KR20040064461A - Anticancer Material Comprising Natural-drug Compound Extracted From Sargassum fulvellum - Google Patents
Anticancer Material Comprising Natural-drug Compound Extracted From Sargassum fulvellum Download PDFInfo
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Abstract
Description
본 발명은 모자반 (Sargassum fulvellum)으로부터 추출한 생약성분 의 항암제에 관한 것으로 보다 상세하게는 영일만을 중심으로한 한국 동해연안 감포연해 청정해역에 식생하는 모자반으로부터 에탄올을 용매로 원액 및 주성분(Folic Acid 외 16종 성분)을 추출하고 상기 에탄올 추출액(Ethanol-extracts)의 암세포(Hela cell)에 대한 항암효능을 검증한 모자반으로부터 추출한 생약성분의 항암제에 관한 것이다.The present invention relates to an anticancer agent of a herbal ingredient extracted from Sargassum fulvellum. More specifically, the ethanol is used as a solvent and the main ingredient (Folic Acid et al. Species component) and the ethanol extract (Ethanol-extracts) relates to an anticancer agent of the herbal ingredient extracted from the mothban that has been tested for anticancer efficacy against the cancer cells (Hela cells).
모자반(Sargassum fulvellum)은 해조류 갈조류 갈조식물문 이형세대강 원포자강 모자반목 모자반과에 속하는 1년생 해조이다. 체색은 적색을 띤 황색이며 저조선부근의 바위에 분포한다. 우리나라에서는 동해 구룡포, 감포, 울릉도, 부산 거제도, 제주도 등지의 전 연안에 분포하며 식용 및 사료로 이용된다. 뿌리는 가반상이고 줄기는 단조(單條)이며 긴 가지를 많이 낸다. 가지는 삼릉주(三稜柱) 또는 삼각형이며 비틀어져 있다. 큰 것은 높이 수 m에 달한다.Sargassum (Sargassum fulvellum) is a freshman seaweed and algae belonging to brown algae plants galjo door yihyeongse Rivers won spores River hats antagonism Sargassum. Body color is reddish yellow and distributed in rocks near low shipbuilding. In Korea, it is distributed all over the coast of Guryongpo, Gampo, Ulleungdo, Busan Geoje, Jeju Island, etc. and is used for food and feed. Roots are plaques, stems are monotonous, and long branches are produced. Branches are three or three triangular and twisted. The large reaches several meters in height.
잎은 아래로 반곡(反曲)하고 주걱꼴 또는 타원형이며 잎의 중앙까지 약한 중륵(中肋)이 있고 상부(上部)의 잎은 피침형(披針形)이며 톱니가 있고 중륵(中肋)은 없다. 색은 암황갈(暗黃褐)색이며 연하고 잎면(面)에 검은 점이 있다.Leaves are bent downward, spatula or oval, weak to middle of leaf, upper leaf is lanceolate, sawtoothed, middle is none. The color is dark yellow (暗 黃褐), light and has black spots on the leaf surface.
기포(氣胞)는 다수 있고 위치에 따라 모양이 다르며 가지의 기부(基部)에 있는 것은 상부(上部)에 있는 것보다 크다. 모양은 도란형(倒卵形) 또는 타원형이며 더러는 가지모양이며 짧은 자루가 있다. 길이는 5∼12mm 폭은 8∼9mm이다. 꼭대기는 보통 둥글고 때로는 미철두(微凸頭) 또는 소엽(小葉)이 있다.There are many bubbles and the shape varies depending on the position, and the one on the base of the branch is larger than the one on the upper part. Its shape is obovate or elliptical, some are branched, and have a short bag. The length is 5-12 mm and the width is 8-9 mm. The apex is usually round and sometimes has a small iron head or lobule.
생식기탁(生殖器托)은 긴 원주상(圓柱狀)이며 짧은 자루가 있고 총상 (總狀)으로 배열(配列)한다.Reproductive deposits are long, columnar, with short sacks, arranged in gunshots.
생육(生育)은 저조선(低潮線)부근의 암상(岩上)에 한한다.Growth is limited to rock formations in the vicinity of low shipbuilding.
일본, 오스트레리아, 인도, 말레이시아, 중국, 멕시코 연해의 조간대 암 상(岩上)· 사르기소 해(Sargasoo-sea)의 부유종(浮遊種)으로도 서식 분포한다.It is also distributed in the intertidal rocks of the coasts of Japan, Australia, India, Malaysia, China, and Mexico, and in the Sargasoo-sea.
한편, 공지의 항암제로는On the other hand, as a known anticancer agent
a) 피토폴리사카라이드가 항암력이 있음을 실험한 바 있다. (Chang K. M. 1985년 Arch. Pharm. Res. 8 (1), p. 42-44)a) Phytopolysaccharides have been tested for anticancer activity. (Chang K. M. 1985 Arch. Pharm. Res. 8 (1), p. 42-44)
b) 율무(Coix agrestis)가 암세포 사코마 180에 치료 효과가 있음을 보고 한 바 있다. (Jung B. S. 1970년 J. Pharm. Soc. Kor. 14, p. 51-53)b) Coix agrestis has been reported to have therapeutic effects on cancer cell Sacoma 180. (Jung BS 1970 J. Pharm. Soc.Kor. 14, p. 51-53)
c) β-(1-메틸-1-히드록시 메틸)에틸아미노-2,4-히드록시-5-니트로프로 피오-페논(v)이 암 종양세포 사코마-180의 치료에 효능이 있음을 시험한 바 있다. (Jung W. K. 1973년 J. Pharm. Soc. Kor. 17, p.115-121)c) β- (1-methyl-1-hydroxy methyl) ethylamino-2,4-hydroxy-5-nitropropio-phenone (v) is effective in the treatment of cancer tumor cell Sacoma-180. I have tested it. (Jung W. K. 1973 J. Pharm. Soc.Kor. 17, p. 115-121)
d) 5-프루오로프라실, 미토마이신, 아니그달린 카르복시 에트게르마늄 세 스구옥사이드(GE 132)가 사코마-180 유발 생쥐의 생존 일수를 연장시키고 적혈구 수를 증가시킴을 보고한 바 있다. (Na D. J. 1985년 Jung Ang Univ.)d) 5-Fluoroprasyl, mitomycin, and anigdaline carboxy ethgermanium sesquioxide (GE 132) have been reported to prolong the survival days and increase the number of red blood cells in Sacoma-180 induced mice. (Na D. J. 1985 Jung Ang Univ.)
e) 조각자가 유종의 5, 흉복부 적취에 1, 피보 옹저(癰疽)에 4, 오장 옹저에 2인 치료 기대 출현빈도를 나타냄을 지적한 바 있다. (Cha S. M. 1977년 Kor. J. Pharmacog. Vol. 8, No. 1, p. 1-15)e) The sculptor pointed out the frequency of treatment expected to be 5, 5 for chest abdominal ablation, 4 for Pivot and 2 for ileum. (Cha S. M. 1977 Kor. J. Pharmacog. Vol. 8, No. 1, p. 1-15)
f) 메토트레세이트 6×10-3M 농도가 항암제로서 미세 피라멘트 생성 억제 효능이 있으며 과립 백혈구의 억제 효능이 있음을 보고한 바 있다. (Park S. H. 1989년 Yeon Sei Univ.)f) Methotrecetate 6 × 10 −3 M concentration has been reported to be effective as an anticancer agent for inhibiting the production of fine filaments and to inhibit granulocytes. Park SH 1989 Yeon Sei Univ.
g) 복수암(Sarcoma-180)에 감염된 쥐에 복령 추출액을 투여한 결과 투여 군이 대조군에 비해 수명 연장 효과가 있었다고 보고한 바 있다. (Lee B. I. 1989년 Dong Kuk Univ.)g) As a result of administering the extract of Bokryeong to rats infected with ascites cancer (Sarcoma-180), it was reported that the administration group had a longer life effect than the control group. Lee B. I. 1989 Dong Kuk Univ.
h) 복수암(Sarcoma-180) 투여군에 비해 아미그달린 투여군이 생존 일수가 연장 되었음을 시험한 바 있다. (Na D. J. 1985년 Jung Ang Univ.)h) We have tested that the amidaline group has a longer survival compared to the Salcoma-180 group. (Na D. J. 1985 Jung Ang Univ.)
i) 플루다라빈(Fludarabine)은 50-150 mg/m2로 5-7회 투여시 실명 및 탈수초(Demyelination)이 일어나고 25-30 mg/m2로 주 5일간 투여시 만성 임파구 백혈병(Chronic Lymphocytic Leukemia)에 유효함을 보고한 바 있다. (Lee K. H. 1999년 ULSAN Univ. Med. Coll.)i) Fludarabine causes blindness and demyelination when administered 5-7 times at 50-150 mg / m 2 and chronic lymphocytic leukemia when administered at 25-30 mg / m 2 for 5 days a week. It has been reported to be effective for lymphocytic leukemia. (Lee KH 1999 ULSAN Univ. Med. Coll.)
j) 토포테칸(Topotecan)은 세포핵내 효소 토포아이소머라아제(Topoisomerase)를 억제하며 최대 내용량은 0.68mg/m2이며 탈모, 점막염, 구토 등의 부작용이 경미 하였다고 보고한 바 있다. (Smith. K. L. Beecham 2000. 2.)j) Topotecan inhibits the enzyme Topoisomerase in the cell nucleus and has a maximum content of 0.68 mg / m 2 and reported side effects such as hair loss, mucositis and vomiting. (Smith.KL Beecham 2000. 2.)
k) 73%의 종양조직에서 과메틸화(hypermethylation)이 관찰되었으며 그중 81%의 환자에서는 혈청, 혈장에서도 이상메틸화(aberrant methylation)가 관찰되었음을 보고하였다. (Wong et all 1999년 Cancer Res. 59)k) Hypermethylation was observed in 73% of tumor tissues, and 81% of patients reported aberrant methylation in serum and plasma. (Wong et all 1999 Cancer Res. 59)
l) HMG-1 단백질은 위암의 발암기전 및 분화과정 기능에 관여함을 보고한 바 있다. (Park D. I. 2000년 J. Kor Gastroent, Vol.36, No. 1, p. 7)l) HMG-1 protein has been reported to be involved in the carcinogenesis and differentiation process of gastric cancer. (Park D. I. 2000 J. Kor Gastroent, Vol. 36, No. 1, p. 7)
m) 위암 환자에서는 120 KDa 세포독성 연관(Cag-A) 단백질에 대한 항체가 많았음을 보고한 바 있으며, (Crabtree et al. 2000) 87 KDa 액포화 독소(vacuolating toxin)에 대한 항체가 96%의 고 빈도로 검출된바 있다고 보고 하였다. (Hirai et al. 2000)m) Patients with gastric cancer reported high levels of antibodies to 120 KDa cytotoxicity-associated (Cag-A) proteins (Crabtree et al. 2000) and 87% of antibodies against 87 KDa vacuolating toxins. It was reported that high frequency was detected. (Hirai et al. 2000)
n) 간세포 암종의 발생에 있어 DNA 복제시 유전자 결함이 일어나고 변 이 세포가 클론으로 선택 촉진 성장되어 효소 병변으로 암발생 기전이 된다고 보고한 바 있다. (Lee D. H. 2000년 J. Kor. Cancer 6-11)n) In the development of hepatocellular carcinoma, it has been reported that gene defect occurs when DNA replication occurs, and that mutant cells are selectively promoted to grow into clones, which is an enzyme lesion, which causes cancer. (Lee D. H. 2000 J. Kor. Cancer 6-11)
o) 쥐의 간암 발생시 단계 특이적 유전자 발현(Stage specific gene expression)이 생겨남을 보고한 바 있다. (Pitot H. C. 1996년 J. Cancer Res. Clin Oncol. 122: 257-265)o) It has been reported that stage specific gene expression occurs in rat liver cancer. (Pitot H. C. 1996 J. Cancer Res. Clin Oncol. 122: 257-265)
p) C-myc 및 C-myc/TGFa 전이에 의한 간암 발생 진행시 세포사멸(apoptosis)과 세포주기(Cell cycle)에 현격 교번 현상이 발생하였음을 보고한 바 있다. (Samtoni-Rugiu E., Jensen MR. 1977년 Am. Assoc. Cancer Res.38 : 279-280)p) It has been reported that apoptosis and cell cycle have occurred in the course of liver cancer development by C-myc and C-myc / TGFa metastasis. (Samtoni-Rugiu E., Jensen MR. 1977 Am. Assoc. Cancer Res. 38: 279-280)
q) 인체(人體) 눈물의 Lg 단백질은 전립선암증의 검증 기준이 된다고 보 고한바 있다. (Kochs 2000 New South Weilz Univ. Optics Res. Centr.)q) Lg protein in human tears has been reported to be a criterion for prostate cancer. (Kochs 2000 New South Weilz Univ.Optics Res. Centr.)
r) 비리온 파마슈티컬 살모넬라(Virion Pharmaceutical Salmonella)를 암 발생 쥐에 투입하면 암세포 수를 감소시킨다고 보고 하였다. (Bermudes 2000 Yale Univ.)r) Virion Pharmaceutical Salmonella was reported to reduce the number of cancer cells in cancer-causing mice. (Bermudes 2000 Yale Univ.)
s) 염산 셉틴(Ceptin)와 Her 2 단백질의 복합체는 암세포 성장을 억제한다고 보고하였다. (Gene Tech 2000)s) A complex of Septin hydrochloride (Ceptin) and Her 2 protein has been reported to inhibit cancer cell growth. (Gene Tech 2000)
t) 인체 암세포 발생의 분지 효능은 콜터 입자(Coulter Particle) 측정 장치를 사용하여 측정이 가능하다고 보고 하였다. (Hazelton B. J. et. al. 1984 J.Natl. Cancer Inst. 73(3) : 5555-563)t) It was reported that the branching efficacy of human cancer cell development can be measured using a Coulter Particle measuring device. (Hazelton B. J. et. Al. 1984 J. Natl. Cancer Inst. 73 (3): 5555-563)
u) 마크로모마이신-1에 의해 헬라 S3세포의 DNA 복제 저지 기능이 있 음을 보고한 바 있다. (Woynarowski, J. M., 1980, Mol. Pharmacol. 18(3): 491-496)u) Macromomycin-1 has been reported to inhibit DNA replication of HeLa S 3 cells. (Woynarowski, JM, 1980, Mol. Pharmacol. 18 (3): 491-496)
v) 인체 간암세포의 재발 유전 불평형은 비교 유전 인자의 잡종번식에 의해 그 패턴이 검증되었다고 보고하였다. (Zimonjik DB et. al., 1999, Hepatology 29: 1208-1214)v) Recurrent genetic imbalance of human liver cancer cells reported that the pattern was verified by hybrid breeding of comparative genetic factors. (Zimonjik DB et. Al., 1999, Hepatology 29: 1208-1214)
w) 암 환자의 91.6% 시료에서 단백질 사이클린 DI가 유전자 표시자로 확인 되었다고 보고한 바 있다. (Kamal B.-H. 2000)w) Protein cyclin DI has been identified as a gene marker in 91.6% of cancer patients. (Kamal B.-H. 2000)
x) (ⅰ) 소마토스타틴 호르몬이 위암종 발생을 억제한다고 한 바 있으 며, (Paola Tomassetti 2000)x) (v) Somatostatin hormone has been shown to inhibit the development of gastric carcinoma, (Paola Tomassetti 2000)
(ⅱ) 레티노익 산 RAR-베타가 흉부적 육종의 억제 효과가 있다고 보고한 바 있고, (A. F. Gazdar 2000)(Ii) Retinoic acid RAR-beta has been reported to have an inhibitory effect on thoracic sarcoma (A. F. Gazdar 2000)
(ⅲ) 유전적 알코올 증후군에 온단 센트론(Ondan Cetron)이 유효함을 보고한 바 있다. (Ben. Coll. J. 2000)(Iii) Ondan Cetron has been reported to be effective for hereditary alcohol syndrome. (Ben. Coll. J. 2000)
y) 사가섬(Sargassum) 종들 중의 니트민이 임파, 육종(肉腫), 백혈병(白血病) 담즙 암(Gall cancer)의 정지, 핵 이상 증식 억제력을 가진다고 보고한 바 있다. (J. Tokida 1958 Bull. of Jap. Soc. Phycol. Vol. Ⅵ, No. 3, p. 93-99)y) Nitmin in Sargassum species has been reported to have the ability to stop lymphatic, sarcoma, leukemia gall cancer, and nuclear proliferation. (J. Tokida 1958 Bull. Of Jap. Soc. Phycol.Vol. VI, No. 3, p. 93-99)
상기 중 a) c) d)의 경우 몇 가지 폴리사카라이드, β-(1-메틸-1-히드록시메틸)에틸아미노-2, 4-디히드록시-5-니트로프로피오페논(v) 및 5-플루오로우라실, 미토마이신, 아미그달린 카르복시 에티게르마늄 세스구옥 사이드(GE132)등의 복합 화합물이 생존일수율 0.304배 증가시키고, 사코미어-180 암세포의 수를 근소 수치 감소시킨 효능이 있다고 하였다.A) c) d) above, several polysaccharides, β- (1-methyl-1-hydroxymethyl) ethylamino-2, 4-dihydroxy-5-nitropropiophenone (v) and Complex compounds such as 5-fluorouracil, mitomycin, and amidaline carboxy etigermanium sesgucoxide (GE132) increased the number of days of survival by 0.304 fold and slightly reduced the number of sacomere-180 cancer cells.
b) d)는 율무와 조각자의 고농도 추출액의 투여에 의한 초기 제암력 실험 및 처방 출현 빈도만 지적하고 있다.b) d) points out only the initial anticancer activity and the frequency of prescription by the administration of Yulmu and high concentration extract of engraver.
f) h)는 각 메토트레세이트 6×10-3M 및 아미그달린의 투여로 극소 생존일수의 연장 효능이 있음을 시험한 것이다.f) h) was tested for the prolonged efficacy of minimizing survival by administration of each methotrexate 6 × 10 −3 M and amigdaline.
g)의 경우는 생약복령(Paephyma Hoelen Rumphius)투여군이 대조군에 비해 단기 생존 일수의 연장 효능이 확인된 것에 한하고 있다.In the case of g), the group receiving the herbal medicine (Paephyma Hoelen Rumphius) showed the extended efficacy of short-term survival days compared to the control group.
i)의 경우는 탈수초(Demyelination)이 발생하는 경우이며,i) is when demyelination occurs,
k)의 경우는 과메틸화(hypermethylation)이 73% 종양조직에서 발생하는 경우이다.In case k), hypermethylation occurs in 73% of tumor tissues.
l) m) q) s)의 경우는 HMG-1 단백질, Lg 단백질, Her 2 단백질, 사이클린 DI 단백질 등의 단백질 이상 유형 발생으로 암이 발전기전을 가지는 경우이다.l) m) q) s) is a case where the cancer has dysgenesis due to the development of protein abnormal types such as HMG-1 protein, Lg protein, Her 2 protein, and cyclin DI protein.
x)의 (ⅰ) (ⅱ) 경우는 소마토스테이션 단백질(Somatostation protein) 및 레티노익 산(Retinoic acid)이 육종 생성 저지 효능이 있음을 스크링한 경우이다.In case of (iii) (ii) of x), Somatostation protein and Retinoic acid are screened for suppressing sarcoma production.
y)의 경우는 암세포의 이상증식 조직 저지력을 미레란 일반 세포 독(Myleran General cell poison)으로 기능하는 경우와 같다.In the case of y) is the same as the case of acting as a myleran general cell poison (myleran general cell poison) of the cancer cell dysplasia.
종래의 항균제는 개발 치료기관의 시간 지체 때문에 유해세균의 면역력을 증가시켰고, 합성비용은 과다하지만 치료효능은 낮았으며, 항균제가 인체 세포성분의 일반 성분이 아닌 특수 단백질 화합물이므로 쇼크사 등의 부작용이 많았다.Conventional antimicrobial agents increased the immunity of harmful bacteria due to time delays in development and treatment institutions, the synthesis cost was excessive, but the therapeutic efficacy was low. .
종래의 항암제인 니트로 프로피오페논 및 아미그달린 카르복시 에티게르마늄 등의 화합물 등은 5%내외의 생존일수 연장 등의 암치료 효능이 있었고, 암치료제 투여로 인한 소화, 순환, 호흡, 배설, 내분비의 대사 저해증을 유발한 사례 빈도가 높았다. 둥근 바위솔(Orostachys aggregeatus: 와송)등으로 현재, 동물 및 임상 시험 중인 생약 항암제는 10%내외의 생존 일수 연장 및 1.5%내외의 백혈구 수의 증가 등 치료효능은 인정되고 있으나 제암력의 제고가 요구되고 있다.Conventional anticancer drugs, such as nitro propiophenone and amigdaline carboxy etigermanium, were effective in treating cancers such as prolonging the survival period of about 5%, and inhibiting digestion, circulation, respiration, excretion, and endocrine metabolism due to the administration of cancer treatment agents. There was a high frequency of cases that caused the disease. Herbal anticancer drugs, currently being used in animal and clinical trials, such as round rock brush (Orostachys aggregeatus) are recognized for their therapeutic efficacy, including prolonged survival of about 10% and increased white blood cell counts of about 1.5%. have.
미국의 BMS가 개발한 택솔, 그래스태틴, 엔도스태틴 영국의 CRC가 개발중인 가시불가사리, 라미부틴, 캄토테신, 선프라, 하이루비신 및 제넥솔 등은 인체장기, 피부암 등에 일정비율의 제암력은 있으나 소화순환, 배설, 뇌 신경대사 저해증을 유발한 사례 빈도가 있으며 독성 부작용 등이 제기되고 있으며 버섯의 MGI114는 부작용은 경미하나 제암력이 비교적 낮았다.Taxol, grassstatin, and endostatin developed by BMS in the United States, thorn starfish, lamibutin, camptothecin, sunpla, hyrubicin, and genexol, which are being developed by CRC in the UK, have a certain ratio of anticancer activity to human organs and skin cancer. However, there are frequent cases of digestive circulation, excretion, and brain metabolism inhibition, and toxic side effects are being reported. MGI114 of mushroom has mild side effects but relatively low anticancer activity.
도 1. 모자반 에탄올 추출액 처리시, 헬라 육종 세포의 상대 생존율.Relative viability of HeLa sarcoma cells when treated with Maban ethanol extract.
본원 발명은 모자반(Sargassum fulvellum)을 채집, 수집, 선정하고, 기원을 감정한 후 동정하고, 대한 약전 시험법에 따라, 수집한 모자반 생약을추출장치에 넣고, 증류수를 더해 가열 추출하여 감압 여과한 후 얻어지는 모자반으로부터 추출한 생약성분인 것을 특징으로 하는 항암제 및 상기 채집 수득된 모자반 생약을 건조하여 마쇄한 순수 모자반 분말시료를 에탄올과 함께 수욕상에서 추출, 여과한 정제 시료로 제조하고 증류수 및 DMSO에 상 분리시료를 희석 전환 정제하여 얻어지는 모자반으로부터 추출한 생약성분인 것을 특징으로 하는 항암제에 관한 것이다.The present invention collects, collects, selects, and determines the origin of Sargassum fulvellum, and after identifying the origins, according to the pharmacopeia test method, the collected half-covered herbal medicines are put in an extraction device, and distilled water is added to extract the heat and filtered under reduced pressure. The anticancer agent characterized in that it is a herbal ingredient extracted from the mother-child obtained after harvesting, and the pure mother-hood powder sample obtained by drying the harvested mother-child medicine obtained by drying is prepared as a purified sample extracted and filtered in a water bath with ethanol and phase separated in distilled water and DMSO. The anticancer agent characterized in that it is a herbal ingredient extracted from the mother board obtained by dilution conversion purification of a sample.
본 출원인의 실험에 의하면 모자반(Sargassum fulvellum) 추출 검액 1,600μg/ml를 암세포 헬라 세포에 투여한 결과 6일후 100% 기준대비 암세포의 생존율은 0.6%이여서 모자반(Sargassum fulvellum) 추출액의 제암력이 매우 높았음이 검증 확인되었다.According to the experiments of the applicants, 1,600 μg / ml of Sargassum fulvellum extract sample was administered to cancer cell HeLa cells, and as a result, the cancer cell survival rate was 0.6% compared to 100% after 6 days, and the anticancer activity of the Sargassum fulvellum extract was very high. Yin was verified.
한국 동해 영일만을 중심한 감포연해 청정해역에서 모자반을 채집, 수집, 선정하고 기원을 감정 동정한 생시료를 증류수에 5회 세척하고 음건한 후 이를 45∼50℃에서 3일간 건조한다. 이를 1차로 대한 약전 시험법에 따라 생약을 세절하고 다시 80mesh로 마쇄한후 그중 수분 함량 3.5∼5.0%인 분말시료 5g을 수득 이를 75% 에탄올 100ml와 함께 수욕상에서 1시간 추출 여과 풍건한 후 2차로 그중 1.2g만을 분리하여 이차증류수 19.0 ml 및 DMSO 1.0 ml에 희석 최종농도 57 μg/μl 가 되도록 제조한 모자반 추출물 주성분에는 포릭산, 니트로민, 메탄티올, 리나롤, 미레란, 디메틸 설파이드, 이노시톨, 바이오틴, 제토니올, 리보프라빈, 카이닉산, 알라닌, 아스팔틱산, 구루타민산, 프로린, 트레오닌, 바린, 시스틴의 18가지 주요 성분이 있다. 모자반으로부터 추출한 생약성분으로 된 항암효능은 헬라 세포에 제암력이 99.4%이며, 인체내 10종 유해세균에 대한 항균력은 높아 소화순환 호흡 배설 대사율이 양호하며 독성이 경미하여 종래 항암제에 비해 생리 및 약리 효능이 월등 우수함이 검증되고 있다.In the clean waters of Gampo Yeonhae centered on Yeongil Bay, Korea, the mother and child are collected, collected and selected, and the raw samples having been identified for their origin are washed with distilled water five times, dried and dried at 45 ~ 50 ℃ for 3 days. According to the pharmacopeia test method for the first time, the crude medicine was chopped and ground again to 80 mesh to obtain 5 g of a powder sample having a water content of 3.5 to 5.0% therein. The extract was air dried for 1 hour in a water bath with 100 ml of 75% ethanol, and then secondly. The main components of Maban extract, which were prepared by separating only 1.2 g of the distilled water and diluting it in 19.0 ml of secondary distilled water and 1.0 ml of DMSO to a final concentration of 57 μg / μl, included formic acid, nitromin, methanethiol, linarol, mireran, dimethyl sulfide, inositol, There are 18 main ingredients: biotin, zetoniol, riboprabin, carboxylic acid, alanine, asphatic acid, gurutamic acid, proline, threonine, varin, and cystine. The anticancer effect of herbal medicine extracted from Maban has 99.4% anti-cancer activity in HeLa cells, and the antibacterial activity against 10 harmful bacteria in human body is high. It is proven that the efficacy is excellent.
상기와 같은 검액 성분을 추출 규명하는 방법으로는 다음과 같은 방법이 있다.As a method of extracting and identifying the sample components as described above, there are the following methods.
(가) DMSO에 의한 모자반 검액 성분 포릭산 추출법(A) Extraction of malic acid sample component foric acid by DMSO
(ⅰ) 에탄올에 의한 포릭산 추출법(Iii) Extraction of Foric Acid by Ethanol
한국 동해 영일만을 중심으로한 감포연해 청정해역에서 모자반을 채집, 수집, 선정하고 기원을 감정 동정한 모자반(Sargassum fulvellum)의 엽, 경, 부착부를 건조후 세절하고, 80 mesh로 마쇄한후 수분함량 3.5∼5.0% 인 분말시료 5g을 수득하고 이를 75% 에탄올 100ml와 함께 수욕상에서 1시간 추출, 여과, 풍건한 후 그중 1.2g만을 분리 이차증류수 19ml 및 DMSO 1.0ml에 희석 최종농도가 57μg/μl 가 되도록 제조한 것을 검액 성분으로 하였다.Collected, collected, selected, and collected the leaves, diameters, and attachments of the sargassum fulvellum in the Gampo Yeonhae clean water centered on Yeongil Bay, Korea. Obtain 5g of 3.5 ~ 5.0% of powder sample, extract with 100ml of 75% ethanol for 1 hour in water bath, filter, and air dry, and then separate only 1.2g of them. Dilute final distilled water in 19ml and DMSO 1.0ml. What was prepared so that it might be set as the sample liquid component.
검증에 사용된 배지는 라이프 테크놀로지사제 D-MEM(Dulbecco's Modified Eagle Medium, powder)을 3차 증류수에 녹여 여과한 후, 같은 회사제의 Serum(혈청) 10% 첨가하여 사용하였다. 그 조성은 이차증류수 1l당 D-MEM 분말 13.4g NaHCO33.7g 소혈청(Fetal Bovine Serum) 112ml로 하였다.The medium used for verification was D-MEM (Dulbecco's Modified Eagle Medium, powder) manufactured by Life Technologies, dissolved in tertiary distilled water, filtered, and then used by adding 10% of Serum (serum) manufactured by the same company. The composition was 112 ml of D-MEM powder 13.4 g NaHCO 3 3.7 g bovine serum (Fetal Bovine Serum) per 1 l of secondary distilled water.
미생물에 의한 오염을 막기 위하여 사용한 항생제는 시그마사제 복합항생제 안티마이코틱(Antimycotic)을 여과하여 사용하였으며 그 조성은 복합항생제 1ml 당 페니실린 10,000단위, 스트랩토마이신 10mg, 암포테리신 25mg, 배지 100ml로 하였다.Antibiotics used to prevent contamination by microorganisms were used by filtration of antimicrobial antimicrobial (Sigma Co., Ltd.) and the composition was 10,000 units of penicillin, 10 mg of strapomycin, 25 mg of amphotericin, 100 ml of medium per 1 ml of the combined antibiotics. .
배양한 암세포(HeLa Cell)의 세척에는 PBS(Phosphate Buffered Solution)를 멸균하여 사용하였으며 그 조성은 이차증류수 1l당 Nacl 8g, Na2HPO4·12H2O 2.89g, KH2HPO40.2g, KCl 0.2g으로 하였다.PBS (Phosphate Buffered Solution) was sterilized and used for washing cultured cancer cells (HeLa Cell) .The composition was composed of Nag 8g, Na 2 HPO 4 · 12H 2 O 2.89g, KH 2 HPO 4 0.2g, KCl It was 0.2 g.
배양한 암세포의 수확에는 라이프 테크놀로지사제 트립신(0.5% 10× solution)용액 20ml을 PBS 980ml에 섞어 여과하여 사용하였다.For harvesting the cultured cancer cells, 20 ml of trypsin (0.5% 10 × solution) solution manufactured by Life Technologies was mixed with 980 ml of PBS and used by filtration.
세포수 측정용 측정 완충액으로는 콜터 일렉트로닉(Coulter Electronics)사제 이소톤 Ⅲ 균형 전해용질 용액을 사용하였으며, 세포의 고정과 염색에는 준세이(Junsei)사제 크리스탈 바이올렛 분말 0.6g을 이차증류수 100ml에 섞어 0.6% 저장 용액을 만들고 이것으로 다시 실시 용액(Working solution)을 만들어 사용하였다. 검액(檢液)의 용해에는 시그마(Sigma)사제 DMSO(Dimethyl sulfoxide)를 사용하였다.Isotone III balanced electrolytic solute solution, manufactured by Coulter Electronics, was used as the measurement buffer for cell number measurement.For fixation and staining of cells, 0.6 g of crystal violet powder made by Junsei was mixed with 100 ml of secondary distilled water and 0.6% A stock solution was made and used again as a working solution. DMSO (Dimethyl sulfoxide) manufactured by Sigma was used for dissolution of the sample solution.
(ⅱ) 검증 세포주(Cell line) 및 배양 조건(Ii) Cell lines and culture conditions
검증에 사용된 암세포주는 헬라 세포로 세포주는 경상대학교 의과대학 생화학 실험실로부터 분양받아, 동국대학교 생물학과 세포 유전학실험실에서 배양중인 세포주를 사용하였다. (Woynarowski, J.M. and Beerman, T. A., 1980)The cancer cell line used for the verification was a HeLa cell, and the cell line was distributed from the Biochemistry Laboratory, College of Medicine, Gyeongsang National University, and the cell line was cultured in the Department of Biology and Genetics, Dongguk University. (Woynarowski, J.M. and Beerman, T. A., 1980)
암세포배양에 필요한 환경 조건은 온도 37℃, 습도 100%, CO2농도 10%를 유지하였다. (Freshney, R. L., 1994)Environmental conditions required for cancer cell culture were maintained at a temperature of 37 ℃, 100% humidity, 10% CO 2 concentration. (Freshney, RL, 1994)
(ⅲ) 검증 기기 및 장치(Iii) verification equipment and devices
암세포를 배양하기 위해서 CO2인큐베이터(Sheldon manufacturing Co. , Model: 2300), 배양한 암세포 수를 파악하기 위하여 콜터 카운터(Coulter Electronics Co. , Model: Z-1 cell counter, Aperture tube pore size: 100μm, 설정 세포 크기 : 5-15μm)를 사용하였다. (Hazelton, B. J. et al. 1984)CO 2 incubator for culturing cancer cells (Sheldon manufacturing Co., Model: 2300), Coulter counter (Coulter Electronics Co., Model: Z-1 cell counter, Aperture tube pore size: 100μm, Set cell size: 5-15 μm). (Hazelton, BJ et al. 1984)
암세포 배양 용기로는 코닝(Corning)사제 25cm2일회용 멸균 조직 배양 플라스크(Polystyrene)를 사용하였고, 실험에 쓰인 용기는 같은 회사제 24-웰 플레이트(with lid, flat bottom, well diameter 16mm, polystyrene)를 사용하였다. (Dealtry, G. B and Rickwood, D. , 1992)As a cancer cell culture vessel, a 25 cm 2 disposable sterile tissue culture flask (Polystyrene) manufactured by Corning was used, and the container used for the experiment was a 24-well plate (with lid, flat bottom, well diameter 16 mm, polystyrene) manufactured by the same company. Used. (Dealtry, G. B and Rickwood, D., 1992)
배지 여과를 위하여 여과기(Millipore Co., Pore size : 0.2μm)을 항생제의 여과를 위하여 주사기 필터(Pore size : 0.2μm)를 사용하였다.A filter (Millipore Co., Pore size: 0.2 μm) was used for filtration of media and a syringe filter (Pore size: 0.2 μm) was used for filtration of antibiotics.
배양중인 암세포의 관찰에는 니콘사제 위상차현미경을 사용, 검경하였고, 염색된 세포의 콜로니 관찰에는 같은 회사의 SMZ-1 해부현미경을 사용하였다.The observation of cancer cells in culture was carried out using a Nikon retardation microscope, and SMZ-1 dissecting microscope of the same company was used to observe the colonies of the stained cells.
(ⅳ) 세포 도말 방법(Iii) Cell smearing method
검액 약제 투여 전일, 24-웰 플레이트에 혈청 10% D-MEM을 각 웰 마다 1.0ml씩 넣은 다음, 배양중의 세포를 트립신 처리하여 수확한 후 세포수 측정기로 암세포 농도를 계산하였다. D-MEM으로 농도별로 희석하여 암세포 수를 달리한 다음 각각의 웰에 도말하였다.On the day before the administration of the test agent, 1.0 ml of serum 10% D-MEM was added to each well in a 24-well plate, and the cells in the culture were trypsinized and harvested. Diluted by concentration with D-MEM to vary the number of cancer cells and then plated in each well.
측정 방법은 8회 측정하여 최고치와 최저치를 뺀 나머지의 평균을 구 하였다.The measurement method was measured 8 times and the average of the remaining minus the highest and lowest values was obtained.
(ⅴ) 모자반 추출 검액(檢液)의 투여(Iv) Administration of Mother-and-Child Extraction Sample Solution
세포 도말 18시간 후, 배지를 제거한 후, 혈청이 없는 D-MEM을 웰당 1.0ml씩 넣고, 검액 약제를 혈청 없는 D-MEM으로 농도별 희석하여 각 웰에 투여하였다. 투여 4시간 후, 배지를 모두 제거한 후 다시 혈청 10% D-MEM을 웰당 2.0ml씩 넣었다. (Piovella, F. M. et al., 1984)After 18 hours of cell smearing, the medium was removed, and then 1.0 mL of D-MEM without serum was added to each well, and the sample solution was diluted to D-MEM without serum and administered to each well. After 4 hours of administration, the medium was completely removed, and then serum 10% D-MEM was added 2.0 ml per well. (Piovella, F. M. et al., 1984)
(ⅵ) 잔류 암세포계수 방법(Iii) Residual Cancer Cell Counting Method
검액 투여 6일 후, 배지를 모두 제거하고 크리스탈 바이올렛으로 3분간 염색하여 건조시킨 후, 해부현미경으로 관찰, 암세포 수를 파악한 후 도말한 세포 수와 비교하여 생존율을 계산하였다. (Satish, J. et al. , 1985)Six days after the administration of the sample solution, all of the medium was removed, stained with crystal violet for 3 minutes, dried, and observed under a dissecting microscope to determine the number of cancer cells, and the survival rate was calculated by comparing with the number of smeared cells. (Satish, J. et al., 1985)
(나) 모자반 추출 검액 투여 암세포(HeLa Cell) 평균 생존율 검증법(B) Average test for survival rate of HeLa Cells
모자반 추출 검액 투여 6일 후 암세포의 평균 생존율을 대조군(100%)과 비교한 결과 최저치는 1,600 μg/ml의 추출 검액 투여시 0.6%의 생존율을 보여 높은 제암력(制癌力)을 나타내었다.When the average survival rate of cancer cells was compared with the control group (100%) 6 days after the administration of the mother extract, the lowest value was 0.6% when the 1,600 μg / ml extract was administered, indicating high anticancer activity.
* 본 발명의 상품화 방안은 다음과 같다.* Commercialization method of the present invention is as follows.
5g의 모자반 추출액 10±6.6 저농도 희석액을 51g/kg 투여량 기준 (1.2∼ 1.6g)의 가) 캡슐제 나)주사제 다)산제 (0.9∼1.6g) 라)환제 마)도포제를 상품화 한다.5g Mabanban extract 10 ± 6.6 low concentration diluent 51g / kg dose (1.2 ~ 1.6g) a) capsules b) injections c) powders (0.9-1.6 g) d) pills E) coating agent is commercialized.
* 본 발명을 다음 실시예로 좀더 상세히 설명하지만 본 발명이 다음의 실 시 예의 범위에만 국한되는 것은 아니다.* The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the scope of the following examples.
실시 예 1Example 1
모자반을 한국 동해 영일만을 중심으로 한 감포 연해 청정해역에서 채집, 수집, 선정한후 기원을 감정하고 동정한 생시료를 증류수에 5회 세척하고 불순물을 제거 음건한 후 45∼50℃에서 3일간 건조 1차로 대한 약전 시험법에 따라 세절하고 다시 80mech로 마쇄한 후 그중 수분 함량 3.5∼ 5.0%인 분말 시료 5g만을 수득하고 이를 75% 에탄올 100ml와 함께 수욕 상에서 1시간 추출, 여과, 풍건 하였다. After collecting, collecting and selecting the mother and child in the Gampo coastal clean water centered on Yeongil Bay, South Korea, the origin was assessed and the identified raw samples were washed 5 times in distilled water and dried to remove impurities for 3 days and then dried at 45 ~ 50 ℃ for 3 days. After crushing according to the pharmacopoeia test method and pulverized again to 80mech, only 5g of a powder sample having a water content of 3.5-5.0% was obtained, which was extracted, filtered and air dried for 1 hour in a water bath with 100ml of 75% ethanol.
2차로 그중 1.2g만을 평량 분리하여 이차증류수 19.0ml 및 DMSO 1.0ml로 희석하여 최종농도가 57μg/μl 가 되도록 하여 제조한 모자반으로부터 추출한 포릭산 등 16종 생약성분의 항암제에 관한 것으로서 암세포(HeLa Cell)에 항암효과가 있음을 특징으로 한다.Secondly, the anticancer agent of 16 kinds of herbal medicines such as poric acid extracted from Maban prepared by dividing only 1.2 g of the basis weight and diluting it with 19.0 ml of secondary distilled water and 1.0 ml of DMSO to have a final concentration of 57 μg / μl. ) Has an anticancer effect.
이때 실험에 사용된 배지는 라이트 테크놀로지사제 D-MEM(Dulbecco's Modified Eagle Medium, powder)을 3차 증류수에 녹여 여과한 후, 같은 회사제의 혈청을 10% 첨가하여 사용하였다.At this time, the medium used in the experiment was dissolved D-MEM (Dulbecco's Modified Eagle Medium, powder) manufactured by Light Technology in tertiary distilled water and filtered, and then used by adding 10% serum of the same company.
미생물에 의한 오염을 막기 위하여 사용한 항생제는 시그마사제 복합항생제 안티마이코틱을 여과하여 사용하였으며 그 조성은 배지 1ml당 페니실린 10,000 유니트, 스트렙토마이신 10mg, 암포테리신 25mg, 배지 100ml로 하였다.Antibiotics used to prevent contamination by microorganisms were used by filtering antimicrobial antimicrobial compound made by Sigma, the composition was 10,000 units of penicillin per 10ml, streptomycin 10mg, amphotericin 25mg, 100ml medium.
배양한 세포의 세척에는 PBS(Phosphate Buffered Solution)를 멸균하여 사용하였으며 그 조성은 이차증류수 1l당 Nacl 8g, Na2HPO4·12H2O 2.89g KH2HPO40.2g KCl 0.2g으로 하였다.Washing the cultured cells have been used to sterilize (Phosphate Buffered Solution) PBS its composition was as per 1l distilled water Nacl 8g, Na 2 HPO 4 · 12H 2 O 2.89g KH 2 HPO 4 0.2g KCl 0.2g.
배양한 암세포의 수확에는 라이프 테크놀로지사제 트립신(0.5% 10× solution)용액 20ml을 PBS 980ml에 섞어 여과하여 사용하였다.For harvesting the cultured cancer cells, 20 ml of trypsin (0.5% 10 × solution) solution manufactured by Life Technologies was mixed with 980 ml of PBS and used by filtration.
세포수 측정용 측정 완충액으로는 콜터 일렉트로닉사제 아이소톤 Ⅲ 균형 전해용질 용액을 사용하였으며, 세포의 고정과 염색에는 준세이사제 크리스탈 바이올렛 분말 0.6g을 이차증류수 100ml에 섞어 0.6% 저장 용액을 만들고 이것으로 다시 실시 용액(Working solution)을 만들어 사용하였다. 모자반 검액의 용해에는 시그마사제 DMSO(Dimethyl sulfoxide)를 사용하였다.Isotone III balanced electrolysate solution from Coulter Electronics was used as the measurement buffer for cell number measurement. For fixation and staining of cells, 0.6 g of Crystal Violet powder manufactured by Junsei Co., Ltd. was mixed with 100 ml of secondary distilled water to make 0.6% stock solution. Working solution was made and used. DMSO (Dimethyl sulfoxide) manufactured by Sigma Co., Ltd. was used for dissolution of the mother plaque.
실시 예 2Example 2
상기 실시 예 1에서 얻은 모자반 검증 용액으로 검증 대상에 사용된 암세포주는 헬라 세포이며 세포주는 경상대학교 의과대학 생화학 실험실로부터 분양받아, 동국대학교 생물학과 세포 유전학 실험실에서 배양중인 세포주를 사용하였다. (Woynarowski, J.M. and Beerman, T. A. , 1980)The cancer cell line used for the test subject with the Maban verification solution obtained in Example 1 was a HeLa cell and the cell line was distributed from the Biochemistry Laboratory of the College of Medicine, Gyeongsang National University, and the cell line was cultured in the Department of Biology and Cytogenetics, Dongguk University. (Woynarowski, J.M. and Beerman, T. A., 1980)
암세포배양에 필요한 환경 조건은 온도 37℃, 습도 100%, CO2농도 10%를 유지하였다. (Freshney, R. L., 1994)Environmental conditions required for cancer cell culture were maintained at a temperature of 37 ℃, 100% humidity, 10% CO 2 concentration. (Freshney, RL, 1994)
암세포를 배양하기 위해서 CO2인큐베이터(Sheldon Manufacturing Co., Model: 2300), 배양한 암세포 수를 산정하기 위하여 콜터 카운터(Coulter Electronics Co. , Model: Z-1 cell counter, Aperture tube pore size: 100μm, 설정 세포 크기: 5-15μm)를 사용하였다. (Hazelton, B. J. et al. 1984)CO 2 incubator (Sheldon Manufacturing Co., Model: 2300) to culture cancer cells, Coulter counter (Coulter Electronics Co., Model: Z-1 cell counter, Aperture tube pore size: 100μm, Set cell size: 5-15 μm). (Hazelton, BJ et al. 1984)
암세포 배양 용기로는 코닝사제 25cm2일회용 멸균 조직 배양 플라스크(Polystyrene)와 24-웰 플레이트(뚜껑있음, 평평한 바닥, 웰 직경 16mm, 폴리스티렌)를 사용하였다. (Dealtry, G. B and Rickwood, D. , 1992)As a cancer cell culture vessel, a 25 cm 2 disposable sterile tissue culture flask (Polystyrene) manufactured by Corning Corporation and a 24-well plate (with a lid, flat bottom, well diameter 16 mm, and polystyrene) were used. (Dealtry, G. B and Rickwood, D., 1992)
암세포 배지 여과를 위하여 여과기(Millipore Co. , Pore size : 0.2μm)를 항생제의 여과를 위하여 주사기 필터(구멍 크기: 0.2μm)를 사용하여 검증 하였다.A filter (Millipore Co., Pore size: 0.2 μm) for cancer cell medium filtration was verified using a syringe filter (pore size: 0.2 μm) for filtration of antibiotics.
모자반 추출 검액 투여 24시간 전 24-웰 플레이트에 혈청 10% D-MEM을 각 웰마다 1.0ml씩 넣은 다음 배양중의 세포를 트립신 처리 수확하여 세포수 측정 장치로 암세포 농도를 측정하였다.Serum 10% D-MEM was added to each well for 24 hours 24 hours prior to the administration of the mother extract test solution, and then the cells in the culture were trypsinized and cultured, and the cancer cell concentration was measured by a cell count measuring device.
D-MEM으로 농도별로 희석하여 암세포 수를 달리한 후 각각의 웰에 도말하였다. 측정 방법은 8회 측정하여 최고치와 최저치를 뺀 나머지의 평균을 구하였다. 세포 도말 18시간 후, 배지을 제거한 후, 혈청이 없는 D-MEM을 웰당 1.0ml씩 넣고, 검액을 혈청이 없는 D-MEM으로 농도별 희석하여 각 웰에 투여하였다. 투여 4시간 후, 배지를 모두 제거하고 다시 혈청 10% D-MEM을 웰당 2.0ml씩 넣었다. (Piovella, F. M. et al., 1984)Diluted by concentration with D-MEM to vary the number of cancer cells and plated in each well. The measurement method measured eight times and calculated | required the average of the remainder which subtracted the highest value and the lowest value. After 18 hours of cell smearing, the medium was removed, and then 1.0 mL of D-MEM without serum was added to each well, and the test solution was diluted to D-MEM without serum and administered to each well. After 4 hours of administration, the medium was removed, and again, 2.0 ml of serum 10% D-MEM was added per well. (Piovella, F. M. et al., 1984)
실시 예 3Example 3
상기 실시 예 2에서 얻은 암세포 (Serum 10% D-MEM을 웰당 2.0ml 씩 넣은 암세포)에다 모자반 검액을 투여한 6일 후, 배지를 모두 제거하고 크리스탈 바이올렛으로 3분간 염색하여 건조시킨 후, 해부현미경으로 암세포 수를 산정 하고 이를 도말한 암세포 수와 비교하여 생존율을 계산(Satish, J. et al., 1985)한 결과 암세포의 평균 생존율을 대조군(100%)과 비교한 결과 생존율 최저치는 1,600 μg/ml의 모자반 추출물 투여시 0.6% 로 99.4%의 양호한 제암력을 나타내었다.After 6 days of administration of the mother cell sample to the cancer cells obtained in Example 2 (cancer cells containing 2.0 ml per well of Serum 10% D-MEM), all of the medium was removed, stained with crystal violet for 3 minutes, and dried, followed by a dissecting microscope. The survival rate was calculated by comparing the number of cancer cells with the number of cancer cells (Satish, J. et al., 1985), and compared with the control group (100%). The lowest survival rate was 1,600 μg / Administration of ml of Mabanban extract showed good anticancer activity of 0.6% to 99.4%.
실시 예 4Example 4
모자반을 한국 동해 영일만을 중심으로 한 감포 연해 청정해역에서 채집, 수집, 선정한 후 기원을 감정하고 동정한 후 3개의 부위별로 각 50g씩 평량하여 둥근 플라스크 (2중 추출장치: EYELA Evaporator Aspirator A-3s: Tokyo Rikakikai Co.)에 넣고 증류수 1,500ml를 더해 4.5시간 가열 추출하여 감압 여과한 후 150g의 검액을 수득 하였다.Mabuban is collected, collected and selected in the Gimpo coastal clean sea area centered on Yeongil Bay, Korea.Then, the origin is assessed and identified, followed by a round flask weighing 50g for each of the three parts (double extraction device: EYELA Evaporator Aspirator A-3s). : Tokyo Rikakikai Co.) and 1,500ml of distilled water were added and extracted by heating for 4.5 hours to obtain 150g of sample solution.
상기 검액을 증류수로 1000±180배로 희석하였다.The test solution was diluted 1000-180 times with distilled water.
알라닌, 아스타트 산, 글루탐산, 프로린, 쓰레오닌, 발린, 리나룰, 시스테인의 추출은 U. O. Lee (1965, 1975, 1976,1978, 1979), Do. I (1953), Kitajawa (1953), Matsushima (1928), Nakami (1909), Takao(1980, 1981, 1982), W. C. Cutting (1971)법에 의했다.Extraction of alanine, aspartic acid, glutamic acid, proline, threonine, valine, linalul and cysteine is described in U. O. Lee (1965, 1975, 1976, 1978, 1979), Do. I (1953), Kitajawa (1953), Matsushima (1928), Nakami (1909), Takao (1980, 1981, 1982), and W. C. Cutting (1971).
실시 예 5Example 5
항암효과 입증 실험Anti-cancer effect proved experiment
1차로 45∼50℃에서 3일간 건조하여 80 mesh로 마쇄한 5g 순수 모자반 분말시료를 75% 에탄올 100ml와 함께 수욕상에서 1시간 추출, 여과한 정제 시료로 제조하고 그중 1.2g만을 평량 분리 하여 2차로 이차증류수 19.0ml 및 DMSO 1.0ml에 상 분리시료를 희석 57μg/μl로 농도조정 검액한 후 1,600μg/ml로 최종 조절 검액으로 전환 정제하여 암세포(HeLa Cell)에 투여 했다. 투여 결과 6일후 표준 생존율 100% 기준 대비 암세포(HeLa Cell)의 생존율은 0.6%로 검증 되었다. 즉, 모자반으로부터 추출한 생약물질의 제암력은 99.4%임이 확인되었다.Firstly, a 5g pure Maban powder sample, dried at 45-50 ° C. for 3 days and ground in 80 mesh, was prepared as a purified sample extracted for 1 hour in a water bath with 100 ml of 75% ethanol. Phase separation samples were diluted in 19.0 ml of secondary distilled water and 1.0 ml of DMSO, diluted to 57 μg / μl, and then converted to final controlled samples at 1,600 μg / ml and administered to HeLa Cell. After 6 days, the survival rate of HeLa cell was 0.6% compared to the standard survival rate of 100%. That is, it was confirmed that the anticancer power of the herbal medicine extracted from the mother board was 99.4%.
본 출원인의 실험에 의하면 모자반(Sargassum fulvellum) 추출물 1.2g을 이차증류수 19.0ml가 되도록 DMSO 1.0ml로 희석하여 1차 최종농도가 57μg/μl 가 되도록 제조한 시료 검액을 2차 검액 1,600μg/ml로 제조하여 첨가하였을 때 투여결과 6일후 표준 100% 기준 대비 암세포 헬라 세포의 생존율은 0.6%이였으므로 제암력은 99.4%로 확인되었다.According to the applicant's experiment, 1.2 g of Sargassum fulvellum extract was diluted with 1.0 ml of DMSO to make 19.0 ml of secondary distilled water, and the sample sample prepared so as to have a final final concentration of 57 μg / μl to 1,600 μg / ml of secondary sample solution. When prepared and added, the cancer cell HeLa cell survival rate was 0.6% compared to the standard 100% standard after 6 days of administration, and the anticancer activity was confirmed as 99.4%.
본 발명의 이점은 모자반(Sargassum fulvellum)의 주요성분 중 포릭산, 바이오틴, 이노시톨, 아스탈틱산, 알라닌은 혈구 생성, 생식선 조성, 효소 발육, 뇌세포 활성, 항생작용, 바시트란 기능조성, 유해 세균 세포층 페프트 글리칸 용해기능, 헬라 세포 주효소 저해기능이 있으며 글루타민산, 프로린, 트레오닌, 발린, 시스틴은 항체 생성 기능, 틸라코이드 구성대사, 해독·항균 작용, 헬라 세포 분열 대사효소 억제기능이 있으며 제약비용이 극히 저렴하다. 종래의 둥근 바위솔(Orostachys aggregeatus) 와송 등 현재 동물 및 임상시험 중인 생약 함암제의 치료 효능이 10% 내외의 생존일수 연장 효능이 있는 것에 비교하면 본 발명의 모자반(S. fulvellum) 에탄올 추출액이 0.6% 암세포 헬라 세포의 생존율을 나타내게 됨으로서 높은 항암력이 검증되었음을 알 수 있다.Advantages of the present invention are the main components of Sargassum fulvellum, foric acid, biotin, inositol, acetic acid, alanine are blood cell production, gonad composition, enzyme development, brain cell activity, antibiotic activity, vacitlan function composition, harmful bacteria Cell layer-peptide glycan lysis, Hela cell protease inhibitory function, glutamic acid, proline, threonine, valine, cystine have antibody production function, thylakoid constituent metabolism, detoxification and antibacterial activity, inhibition of HELL cell division metabolic enzyme and pharmaceutical cost This is extremely cheap. Compared to the conventional animal and clinically tested herbal anticancer drugs , such as Orostachys aggregeatus Wasong , which have a 10-year prolonged survival time, the S. fulvellum ethanol extract of the present invention is 0.6% cancer cells. By showing the survival rate of HeLa cells, it can be seen that high anticancer activity has been verified.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006129901A1 (en) * | 2005-06-01 | 2006-12-07 | Blc Bio Tech Inc. | Anticancer agent, food and manufacturing method of anticancer agent and food |
KR101239175B1 (en) * | 2009-08-28 | 2013-03-06 | 박헌경 | Method for manufacturing fodder with blc powder |
-
2003
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006129901A1 (en) * | 2005-06-01 | 2006-12-07 | Blc Bio Tech Inc. | Anticancer agent, food and manufacturing method of anticancer agent and food |
KR101239175B1 (en) * | 2009-08-28 | 2013-03-06 | 박헌경 | Method for manufacturing fodder with blc powder |
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