량은 2219 (도 6A)였고, Chen 등2)에서 분리한 Julibroside I (MW; C102H164O48), Julibroside II(MW; C108H174O53), Julibroside III(MW; C103H164NO48)와 비교 결과 분리 정제한 saponine(Ha75F1)은 Julibroside III와 일치한다는 것을 발견하였다 (도 6B). 이로써, 본 발명에서는 Julibroside III의 순수 분리 방법도 확립되었다. Purity of the purified sample (Ha75F1) of the silk bark extract obtained in Preparation Example 2 was measured by TLC. That is, TLC performed RP-C18 TLC in a developing solvent system (MeOH / MeCN / H 2 O = 80/20/42). In addition, 1 H-NMR and 13 C-NMR spectra were used for the structural analysis of the purified sample Ha75F1, and JEOL JNM-ECR spectra were used, and HMQC and HMBC spectra were recorded by JEOL LNM-EPC using wavelengths in the gradient range. In addition, 1 H-NMR, 13 C-NMR, and HMQC spectra were performed to determine the structure of purified Ha75F1. Table 2 shows the results of 1 H-NMR and 13 C-NMR, and the results of analysis of heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC). The mass spectrum for the molecular weight measurement was used with the Normal Ion [MF-Linear] spectrum type. As a result, the molecular weight was 2219 (Fig. 6A), Chen, etc. 2) Julibroside I (MW isolated from; C 102 H 164 O 48) , Julibroside II (MW; C 108 H 174 O 53), Julibroside III (MW; C 103 H 164 NO 48 ), and found that the purified saponine (Ha75F1) is consistent with Julibroside III (Fig. 6B). Thus, the pure separation method of Julibroside III was also established in the present invention.
<< 실험예Experimental Example 3> 자귀나무 껍질 추출물과 3> Albizia bark extract 독소루비신의Doxorubicin 병용 효과 Combination Effect
제조예 2에서 정제된 Julibroside III와 독소루비신의 병용 효과를 Jurkat T 세포로 실험하였다. Canfield등이 기술한 방법11 )을 약간 변형하여 수행하였다. 즉, 독소루비신과 Juribroside III가 함유된 96-웰 마이크로플레이트에 Human Jurkat T세포 (2x105 cells/ml)를 접종하여 10% FBS가 함유된 RPMI 1640에서 CO2 incubator (5% CO2)에서 37℃, 48 시간 배양시킨 후 MTT assay로 세포독성을 측정하고, 독소루비신과 Juribroside III에 대한 분할 저해 농도 (FIC:fractional inhibitory concentrations)와 ΣFIC를 산출하여 이들 Julibroside III와 독소루비신의 상호작용을 검토하였다. 이때 상기 물질은 각각 10ug/ml로 사용하였다. 세포 독성 시험은, Ciapetti 등이 기술한 방법10 )을 약간 변형시켜서 수행하였다. 즉, Jurkat T세포에 대한 독성시험은 96-웰 마이크로플레이트의 각 웰에 RPMI 1640배지(100㎕)를 넣고, 별도로 시료(10mg)는 Dimethylsufoxide (DMSO) 1㎖에 녹여서 스톡 용액 (stock solution)으로 조제한 후 10% FBS와 1% 글루타민이 함유된 RPMI1640배지로 시료(1mg/㎖)를 희석시켰다. 배지가 함유된 플레이트의 각 웰에 시료농도가 500?g/㎖에서 0.25?g/㎖이 되도록 연속적으로 2배씩 희석시켰다. 그 후, Human Jurkat T (2x105 cells/ml)를 각 well에 접종한 후 48시간 동안 CO2항온기(37℃)에서 세포를 배양한 뒤 3(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide 방법 (MTT assay)으로 세포의 생육을 측정하여 세포 독성 시험을 수행하였다. 본 실험예에서 사용된 Human Jukat T(Jurkat E6-1)세포는 경북대학교 김 영호 교수에게서 분양받은 것을 사용하였으며, 이 암세포의 배양은 HEPES (Sigma, H-3784), 겐타마이신 (Sigma, G-1264), 탄산수소나트륨 (Sigma, S-5761), β-머캅토에탄올과 10% FBS (Fetal bovine serum: 소태야혈청)가 포함된 RPMI1640 (Sigma, R-6504)배지를 사용하였다.
The combined effect of Julibroside III and doxorubicin purified in Preparation Example 2 was tested with Jurkat T cells. Method 11 ) described by Canfield et al. Was carried out with a slight modification. Human Jurkat T cells (2x10 5 cells / ml) were inoculated into 96-well microplates containing doxorubicin and Juribroside III and 37 ° C in a CO 2 incubator (5% CO 2 ) at RPMI 1640 containing 10% FBS. After 48 hours of incubation, cytotoxicity was measured by MTT assay, and fractional inhibitory concentrations (FIC) and ΣFIC for doxorubicin and Juribroside III were calculated to examine the interaction of Julibroside III with doxorubicin. At this time, the material was used in each 10ug / ml. Cytotoxicity tests, methods such as a technique Ciapetti 10) was carried out by the slightly modified. In other words, to test the toxicity of Jurkat T cells, 1640 RPMI medium (100 µl) was added to each well of a 96-well microplate, and a separate sample (10 mg) was dissolved in 1 ml of Dimethylsufoxide (DMSO) as a stock solution. After preparation, samples (1 mg / ml) were diluted with RPMI 1640 medium containing 10% FBS and 1% glutamine. Each well of the plate containing the medium was continuously diluted 2-fold so that the sample concentration was from 500 g / ml to 0.25 g / ml. Human Jurkat T (2x10 5 cells / ml) was then inoculated into each well, followed by incubation of the cells in a CO 2 thermostat (37 ° C) for 48 hours, followed by 3 (4,5-dimethylthiazol-2-yl) -2. The cytotoxicity test was performed by measuring the growth of cells by the, 5-diphenyl-tetrazolium bromide method (MTT assay). Human Jukat T (Jurkat E6-1) cells used in this experiment were obtained from Professor Kim Young-ho of Kyungpook National University. The culture of these cancer cells was HEPES (Sigma, H-3784) and gentamicin (Sigma, G- 1264), RPMI1640 (Sigma, R-6504) medium containing sodium bicarbonate (Sigma, S-5761), β-mercaptoethanol and 10% FBS (Fetal bovine serum) was used.
실험 결과, 독소루비신을 단독으로 처리한 경우에는 IC50가 0.31㎍/ml, Julibroside III을 단독으로 처리한 경우의 IC50는 2.50㎍/ml인데 반하여, 상기 두 가지 물질을 병용처리한 경우에는 독소루비신과 Julibroside III의 IC50가 각각 0.16㎍/ml, 0.63㎍/ml로 Jurkat T 세포에 대한 독성에 있어서 강한 상승작용을 나타냈다 (표 3). 이상의 결과로, 자귀나무 껍질에서 순수 분리한 Julibroside III를 독소루비신과 함께 Jurkat T 세포에 처리했을 때, 아주 강한 세포독성 효과를 나타낸다는 것을 발견하였다. The experimental results, when the case of single treatment with doxorubicin is inde IC 50 in the case where the IC 50 as a single treatment 0.31㎍ / ml, Julibroside III is 2.50㎍ / ml In contrast, the combination of two materials, the treatment with doxorubicin The IC 50 of Julibroside III was 0.16 μg / ml and 0.63 μg / ml, respectively, indicating strong synergy in toxicity against Jurkat T cells (Table 3). As a result, it was found that Julibroside III purely isolated from bark of bark tree showed very strong cytotoxic effect when treated with Jurkat T cells with doxorubicin.
또한, 이 두 가지 약제에 대한 ∑FIC (sum FIC: fractional inhibitory concentrations)를 측정 결과값은 0.75였다. Canfield 등11 )이 기술한 바에 의하면, ∑FIC 값이 <1 이면 서로 상승작용(synergistic)을 나타내고, ∑FIC 값이 >1이면 서로 저해작용(antagonistic)을 나타내며, ∑FIC가 1과 동일하면 서로 무관하게 작용(indifferent)하는 것이다. 따라서 Julibroside III는 독소루비신과 병용 사용하는 경우, Jurkat T세포의 48시간 동안 배양한 결과에서 독소루비신과 Julibroside의 비율이 약 1.57/6.25나 1.57/12.5에서 ∑FIC의 값이 1보다 작았음으로 약 1/4-1/8의 혼합액을 사용하였을 때가 이들 약제를 단독으로 사용했을 때보다 Jurkat T세포에 대한 세포독성이 더욱 강하다는 것을 알 수 있다 (표 3). 이는 전보7 )에서 기술한 바와 같이 자귀나무의 BuOH 추출물에 함유된 Julibroside는 Jurkat T 세포에 대한 독성(IC50=2.5ug/ml)이 인간의 PBMC(IC50=52ug/ml)에 대한 독성 보다 크다는 것을 알 수 있다. 마우스의 육종 (sarcoma)에 대한 생체실험에서 Juribroside를 400mg/kg의 량까지 주입하여도 마우스의 간이나 신장에 조직학적으로 독성을 나타내는 징후가 전혀 없었다는 것을 고려할 때, Julibroside와 독소루비신의 혼합물을 사용하는 경우 목적하는 항암제 사용 효과를 동등한 수준으로 얻으면서, 동시에 독소루비신에 의한 부작용(cardiomyopathy)을 최소한으로 줄이는 것이 가능함을 알 수 있다. In addition, the result of measuring fractional inhibitory concentrations (sum FIC) for these two drugs was 0.75. Canfield et al. 11 ) described that ∑FIC values <1 indicate synergistic interactions; ∑FIC values> 1 indicate antagonistic interactions, and ∑FIC equals 1 It is independent. Therefore, when Julibroside III was used in combination with doxorubicin, the ratio of doxorubicin and Julibroside was about 1.57 / 6.25 or 1.57 / 12.5, which was less than 1 at about 1.57 / 6.25 or 1.57 / 12.5 when incubated for 48 hours with Jurkat T cells. It can be seen that the use of the mixed solution of 4-1 / 8 is more cytotoxic to Jurkat T cells than when these agents are used alone (Table 3). This suggests that Julibroside contained in BuOH extract of Albizia julibrissin, as described in Telegram 7 ) , was more toxic to Jurkat T cells (IC 50 = 2.5ug / ml) than human PBMC (IC 50 = 52ug / ml). You can see that it is large. Using a mixture of Julibroside and doxorubicin, in vivo, in vivo sarcoma studies showed no evidence of histologically toxic signs of mouse liver or kidney even when Juribroside was injected up to 400 mg / kg. In this case, it can be seen that it is possible to obtain the desired effect of using anticancer drugs at the same level, and at the same time, to minimize the cardiomyopathy caused by doxorubicin.
<실험예 4> 자귀나무 껍질 추출물과 독소루비신의 암세포 억제 효과Experimental Example 4 Inhibitory Effect of Alder Bark Extract and Doxorubicin on Cancer Cells
제조예 2에서 정제된 Julibroside III와 독소루비신의 바람직한 혼합 비율을 알아보기 위하여 다양한 비율로 혼합하여 세포 증식 억제 효과를 실험하였다. 실험 결과는 하기의 표 4에 나타냈다. 세포는 Jurkat T 세포를 사용하였다. In order to determine the preferred mixing ratio of Julibroside III and doxorubicin purified in Preparation Example 2, the cell growth inhibition effect was tested by mixing in various ratios. The experimental results are shown in Table 4 below. The cells used Jurkat T cells.
상기 결과에서 알 수 있는 바와 같이, 독소루비신과 Julibroside III를 병용 사용시 Jurkat T 세포의 증식을 효과적으로 억제할 수 있음을 알 수 있다. 독소루비신과 Julibroside III은 5~15: 2~40, 그 중에서도 1:1의 비율로 혼합하여 사용할 때, 약물의 상승 효과가 가장 우수하였다. As can be seen from the above results, it can be seen that the combination of doxorubicin and Julibroside III can effectively inhibit the proliferation of Jurkat T cells. Doxorubicin and Julibroside III had the highest synergistic effect of the drug when mixed in a ratio of 5 to 15: 2 to 40, and 1: 1.
또한, Julibroside III의 사용으로 인하여 기존의 항암제, 그 중에서도 독소루비신의 투여 용량을 어느 정도까지 줄일 수 있는지 알아보기 위하여 다양한 투여용량으로 독소루비신과 Julibroside III를 혼합하고 그에 대한 세포 증식 억제 효과를 실험하였다. 실험 결과는 하기의 표 5에 나타냈다. In addition, to determine the extent to which the dose of the existing anticancer agent, especially doxorubicin, can be reduced by the use of Julibroside III, doxorubicin and Julibroside III were mixed at various doses and tested for cell proliferation. The experimental results are shown in Table 5 below.
상기 결과에서 알 수 있는 바와 같이, 독소루비신과 Julibroside III를 병용 사용시 독소루비신의 투여량을 약 1/4까지 줄일 수 있다는 것을 알 수 있었다. As can be seen from the above results, when doxorubicin and Julibroside III are used in combination, it can be seen that the dose of doxorubicin can be reduced to about 1/4.
<< 실험예Experimental Example 3> 3> 육종암에On sarcoma cancer 대한 About JulibrosideJulibroside IIIIII 와 Wow 독소루비신의Doxorubicin 항암 효과 Anticancer effect
Julibroside III는 사포닌임으로 혈관 주사하면 적혈구가 파괴됨으로, 1%의 Na-CMC 용액에 Julibroside III를 혼합하여 경구투여하였다. 동시에 독소루비신은 마우스 (mouse) 체중 kg당 5mg이하의 양으로 복강 주사하여, Julibroside III와 독소루비신의 마우스 육종암(sarcoma 180)에 대한 항암효과를 조사하였다. 왼쪽 대퇴부에 sarcoma 180 세포 (1.3x106 cell/200ul)을 접종한 후 대퇴부에 암조직이 형성된 것이 확인된 마우스를 상기 실험에 사용하였다(약 3주간). 각 group당 10마리의 마우스를 사용하였으며, 1주일 간격으로 4회 투여한 후 1주일이 지난 후에 마우스를 해부하여 암조직의 무게를 측정하였다. 측정 결과는 하기의 표 6에 나타냈다.Julibroside III is saponin, and red blood cells are destroyed by vascular injection. Julibroside III was orally administered with 1% Na-CMC solution. At the same time, doxorubicin was injected intraperitoneally at an amount of less than 5 mg / kg of mouse weight to investigate the anticancer effect of Julibroside III and doxorubicin on mouse sarcoma (sarcoma 180). After inoculating sarcoma 180 cells (1.3 × 10 6 cells / 200 ul) in the left thigh, mice were confirmed to have formed cancer tissue in the thigh (about 3 weeks). Ten mice were used in each group, and four weeks at weekly intervals, one week later, the mice were dissected to measure the weight of the cancer tissue. The measurement results are shown in Table 6 below.
상기에서 살펴본 바와 같이, 본 발명에 의해서 암세포에 가장 강한 세포 독성을 나타내는 자귀나무 Julibroside III의 분리/정제 방법이 확립되었다. 또한, 본 발명의 자귀나무 껍질 추출물의 분획물인 Juribroside III은 항암제(독소루비신)와 함께 병용 사용시, 항암제의 항암 활성을 더욱 상승시킨다는 작용을 한다는 것을 알 수 있다. 따라서, 자귀나무 껍질 추출물은 기존의 항암제와 함께 사용할 수 있으며, 병용 사용시 항암제의 사용량을 현저히 줄일 수 있으므로 기존에 사용되던 항암제의 다량 투여로 인한 부작용 및 독성도 줄일 수 있는 바, 항암 또는 항암 보조를 위한 보조 약품 또는 기능성 식품 등으로 광범위하게 사용될 수 있어서 유용하다. As described above, according to the present invention, a method for isolating / purifying the silk tree Julibroside III, which exhibits the strongest cytotoxicity to cancer cells, has been established. In addition, it can be seen that Juribroside III, a fraction of the bark extract of the present invention, when used in combination with an anticancer agent (doxorubicin), further increases the anticancer activity of the anticancer agent. Therefore, the bark extract can be used together with the existing anticancer drugs, and when combined use can significantly reduce the amount of anticancer drugs used to reduce the side effects and toxicity caused by the large dose of conventional anticancer drugs, anti-cancer or anticancer aid It is useful because it can be widely used as an auxiliary medicine or a functional food.
참고 문헌references
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9) Joshi G, Sukhsana R, Tangpong J, Cole MP, Clair ST, Vore M., Estus S, Butterfield DA. Free radical mediated oxidative stress and toxic side effects in brain induced by the anti cancer adriamycin: Insight into chemobrain. Free Radical Research. 39, 1147-1154, 2005.9) Joshi G, Sukhsana R, Tangpong J, Cole MP, Clair ST, Vore M., Estus S, Butterfield DA. Free radical mediated oxidative stress and toxic side effects in brain induced by the anti cancer adriamycin: Insight into chemobrain. Free Radical Research. 39, 1147-1154, 2005.
10) Ciapetti G, Cenni E., Pratelli L, and Pizzoferrato A. In vitro valuation of cell/biomaterial interaction by MTT assay. Biomaterials. 14, 359-364, 1993. 10) Ciapetti G, Cenni E., Pratelli L, and Pizzoferrato A. In vitro valuation of cell / biomaterial interaction by MTT assay. Biomaterials. 14, 359-364, 1993.
11) Canfield CJ, Pudney M, Gutteridge WE. Interaction of Atovaquone with other antimalarial drugs against Plasmodium flaciparum in vitro. Experimental Parasitology. 80, 373-381, 1995. 11) Canfield CJ, Pudney M, Gutteridge WE. Interaction of Atovaquone with other antimalarial drugs against Plasmodium flaciparum in vitro. Experimental Parasitology. 80, 373-381, 1995.
12) Kim TJ, Korean resources plants, II, Seoul National University Press, Korea, pp.194-195, 1996.12) Kim TJ, Korean resources plants, II, Seoul National University Press, Korea, pp.194-195, 1996.
13) Woo WS, Lee EB, Shin KH. A review of research on plants for fertility regulation in Korea. Korean Journal of Pharmacology. 12, 153-170, 1981. 13) Woo WS, Lee EB, Shin KH. A review of research on plants for fertility regulation in Korea. Korean Journal of Pharmacology . 12, 153-170, 1981.
14) Yoo JS, Jang JS, Lee TH, Son KH, Suh HW, Song DK, Kim YH. Inhibitory effect of extracts form traditional herbal drugs on 5-Hydroxytryptophan- induced diarrhea in mice. Korean Journal of Pharmacology. 26, 355-359, 1995. 14) Yoo JS, Jang JS, Lee TH, Son KH, Suh HW, Song DK, Kim YH. Inhibitory effect of extracts form traditional herbal drugs on 5-Hydroxytryptophan- induced diarrhea in mice. Korean Journal of Pharmacology. 26, 355-359, 1995.
15) Kang TH, Jeong ST, Kim NY, Higuchi R, Kim YC, Sedactive activity of two flavonol glycosides isolated from the flower of Albizza julibrissin Durazz, Journal of Ethnopharmacology. 71, 321-323, 2000. 15) Kang TH, Jeong ST, Kim NY, Higuchi R, Kim YC, Sedactive activity of two flavonol glycosides isolated from the flower of Albizza julibrissin Durazz, Journal of Ethnopharmacology. 71, 321-323, 2000.
16) Jung JW, Cho JH, Ahn NY, Oh HR, Kim SY, Jang CG, Ryu JH. Effect of chronic Albizzia julibrissin treatment on 5-hydroxytryptamine1A receptors in rat brain. Pharmacology , Biochemistry and Behavior . 81, 205-210, 2005.16) Jung JW, Cho JH, Ahn NY, Oh HR, Kim SY, Jang CG, Ryu JH. Effect of chronic Albizzia julibrissin treatment on 5-hydroxytryptamine 1A receptors in rat brain. Pharmacology , Biochemistry and Behavior . 81, 205-210, 2005.
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| KR20220153547A (en) | 2020-07-10 | 2022-11-18 | 한국화학연구원 | Medicament reaction analyzing method using medicament reaction container |
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