KR100537437B1 - Anticancer drug composition containing swellfish extract - Google Patents

Anticancer drug composition containing swellfish extract Download PDF

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KR100537437B1
KR100537437B1 KR10-2002-0056318A KR20020056318A KR100537437B1 KR 100537437 B1 KR100537437 B1 KR 100537437B1 KR 20020056318 A KR20020056318 A KR 20020056318A KR 100537437 B1 KR100537437 B1 KR 100537437B1
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cancer
pufferfish
egg extract
extract
blowfish
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김익수
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56Materials from animals other than mammals
    • A61K35/60Fish, e.g. seahorses; Fish eggs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

본 발명은 복어 알의 수성 추출물을 유방암, 위암, 간암, 난소암 등 다양한 암을 치료할 수 있는 항암제 조성물로 이용하고자 하는 것이다. 이러한 본 발명은 복어 알 1 중량부에 대하여 3 중량부의 물을 가하여 가열 추출한 후 여과 또는 원심 분리하여 얻은 복어 알 추출물을 주성분으로 함유하는 항암제 조성물로서 암환자에게 투여 시 항암 효과를 얻을 수 있고, 암환자의 통증을 제어하는 효과도 탁월하여 통증을 호소하는 암환자에게 투여 시 항암 치료효과는 물론 진통효과까지도 함께 얻어지는 장점을 가지고 있다.The present invention is intended to use the aqueous extract of pufferfish eggs as an anticancer composition that can treat a variety of cancers, such as breast cancer, stomach cancer, liver cancer, ovarian cancer. The present invention is an anticancer composition containing a blowfish egg extract obtained by filtration or centrifugation after heating by adding 3 parts by weight of water to 1 part by weight of pufferfish eggs to obtain an anticancer effect when administered to cancer patients. The effect of controlling the pain of the patient is also excellent, and when administered to cancer patients complaining of pain, the anti-cancer treatment effect as well as the analgesic effect is obtained.

그 작용 기작은 암세포만을 특이적으로 선별하여 세포고사 기작을 통한 세포사멸로 유도하므로 정상 세포에 대한 독성이 낮은 안전한 항암제로 개발될 수 있는 것이다.The mechanism of action can be developed as a safe anticancer agent with low toxicity to normal cells because it specifically selects only cancer cells and induces apoptosis through apoptosis mechanism.

Description

복어 알 추출물을 함유하는 항암제 조성물{ANTICANCER DRUG COMPOSITION CONTAINING SWELLFISH EXTRACT}Anticancer agent composition containing blowfish egg extract {ANTICANCER DRUG COMPOSITION CONTAINING SWELLFISH EXTRACT}

본 발명은 복어 알 추출물에 함유된 항암제 조성물에 관한 것으로, 더욱 상세하게는, 복어 알의 수성 추출물을 유방암, 간암, 난소암 등 다양한 암을 치료함과 아울러 말기 암 환자의 통증을 제거할 수 있는 항암제 조성물로 이용하고자 하는 것이다.The present invention relates to an anticancer composition contained in a blowfish egg extract, and more particularly, the aqueous extract of blowfish eggs can be used to treat various cancers such as breast cancer, liver cancer and ovarian cancer as well as to remove the pain of terminal cancer patients. It is intended to be used as an anticancer composition.

일반적으로, 복어 알 추출물이 항암효과가 있다는 과학적인 자료는 미약하고, 다만 민간 요법으로 암 치료에 이용하고 있을 뿐이다. 그러나 복어 알에는 독성이 강한 테트로도톡신(tetrodotoxin: TTX) 성분이 함유되어 있다는 것이 오랫동안 알려져 왔고, 그 복어 독의 화학적인 구조와 약리작용에 대해 비교적 상세히 밝혀져 있다. In general, the scientific data that fugu egg extract has anti-cancer effect is weak, but only folk remedies are used to treat cancer. However, it has long been known that blowfish eggs contain the highly toxic tetrodotoxin (TTX) component, and the chemical structure and pharmacological action of the blowfish poison are relatively detailed.

복어 독의 화학적 성분은 극독성의 비단백 신경독소로서, 일종의 아미노산 수산퀴놀린형의 구조를 갖는 화합물이다. 복어 독은 활무늬 반점이 있는 복어(궁반동 방동), 벌레무늬가 있는 복어(충문동 방돈) 및 암색동 방돈의 고환, 난소, 간장, 비장, 안구 및 혈액 내에 존재하고, 그 작용으로 「독약본초」에는 복어 독이 대독하며 진통, 항심율실상, 혈압강하 등을 치료하고 문둥병으로 인한 동통을 치료하는 효능이 있다고 적혀있다.The chemical component of the pufferfish poison is a highly toxic nonprotein neurotoxin, which is a compound having a structure of an amino acid quinoline type. Pufferfish poison is present in the testes, ovaries, soy, spleen, eyeballs and blood of pufferfish (bowungdongdong), insect patterned pufferfish (chongmundong), and dark sakdong piglets. The herbal medicine says that pufferfish poison is toxic and it is effective in treating pain, real heart rate, lowering blood pressure and treating pain caused by leprosy.

복어 독의 약리 작용에 대해 많은 연구가 이루어져 있으나 본 발명의 목적에 부합되는 대표적인 약리작용은 다음과 같다.Although many studies have been made on the pharmacological action of pufferfish poison, the representative pharmacological actions in accordance with the purpose of the present invention are as follows.

첫째, 세포분열에 대한 영향으로 복어 독은 작은 쥐의 골수세포 미핵 및 분열지수의 산생에 현저한 영향을 주며, 장춘(長春)(Vincristine Siltate, Vincristine. Leurocristine. On-cavin. Vcr. Nsc-67574. Lcr. Leucicl. Kyocristine)과 비교할 때, 미핵지표상으로는 장춘의 수준에 달하지 못하지만 분열지수상으로는 장춘을 능가하는 효과가 있어 비교적 강한 세포유사분열을 억제하는 작용이 있다.Firstly, blowfish poison has a significant effect on the production of the micronucleus and division index of bone marrow cells in small rats due to its effect on cell division. Changchun (Vincristine Siltate, Vincristine. Leurocristine. On-cavin. Vcr. Nsc-67574. Compared with Lcr. Leucicl. Kyocristine), it does not reach the level of Changchun on U.S. nucleus index, but it has the effect of exceeding Changchun on the division index.

둘째, 진통 효과에 관한 것으로, 복어 간 중에서 증류시켜 뽑아낸 복어 독 산(酸) 주사액은 그 진통 효과가 모르핀, 두령정(杜令丁, Pethidine Hydrochoride)에 상당하여 둔통이나 예통에 대하여 완화 작용을 할 수 있다.Secondly, it relates to analgesic effect. Pufferfish poisonous acid extract extracted from the pufferfish liver has an analgesic effect equivalent to morphine and durinjeong (Petryin, Pethidine Hydrochoride), which alleviate the effect of blunt pain and cramps. can do.

셋째, 복어 독은 국소 마취약과 함께 사용하였을 때 마취시간을 연장시키는 작용 등이 알려져 있다.Third, blowfish poison is known to prolong anesthesia when used in conjunction with local anesthetics.

본 발명의 목적은 복어 알에서 항암제 역할을 하면서 동시에 암 환자의 통증을 장기적으로 제어하기 위한 항암제 조성물을 제공하는 것이다. 진통효과는 복어 독인 테트로도톡신(tetrodotoxin: TTX)에 의한 것임이 분명하나 항암효과는 TTX라기 보다는 TTX 이외의 성분으로 사료되고, TTX는 강력한 호흡마비 작용 때문에 적당량 이상으로 투여될 때 치명적인 결과를 초래할 수 있다. 따라서 본 발명은 이 두 물질을 분리정제하고 다시 재조합하는 과정을 통하여 항암효과를 증대시키면서 진통효과를 조절하는 것이 당면 과제이나 궁국적으로는 항암효과를 갖는 물질의 구조를 밝히는 것이다. It is an object of the present invention to provide an anticancer composition for acting as an anticancer agent in pufferfish eggs for the long term control of pain in cancer patients. The analgesic effect is obviously due to the pufferfish poison tetrodotoxin (TTX), but the anticancer effect is considered to be a component other than TTX rather than TTX, and TTX can have fatal effects when administered in an appropriate amount due to its strong respiratory paralysis. . Therefore, the present invention is to identify the structure of a substance having an anticancer effect, but ultimately to control the analgesic effect while increasing the anticancer effect through the process of separating and refining and recombining these two substances.

상기 목적을 달성하기 위하여 본 발명은, 복어 알 추출물을 활성성분으로 함유하는 항암제 조성물을 제공한다.In order to achieve the above object, the present invention provides an anticancer composition containing a blowfish egg extract as an active ingredient.

여기에서, 본 발명의 복어 알 추출물은 복어 알 1 중량부에 대하여 2∼3 중량부의 물을 가하여 가열 추출한 후 여과 또는 원심분리하여 얻은 여액인 것이 바람직하다.Here, the pufferfish egg extract of the present invention is preferably a filtrate obtained by heating and extracting by adding 2-3 parts by weight of water to 1 part by weight of pufferfish eggs.

구체적으로, 본 발명의 복어 알 추출물은 복어 알 1 중량부(1 Kg)에 대하여 3 중량부의 물(3,000 mL)을 가하여 약 6 시간 동안 100℃로 가열하면서 추출하고, 추출물을 여과 또는 원심분리한 후 상등액을 취하여 얻을 수 있다.Specifically, the pufferfish egg extract of the present invention is added to 3 parts by weight of water (3,000 mL) with respect to 1 part by weight (1 Kg) of pufferfish eggs and extracted while heating to 100 ℃ for about 6 hours, the extract is filtered or centrifuged It can then be obtained by taking the supernatant.

상기와 같이 제조된 본 발명의 복어 알 추출물에는, 복용상의 편의를 위하여 약학적으로 허용되는 첨가제, 예를 들어 부형제, 안정화제, 방향제, 교미제 등을 첨가하여, 통상의 산제, 과립제, 캅셀제 및 정제의 제법에 따라 제형화될 수 있다.To the puffer fish egg extract of the present invention prepared as described above, pharmaceutically acceptable additives, for example, excipients, stabilizers, fragrances, copulation agents, etc. for the convenience of taking, to add conventional powders, granules, capsules and It may be formulated according to the preparation of the tablet.

본 발명의 항암제 조성물은 복어 알 추출물 외에 활성성분으로서 5-플루오로우라실, 메토트렉세이트, 아드리아마이신 및 탁솔로 이루어진 군으로부터 선택된 적어도 하나의 항암제를 더욱 포함할 수 있다.The anticancer agent composition of the present invention may further include at least one anticancer agent selected from the group consisting of 5-fluorouracil, methotrexate, adriamycin and taxol as an active ingredient in addition to the pufferfish egg extract.

본 발명의 복어 알 추출물에 더욱 포함될 수 있는 항암제에는 특별한 제한이 없고, 단독 또는 이들의 혼합물이 사용될 수 있다. 본 발명의 복어 알 추출물에 이들 항암제로 공지된 다른 활성 성분이 첨가됨으로써 상승적 항암 효과를 얻을 수 있다.There is no particular limitation on the anticancer agent that may be further included in the pufferfish egg extract of the present invention, alone or a mixture thereof may be used. Synergistic anticancer effect can be obtained by adding the other active ingredients known as these anticancer agents to the blowfish egg extract of this invention.

본 발명의 복어 알 추출물을 항암제로 사용하는데 있어 항암성분과 복어 독인 TTX가 함께 포함되어 있으므로 투여에 있어 세심한 주의를 요한다. 따라서 성인의 경우 1 일 50∼150 ㎖ 의 양을 1∼3 회에 나누어 투여할 수 있으며, 투여 경로는 경구로 한다.In using the pufferfish egg extract of the present invention as an anticancer agent, since the anticancer component and the pufferfish poison TTX are included together, careful care is required in administration. Therefore, the adult can be administered by dividing the amount of 50 ~ 150 ㎖ daily 1 to 3 times, the route of administration is oral.

본 발명에 따른 복어 알 추출물을 항암제로 사용할 수 있으려면, 정상 세포에 대해서는 세포사멸의 작용이 미약하고, 암 세포에 대해서는 강력한 세포사멸의 효과를 나타내고 임상에서 사용될 수 있을 정도로 독성이 적어야 할 것이다. 이에 따라, 본 발명의 복어 알 추출물의 정상 세포와 암 세포에 대한 세포사멸 효과를 검색하기 위하여 정상 세포로는 임파구(lymphocyte) 및 성상세포(astrocyte)와 이에 상응하는 암세포 주로는 각각 HL-60과 U937, 그리고 C6 glioma 세포 등을 배양하면서 복어 알 추출물을 첨가하여 세포생존율 시험(viability test)을 실시하였다.To be able to use the pufferfish egg extract according to the present invention as an anticancer agent, the effect of apoptosis on the normal cells is weak, the effect of strong apoptosis on the cancer cells should be low enough to be used in the clinic. Accordingly, in order to detect the apoptosis effect of the pufferfish egg extract of the present invention on normal cells and cancer cells, lymphocytes (astmphocytes) and astrocytes (astrocytes) and corresponding cancer cells are mainly HL-60 and The viability test was performed by adding puffer egg extract while culturing U937 and C6 glioma cells.

또한, 본 발명의 복어 알 추출물이 암세포를 사멸시키는 작용이 세포고사에 의한 것인지를 분석하기 위하여 암세포 주를 배양하면서 복어 알 추출물을 첨가하여 세포고사의 전형적인 현상인 핵 분절현상, 미토콘드리아의 손상과 이에 따른 세포질 내의 분포를 플로우 사이토메트리(flow cytometry)에 의해 분석하였고, 카스파제프로테아제류의 활성변화, 이 효소에 의한 손상된 DNA 복구에 필요한 PARP의 절단, 그리고 카스파제 활성에 영향을 미치는 Bid의 절단 등을 관찰하였다. 그 결과 본 발명의 복어 알 추출물은 암세포를 특이적으로 선별하여 세포고사 기작을 통해 암세포를 사멸로 유도하고 있음을 알 수 있었다.In addition, the blowfish egg extract of the present invention by adding a blowfish egg extract while culturing the cancer cell line to analyze whether the action of killing cancer cells by the cell death, nuclear fragmentation, the damage of mitochondria and the typical phenomenon of cell death The distribution in the cytoplasm was analyzed by flow cytometry and changes in the activity of caspase proteases, the cleavage of PARP required for repair of damaged DNA by this enzyme, and the cleavage of Bids affecting caspase activity. Etc. were observed. As a result, the blowfish egg extract of the present invention was specifically selected to induce cancer cells by killing the cancer cells through apoptosis mechanism.

이하, 실시예를 통하여 본 발명에 따른 복어 알 추출물을 포함하는 항암제 조성물의 제조 및 작용 효과를 상세히 설명한다.Hereinafter, the preparation and action effects of the anticancer agent composition comprising a blowfish roe extract according to the present invention will be described in detail through Examples.

제조예 1: 복어 알 추출물의 제조 Preparation Example 1 Preparation of Blowfish Egg Extract

복어를 개복하여 알 1 kg을 취한 후 물 3 ℓ를 넣고 약 100 ℃로 약 3 시간 동안 가열하면서 추출하고, 20,000 x g에서 20분 동안 원심분리하여 그 상등액 2.850 ㎖ 취하였다. 이때 본 발명의 복어 알 추출물의 수득율은 약 95% 였다.Pufferfish was opened, 1 kg of eggs were taken, 3 liters of water was added thereto, and the extract was heated to about 100 ° C. for about 3 hours, followed by centrifugation at 20,000 × g for 20 minutes, and 2.850 ml of the supernatant was taken. At this time, the yield of blowfish egg extract of the present invention was about 95%.

실시예 1: 복어 알 추출물에 의한 정상세포와 암세포의 세포사멸 작용 Example 1 Apoptosis of Normal and Cancer Cells by Blowfish Egg Extract

실험에 사용한 정상 임파구(lymphocytes)는 성인남자 말초 혈액 50㎖를 채취하여 임파구 분리 멸균용액인 Ficoll paque TM plus(Amersham pharmacia biotech)을 이용하여 정상 임파구만을 분리하여 사용하였고, 정상 성상세포(astrocytes)와 HL-60, U937, C6 glioma 등 실험에 사용한 모든 암세포 주는 원광대학교 의과대학 미생물학 교실에 보관중인 세포 주를 사용하였다.Normal lymphocytes (lymphocytes) used in the experiment were collected from 50 ml of adult male peripheral blood and separated from normal lymphocytes using Ficoll paque TM plus (Amersham pharmacia biotech), which is a sterile solution for lymphocyte separation. All cancer cell lines used in the experiments, such as HL-60, U937, and C6 glioma, were used in the microbiology department of Wonkwang University School of Medicine.

1-1. 세포 생존율 측정(Viability Test)1-1. Cell Viability Test

세포사멸은 세포생존율로 측정하였으며 그 세포생존율 측정을 위한 MTT assay는 다음과 같이 시행하였다. 먼저, 실험에 사용할 세포를 24-웰(well) 세포 배양판에 1×105 cells/㎖씩 분주하여 3시간 안정화시키고, 안정화된 세포에 본 발명의 복어 알 추출물을 농도별로 처리하여 일정시간 후에 MTT(0.5㎎/㎖)와 3시간 반응시켰다. 살아있는 세포에 의해 MTT로부터 생성된 보라색 불용성 formazan은 10% SDS로 용해하여 540㎚ 파장에서 ELISA reader(THERMA max, U.S.A)로 흡광도를 측정하였다. 측정한 formazan 생성정도는 정상적인 세포의 값과 비교하여 백분율(%)로 표시하였다.Apoptosis was measured by cell viability, and MTT assay for cell viability was performed as follows. First, the cells to be used for experiments were stabilized for 3 hours by dispensing 1 × 10 5 cells / ml in a 24-well cell culture plate, and the pufferfish egg extract of the present invention was treated to stabilized cells by concentration. It was reacted with MTT (0.5 mg / ml) for 3 hours. Purple insoluble formazan generated from MTT by viable cells was dissolved in 10% SDS and absorbance was measured by ELISA reader (THERMA max, USA) at 540 nm wavelength. Measured formazan production was expressed as a percentage (%) compared to the normal cell value.

1-2. 정상세포와 암세포에서의 세포사멸1-2. Apoptosis in Normal and Cancer Cells

정상 임파구(lymphocytes)와 이에 상응하는 백혈병 암세포 주인 HL-60, 및 U937, 그리고 정상 성상세포(Astrocyte)와 이에 상응하는 신경교종(C6 glioma)세포에 본 발명의 복어 알 추출물을 전체 배지용적의 10, 20, 30, 40, 50 %가 되도록 처리하고, 24시간 후 MTT assay를 이용하여 세포생존율을 측정하여 사멸되는 정도를 측정하였다. 도 1에서, (A)는 정상 임파구(lymphocytes)에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타낸다. (A)와 (C)는 정상 임파구(lymphocytes)에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타낸다. (B)는 암세포 주 HL-60에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타내며, (D)는 암세포 주 U937에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타낸다. 그리고 도 2에서, (A)는 정상 성상세포(Astrocyte)에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타낸다. (B)는 암세포 주 C6 glioma에 본 발명의 복어 알 추출물을 농도별로 처리한 결과 세포생존율을 도시한 그래프로서, 횡축은 복어 알 추출물의 농도를 나타내고 종축은 생존율(Viability:%)을 나타낸다. The pufferfish egg extract of the present invention was applied to normal lymphocytes and corresponding leukemia cancer cell owners HL-60, and U937, and to normal astrocytes and corresponding glioma cells. , 20, 30, 40, 50% was treated, and after 24 hours using a MTT assay to measure the cell viability to determine the degree of death. In Figure 1, (A) is a graph showing the cell viability as a result of treatment of the pufferfish egg extract of the present invention to normal lymphocytes (lymphocytes) by concentration, the horizontal axis represents the concentration of pufferfish egg extract and the vertical axis represents the viability (%) ). (A) and (C) is a graph showing the cell viability as a result of treatment of the pufferfish egg extract of the present invention to the normal lymphocytes (lymphocytes) by concentration, the horizontal axis represents the concentration of puffer egg extract and the vertical axis represents the viability (%) ). (B) is a graph showing the cell viability as a result of treatment of the blowfish egg extract of the present invention to the cancer cell line HL-60 by concentration, the horizontal axis represents the concentration of puffer egg extract, the vertical axis represents the viability (%), (D) is a graph showing the cell viability as a result of treatment of the pufferfish egg extract of the present invention to cancer cell line U937 by concentration, wherein the horizontal axis represents the concentration of puffer egg extract and the vertical axis represents viability (%). In Figure 2, (A) is a graph showing the cell viability as a result of treatment of the pufferfish egg extract of the present invention to the normal astrocytic cells (Astrocyte), the horizontal axis represents the concentration of puffer egg extract and the vertical axis represents the viability (Viability :%). (B) is a graph showing the cell viability as a result of treatment of the blowfish egg extract of the present invention to the cancer cell line C6 glioma by concentration, the horizontal axis represents the concentration of puffer egg extract and the vertical axis represents the viability (%).

도 1 및 도 2를 참조하면, 정상세포는 영향을 받지 아니하나 암세포에서는 복어 알 추출물의 농도 의존적으로 세포생존율이 현저히 감소됨을 보이고 있다. 더욱이 백혈병 암세포 주에서는 세포사멸의 정도가 더 현저함을 알 수 있다.Referring to Figures 1 and 2, normal cells are not affected, but cancer cells have been shown to significantly reduce the cell survival rate depending on the concentration of pufferfish egg extract. Moreover, the degree of apoptosis is more pronounced in leukemia cancer cell lines.

1-3. 다른 암세포에의 세포사멸1-3. Apoptosis on Other Cancer Cells

다른 암세포들로서 폐암(Lung cancer), 전립선암(Prostate cancer), 간암세포(Hepatoma), 및 자궁경부암(Cervical cancer)에 대해서도 세포사멸에 대한 동일한 실험을 시행하였다. 도 3은의 (A)는 폐/전립선암 세포주인 A549, DU145, LNCap 및 PC-3에 상기 복어 알 추출물을 농도별로 처리하여 얻은 각각의 세포생존율을 나타낸 것이고, (B)는 경부암 세포주인 HeLa에 상기 복어 알 추출물을 농도별로 처리하여 얻은 각각의 세포생존율을 나타낸 것이다. 도 3을 참조하면, 본 발명의 복어 알 추출물이 효과적으로 모든 암세포를 사멸로 유도하고 있음을 나타내고 있다.Lung cancer, prostate cancer, hepatoma, and cervical cancer were the same experiments for apoptosis as other cancer cells. Figure 3 (A) shows the cell survival rate obtained by treating the pufferfish egg extract concentrations in lung / prostate cancer cell lines A549, DU145, LNCap and PC-3, (B) is in HeLa, a cervical cancer cell line It shows the cell survival rate obtained by treating the blowfish egg extract for each concentration. Referring to Figure 3, the blowfish egg extract of the present invention has been shown to effectively induce all cancer cells to death.

실시예 2. 복어 알 추출물의 암세포 세포고사 작용Example 2 Cancer Cell Apoptosis of Pufferfish Egg Extract

본 발명의 복어 알 추출물의 세포사멸 작용이 세포고사를 유도하여 일어나는 것인지 여부를 알아보기 위하여 상술한 세포고사의 전형적인 현상을 검색한 결과 암세포의 세포사멸 작용이 세포고사에 의함을 확인하였다.In order to determine whether the apoptosis action of the pufferfish egg extract of the present invention is induced by apoptosis, a typical phenomenon of the above-described apoptosis was searched, and it was confirmed that the apoptosis action of cancer cells was caused by apoptosis.

2-1. 핵 및 미토콘드리아에 미치는 영향2-1. Impact on the Nucleus and Mitochondria

세포고사와 연계되어 핵과 미토콘드리아가 손상되기 때문에 암 세포(HL-60)에 복어 알 추출물을 24시간 처리하고 핵은 핵 염색 염료인 Hoechst 33258로, 그리고 미토콘드리아는 미토콘드리아만을 특이적으로 염색하는 Rhodamine 123으로 염색하고 역상 형광 현미경을 이용하여 관찰하였다. 도 4의 (A)와 (B)는 HL-60의 핵을 나타내는 염색 사진으로서, (A)는 복어 알 추출물로 처리하지 않은 경우를 보여주고, (B)는 복어알 추출물로 처리한 경우를 나타낸다. 도 4의 (A)와 (B)를 비교해보면, 균일한 타원형의 핵모양을 나타내는 왼쪽의 대조군(A)과는 달리 복어 알 추출 물을 50% 농도 처리한 실험군(B)에서는 세포고사의 전형적인 현상인 핵 분절현상(nuclear fragmentation)이 관찰되었다.The nucleus and mitochondria are damaged in connection with apoptosis, so the blowfish egg extract is treated with cancer cells (HL-60) for 24 hours, the nucleus is a nuclear dye dye Hoechst 33258, and the mitochondria is specifically Rhodamine 123, which stains only mitochondria. Stained and observed using reversed-phase fluorescence microscopy. Figure 4 (A) and (B) is a stained photograph showing the nucleus of HL-60, (A) shows a case that is not treated with blowfish egg extract, (B) is a case of treatment with a blowfish egg extract Indicates. Comparing (A) and (B) of Figure 4, unlike the control group (A) on the left showing a uniform elliptical nucleus, the experimental group (B) treated with 50% concentration of blowfish egg extract is typical of cell death. Nuclear fragmentation was observed.

그리고 도 4의 (C)와 (D)는 HL-60의 미토콘드리아를 나타내는 염색 사진으로서, (C)는 복어 알 추출물로 처리하지 않은 경우를 보여주고, (D)는 복어 알 추출물로 처리한 경우를 나타낸다. 도 4의 (C)와 (D)를 비교해 보면, 정상 대조군(C)에서는 세포질 일부에 정상적으로 분포되어 있지만 50% 처리군(D)에서는 세포질 전체에 퍼져있어 미토콘드리아가 치명적인 손상을 입고 있음을 보이고 있다.And (C) and (D) of Figure 4 is a stained photograph showing the mitochondria of HL-60, (C) shows the case not treated with pufferfish egg extract, (D) is treated with pufferfish egg extract Indicates. Comparing (C) and (D) of FIG. 4, the normal control group (C) is normally distributed in a part of the cytoplasm, but 50% treated group (D) is spread throughout the cytoplasm showing that the mitochondria are fatally damaged. .

즉, 도 4를 참조하면, 상단 사진(A,B)은 복어 알 추출물의 핵 분절효과를 검색하기 위하여 복어 알 추출물을 2시간 처리한 후 핵 염색연료인 Hoechst 33258을 10uM로 희석하여 10분 염색하고 다시 PBS로 세척한 후 역상 형광 현미경(Nikon Eclipse TE 300, Japan)을 이용하여 핵 형태를 관찰하면서 10 x 10의 배율로 찍은 사진이다.That is, referring to Figure 4, the upper photo (A, B) is 10 minutes after diluting the nuclear dye fuel Hoechst 33258 to 10uM after treatment of the pufferfish egg extract for 2 hours to detect the nuclear segmentation effect of pufferfish egg extract After washing with PBS again, the image was taken at a magnification of 10 × 10 while observing the nuclear morphology using a reversed-phase fluorescence microscope (Nikon Eclipse TE 300, Japan).

하단의 사진(C,D)은 복어알 추출물의 미토콘드리아에 대한 손상여부를 관찰하기 위하여 복어알 추출물을 24시간 처리한 다음 미토콘드리아만을 염색하는 염료인 Rhodamine 123이 포함된 배양액에서 30분 반응시킨 후 배양액을 제거하고 세포표면을 PBS로 3회 세척하였다. PBS를 완전히 제거한 뒤 살아있는 세포상태를 유지하기 위하여 슬라이 글라스로 세포표면을 조심스럽게 세척한 다음 역상 형광 현미경을 이용하여 관찰하고 10 x 10의 배율로 찍은 사진이다.The picture (C, D) at the bottom shows the blowfish roe extract for 24 hours to observe whether the blowfish roe extract is damaged against mitochondria, and then reacted for 30 minutes in a culture solution containing Rhodamine 123, a dye that stains only mitochondria. The cell surface was washed three times with PBS. After the PBS is completely removed, the surface of the cells is carefully washed with a slice glass in order to maintain a living cell state, which is observed using a reversed-phase fluorescence microscope and photographed at a magnification of 10 × 10.

2-2 플로우 사이토메트리에 의한 미토콘드리아 분포분석2-2 Analysis of Mitochondria Distribution by Flow Cytometry

복어 알 추출물에 의한 미토콘드리아의 손상정도를 보다 확실히 하기 위하여 미토콘드리아만을 특이적으로 염색하는 염료인 JC-1으로 염색한 다음 플로우 사이토메트리로 세포질 내의 분포를 검색하였다.In order to more clearly confirm the damage of mitochondria by pufferfish egg extract, dyes were stained with JC-1, a dye that specifically stains only mitochondria, and the distribution in the cytoplasm was searched by flow cytometry.

도 5a는 정상 임파구에 대한 대조군(A)과 복어 알 추출물을 처리한 실험군(B)을 비교한 것으로, 두 군데 모두에서 별다른 차이없이 한 곳에 분포되어 있다. 그러나 동일한 시험을 암세포 HL-60를 이용한 결과인 도 5b를 보면 정상분포를 보이는 대조군(A)과는 달리 복어 알 추출물을 처리한 실험군(B)에서 손상을 받아 세포질 내에 널리 분포되어 있음을 보이고 있어 미토콘드리아가 본 발명의 복어 알 추출물에 의해 치명적인 손상을 받고 있음을 확인하였다.FIG. 5a compares the control group (A) and the experimental group (B) treated with blowfish roe extract for normal lymphocytes, and are distributed in one place without much difference in both places. However, when the same test using cancer cell HL-60 is shown in FIG. It was confirmed that the mitochondria are fatally damaged by the blowfish egg extract of the present invention.

2-3. 카스파제 프로테아제류의 활성에 미치는 영향2-3. Effect on Caspase Protease Activity

카스파제 프로테아제류( Caspase protease family)는 염증반응 및 세포고사 유도에 핵심적인 역할을 하는 효소로서, 현재 14 종류의 효소가 알려져 있다. 이중 카스파제-3는 카스파제 캐스케이드의 하방에 위치하여 상방에 위치한 카스파제-8과 카스파제-9의 활성신호와 합류하는 효소로서 손상된 DNA복구에 관련된 단백질인 PARP를 절단하여 세포고사를 유도한다. 따라서 암세포(HL-60, U937 및 Molt-4)를 배양하면서 복어 알 추출물을 처리하고 배양시간에 따라 카스파제-3, -8 및 -9의 활성을 각각 측정하였다. 도 6에서 (A)는 HL-60를 배양하면서 복어 알 추출물을 처리하여 배양시간에 따라 카스파제-3, -8 및 -9의 활성을 각각 측정한 그래프이고, (C)는 U937을 배양하면서 복어 알 추출물을 처리하여 배양시간에 따라 카스파제-3, -8 및 -9의 활성을 각각 측정한 그래프이며, (C)는 Molt-4를 배양하면서 복어 알 추출물을 처리하여 배양시간에 따라 카스파제-3, -8 및 -9의 활성을 각각 측정한 그래프이다. 도 6의 그래프를 참조하면, 카스파제-8을 제외하고, 카스파제-3과 -9가 유의하게 증가됨을 알 수 있다.The caspase protease family is an enzyme that plays a key role in inducing inflammatory reactions and apoptosis. Currently, 14 kinds of enzymes are known. Caspase-3 is an enzyme that joins the activation signals of caspase-8 and caspase-9 located below the caspase cascade and induces apoptosis by cleaving PARP, a protein involved in damaged DNA repair. . Therefore, while culturing cancer cells (HL-60, U937 and Molt-4) was treated pufferfish egg extract and the activity of caspase-3, -8 and -9 was measured according to the culture time. In Figure 6 (A) is a graph measuring the activity of caspase-3, -8 and -9 according to the incubation time by treating the blowfish egg extract while culturing HL-60, (C) is a culture of U937 Treated blowfish egg extract to measure the activity of caspase-3, -8 and -9 according to the incubation time, (C) is a caspa according to the incubation time by treating blowfish egg extract while culturing Molt-4 It is a graph which measured the activity of -3, -8, and -9, respectively. Referring to the graph of FIG. 6, except for caspase-8, it can be seen that caspase-3 and -9 are significantly increased.

즉, 도 6은 카스파제 프로테아제류 활성도 변화를 도시한 그래프로서, 암세포(HL-60, U937, 및 Molt-4)를 50% 농도의 복어 알 추출물을 함유하는 배지에서 배양하면서 시간에 따라 카스파제-3, -8, 및 -9의 활성변화를 측정하였다.That is, Figure 6 is a graph showing the change in caspase protease activity, caspase over time while culturing cancer cells (HL-60, U937, and Molt-4) in a medium containing 50% concentration of blowfish egg extract Changes in activity of -3, -8, and -9 were measured.

실험조건의 세포를 4℃에서 세포 파쇄용액(1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1mM PMSF, 1 ug/ml leupeptin, 2 mM DTT, 10mM Tris/HCI, pH 8.0)으로 파쇄하여 14,000rpm으로 15분간 원심분리하고, 이때 얻어진 상층액은 540 nm에서 정량하였다. 정량하여 얻어진 100 ug의 세포 파쇄액은 caspase assay buffer(100 mM HEPES, 10% sucrose, 0.1% chaps, 1mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin, 2 mM DTT, pH 7.5)에 희석되어 형광 표지된 기질과 37℃에서 30분간 반응시킨 후 fluorpmeter로 caspase activity(caspase-3: AMC-DEVD, caspase-8: Z-IETD-AFC, caspase-9: Ac-LEHD-AFC)를 측정하였다. 이때의 파장은 excitation wavelength(380nm)와 emission wavelength(460 nm)를 사용하였다.The cells of the experimental conditions were crushed with a cell disruption solution (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 1 mM PMSF, 1 ug / ml leupeptin, 2 mM DTT, 10 mM Tris / HCI, pH 8.0) at 4 ° C. The mixture was centrifuged at 14,000 rpm for 15 minutes, and the obtained supernatant was quantified at 540 nm. Quantification of 100 ug of cell lysate was diluted in caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% chaps, 1 mM PMSF, 1 ug / ml aprotinin, 1 ug / ml leupeptin, 2 mM DTT, pH 7.5). After reacting with a fluorescently labeled substrate at 37 ° C. for 30 minutes, caspase activity (caspase-3: AMC-DEVD, caspase-8: Z-IETD-AFC, caspase-9: Ac-LEHD-AFC) was measured with a fluorpmeter. . At this time, excitation wavelength (380 nm) and emission wavelength (460 nm) were used.

2-4. PARP 및 Bid의 절단에 미치는 영향2-4. Effect on the cleavage of PARP and Bid

PARP는 손상된 DNA복구와 연관된 단백질로서 카스파제-3에 의해 절단되고, Bid는 절단되면 카스파제-9을 활성화하여 결국은 이 효소의 캐스케이드의 하방에 위치한 카스파제-3을 활성화시키므로서 세포고사를 유도한다. 도 7은 Western bolt를 시행하여 암세포(HL-60, U937, Molt-4)에서 복어 알 추출물에 의해 이들 단백질이 절단되는지 여부를 관찰한 것으로, 위로부터 PARP, Bid, Bcl2, BclXL/S, p53, Fas를 각각 관찰한 사진이다.PARP is a protein associated with damaged DNA repair that is cleaved by caspase-3, which, when cleaved, activates caspase-9, which in turn activates caspase-3, located below the cascade of this enzyme. Induce. FIG. 7 shows whether these proteins are cleaved by blowfish egg extract in cancer cells (HL-60, U937, Molt-4) by Western bolt. PARP, Bid, Bcl 2 , Bcl XL / S from above , p53 and Fas, respectively.

도 7을 참조하면, 몇가지 관련 단백질 중에서 115 kDa의 PARP가 85kDa으로 절단되었고, 또한 Bid도 복어 알 추출물에 의해 절단됨을 관찰하였다. 이때, PARP와 Bid 등 여러 단백질의 절단여부를 알아보기 위한 western blotting은 다음과 같이 시행하였다. 정상세포와 실험조건의 세포를 포집하여 세포 파쇄용액으로 4 ℃에서 파쇄한 후 BCA용액으로 동량이 되게 하였다. 두 배의 sample buffer(5mM EDTA, 4% sodium dodesyl sulphate(SDS), 20% glycerol, 299 mM Tris, pH 6.8 0.06% bromophenol blue)를 혼합한 후 100℃에서 3분 가열하여 단백질 변성을 유도하고 10% SDS-PAGE를 시행하였다. 전기영동을 마친 gel의 단백질을 0.8 mA/Cm2의 전기를 걸어주어 nitrocellulose membrane으로 이동시킨 다음 5% skin milk로 만들어진 blocking buffer를 이용하여 상온에서 1시간 반응시켜 비 특이적인 항체만을 억제시켰다. 일차 항체를 TBS-T에 1:1,000으로 희석하여 ntrocelluose membrane과 상온에서 2시간 반응시키고, TBS-T로 10분간 3번 세척한 후 이차 항체인 anti-rabbit lgG conjugated HRP(TBS-T로 1:3,000으로 희석, Amersham Co., England)와 nitrocellulose membrane을 상온에서 1시간 반응시켜 ECL kit(Amersham Co., England)를 이용하여 필름에 노출시켰다.Referring to FIG. 7, it was observed that among several related proteins, 115 kDa PARP was cleaved to 85 kDa, and Bid was also cleaved by blowfish egg extract. At this time, western blotting was performed as follows to determine whether various proteins such as PARP and Bid were cleaved. Normal cells and cells under experimental conditions were collected, crushed at 4 ° C. with a cell crushing solution, and then equalized with BCA solution. After mixing twice the sample buffer (5mM EDTA, 4% sodium dodesyl sulphate (SDS), 20% glycerol, 299 mM Tris, pH 6.8 0.06% bromophenol blue) and heated for 3 minutes at 100 ℃ to induce protein denaturation 10 % SDS-PAGE was performed. After electrophoresis, the gel protein was transferred to the nitrocellulose membrane by applying 0.8 mA / Cm 2 of electricity, and then reacted at room temperature for 1 hour using a blocking buffer made of 5% skin milk to inhibit only nonspecific antibodies. The primary antibody was diluted 1: 1,000 in TBS-T, reacted with the ntrocelluose membrane for 2 hours at room temperature, washed three times with TBS-T for 10 minutes, and then the anti-rabbit lgG conjugated HRP (TBS-T 1 :). Diluted to 3,000, Amersham Co., England) and nitrocellulose membrane was reacted for 1 hour at room temperature and exposed to the film using the ECL kit (Amersham Co., England).

실시예 3. 말기 암환자에 대한 임상시험Example 3 Clinical Trials in Terminal Cancer Patients

극심한 통증을 호소하는 유방암 환자 3명, 위암환자 7명, 폐암환자 2명, 간암환자 3명, 그리고 난소환자 1명이 통증을 호소할 때마다 10-50 ㎖ 정도의 분량을 a 물에 희석하여 음용시켰다. 그 결과 모든 환자에게서 탁월한 진통효과를 얻을 수 있었다.Whenever 3 patients with severe pain, 7 patients with gastric cancer, 2 patients with lung cancer, 3 patients with liver cancer, and 1 patient with ovary complain of pain, dilute 10-50 ml in water and drink it. I was. The result was an excellent analgesic effect in all patients.

위암환자의 경우에는 음식물이 소화되는 정도가 현저히 개선되는 효과를 얻을 수 있었는데, 이는 복어 알 추출물의 항암 효과로 인해 종양의 크기가 줄어든데 기인한 것으로 사료된다.In the case of gastric cancer patients, the degree of digestion of food was remarkably improved, which may be due to the decrease in tumor size due to the anticancer effect of puffer fish extract.

이상 살펴본 바와 같이, 본 발명의 복어 알 추출물은 암 환자에게 투여시 항암 효과를 얻을 수 있고 암 환자의 통증을 제어하는 효과도 함께 얻을 수 있어 통증을 호소하는 암 환자에게 투여시 항암 치료 효과는 물론이고, 탁월한 진통 효과까지도 함께 얻어지는 장점을 가진 것이다.As described above, the pufferfish egg extract of the present invention can obtain an anticancer effect when administered to a cancer patient, and can also obtain the effect of controlling the pain of the cancer patient, as well as the anticancer treatment effect when administered to a cancer patient complaining of pain. And it has the advantage that even excellent analgesic effect is obtained together.

또한, 본 발명의 복어 알 추출물은 암세포만을 특이적으로 선별하여 세포 고사 기작을 통해 세포 사멸로 유도하므로, 정상 세포에 대한 독성이 낮은 안전한 항암-진통제로서 개발될 수 있을 것이다.In addition, the pufferfish egg extract of the present invention specifically selected only cancer cells to induce apoptosis through apoptosis, it can be developed as a safe anti-cancer analgesic with low toxicity to normal cells.

또한, 본 발명의 복어 알 추출물은 암세포만을 특이적으로 선별하여 세포 고사 기작을 통해 세포 사멸로 유도하므로, 정상 세포에 대한 독성이 낮은 안전한 항암제로서 개발될 수 있을 것이다.In addition, the pufferfish egg extract of the present invention specifically selected only cancer cells to induce apoptosis through apoptosis mechanism, it can be developed as a safe anticancer agent with low toxicity to normal cells.

도 1은 정상 임파구와 이에 상응하는 암세포인 HL-60 및 U937를 배양하면서 복어 알 추출물을 농도별로 처리하여 세포생존율을 측정한 결과를 도시한 그래프,1 is a graph showing the results of measuring the cell viability by treatment of pufferfish egg extract concentrations while culturing normal lymphocytes and corresponding cancer cells HL-60 and U937,

도 2는 정상 성상세포(astrocytes)와 이에 상응하는 암세포(C6 glioma)에 복어 알 추출물을 농도별로 처리한 경우의 세포 생존율 검사결과를 나타낸 그래프,Figure 2 is a graph showing the results of cell viability test results when the pufferfish egg extracts were treated to normal astrocytes and corresponding cancer cells (C6 glioma) by concentration,

도 3은 다른 암세포들에 대해 복어 알 추출물을 농도별로 처리한 경우의 세포 생존율 검사결과를 도시한 그래프,Figure 3 is a graph showing the cell viability test results in the case of treatment of blowfish egg extract by concentration for other cancer cells,

도 4는 복어알 추출물에 의해 암세포 주에 나타나는 핵 분절과 미토콘드리아 손상을 관찰하여 촬영한 사진,4 is a photograph taken by observing nuclear fragments and mitochondrial damage appearing in cancer cell lines by blowfish roe extract,

도 5a 및 5b는 복어알 추출물에 의해 처리된 암세포 주를 플로우 사이토메트리에 의해 분석하여 미토콘드리아 분포를 도시한 그래프,5A and 5B are graphs showing the mitochondrial distribution by analyzing cancer cell lines treated by blowfish roe extract by flow cytometry;

도 6은 복어알 추출물에 의해 암세포 주에 나타나는 카스파제 프로테아제류 활성도 변화를 도시한 그래프,Figure 6 is a graph showing the change in caspase protease activity appear in cancer cell lines by puffer fish extract,

도 7은 복어알 추출물에 의해 암세포 주에 나타나는 단백질의 절단 여부를 알아보기 위한 western blotting을 촬영한 사진이다.Figure 7 is a photograph taken western blotting to determine whether the protein cut in the cancer cell line by blowfish roe extract.

Claims (6)

복어 알 추출물을 활성성분으로 함유하는 항암제 조성물에 있어서, 상기 복어 알 추출물은 복어 알 1 중량부에 대하여 2~3 중량부의 물을 가하여 가열 추출한 후 여과 또는 원심분리한 상등액을 취하여 제조되고, In the anticancer agent composition containing the pufferfish egg extract as an active ingredient, the pufferfish egg extract is prepared by adding 2 to 3 parts by weight of water with respect to 1 part by weight of pufferfish eggs, followed by filtration or centrifugation of the supernatant. 5-플루오르우라실, 메토트렉세이트, 아드리아마이신 및 탁솔로 이루어진 군으로부터 선택된 적어도 하나의 항암제를 더 포함하며,At least one anticancer agent selected from the group consisting of 5-fluorouracil, methotrexate, adriamycin and taxol, 부형제, 안정화제, 방향제, 교미제 등을 첨가하여, 산제, 과립제, 캅셀제 및 정제의 제법에 따라 제형된 것을 특징으로 하는 항암제 조성물.An anticancer composition characterized in that it is formulated according to the preparation of powders, granules, capsules and tablets by adding excipients, stabilizers, fragrances, copulation agents and the like. 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete
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