KR101477966B1 - A composition comprising the extract of pine tree leaf for the prevention and treatment of cervical cancer - Google Patents
A composition comprising the extract of pine tree leaf for the prevention and treatment of cervical cancer Download PDFInfo
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- KR101477966B1 KR101477966B1 KR1020120058332A KR20120058332A KR101477966B1 KR 101477966 B1 KR101477966 B1 KR 101477966B1 KR 1020120058332 A KR1020120058332 A KR 1020120058332A KR 20120058332 A KR20120058332 A KR 20120058332A KR 101477966 B1 KR101477966 B1 KR 101477966B1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/13—Coniferophyta (gymnosperms)
- A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
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Abstract
본 발명은 솔잎 추출물을 유효성분으로 함유하는 조성물에 관한 것으로, 구체적으로 본 발명의 솔잎 추출물은 인체 파필로마바이러스(human papillomavirus, HPV)를 억제하여 상기 바이러스 감염으로 기인한 자궁경부암 예방 및 치료용 약학조성물 및 건강기능식품에 유용하게 이용될 수 있다.The present invention relates to a composition containing pine needle extract as an active ingredient. Specifically, the pine needle extract of the present invention inhibits human papillomavirus (HPV) and is useful as a medicine for prevention and treatment of cervical cancer caused by the above- Compositions and health functional foods.
Description
본 발명은 인체 파필로마바이러스(human papillomavirus, HPV)를 억제함으로써, 자궁경부암 억제활성을 가지는 솔잎 추출물을 포함하는 자궁경부암 예방 및 치료용 조성물을 제공함을 목적으로 한다.It is intended to provide a composition for the prevention and treatment of cervical cancer comprising a pine needle extract having a cervical cancer inhibitory activity by inhibiting human papillomavirus (HPV).
[문헌 1] Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85(13), 1028-1029(1993)[0005] Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85 (13), 1028-1029 (1993)
[문헌 2] Munoz, N. and Bosch, F. X., Epidemiology of cervical cancer. In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, F. X. and Jensen, O. M. IARC Scientific Publications, Lyon, 9-40(1989)[Document 2] Munoz, N. and Bosch, F. X., Epidemiology of cervical cancer. In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, F. X. and Jensen, O. M. IARC Scientific Publications, Lyon, 9-40 (1989)
[문헌 3] Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer cells 4/DNA tumor viruses, Cold Spraing Harbor Laboratory, USA, 17-36(1989)[Patent Document 3] Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer
[문헌 4] Durst, M. et al., Papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographical regions, Proc. Natl. Acad. Sci., USA, 80. 3812-9-2 1019950024886, 3815(1983)[4] Durst, M. et al., Papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographical regions, Proc. Natl. Acad. Sci., USA, 80. 3812-9-2 1019950024886, 3815 (1983)
[문헌 5] Hausen, H., Viruses in human cancers, Science, 254, 1173-1187(1991)[Literature 5] Hausen, H., Viruses in human cancers, Science, 254, 1173-1187 (1991)
[문헌 6] Galloway, D. A. et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171(1990)[Literature 6] Galloway, D. A. et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171 (1990)
[문헌 7] Lorincz, A. T. et al., Human Papillomavirus Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337(1992)[Literature 7] Lorincz, A. T. et al., Human Papillomavirus Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337 (1992)
[문헌 8] Kim, T.J., Korean Resources Plants . II , pp194-195, 1996[Document 8] Kim, TJ, Korean Resources Plants . II , pp194-195, 1996
[문헌 9] Kang, T.H., Jeong, S.T., et al., Journal of Ethnopharmacology , 71, pp321-323, 2000[Literature 9] Kang, TH, Jeong, ST, et al., Journal of Ethnopharmacology , 71 , pp 321-323, 2000
[문헌 10] Jung, M.J., Chung, H.Y. et al., Archives of pharmacal research, 26(6), pp458-462[Literature 10] Jung, MJ, Chung, HY et al., Archives of pharmacal research , 26 (6) , pp 458-462
[문헌 11] Kaneta M., Hikichi H. et al., Agricultural and Biological Chemistry, 44, pp1407, 1980[Document 11] Kaneta M., Hikichi H. et al. Agricultural and Biological Chemistry , 44 , pp1407, 1980
[문헌 12] Woo W.S., Kang S.S. et al., Journal of Natural Products, 49, pp547-549, 1984[Document 12] Woo WS, Kang SS et al., Journal of Natural Products , 49 , pp 547-549, 1984
[문헌 13] Woo W.S., Lee E.B. et al., Kor . J. Pharmacogn , 12, pp153, 1981[Literature 13] Woo WS, Lee EB et al., Kor . J. Pharmacogn. , 12, pp 153, 1981
[문헌 14] Moon C.K., Lee B.G. et al., Archives of Pharmacal Research, 8, pp277, 1985[14] Moon CK, Lee BG et al., Archives of Pharmacal Research , 8 , pp 277, 1985
[문헌 15] Yang M.S., Ha Y.L. et al., Agricultural Chemistry and Biotechnology, 38, pp584, 1995[Literature 15] Yang MS, Ha YL et al., Agricultural Chemistry and Biotechnology, 38 , pp 584, 1995
[문헌 16] Yoo J.S., Jang J.S. et al., KOR . J. Pharmacogn , 26, pp355, 1995[16] Yoo JS, Jang JS et al., KOR . J. Pharmacogn , 26 , pp 355, 1995
[문헌 17] Zou K., Zhao Y. et al., Carbohydrate Research , 324, pp182-188, 2000[Literature 17] Zou K., Zhao Y. et al., Carbohydrate Research , 324 , pp 182-188, 2000
[문헌 18] 赤松金芳저, 신정 화한약, pp664-665, 1980 [Literature 18] Kim, Akamatsu, Shin Jeonghwa Chinese medicine, pp664-665, 1980
[문헌 19] 御影雅幸저, 日本生藥學雜誌, p 336, 1991[Literature 19] Masayoshi Ogaga, Journal of the Japanese Institute of Pharmaceutical Sciences, p 336, 1991
[문헌 20] 박종갑 저, 한방대의전, p134, 1984; 문화방송편저, 한국민간요법대전, p21, 1988[Literature 20] Park, Jong-gap, I, pp. 134, 1984; Cultural Broadcasters, Korean Folk Therapy, p21, 1988
[문헌 21] Harborne J.B., Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed ., pp6-7, 1998[Document 21] Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed . , pp6-7, 1998
[문헌 22] 대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989[Literature 22] Korean Pharmacopoeia Commentary, Mun Sung-sa, Korea Pharmacy College Council, 5th edition, p33-48, 1989
[문헌 23] Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 13; 857-861.
[23] Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 13; 857-861.
자궁경부암은 자궁암의 90% 이상을 차지하는 악성종양 유발 바이러스로 한국 여성에 있어서 발생 및 사망 빈도가 가장 높은 암 질환인데, 최근 들어 인체 파필로마바이러스(human papillomavirus, HPV)의 감염이 발암 기전에서 가장 중요하게 작용하는 인자인 것으로 밝혀졌다.Cervical cancer is a malignant tumor-causing virus that accounts for more than 90% of uterine cancer. It has the highest incidence and mortality rate in Korean women. In recent years, infection of human papillomavirus (HPV) Which is known to be a causative agent.
자궁암은 현재 개발도상국에 있어 여성의 암중 발생률이 가장 높으며(Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85(13), 1028-1029(1993)), 매년 새로 발생하는 환자의 수가 약 50 만명에 이르는 것으로 보고된 바 있다(Munoz, N. and Bosch, F. X., Epidemiology of cervical cancer. In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, F. X. and Jensen, O. M. IARC Scientific Publications, Lyon, 9-40(1989)). 서울대 의대 연구팀(김진복과 안윤옥, 1993)의 우리나라 서울 지역에서의 1991년 7월부터 1993년 6월까지 2년간의 암 발생률 조사 결과에 의하면, 여성은 10만 명당 123명이 연간 암에 걸리며 이중 자궁암이 22%, 위암 18%, 유방암 12.9%로 우리나라에서도 자궁암 발생률이 가장 높은 것으로 나타났다.
Cervical cancer is currently the most common cancer incidence in developing countries (Vanchieri, C., IARC Publishes Data on Worldwide Cancer Cases, Journal of the National Cancer Institute, 85 (13), 1028-1029 The number of patients has been reported to reach about 500,000 (Munoz, N. and Bosch, FX, Epidemiology of cervical cancer. In: Human Papillomaviruses and Cervical Cancer, eds. Munoz, N., Bosch, FX and Jensen , OM IARC Scientific Publications, Lyon, 9-40 (1989)). According to the results of a 2-year cancer incidence study conducted between July 1991 and June 1993 in the Seoul area of Seoul National University Medical Research Team (Kim, Jin-bok, and An Yeon-oh, 1993), 123 women per 100,000 women per year had cancer, 22%, gastric cancer 18%, breast cancer 12.9%, the highest incidence of cervical cancer in Korea.
파필로마바이러스는 동물의 여러조직의 상피세포에 감염하여 손, 발, 피부, 후두 등에 흔히 사마귀로 불리우는 양성종양을 유발하는 것으로 알려져 왔다. 이들 파필로마바이러스는 현재 인간에 있어서 70종류 정도의 유전자형이 밝혀져 있는데, 여러 유전자형이 그 감염조직에 대해 특이성을 나타내고 다양한 질병을 유발하는 것으로 보고되어 있다(Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer cells 4/DNA tumor viruses, Cold Spraing Harbor Laboratory, USA, 17-36(1989)).Papilloma viruses have been known to infect epithelial cells of various tissues of animals and cause benign tumors, often called warts, on the hands, feet, skin, and larynx. These papilloma viruses are now identified in about 70 genotypes in humans, and several genotypes have been reported to be specific for the infected tissue and cause a variety of diseases (Broker et al., Papillomaviruses: Retrospectives and Prospectives, Cancer
이러한 여러 유전자형의 파필로마바이러스 대부분은 치료가 잘 이루어지지 않아서 감염 환자에게 큰 고통을 주기는 하지만 치명적인 질병을 유발하지는 않았기 때문에 주목되지 않았다. 그러나, 최근 들어 이 바이러스의 특정형, 특히 인체 파필로마바이러스 16형과 18형은 남녀 생식기, 구강, 피부 등에서의 악성 종양과 관련되며 여성 자궁암의 90% 이상을 차지하는 자궁경부암을 일으키는 주요인자로 작용할 뿐 아니라, 6b형과 11형은 곤지름(condyloma acuminata)으로 일컬어지는 남녀 생식기의 양성 종양을 유발하는 것으로 밝혀졌다. 또한, 역학조사 결과 자궁암이 주로 성적접촉에 의해 전파되는 인자에 의해 발생되는 것으로 제시되었으며(Durst, M. et al., Papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographical regions, Proc. Natl. Acad. Sci., USA, 80. 3812-9-2 1019950024886, 3815(1983)), 자궁내막상피이상증(cervical intraepithelial neoplasm, CIN)으로 명명된 전구암 병소의 85 내지 100%에 파필로마바이러스가 감염되어 있다는 것 등, 파필로마바이러스가 자궁암 유발에 관련된다는 많은 증거들이 밝혀짐으로 해서(Hausen, H., Viruses in human cancers, Science, 254, 1173-1187(1991)), 파필로마바이러스의 발암 기작에 대한 연구는 물론 이를 이용한 진단제 및 치료제의 개발이 필요하게 되었다(Galloway, D. A. et al., Human papillomaviruses and carcinomas, Adv. Virus Res., 37, 125-171(1990)).Many of these genotypic papillomaviruses were not treated because they were not well treated and did not cause a fatal disease, although they inflicted great distress on infected patients. However, in recent years, certain types of the virus, especially
한편, 자궁암 환자에게 인체 파필로마바이러스 16형과 18형의 감염율은 일반적으로 16형이 50 내지 70% 정도로 이르고 18형은 15 내지 25%에 이르는 것으로 보고되어 있다. 그러나 전이암의 경우에는 16형에 감염된 암환자의 25%, 그리고 18형에 감염된 암환자의 50% 이상이 전이암 환자인 것으로 조사되었다 (Lorincz, A. T. et al., Human Papillomavirus Infection of the Cervix: Relative Risk Associations of 15 Common Anogenital Types, Obsterics and Gynecology, 79, 328-337(1992)).
On the other hand, infection rates of
국내에 자생하는 식물 중에 솔잎은 한국에 널리 분포하고 있으며, 예로부터 불면증, 강장, 흥분, 이뇨, 진통, 구충제 등의 민간약제로 사용되어 왔다 (Kim, T.J., Korean Resources Plants . II , pp194-195, 1996). Among the plants that are native to Korea, pine leaves are widely distributed in Korea and have been used as a medicinal agent for insomnia, tension, excitement, diuretic, analgesic and insect repellent (Kim, TJ, Korean Resources Plants . II , pp194-195, 1996).
최근 솔잎에서 많은 화학적 성분이 분리되고 그 구조가 밝혀지고 있다 (Kang, T.H., Jeong, S.T., et al., Journal of Ethnopharmacology , 71, pp321-323, 2000; Jung, M.J., Chung, H.Y. et al., Archives of pharmacal research, 26(6), pp458-462). 예를 들면 솔잎 성분에 관한 연구는 껍질에서 플라보노이드 (flavonoid) 인 퀴르세틴-3-O-갈락토사이드 (quercetin 3-O-galactoside), 퀴르세틴-3-O-람노사이드(quercetin 3-O- rhamnoside), 3,4,7- 트리하이드록시 플라본 (trihydroxyflavone)과 알파 -스피나터리 배당체 (a-spinastery glucoside), 수종의 트리테르페노이드 (triterpenoid), 사포닌 (saponin) 및 리그난 (lignan) 성분 등이 분리되었으며 (Kaneta M., Hikichi H. et al., Agricultural and Biological Chemistry, 44, pp1407, 1980; Woo W.S., Kang S.S. et al., Journal of Natural Products, 49, pp547-549, 1984), 이외에도 꽃에서 플라보놀 배당체 (flavonol glycosides)인 퀘르시트린 (quercitrin)과 이소퀘르시트린 (isoquercitrin) 이 분리되었다. 또한 솔잎 성분의 약리작용에 관한 연구는 사포닌 분획(saponin fraction)에서 자궁수축작용 증가 (Woo W.S., Lee E.B. et al., Kor . J. Pharmacogn , 12, pp153, 1981), 다당체 분획(polysaccharide fraction)의 사코마 180 고체 상 (sarcoma 180 solid form)에 대한 항암활성(Moon C.K., Lee B.G. et al., Archives of Pharmacal Research, 8, pp277, 1985), 항균활성(Yang M.S., Ha Y.L. et al., Agricultural Chemistry and Biotechnology, 38, pp584, 1995), 설사억제 효과(Yoo J.S., Jang J.S. et al., KOR . J. Pharmacogn , 26, pp355, 1995), 및 플라보놀 배당체 (flavonol glycoside)에 의한 수면 효과등이 있다. 이들 화합물 중에서 사포닌이 암세포주에 강한 치사활성을 나타낸다고 알려져 있으나 (Zou K., Zhao Y. et al., Carbohydrate Research , 324, pp182-188, 2000), 분자 수준에서 세포독성 기작은 아직 규명하지 못했다. Recently, many chemical components have been isolated from pine needles and their structure has been revealed (Kang, TH, Jeong, ST, et al., Journal of Ethnopharmacology , 71 , pp 321-323, 2000; Jung, MJ, Chung, HY et al., Archives of pharmacal research , 26 (6) , pp 458-462). For example, studies on pine needle components have shown that the flavonoids quercetin 3-O-galactoside, quercetin 3-O-galactoside, rhamnoside, 3,4,7-trihydroxyflavone and a-spinastery glucoside, several kinds of triterpenoid, saponin and lignan components (Kaneta M., Hikichi H. et al., &Quot; Agricultural and Biological Chemistry , 44 , pp1407, 1980; Woo WS, Kang SS et al., Journal of Natural Products , 49 , pp 547-549, 1984). In addition, fragonol glycosides quercitrin and isoquercitrin were isolated from flowers. In addition, studies on the pharmacological action of pine needle components have shown that the saponin fraction increases the uterine contraction (Woo WS, Lee EB et al., Kor . J. Pharmacogn , 12, pp 153, 1981), polysaccharide fraction (Moon CK, Lee BG et al., Archives of Sarcoma 180 solid form) of Pharmacal Research , 8 , pp 277, 1985), antimicrobial activity (Yang MS, Ha YL et al., Agricultural Chemistry This and Biotechnology, 38, pp584, 1995 ), such as diarrhea inhibitory effect (Yoo JS, JS Jang et al., KOR. J. surface effect by Pharmacogn, 26, pp355, 1995) , and flavonol glycosides (flavonol glycoside) have. Among these compounds, saponin is known to exhibit strong lethal activity in cancer cell lines (Zou K., Zhao Y. et al., Carbohydrate Research , 324 , pp182-188, 2000), but the cytotoxic mechanism at the molecular level has not yet been elucidated.
소나무는 피너스 (Pinus)속으로 세계에 약 80-90종이 있으나 이들중 피너스 팔루스트리스 밀러 (P. palustris Miller : 북미), 피너스 피나스터에이톤 (P. pinaster Aiton : 프랑스), 피너스 실베스트리스 (P. sylvestris L. : 유럽전역), 피너스 라리시드 포이렛 (P. laricid Poiret : 오스트리아), 피너스 론기폴리아 룩스버그 (P. longifolia Rocvurgh : 인도), 피너스 덴시플로라 시엡 (P. densiflora Sieb. et Zucc. : 한국, 일본), 피너스 던베리 팔라토레 (P. thunberii Palatore : 해송, 일본) 등에서 채취한 테르펜등이 주로 산업에 이용되고 있다.There are about 80-90 species of pines in the world, among them Pinus. Among them, P. palustris Miller (North America), P. pinaster Aiton (France), Pinus P. longifolia Rocvurgh (India), Pinus densiflora cepab (P. sylvestris L.), P. laricid poiret (Austria), P. longifolia Rocvurgh densiflora Sieb. et Zucc.: Korea, Japan), and P. thunberii Palatore (Haesong, Japan).
솔잎의 주요성분은 테르펜틴 오일 (Terpentine oil), 시네올 (Cineole), 살리니그린 (Salinigrin), 코니페린 (Coniferin), 피-사이멘 (P-Cymen), 덴시피마릭산 (Densipimaric acid), 레텐 (Retene) 등과 엽록소, 단백질, 노지방, 인, 철분, 효소, 미네랄, 지용성 비타민 A 및 비타민 C 등이며, 주요성분은 종과 채취한 계절에 따라서 다소 차이가 있다.The main components of pine needles are terpentine oil, Cineole, Salinigrin, Coniferin, P-Cymen, Densipimaric acid, Protein, fat, phosphorus, iron, enzymes, minerals, fat-soluble vitamin A and vitamin C. The major components are somewhat different depending on species and season.
피너스 덴시플로라 (Pinus densiflora Sieb. et Zucc.)의 잎(Needle)에서 얻은 정유에는 알파-피넨(α-Pinene), 베타-피넨(β-Pinene), 캄페네 (camphene), 펠란드렌 (phellandrene), 보르네올 (borneol), 보르닐아세테이트 (bornylacetate), 카리오필렌 (caryophyllene), 카디넨 디테르펜 (cadinene diterpene), 세스퀴테르펜 (sesquiterpen), 세스퀴테르펜알콜 (sesquiterpenalcohol), 세릴알콜 (cerylalcohol), 밀납에는 쥬니퍼산 (juniperic acid), 사비니산 (sabinic acid), 헥사데칸디올 (hexadecane-diol), 프리아콘타놀 (triacontan-1-ol) 등이 함유되어 있으며, 그 외 마츄스테린 (matsusterin), 피토스테린 (phytosterin), 시닉산 (chinic acid), 시키믹산 (shikimic acid), 퀘르세틴 (quercetine), 캄페롤 (kaempferol) 등이 함유되어 있다 (赤松金芳저, 신정 화한약, pp664-665, 1980 ).Essential oils obtained from the needles of Pinus densiflora Sieb. Et Zucc. Include alpha-pinene, beta-pinene, camphene, pellandrene, borneol, bornylacetate, caryophyllene, cadinene diterpene, sesquiterpen, sesquiterpenalcohol, cerylalcohol, ), Wax contains juniperic acid, sabinic acid, hexadecane-diol, triacontan-1-ol, etc., and matsusterin ), Phytosterin, chinic acid, shikimic acid, quercetine, kaempferol, etc. (Akamatsu Kimyoji, Shinjeonghwa Chinese medicine, pp664- 665, 1980).
우리나라에서 자생하고 있는 소나무과 중에서 잣나무 잎은 민간요법에서 임질과 매독의 치료약으로 사용되고 있으나 (御影雅幸저, 日本生藥學雜誌, p 336, 1991), 솔잎의 여러 가지 약리작용에 대해서는 알려져 있지 않으며 단지 동상 및 피부의 타박상부위에 대해서 응혈된 피부를 빨리 원상으로 회복 시켜주는 효과가 있으며, 습진, 옴 및 땀띠 등을 치유하는 효과가 있는 것으로 알려지고 있다 (박종갑 저, 한방대의전, p134, 1984; 문화방송편저, 한국민간요법대전, p21, 1988). 또한 명의별록에는 모발이 희어지는 것을 방지하는 효과도 있다고 언급되어있으며, 목초강목에는 부스럼, 모발개선, 내장을 튼튼하게 하여주고 수명을 연장한다고 언급되어 있다. 소취효과, 불면증 치유효과 및 피부 미용효과등도 있으나, 상기 문헌 어디에도 솔잎이 자궁암에 대한 치료 효과를 가진다는 것에 대해서는 교시되거나 개시된 바가 없다.Among the pine trees native to Korea, the pine tree leaves are used as a remedy for gonorrhea and syphilis in folk remedies (御 賀 雅 幸 じ ゃ, Nihon Scientific Journal, p 336, 1991), but the various pharmacological actions of pine leaves are not known, It is known that it has the effect of quickly recovering the blood clotted skin against the bruising area of the skin, and has an effect of healing the eczema, the omphalus and the heat rash (Park Jong-Kap, Broadcasting, Korean Folk Therapy, p21, 1988). In addition, it is said that there is an effect to prevent the hair from whitening, and it is mentioned that the grass of the grass is strengthening the swelling, the hair improvement, the interior decoration and lengthening the life span. Deodorant effect, insomnia healing effect, skin cosmetic effect, etc. However, none of the above documents have taught or disclosed that pine needle has therapeutic effect on uterine cancer.
이에, 본 발명자들은 솔잎 추출물 또는 정제물에 대한 인체 파필로마바이러스(human papillomavirus, HPV) 억제효과를 확인함으로서, 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by confirming the inhibitory effect of human papillomavirus (HPV) on pine needle extract or purified water.
상기 목적에 따라, 본 발명은 솔잎 추출물을 유효성분으로 하는 자궁경부암 예방 및 치료용 약학 조성물을 제공한다.According to the above object, the present invention provides a pharmaceutical composition for preventing and treating cervical cancer comprising pine needle leaf extract as an active ingredient.
본원에서 정의되는 추출물은 조추출물, 분획 추출물 또는 정제물을 포함한다.Extracts as defined herein include crude extracts, fractionated extracts or tablets.
본원에서 정의되는 조추출물은 메탄올, 에탄올 및 부탄올과 같은 저급알코올 또는 이들의 혼합용매, 바람직하게는 50 내지 70 % 물 및 C1 내지 C4의 저급알콜의 혼합용매, 보다 바람직하게는 60 % 에탄올에 가용한 추출물을 포함한다.The crude extract as defined herein is soluble in a lower alcohol such as methanol, ethanol and butanol or a mixed solvent thereof, preferably a mixed solvent of 50 to 70% water and a lower alcohol of C1 to C4, more preferably 60% ethanol One extract.
본원에서 정의되는 분획 추출물은 비극성용매 가용 분획 추출물 및 극성용매 가용 분획 추출물을 포함한다.The fraction extracts defined herein include nonpolar solvent soluble fraction extracts and polar solvent soluble fraction extracts.
본원에서 정의되는 비극성용매 가용 분획 추출물은 상기 조주출물을 물로 현탁한 후에 헥산, 메틸렌틀로리드, 디클로로메탄, 클로로포름 또는 에틸 아세테이트로부터 선택된 비극성용매로, 바람직하게는 헥산, 메틸렌 클로라이드 분획, 또는 에틸 아세테이트, 보다 바람직하게는 메틸렌클로리드 비극성 유기용매로 분획하여 얻은 비극성용매에 가용한 분획 추출물을 포함하고; 극성용매 가용 분획 추출물은 상기 비극성용매 가용정제물을 제거하고 남은 조추출물을 부탄올 또는 물로 분획을 수행하여 얻은 극성용매에 가용한 분획 추출물을 포함한다.
The nonpolar solvent soluble fraction extract as defined herein is prepared by suspending the crude extract in water followed by treatment with a nonpolar solvent selected from hexane, methylene chloride, dichloromethane, chloroform or ethyl acetate, preferably hexane, methylene chloride fraction, or ethyl acetate , More preferably a fraction extract which is soluble in a non-polar solvent obtained by fractionation with a methylene chloride nonpolar organic solvent; The polar solvent-soluble fraction extract includes a fraction extract obtained by removing the nonpolar solvent-soluble tablet and allowing the remaining crude extract to be fractionated with butanol or water.
본원에서 정의되는 정제물은 상기 조추출물에 약 1 내지 30 배량(w/w), 바람직하게는 약 5 내지 15 배량(w/w), 보다 바람직하게는 약 8 내지 12 배량(w/w)의 정제수를 넣고 상기 정제수의 중량과 동일 중량의 수용성 물질의 분리정제에 사용되는 SP207, HP20SS, Diaion HP 20, SP-850 resin, 활성탄, Amberlite XAD-2,4, 바람직하게는 Diaion HP 20, SP-850 resin, Amberlite XAD-2,4 등의 흡착성 수지(resin)를 이용한 흡착크로마토그래프의 방법을 이용하여 추가 정제과정을 수행하고 이동상을 순차적으로 극성을 낮추는 에탄올, 아세톤 또는 메틸렌클로리드 용매로 용출시켜 차례로 용출시켜 얻은 물 용출 정제물(이하, S11-HPO 라 함), 30% 에탄올 용출 정제물(이하 S11-HP30라 함), 50% 에탄올 용출 정제물(이하, S11-HP50이라 함), 70% 에탄올 용출 정제물(이하 S11-HP70이라 함), 95% 에탄올 용출 정제물(이하, S11-HP95이라 함), 아세톤 및 메틸렌클로리드 용출 정제물(이하 S11-HPAM이라 함)들을 포함한다.(W / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w), to the crude extract, Of SP207, HP20SS,
본원에서 정의되는 솔잎은 적송 (Pinus densiflora Sieb. et Zucc.), 리기다 송(Pinus rigida), 테에다송(Pinus taeda), 해송 (Pinus thunbergii parlatore) 또는 잣나무(Pinus koraiensis Sieb. et Zucc) 등의 피누스 속(Pinus)의 잎, 바람직하게는 적송 (Pinus densiflora Sieb. et Zucc.), 리기다 송(Pinus rigida), 또는 테에다송(Pinus taeda)의 잎을 포함한다. Pine needles, as defined herein, are selected from the group consisting of Pinus densiflora Sieb. Et Zucc., Pinus rigida, Pinus taeda, Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc The leaves of Pinus, preferably Pinus densiflora Sieb. Et Zucc., Pinus rigida, or Pinus taeda.
상기 자궁경부암은 자궁경부암, 바람직하게는 파필로마바이러스(human papillomavirus, HPV) 감염으로 기인한 자궁경부암을 포함한다.The cervical cancer includes cervical cancer, preferably cervical cancer caused by infection with human papillomavirus (HPV).
본원에서 정의되는 추출물은 조추출물 또는 비극성용매 가용 추출물을 포함하며 하기와 같은 제조공정으로 제조가능하다. The extracts defined herein include crude extracts or non-polar solvent soluble extracts and can be prepared by the following process.
예를 들어, 본원발명의 조추출물은 솔잎 시료 총 중량의 약 1배 내지 200배(w/w), 바람직하게는 10배 내지 100배(w/w)의 정제수를 포함한 물, 주정, 탄소수 1 내지 4의 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물 또는 물 및 에탄올 혼합용매, 보다 바람직하게는 물 또는 50-99% 물 및 에탄올 혼합용매를 가하여 12시간 내지 1주일, 바람직하게는 48시간 내지 72시간 동안, 10℃ 내지 150℃, 바람직하게는 20℃ 내지 100℃, 보다 바람직하게는 실온에서 냉침추출, 열수추출, 초음파 추출, 환류냉각 추출 등의 추출방법, 바람직하게는, 냉침추출법 또는 열수 추출법을 수행하여 추출물을 수득하는 제 1단계 공정을 통하여 수득가능하다.For example, the crude extract of the present invention may contain water, alcohol, and water containing 1 to 200 times (w / w), preferably 10 to 100 times (w / w) To 4% by weight of a lower alcohol or a mixed solvent thereof, preferably water or a mixed solvent of water and ethanol, more preferably water or a mixed solvent of 50% -99% water and ethanol, for 12 hours to 1 week, preferably 48 hours The extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction at a temperature of from 10 to 150 ° C, preferably from 20 to 100 ° C, more preferably at room temperature, preferably, And then conducting a hydrothermal extraction method to obtain an extract.
예를 들어, 본원발명의 비극성용매 및 극성용매 가용 추출물은 상기에서 얻은 조추출물, 바람직하게는 60 내지 90% 에탄올 조추출물 중량의 약 0.0005 내지 0.005배, 바람직하게는 0.05 내지 0.5배 부피 (v/w%)의 물을 가한 후, n-헥산, 메틸렌 클로라이드, 에틸 아세테이트 및 부탄올을 이용한 통상적인 분획과정을 수행하여 n-헥산, 메틸렌 클로라이드, 에틸 아세테이트 등의 비극성 용매에 가용한 비극성 용매 가용 추출 정제물; 및 부탄올, 물 등의 극성용매에 가용한 극성용매 가용 추출 정제물을 각각 수득할 수 있다.For example, the non-polar solvent and polar solvent-soluble extract of the present invention may be used in an amount of about 0.0005 to 0.005 times, preferably 0.05 to 0.5 times (v / v) of the crude extract, preferably 60 to 90% w%) of water, followed by a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol to obtain nonpolar solvent-soluble extraction tablets (n-hexane, water; And a polar solvent-soluble extracted and purified product soluble in polar solvents such as butanol and water.
예를 들어, 본원발명의 정제물은 상기 조추출물에 약 1 내지 30배량(w/w), 바람직하게는 약 5 내지 15배량(w/w), 보다 바람직하게는 약 8 내지 12배량(w/w)의 정제수를 넣고 상기 정제수의 중량과 동일 중량의 수용성 물질의 분리정제에 사용되는 SP207, HP20SS, Diaion HP 20, SP-850 resin, 활성탄, Amberlite XAD-2,4, 바람직하게는 Diaion HP 20, SP-850 resin, Amberlite XAD-2,4 등의 흡착성 수지(resin)를 이용한 흡착크로마토그래프의 방법을 이용하여 추가 정제과정을 수행하고 이동상을 순차적으로 극성을 낮추는 에탄올, 아세톤 또는 메틸렌클로리드 용매로 용출시켜 차례로 물 용출액(이하, S11-HPO 라 함), 30% 에탄올 용출액(이하 S11-HP30라 함), 50% 에탄올 용출액(이하, S11-HP50이라 함), 70% 에탄올 용출액(이하 S11-HP70이라 함), 95% 에탄올 용출액(이하, S11-HP95이라 함), 아세톤 및 메틸렌클로리드 용출액(이하 S11-HPAM이라 함)을 각각 얻을 수 있다.For example, the purified product of the present invention may be added to the crude extract in an amount of about 1 to 30 times (w / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times / w), and SP207, HP20SS,
따라서, 본원 발명은 솔잎 시료 총 중량의 약 1배 내지 200배(w/w), 바람직하게는 10배 내지 100배(w/w)의 정제수를 포함한 물, 주정, 탄소수 1 내지 4의 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물 또는 물 및 에탄올 혼합용매, 보다 바람직하게는 물 또는 50-99% 물 및 에탄올 혼합용매를 가하여 12시간 내지 1주일, 바람직하게는 48시간 내지 72시간 동안, 10℃ 내지 150℃, 바람직하게는 20℃ 내지 100℃, 보다 바람직하게는 실온에서 냉침추출, 열수추출, 초음파 추출, 환류냉각 추출 등의 추출방법, 바람직하게는, 냉침추출법 또는 열수 추출법을 수행하여 추출물을 수득하는 제 1단계; 상기 조추출물에 약 1 내지 30배량(w/w), 바람직하게는 약 5 내지 15배량(w/w), 보다 바람직하게는 약 8 내지 12배량(w/w)의 정제수를 넣고 상기 정제수의 중량과 동일 중량의 수용성 물질의 분리정제에 사용되는 SP207, HP20SS, Diaion HP 20, SP-850 resin, 활성탄, Amberlite XAD-2,4, 바람직하게는 Diaion HP 20, SP-850 resin, Amberlite XAD-2,4 등의 흡착성 수지(resin)를 이용한 흡착크로마토그래프의 방법을 이용하여 추가 정제과정을 수행하고 이동상을 순차적으로 극성을 낮추어 에탄올, 아세톤 또는 메틸렌클로리드 용매로 용출시키는 제 2단계 정제 단계를 포함하는 물 용출 정제물(이하, S11-HPO 라 함), 30% 에탄올 용출 정제물(이하 S11-HP30라 함), 50% 에탄올 용출 정제물(이하, S11-HP50이라 함), 70% 에탄올 용출 정제물(이하 S11-HP70이라 함), 95% 에탄올 용출 정제물(이하, S11-HP95이라 함), 아세톤 및 메틸렌클로리드 용출 정제물(이하 S11-HPAM이라 함)을 각각 얻는 제조방법을 제공한다.Accordingly, the present invention relates to water, alcohol, lower alcohol having 1 to 4 carbon atoms containing purified water of about 1 to 200 times (w / w), preferably 10 to 100 times (w / w) Or a mixed solvent thereof, preferably water or a water and ethanol mixed solvent, more preferably water or a mixed solvent of 50-99% water and ethanol, for 12 hours to 1 week, preferably 48 hours to 72 hours, The extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction, preferably the cold extraction extraction method or the hot water extraction method, is performed at 10 to 150 ° C, preferably 20 to 100 ° C, more preferably at room temperature A first step of obtaining an extract; (W / w), preferably about 5 to 15 times (w / w), more preferably about 8 to 12 times (w / w) of purified water is added to the crude extract and the purified water SP207, HP20SS, Diaion HP 20, and SP-850 resin, which are used for the separation and purification of water- Activated carbon, Amberlite XAD-2,4, preferably Diaion HP 20, SP-850 resin, Amberlite XAD-2,4, and the second step of eluting the mobile phase with a polarity lowering solvent such as ethanol, acetone or methylene chloride solvent by sequentially performing an additional purification process using an adsorption chromatograph method using an adsorption resin such as Amberlite XAD- (Hereinafter referred to as S11-HPO), 30% ethanol eluted tablets (hereinafter referred to as S11-HP30), 50% ethanol eluted tablets (hereinafter referred to as S11-HP50) (Hereinafter referred to as S11-HP70), 95% ethanol-eluted tablets (hereinafter referred to as S11-HP95), acetone and methylene chloride-eluted tablets (hereinafter referred to as S11-HPAM) And a manufacturing method thereof.
따라서 본 발명은 상기의 제조공정 및 상기 제조공정으로 얻어진 솔잎 추출물 또는 정제물을 유효성분으로 함유하는 자궁경부암의 예방 및 치료용 약학 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of cervical cancer containing the pine needle extract or the purified product obtained by the above-described manufacturing process and the above-described manufacturing process as an effective ingredient.
또한, 추가로 통상의 분획 공정을 수행할 수도 있다(Harborne J.B., Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed ., pp6-7, 1998). In addition, conventional fractionation processes may be performed (Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed . , pp6-7, 1998).
따라서 본 발명은 상기 제조방법으로부터 얻어진 솔잎 추출물을 유효성분으로 함유하는 자궁경부암 예방 및 치료용 약학 조성물로 제공한다.Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of cervical cancer, which comprises the pine needle extract obtained from the above-mentioned production method as an active ingredient.
상기와 같은 방법으로 얻은 본 발명의 솔잎 추출물 및 정제물의 인체 파필로마바이러스 저해 활성은 HPV16 슈도비리온이 감염된 293TT 세포에서 바이오루미네센스(SEAP) 검색법으로 시험하였으며, 강력한 저해능을 확인하였고, 상기 바이러스에 대한 접촉 저해능이 증가함을 알 수 있었으며, 슈도비리온(Pseudovirion)으로 유도시킨 C57BL/6 마우스 자궁경부암을 억제함을 생체내 실험으로 확인함으로써, 자궁경부암 예방 및 치료를 위한 조성물로 유용하게 이용될 수 있음을 확인하였다.The human papillomavirus inhibitory activity of the pine needle extract and the purified product of the present invention obtained by the above method was tested by a bioluminescence (SEAP) detection method in 293TT cells infected with HPV16 pseudovirion, It was found that the inhibition of contact with viruses was increased and it was confirmed that the inhibition of C57BL / 6 mouse cervical cancer induced by pseudovirion in vivo is useful as a composition for prevention and treatment of cervical cancer It can be used.
또한, 솔잎은 오랫동안 생약으로 사용되어 오던 약재로서 이로부터 추출된 본 발명의 추출물 역시 독성 및 부작용 등의 문제가 없다. In addition, the pine needle has been used as a herbal medicine for a long time, and the extract of the present invention extracted therefrom has no problems such as toxicity and side effects.
본 발명의 자궁경부암 치료 및 예방을 위한 약학조성물은, 조성물 총 중량에 대하여 상기 추출물 및 정제물을 0.02 내지 50 % 중량백분율로 포함한다. The pharmaceutical composition for the treatment and prevention of cervical cancer according to the present invention comprises 0.02 to 50% of the above extract and purified product, Weight percent.
본 발명의 추출물 및 정제물을 포함하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.Pharmaceutical compositions comprising the extracts and tablets of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.
본 발명의 추출물 및 정제물의 약학적 투여 형태는 이들의 약학적 허용가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다. The pharmaceutical dosage forms of the extracts and tablets of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.
본 발명에 따른 추출물 및 정제물을 포함하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물 및 정제물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition containing the extract and the purified product according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And the like. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.
본 발명의 추출물 및 정제물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명의 추출물 및 정제물은 1일 0.0001 내지 100mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the extract and the purified product of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract and the purified product of the present invention are preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 추출물 및 정제물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내(Intracerebroventricular) 주사에 의해 투여될 수 있다. The extract and the purified product of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.
또한, 본 발명은 솔잎 추출물을 유효성분으로 함유하는 자궁경부암 예방 및 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for prevention and improvement of cervical cancer which contains pine needle leaf extract as an active ingredient.
본원에서 정의되는 "건강기능식품"은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.&Quot; Health functional food "as defined herein means food prepared and processed using raw materials or ingredients having functionality useful to the human body in accordance with Law No. 6727 on Health Functional Foods." Functional " Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.
본 발명의 자궁경부암 예방 및 개선을 위한 건강기능식품은, 조성물 총 중량에 대하여 상기 추출물 및 정제물을 0.01 내지 95 %, 바람직하게는 1 내지 80 % 중량백분율로 포함한다. The health functional food for prevention and improvement of cervical cancer according to the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight, of the extract and the purified product, based on the total weight of the composition.
또한, 자궁경부암 개선 및 예방을 위한 목적으로 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태인 건강기능식품으로 제조 및 가공이 가능하다.For the purpose of improving and preventing cervical cancer, it can be manufactured and processed into a health functional food in the form of tablet, capsule, powder, granule, liquid, ring and the like.
예를 들어, 상기 정제 형태의 건강기능식품은 그대로 또는 부형제, 결합제, 붕해제 또는 다른 첨가제를 넣어 고르게 섞은 것을 적당한 방법으로 과립상으로 한 다음 활택제 등을 넣어 압축성형하여 조제하거나 정제 형태의 건강기능식품을 그대로 또는 부형제, 결합제, 붕해제 또는 다른 적당한 첨가제를 넣어 고르게 섞은 것을 직접 압축성형하여 만들거나 또는 미리 만든 과립에 건강기능식품을 그대로 혹은 적당한 첨가제를 넣어 고르게 섞은 다음 압축성형하여 조제하거나 건강기능식품에 부형제, 결합제 또는 다른 적당한 첨가제를 넣어 고르게 섞은 분말을 용매로 습윤시키고, 습윤된 분말을 저압으로 틀에 넣어서 성형한 후, 적당한 방법으로 건조하여 조제한다. 또한, 상기 정제 형태의 건강기능식품에 필요에 따라 교미제 등을 넣을 수 있으며, 적당한 제피제로 제피 가능하다.For example, the health functional food of the tablet form may be prepared as it is, or it may be prepared by putting an excipient, a binder, a disintegrant, or other additives into the granules by an appropriate method, Functional food may be prepared by directly compressing the mixture of the food or by mixing directly with an excipient, binder, disintegrant or other suitable additive, or by mixing the preformed granules with the proper food additives or the appropriate additives, Add an excipient, binder or other suitable additive to the functional food, wet the powder evenly with a solvent, mold the wet powder into a low-pressure mold, and dry it by a suitable method. In addition, a mating agent or the like may be added to the health functional food in the form of tablets, if necessary.
상기 캅셀 형태의 건강기능식품 중 경질캅셀제는 보통 캅셀에 건강기능식품 또는 건강기능식품에 적당한 부형제 등을 고르게 섞은 것 또는 적당한 방법으로 입상으로 한 것 또는 입상으로 한 것에 적당한 제피제로 제피한 것을 그대로 또는 가볍게 성형하여 충전하여 조제하며, 연질캅셀제는 보통 캅셀에 건강기능식품 또는 건강기능식품에 적당한 부형제 등을 넣은 것을 젤라틴 등 적당한 캅셀기제에 글리세린 또는 소르비톨 등을 넣어 소성을 높인 캅셀기제로 피포하여 일정한 형상으로 성형하여 조제하며, 필요에 따라 상기 캅셀기제에 착색료 보존료 등을 첨가할 수 있다.The hard capsule of the capsule type health functional food is usually prepared by mixing the capsule with an appropriate excipient such as a health functional food or a health functional food or by granulating it in a suitable manner or by granulating it into a granular form suitable for granulation, The soft capsules are usually capsules containing an excipient suitable for a health functional food or a health functional food. The capsules are coated with an appropriate capsule base such as gelatin or the like with glycerin or sorbitol, And if necessary, a coloring agent preservative or the like may be added to the capsule base.
환형태의 건강기능식품은 보통 건강기능식품에 부형제, 결합제, 붕해제 등을 고르게 섞은 다음 적당한 방법으로 구상으로 성형하여 조제하며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.The ring type health functional food is usually prepared by mixing an excipient, a binder and a disintegrant in an ordinary healthy functional food, shaping it into a spherical form by an appropriate method, and then adding a gelatin to starch or other suitable skin care agent, It may also be an objectionable substance.
과립형태의 건강기능식품은 보통 건강기능식품을 그대로 또는 건강기능식품에 부형제, 결합제, 붕해제 등을 넣어 고르게 섞은 다음 적당한 방법으로 입상으로 만들고 될 수 있는 대로 입자를 고르게 한 것이며, 필요에 따라 착향료, 교미제 등을 넣을 수 있다. 과립형태의 건강기능식품은 12호 (1680 μm), 14호 (1410 μm) 및 45호 (350 μm) 체를 써서 다음 입도시험을 할 때에 12호체를 전량 통과하고 14호체에 남는 것은 전체량의 5.0 %이하이고 또 45호체를 통과하는 것은 전체량의 15.0 %이하이어야 한다.The granular health functional foods are usually prepared by mixing the healthy functional food as it is or by adding the excipient, binder, disintegrant, etc. to the health functional food and then making it into granular form by proper method and evenly grinding the granular material as needed. , A mating agent, and the like. In the next granularity test using granules No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm), all of the 12th tongue passed through and 14th And not more than 5.0% of the total amount and not more than 15.0% of the total amount.
본원 발명의 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향료 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 (대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989 ). The definitions of the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, etc. of the present invention are described in documents known in the art and include the same functions or the like (see, for example, , Council of Korean Pharmacy College, 5th ed., P. 33-48, 1989).
본 발명의 솔잎 추출물 및 정제물은 인체 파필로마바이러스(human papillomavirus, HPV)에 대한 억제활성을 나타냄으로써 상기 바이러스 감염으로 기인한 자궁경부암 예방 및 치료용 약학조성물 및 건강기능식품에 유용하게 이용될 수 있다. The pine needle extract and purified product of the present invention exhibit inhibitory activity against human papillomavirus (HPV), and thus can be effectively used in pharmaceutical compositions and health functional foods for the prevention and treatment of cervical cancer caused by the virus infection have.
도 1은 솔잎 추출물, 분획물 및 정제물 시료를 전처리 했을 때의 바이러스 저해능을 나타낸 도이고,
도 2는 솔잎 분획물 및 정제물 시료의 HPV16 슈도비리온을 이용한 공격 접종에 대한 방어능을 비교한 도이며,
도 3는 솔잎 분획물 및 정제물 시료들의 투여 방법에 따른 HPV16 슈도비리온을 이용한 공격 접종에 대한 방어능을 비교한 도이다.Fig. 1 is a graph showing the virus-inhibiting ability when the pine needle leaf extract, the fraction and the purified water sample were pretreated.
FIG. 2 is a graph comparing the protective ability against an attack inoculation using HPV16 pseudovirion of pine needle leaf fraction and purified water sample,
Figure 3 compares the protective ability against attacking inoculation using HPV16 pseudovirion according to the administration method of pine needle leaf fraction and purified water samples.
이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.
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.
실시예 1. 솔잎 에탄올추출물의 제조 Example 1 Preparation of Ethanol Extract of Pine Needle
경동시장에서 구입한 강원도산 솔잎 2kg을 95% 에탄올(주정) 20L로 50℃에서 2회 반복하여 온침으로 추출한 후 감압 농축하여 에탄올 추출물 338.85g (이하 S11-T라 함)을 수득하였다.
2 kg of pine needle purchased from Kyungdong market was repeatedly extracted with 20 L of 95% ethanol (alcohol) at 50 캜 by warming and concentrated under reduced pressure to obtain 338.85 g (hereinafter referred to as S11-T) of an ethanol extract.
실시예Example 2. 솔잎 2. Pine needles 분획물들의Of fractions 제조 Produce
상기 실시예 1의 에탄올 추출물을 증류수 1.8L에 현탁하고 동량의 헥산, 메틸렌 클로라이드, 에틸 아세테이트, n-부탄올로 각각 3회 상법에 따라 차례로 분획하여 헥산 분획(95.18g, 이하 S11-H라 함), 메틸렌 클로라이드 분획(46.11g, 이하 S11-MC라 함), 에틸 아세테이트 분획(19.42g, 이하 S11-E라 함), n-부탄올 분획(57.94g, 이하 S11-B라 함) 그리고 물 분획(90.2g,이하 S11-W라 함)을 수득하였다. The ethanol extract of Example 1 was suspended in 1.8 L of distilled water, and the fraction was sequentially fractionated three times with the same amount of hexane, methylene chloride, ethyl acetate and n-butanol according to the conventional method to obtain a hexane fraction (95.18 g, hereinafter referred to as S11-H) , A methylene chloride fraction (46.11 g, hereinafter referred to as S11-MC), an ethyl acetate fraction (19.42 g, hereinafter referred to as S11-E), an n-butanol fraction (57.94 g, hereinafter referred to as S11- 90.2 g, hereinafter referred to as S11-W).
실시예Example 3. 솔잎 에탄올 추출물의 3. Ethanol extract of pine needles HPHP -20 칼럼을 이용한 -20 column 소분획의Fractional 제조 Produce
실시예 1과 같이 얻어진 솔잎 에탄올 추출물 30g을 30ml의 물에 현탁한 후, DIAION HP-20 칼럼 크로마토그래피를 실시하였다. 먼저 물 2L를 이동상으로 가하여 용출액을 얻고 다시 차례로 30% 에탄올 2L, 50% 에탄올 2L, 70% 에탄올 2L, 95% 에탄올 2L, 아세톤 2L, 메틸렌 클로라이드 1.5L를 가하여 각각의 용출액을 얻었다. 이때 얻어진 용출액 7종 중 아세톤 용출액과 메틸렌 클로라이드 용출액을 혼합하고 감압 농축하여 S11-HP0(물, 7.95g), S11-HP30(30% 에탄올, 3.44g), S11-HP50(50% 에탄올, 2.97g) S11-HP70(70% 에탄올, 1.75g), S11-HP95(95% 에탄올, 6.35g) 그리고 S11-HPAM(아세톤과 메틸렌 클로라이드, 6g)의 소분획을 각각 수득하였다.
30 g of the pine needle ethanol extract obtained as in Example 1 was suspended in 30 ml of water and subjected to DIAION HP-20 column chromatography. First, 2 L of water was added to the mobile phase to obtain an eluate, and 2 L of 30% ethanol, 2 L of 50% ethanol, 2 L of 70% ethanol, 2 L of 95% ethanol, 2 L of acetone and 1.5 L of methylene chloride were added respectively. S11-HP0 (water, 7.95 g), S11-HP30 (30% ethanol, 3.44 g) and S11-HP50 (50% ethanol, 2.97 g) were obtained by mixing acetone eluent and methylene chloride eluent from 7 eluents ) S11-HP70 (70% ethanol, 1.75 g), S11-HP95 (95% ethanol, 6.35 g) and S11-HPAM (acetone and methylene chloride, 6 g) respectively.
참고예 1. 실험 준비 References 1. Experimental preparation
1-1. 세포준비1-1. Cell preparation
: HPV pseudovirus 생산과 in vitro assay를 위해 사용한 293TT (human embryonic kidney cell을 adenovirus E1a가 transform되게 조작하여 만들어진 293T 세포에, SV40 large T antigen를 발현 시킨 세포 주, Schiller Lab으로부터 제공 받음.), 세포는 Dulbecco’s modified Eagles medium (DMEM (SH30243, Hyclone, UT, USA))에 heat inactivated 10% FBS (26140079, Hyclone, UT, USA)를 첨가한 배지를 이용해 배양되었으며 5%의 CO2 가 공급되는 37℃ 조건에서 유지되었다.
: 293TT (human embryonic kidney cell used for HPV pseudovirus production and in vitro assay) was supplied from Schiller Lab, a cell line expressing SV40 large T antigen on 293T cells transformed with adenovirus E1a) Dulbecco's modified Eagles medium (DMEM ( SH30243, Hyclone, UT, USA)) in 10% inactivated FBS was incubated with heat the medium supplemented with (26140079, Hyclone, UT, USA ) in 5% CO 2 Lt; RTI ID = 0.0 > 37 C. < / RTI >
1-2. 1-2. HPVHPV -16 -16 슈도바이러스(pseudovirus)의Pseudovirus 생산 production
1-2-1. 플라스미드 (Plasmid)1-2-1. Plasmid
: In vitro antiviral assay를 위하여 HPV-SEAP 슈도바이러스를 생산하였으며, In vivo challenge test를 위하여 HPV-Luc PV를 생산하였다. HPV-SEAP PV 생산을 위하여 p-SEAP 와 p16L1L2 plasmid, HPV-Luc PV를 생산을 위하여 pc-Luc와 p16L1L2 plasmid를 사용하였다. 각각의 plasmid는 Schiller Lab(Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, USA)으로부터 제공 받았다.: HPV-SEAP pseudovirus was produced for in vitro antiviral assay and HPV-Luc PV was produced for in vivo challenge test. PC-Luc and p16L1L2 plasmids were used to produce p-SEAP and p16L1L2 plasmids and HPV-Luc PV for HPV-SEAP PV production. Each plasmid was provided from Schiller Lab (Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, USA).
1-2-2. 감염 (Transfection) 1-2-2. Transfection
: 293TT cell을 5 X 106개로 75T flask에 seeding 하여 37°C, 5% CO2에서 16시간 incubation 한 후, 19 μg의 p16L1/L2와 19 μg의 pSEAP 또는 pc-Luc plasmid를 Lipofectin Reagent (18292-011, Invitrogen, CA, USA)을 사용하여 co-transfection 하였다. Transfection 6시간 후 complete media로 교환하고 37°C, 48시간 동안 배양 후, 세포를 트립신화(trypsinization)하여 수확하였다. 수확한 세포는 Dulbecco's Phosphate-Buffered Saline (DPBS, 14190-250, Invitrogen, CA, USA)로 washing 하였다.: 293TT cells were seeded in 75T flasks at 5 × 10 6 cells and incubated at 37 ° C and 5% CO 2 for 16 hours. 19 μg of p16L1 / L2 and 19 μg of pSEAP or pc-Luc plasmids were incubated with Lipofectin Reagent (18292-011, Invitrogen, CA, USA). After 6 hours of transfection, the cells were replaced with complete media and incubated at 37 ° C for 48 hours. Cells were harvested by trypsinization. The harvested cells were washed with Dulbecco's Phosphate-Buffered Saline (DPBS, 14190-250, Invitrogen, CA, USA).
1-2-3. 세포수확 및 비리온(virion) 성숙(maturation)1-2-3. Cell harvesting and virion maturation
: DPBS 1ml로 재현탁 후, 5% Triton X-100 (9002-93-1, Sigma, MO, USA), 25mM의 ammonium sulphate (pH 9) (A4418, Sigma-Aldrich, MO, USA) 0.2%의 벤조나제 (9025-65-4, Benzonas, Sigma, UK)를 첨가하여 넣고, 24시간 동안 37°C에서 incubation하여 virus를 성숙(maturation) 시켰다.: 0.2 ml of a 5% Triton X-100 (9002-93-1, Sigma, MO, USA), 25 mM ammonium sulphate (pH 9) (A4418, Sigma-Aldrich, MO, USA) was resuspended in 1 ml of DPBS Benzonase (9025-65-4, Benzonas, Sigma, UK) was added and incubated at 37 ° C for 24 hours to maturation of the virus.
1-2-4. 염추출 (Salt extraction)1-2-4. Salt extraction
: 성숙 비리온(maturated virion)을 5분간 얼음에서 냉각시킨 후, 5N NaCl을 0.17 volume으로 넣고 다시 20분간 얼음에서 배양한다. 이후 비리온(virion) 용액을 모두 모아 e-튜브(tube)로 옮기고 4℃, 12,000rpm에서 10분간 원심분리한 후 상층액 만을 모아 opti-prep ultracentrifugation하거나, -80℃에 보관한다.: The maturated virion is cooled on ice for 5 minutes, then added with 0.17 volume of 5N NaCl and incubated on ice for 20 minutes. Then collect all virion solutions and transfer them to an e-tube. Centrifuge at 4 ° C and 12,000 rpm for 10 min. Collect the supernatant, and store at -80 ° C by opti-prep ultracentrifugation.
1-2-5. 정제1-2-5. refine
: SIGMA density gradient medium 77ml에 23ml DPBS/0.8M NaCl을 혼합하여 46%의 optiprep gradient용액을 만들고, 이를 이용하여 37%, 33%, 39%의 gradient용액을 DPBS/0.8M NaCl 용액으로 만든다 (27% : 9.3ml DPBS/0.8M NaCl+13.2ml 46% Optiprep, 33% : 6.4ml DPBS/0.8M NaCl+16.1ml 46%, 39% : 3.4ml DPBS/0.8M NaCl+19ml 46%). 5ml 의 Beckman ultracentrifuge tube (361625, Beckman Coulter, USA)에 각 1ml 씩의 39%, 33%, 27% gradient 용액을 조심스레 넣어 층이 깨어지지 않게 하고, 그 위에 virion 상층액 1ml을 로딩(loading)한다. Ultracentrifuge (Optima L 90K, Beckman Coulter Ultracentrifuge, USA)를 이용하여, 47,800rpm, 16℃에서 4시간 동안 원심분리하였다. 비리온 분획(virion fraction)을 모아서 -80°C에 보관하였다.: Prepare a 46% optiprep gradient solution by mixing 23 ml of DPBS / 0.8M NaCl in 77 ml of SIGMA density gradient medium and make 37%, 33%, and 39% gradient solution with DPBS / 0.8M NaCl solution (27 %: 9.3 ml DPBS / 0.8 M NaCl + 13.2 ml 46% Optiprep, 33%: 6.4 ml DPBS / 0.8 M NaCl + 16.1 ml 46%, 39%: 3.4 ml DPBS / 0.8 M NaCl + 19 ml 46%). Carefully insert 39 ml, 33%, and 27% gradient solutions of 1 ml each into a 5 ml Beckman ultracentrifuge tube (361625, Beckman Coulter, USA) to prevent the layer from breaking and load 1 ml of virion supernatant onto it. do. And centrifuged at 47,800 rpm, 16 ° C for 4 hours using an Ultracentrifuge (Optima L 90K, Beckman Coulter Ultracentrifuge, USA). The virion fractions were collected and stored at -80 ° C.
1-2-6. HPV PVs의 titration 1-2-6. Titration of HPV PVs
: 293TT cell을 5 × 103씩 96-well plate에 seeding하여 37°C, 5% CO2 에서 16시간 incubation 하였다. HPV PVs를 5-fold serial dilution 후, 각 well의 세포에 infection 하여, 72시간 동안 incubation 하였다. HPV-SEAP PV의 titer 측정은 세포 배양 상층액을 취하여 Great EscAPE™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA)를 이용하여 secreted alkaline phosphatase (SEAP)의 활성을 확인 함으로써 이루어졌다. 또한 HPV-Luc PV 의 titer 측정은 세포 배양 상층액을 취하여, BioLuxGaussia Luciferase Assay Kit (0301008, New England biolabs, MA, USA)를 이용하여 luciferase activity를 확인하였다. Chemiluminescent detection은 Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA)를 이용하여 relative light units (RLU) 값을 얻어, 각각의 HPV PVs titer를 산출하였다.
: 293TT cells were seeded in 5 × 10 3 96-well plates and incubated at 37 ° C and 5% CO 2 for 16 hours. After 5-fold serial dilution of HPV PVs, cells were inoculated into each well and incubated for 72 hours. Titer measurements of HPV-SEAP PV were performed by ascertaining the activity of secreted alkaline phosphatase (SEAP) using the Great EscapE ™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA). In addition, titer measurement of HPV-Luc PV was performed by using BioLuxGaussia Luciferase Assay Kit (0301008, New England biolabs, MA, USA). Chemiluminescent detection was performed using the Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) and the relative light units (RLU) were calculated and the HPV PV titer was calculated.
1-3. 1-3. InIn vitrovitro antiviralantiviral assayassay
: 천연물질에 대한 screening assay와 HPV inhibition assay는 Shaneyfelt의 방법(Shaneyfelt et al., 2006)과 Schiller Lab의 방법(Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002)을 응용하였다. In vitro antiviral assay 시작 전, 293TT cell을 5 × 103씩 96-well plate에 seeding하여 37°C, 5% CO2 incubation하였다. 16시간 후, 배양액을 제거하고 각 extract를 100 μg/ml 또는 50 μg/ml의 농도로 희석하여 100 μl씩 cell에 분주하였다. Extract를 16시간 처리 한 후, 배양액을 제거하고 PBS로 세포를 두 번 세척하였다. 106RLU/ml의 HPV PVs 를100 μl씩 세포에 감염시켜, 48시간 동안 37°C, 5% CO2 incubation하였다. Antiviral activity 측정은 세포 배양 상층액을 취하여 Great EscAPE™ SEAP Chemiluminescence Kit (Clontech, CA, USA)를 이용하여 secreted alkaline phosphatase (SEAP)의 활성을 확인하였다. SEAP activity는 Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA)를 이용하여 relative light units (RLU) 값을 얻었으며, extract를 처리하지 않은 cell과 extract를 처리한 cell에서의 RLU 값을 비교하여, SEAP activity의 감소를 HPV PVs에 대한 inhibition 효과로 간주하였다.
: Screening assays and HPV inhibition assays for natural substances were performed using Shaneyfelt's method (Shaneyfelt et al., 2006) and Schiller's method (Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002). Before the initiation of the in vitro antiviral assay, 293TT cells were seeded on a 96-well plate at 5 × 10 3 and incubated at 37 ° C and 5% CO 2 lt; / RTI > After 16 hours, the culture medium was removed and each extract was diluted to a concentration of 100 μg / ml or 50 μg / ml and dispensed into cells in a volume of 100 μl. After the extract was treated for 16 hours, the culture was removed and the cells were washed twice with PBS. Cells were infected with 100 μl of HPV PVs at 106 RLU / ml and incubated at 37 ° C and 5% CO 2 for 48 hours. To determine antiviral activity, the cell culture supernatant was assayed for secreted alkaline phosphatase (SEAP) activity using the Great EscapE ™ SEAP Chemiluminescence Kit (Clontech, CA, USA). Relative light units (RLU) values were obtained by using a luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA) for SEAP activity, and the RLU values of extract- , And the decrease in SEAP activity was regarded as an inhibition effect on HPV PVs.
1-4. 투여(1-4. administration( AdministrationAdministration ))
: 6주령 Female Balb/C 마우스(Orientbio Co., Korea)를 사용하였으며, 다음과 같이 S11-MC and S11-HP(70-95)를 경구 또는 외용 투여하였다. 경구 투여는 extract를 0.5% methyl cellulose (Sigma, MO, USA)와 혼합하여 300 mg/kg씩 하루에 한번, 5일간 먹였다. 마지막 경구 투여 4시간 후, HPV16 PVs를 복부 피하 부분에 주사하였다. 외용 투여는 extract를 genital tract에 외용제로 사용하였으며, 0.5% methyl cellulose와 혼합하여 150 mg/kg씩 하루에 한번, 3일간 투여하였다. 마지막 경구 투여 4시간 후, HPV16 PVs를 genital tract에 injection 하였다. 음성 대조군으로 extract를 투여하지 않은 마우스를 사용하였다.
: Male Balb / C mice (Orientbio Co., Korea) were used at 6 weeks of age and S11-MC and S11-HP (70-95) were orally or externally administered as follows. For oral administration, extract was mixed with 0.5% methyl cellulose (Sigma, MO, USA) and fed at 300 mg / kg once a day for 5 days. Four hours after the last oral dose, HPV16 PVs were injected into the abdomen subcutaneous area. For external use, extract was used as a topical agent in the genital tract, mixed with 0.5% methyl cellulose and administered at a dose of 150 mg / kg once a day for 3 days. Four hours after the last oral administration, HPV16 PVs were injected into the genital tract. A mouse without extract administration as a negative control was used.
1-5. 1-5. 슈도바이러스Pseudovirus 공격접종 ( Attack inoculation ( challengechallenge ))
: 공격 접종 시험은 HPV PVs를 이용하였으며, luciferase gene을 발현하는 HPV16-Luc PVs를 생산, maturation, extraction, 정제, titration 하여 사용하였다(http://home.ccr.cancer.gov/lco/). 공격 접종 4일 전, 마우스에 3 mg 의 Depot medroxyprogesterone acetate (Depo-Provera™) (Pharmacia, Belgium)를 주사하였고, HPV16-Luc PVs를 challenge 할 때에는 3%의 carboxymethylcellulose (Sigma Aldrich, MO, USA)와 4%의 Nonoxinol-9 (Sigma Aldrich, MO, USA)을 사용하였다 (Roberts et al., 2007). 투여 조건에 따른 공격 접종 방법은, S11-MC와 S11-HP(70-95)를 경구 투여한 마우스는 HPV16-Luc PVs를 5 × 106 RLU씩 복부피하에 주사하였다. 외용 투여한 마우스들은 HPV16-Luc PVs challenge 6시간 전에, 마우스들에게 20㎕의 4% nonoxynol-9을 intravaginally에 처리하였으며, 그 후, 20㎕의 3% carboxymethylcellulose 와 혼합한 HPV16-Luc PVs를 5 × 106 RLU씩 vaginal tract에 주입하였다. 공격 접종 3일 후, 모든 마우스를 마취시켜 경구 투여 마우스는 복강으로 luciferin(caliper, MA, USA)을 7 mg/ml의 농도로 30㎕을 주사하였다. HPV16-Luc PVs는 luciferase gene을 전달하는 plasmid, pLucf가 encapsidate되어 pseudoinfection이 가능하다(Jeff Roberts, 2007). 10분간 방치 한 후, 각각의 마우스는 IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA)을 이용하여 luciferase 발현을 detection 하였으며, image를 비교 분석 하였다. 또한 image는 Living Image 2.20 software (Xenogen, NJ, USA)를 이용하여, luciferase의 발현량을 정량 하였으며, extract의 HPV 방어 효능을 비교 분석 하였다.
: HPV PVs were used for the inoculation test, and HPV16-Luc PVs expressing the luciferase gene were produced, maturation, extraction, purification and titration (http://home.ccr.cancer.gov/lco/). Four weeks before challenge, mice were injected with 3 mg depot medroxyprogesterone acetate (Depo-Provera ™) (Pharmacia, Belgium) and 3% carboxymethylcellulose (Sigma Aldrich, MO, USA) 4% nonoxinol-9 (Sigma Aldrich, MO, USA) was used (Roberts et al., 2007). The mice were orally injected with S11-MC and S11-HP (70-95) at 5 × 10 6 RLU of HPV 16-Luc PVs by subcutaneous injection. Mice were treated intravaginally with 20 μl of 4% nonoxynol-9 6 hours before challenge with HPV16-Luc PVs, and then HPV16-Luc PVs mixed with 20 μl of 3% carboxymethylcellulose was injected into 5 × 10 6 RLU were injected into the vaginal tract. Three days after challenge, all mice were anesthetized and injected intraperitoneally with 30 μl of luciferin (caliper, MA, USA) at a concentration of 7 mg / ml. HPV16-Luc PVs can be pseudoinfection by encapsidating the plasmid, pLucf, which carries the luciferase gene (Jeff Roberts, 2007). After 10 minutes of incubation, luciferase expression was detected in each mouse using IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA), and images were compared and analyzed. In addition, the expression of luciferase was quantified using Living Image 2.20 software (Xenogen, NJ, USA) and the HPV defense efficacy of extract was compared and analyzed.
실험예Experimental Example 1. 바이러스 1. Viruses 저해능Low performance 실험 Experiment
상기 실시예에서 얻은 시료의 루시페라제-함유 HPV 바이러스 접촉 억제능을 확인하기 위하여 문헌에 개시된 방법을 응용하여 하기와 같이 실험하였다 (Shaneyfelt et al., 2006, Roden et al., 1996, Unckell et al., 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002). In order to confirm the ability of the samples obtained in the above Examples to inhibit the contact with luciferase-containing HPV virus, the method described in the literature was applied as follows (Shaneyfelt et al., 2006, Roden et al., 1996, Unckell et al , 1997, Touze et al., 1998, Selinka et al., 2003, and Klasse et al., 2002).
96-웰 플레이트에 293TT cell을 5 x 103으로 접종(seeding)하고 16시간 배양한다. 다음날 시료를 50ug, 100ug/ml의 농도로 배지에 섞어주고 16시간 동안 처리한다. PBS 100ul로 2회 세척한 후, 106 RLU/ml의 HPV 슈도비리온을 100 μl씩 세포에 감염시켜, 48시간 동안 37°C, 5% CO2 배양하였다. 293TT the cell in a 96-well plate inoculated (seeding) with 5 x 10 3, and incubated for 16 hours. The next day, the sample is mixed with the medium at a concentration of 50 ug, 100 ug / ml and treated for 16 hours. After washing twice with
Great EscAPE™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA)의 5X lysis buffer를 1X로 만든 후, 1X lysis buffer 45 ul와 세포 배양액 15 ul 을 취하여 96-웰 플레이트 (3912, Costar, NY, USA)에 넣는다. Great EscAPE™ SEAP Chemiluminescence kit 내의 substrate를 60 ul씩 첨가하고, Luminescence coulter(Micro beta triLux 1450, PerkinElmer, CT, USA)를 이용하여 relative light units (RLU) 값을 얻는다. extract를 처리하지 않은 cell과 extract를 처리한 cell에서의 RLU 값을 비교하여, SEAP activity의 감소를 HPV PVs에 대한 inhibition 효과로 간주하였다.
Well plate (3912, Costar, NY, USA) with 45 μl of 1X lysis buffer and 15 μl of the cell culture medium, and incubated for 1 hour in a 5X lysis buffer of Great EscapE ™ SEAP Chemiluminescence Kit (631738, Clontech, CA, USA) . Add 60 μl of substrate in the Great Escape ™ SEAP chemiluminescence kit and obtain relative light units (RLU) using a Luminescence coulter (Micro beta triLux 1450, PerkinElmer, CT, USA). We compared the RLU values in extract-treated cells versus extract-treated cells and considered the reduction in SEAP activity as an inhibition effect on HPV PVs.
본 실험 결과, 하기 도 1에 나타난 바와 같이, 13종 시료의 바이러스 저해능을 확인할 수 있었다. S11-B, S11-E, S11-M, S11-HP70, 그리고 S11-HP95는 100 μg/ml의 시료 농도에서 50% 이상의 바이러스 저해 효과를 보였으며, 그 중, S11-B와 S11-HP70 시료는 50 μg/ml의 시료 농도에서도 HPV16 PVs를 50% 이상 저해 효과를 보였다.
As shown in FIG. 1, 13 samples were able to confirm the virus-inhibiting ability. S11-B, S11-E, S11-M, S11-HP70, and S11-HP95 showed more than 50% virus inhibitory effect at a sample concentration of 100 μg / Showed 50% inhibition of HPV16 PVs even at a sample concentration of 50 μg / ml.
실험예Experimental Example 2. 전처리 시간에 따른 바이러스 2. Virus according to preprocessing time 저해능Low performance 비교 실험 Comparative experiment
실시예의 13개 extract 중, 바이러스 저해 효과가 높았던 S11-MC와 S11-HP(70-95)를 대표 물질로 선정하여 실험예 2의 In vitro antiviral assay 방법으로, 두 시료의 처리 시간을 다양하게 하여 바이러스 저해능을 측정하였다. Among the extracts of Examples, S11-MC and S11-HP (70-95), which were highly effective in inhibiting viruses, were selected as representative materials, and In In vitro antiviral assay was used to measure the virus inhibition by varying the treatment time of two samples.
각 시료를 100 μg/ml 또는 50 μg/ml의 농도로 희석 후, 293TT 세포에 0, 4, 8, 12, 16시간 동안 처리하였다. 각 well의 배양액을 제거하고 PBS로 2회 세척 후, 106 RLU/ml의 HPV16 PVs를 세포에 감염시켰다. 48시간 후, 각각의 세포 배양액으로부터 SEAP activity를 측정하여 HPV16 PVs의 inhibition 정도를 평가하였다. Each sample was diluted to a concentration of 100 μg / ml or 50 μg / ml, and treated for 29, 4, 8, 12, and 16 hours with 293TT cells. The culture medium was removed from each well, washed twice with PBS, and infected with 10 6 RLU / ml of HPV16 PVs. After 48 hours, the inhibition of HPV16 PVs was assessed by measuring SEAP activity from each cell culture.
도 2는 S11-MC와 S11-HP(70-95)의 HPV16 PVs에 대한 저해 정도를 백분율(%)로 나타낸 것이다. S11-MC는 50 ug/ml의 농도에서 4시간에서 8시간 처리 시 8%, 12시간 처리 시12%, 그리고 16시간 처리 시 80%의 HPV16 PVs 감소를 보였으며, 100 μg/ml의 농도에서 4시간 처리 시 10%, 8시간 처리 시 34%, 12시간 처리 시 68%, 그리고 16시간 처리 시 94%의 HPV16 PVs 감소를 보였다 (도 2(A)). S11-HP(70-95)는 50 μg/ml의 농도에서 4시간에서 12시간 바이러스 감소 효과를 거의 보이지 않았으며(0 - 2%), 100 μg/ml의 농도에서 8시간 처리 시 42%, 12시간 처리 시 47%, 그리고 16시간 처리 시 88%의HPV16 PVs 감소를 보였다 (도 2(B)).Figure 2 shows the percent inhibition of S11-MC and S11-HP (70-95) against HPV16 PVs. S11-MC showed a decrease in HPV16 PVs at 8%, 12% at 12 hours, and 80% at 16 hours treatment at a concentration of 50 ug / ml for 4 hours to 8 hours, and at a concentration of 100 μg /
따라서, HPV16 PVs를 감염 시키기 전, S11-MC 또는 S11-HP(70-95) 두 가지 시료를 16시간 이상 세포에 처리하였을 때에, 효과적으로 HPV16 PVs를 저해 할 수 있었다.
Therefore, HPV16 PVs could be effectively inhibited by treatment with S11-MC or S11-HP (70-95) for 16 hours or more before infection with HPV16 PVs.
실험예Experimental Example 4. 마우스 생체 내 4. Mouse in vivo HPV16HPV16 슈도바이러스Pseudovirus 억제 효능 시험 Inhibitory efficacy test
상기 실시예에서 얻은 시료의 루시페라제-함유 슈도비리온으로 처치된 마우스에서의 자궁경부암에 대한 억제활성을 확인하기 위하여 문헌에 개시된 방법을 응용하여 하기와 같이 실험하였다 (Roberts, J.N., et al., (2007).Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 13; 857-861. ).
In order to confirm the inhibitory activity against cervical cancer in mice treated with the luciferase-containing pseudovirion of the sample obtained in the above example, the method described in the literature was applied as follows (Roberts, JN, et al (2007). Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan, Nat Med 13: 857-861.).
4-1. 시료 처치4-1. Sample treatment
6주령 Female Balb/C 마우스(Orientbio Co., Korea)를 사용하였으며, 하기와 같이 S11-MC 또는 S11-HP(70-95)를 경구 또는 외용 투여하였다.Six-week old female Balb / C mice (Orientbio Co., Korea) were used and S11-MC or S11-HP (70-95) were administered orally or externally as described below.
경구 투여는 extract를 0.5% methyl cellulose (Sigma, MO, USA)와 혼합하여 300 mg/kg씩 하루에 한번, 5일간 먹였다. 마지막 경구 투여 4시간 후, HPV16 PVs를 복부 피하 부분에 주사하였다. For oral administration, extract was mixed with 0.5% methyl cellulose (Sigma, MO, USA) and fed at 300 mg / kg once a day for 5 days. Four hours after the last oral dose, HPV16 PVs were injected into the abdomen subcutaneous area.
외용 투여는 extract를 genital tract에 외용제로 사용하였으며, 0.5% methyl cellulose와 혼합하여 150 mg/kg씩 하루에 한번, 3일간 투여하였다. 마지막 경구 투여 4시간 후, HPV16 PVs를 genital tract에 injection 하였다. 음성 대조군으로 extract를 투여하지 않은 마우스를 사용하였다.For external use, extract was used as a topical agent in the genital tract, mixed with 0.5% methyl cellulose and administered at a dose of 150 mg / kg once a day for 3 days. Four hours after the last oral administration, HPV16 PVs were injected into the genital tract. A mouse without extract administration as a negative control was used.
루시페라제-함유 슈도비리온(Luciferase-containing pseudovirion) 공격 접종 (challenge) 4일 전, 6주령 Female Balb/C 마우스 (Orientbio Co., Korea)에 마리당 3mg의 Depot medroxyprogesterone acetate (Depo-Provera™, Pharmacia, Belgium)을 등 부위에 피하 주사하였다.
Luciferase-containing pseudovirion Four days prior to challenge, Depot medroxyprogesterone acetate (Depo-Provera ™, Pharmacia, Belgium) was injected subcutaneously into the back of a 6-week-old female Balb / C mouse (Orientbio Co., Korea).
4-2. 복부 4-2. stomach challengechallenge 시험 exam
외용 마우스의 경우, 시료를 0.5% methyl cellulose와 혼합하여 150 mg/kg씩 하루에 한번, 3일간 vaginal tract에 외용하였다. 각 마우스는 3일째 되는 날, 외용 투여 4시간 후, HPV16-Luc PVs를 1%의 carboxymethylcellulose (Sigma Aldrich, MO, USA)와 1:1 비율로 혼합하여 복부 피하 부분에 주사하였다. infection 3일 후, 마우스를 마취시켜 복강에 luciferin(caliper, MA, USA)을 7 mg/ml의 농도로 30㎕을 주사하고, 10분 후 IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA)을 이용하여 image를 촬영하였다.For the external mouse, the sample was mixed with 0.5% methyl cellulose and administered once a day at 150 mg / kg for 3 days to the vaginal tract. Each mouse was injected with HPV16-Luc PVs in a 1: 1 ratio of 1% carboxymethylcellulose (Sigma Aldrich, MO, USA) for 4 hours after external administration on the third day. After 3 days, mice were anesthetized and luciferin (caliper, MA, USA) was injected into the peritoneal cavity at a concentration of 7 mg / ml at 30 μl and 10 minutes later, IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA) The images were taken.
경구 투여 마우스의 경우, 시료를 0.5% methyl cellulose와 혼합하여 300 mg/kg씩 하루에 한번, 5일간 경구 투여하였다. 마지막 5일째 되는 날, 경구 투여 4시간 후, HPV16-Luc PVs를 1%의 carboxymethylcellulose (Sigma Aldrich, MO, USA)와 1:1 비율로 혼합하여 복부 피하 부분에 주사하였다. 이후의 imaging 방법은 외용 마우스와 동일하게 수행하였다.
In the case of oral administration mice, the samples were mixed with 0.5% methyl cellulose and orally administered at 300 mg / kg once a day for 5 days. On the
4-3. 질 내 4-3. In vagina challengechallenge 시험 exam
시료를 투여한 마우스들은 HPV16-Luc PVs challenge 6시간 전에, 20㎕의 4% nonoxynol-9 (Sigma Aldrich, MO, USA)을 질 내에 처리하였으며, 그 후, 20㎕의 3% carboxymethylcellulose (Sigma Aldrich, MO, USA)와 혼합한 HPV16-Luc PVs를 5 × 106 RLU씩 질 내에 주입하였다. The mice were treated with 20 μl of 4% nonoxynol-9 (Sigma Aldrich, MO, USA) vaginally 6 hours before challenge with HPV 16-Luc PVs and then with 20 μl of 3% carboxymethylcellulose (Sigma Aldrich, MO, USA) were injected into the vagina at a dose of 5 × 10 6 RLU.
공격 접종 3일 후, 모든 마우스를 마취시켜 경구 투여 마우스는 복강으로 luciferin(caliper, MA, USA)을 7 mg/ml의 농도로 30㎕을 주사하였다. HPV16-Luc PVs는 luciferase gene을 전달하는 plasmid, pLucf가 encapsidate되어 pseudoinfection이 가능하다(Jeff Roberts, 2007). 10분간 방치 한 후, 각각의 마우스는 IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA)을 이용하여 luciferase 발현을 detection 하였으며, image를 비교 분석 하였다. 또한 image는 Living Image 2.20 software (Xenogen, NJ, USA)를 이용하여, luciferase의 발현량을 정량 하였으며, extract의 HPV 방어 효능을 비교 분석 하였다.
Three days after challenge, all mice were anesthetized and injected intraperitoneally with 30 μl of luciferin (caliper, MA, USA) at a concentration of 7 mg / ml. HPV16-Luc PVs can be pseudoinfection by encapsidating the plasmid, pLucf, which carries the luciferase gene (Jeff Roberts, 2007). After 10 minutes of incubation, luciferase expression was detected in each mouse using IVIS 200 bioluminescence imaging system (Xenogen, NJ, USA), and images were compared and analyzed. In addition, the expression of luciferase was quantified using Living Image 2.20 software (Xenogen, NJ, USA) and the HPV defense efficacy of extract was compared and analyzed.
4-4. 4-4. ChallengeChallenge 시험 결과 Test result
음성 대조군으로, extract를 투여하지 않은 6주령 Female Balb/C 마우스를 사용하였으며, 공격접종 방법은 상기 방법과 동일하다. As a negative control, 6-week-old Female Balb / C mice without extract were used, and the method of attack inoculation was the same as described above.
Vaginal tract을 통하여 challenge한 마우스는 luciferase의 발현이 genital 부분에 국한 되어 나타났으며, 복부 피하에 challenge한 마우스는 주사한 부위와 마우스의 몸 전체에서 luciferase의 발현이 나타났다. 따라서, HPV16 PVs 감염에 의하여 luciferase activity가 detection 됨을 확인하였다. 또한, 음성 대조군에서의 luminescence를 확인하였으므로, 실험군에서의 방어 효능을 비교, 확인할 수 있게 되었다. In the mice challenged through the vaginal tract, the expression of luciferase was localized to the genital part. In the subcutaneous adventitious mice, luciferase expression was observed in the injection site and whole body of the mouse. Therefore, luciferase activity was detected by infection with HPV16 PVs. In addition, since luminescence in the negative control group was confirmed, it was possible to compare and confirm the protective effect in the experimental group.
S11-MC를 외용 투여한 마우스는 luminescence가 관찰되지 않았으며, HPV16 PVs를 완벽하게 방어한 것으로 나타났다. 또한, S11-MC를 0.5% methyl cellulose와 혼합하여 300 mg/kg씩 하루에 한번, 5일간 경구 투여하였다. 마지막 5일째 되는 날, 경구 투여 4시간 후, HPV16-Luc PVs를 복부 피하 부분에 주사하였다. 이후의 imaging 방법은 위와 동일하게 수행하였다. S11-MC를 경구 투여한 마우스는 복부 부분에 luminescence가 관찰되어 HPV PVs를 방어하지 못한 것으로 나타났다. The mice treated with S11-MC externally showed no luminescence and showed complete protection against HPV16 PVs. In addition, S11-MC was mixed with 0.5% methyl cellulose and orally administered at 300 mg / kg once a day for 5 days. On the last day of the 5th day, 4 hours after oral administration, HPV16-Luc PVs were injected into the abdomen subcutaneous area. The subsequent imaging method was performed as described above. S11-MC orally administered mice showed that luminescence was observed in the abdominal area, which did not protect HPV PVs.
S11-HP(70-95)를 0.5% methyl cellulose와 혼합하여 150 mg/kg씩 하루에 한번, 3일간 vaginal tract에 외용하여 HPV에 대한 challenge 시험을 수행하였다. S11-HP(70-95)를 외용 투여한 마우스에서도 luminescence가 관찰되지 않아, HPV16 PVs를 완벽하게 방어한 것으로 확인되었다. 또한, S11-HP(70-95)를 0.5% methyl cellulose와 혼합하여 300 mg/kg씩 하루에 한번, 5일간 경구 투여 후, HPV16-Luc PVs를 복부 피하 부분에 주사하여 image를 촬영하였다. S11-HP(70-95)를 경구 투여한 마우스는 복부 부분과 몸 전체에 luminescence가 관찰되어 HPV16 PVs를 전혀 방어하지 못한 것으로 확인되었다. S11-HP (70-95) was mixed with 0.5% methyl cellulose and challenged with HPV by external application to 150 mg / kg once a day for 3 days in the vaginal tract. It was also confirmed that luminescence was not observed even in mice administered S11-HP (70-95) externally, thus completely defending HPV16 PVs. In addition, S11-HP (70-95) was mixed with 0.5% methyl cellulose and administered orally at a dose of 300 mg / kg once a day for 5 days, and HPV16-Luc PVs were injected into the abdomen subcutaneously to image. S11-HP (70-95) mice were orally administered with luminescence in the abdomen and the whole body, and it was confirmed that HPV16 PVs were not defended at all.
Luciferase에 의한 luminescence 양을 정량한 결과, S11-MC와 S11-HP(70-95)를 외용 투여한 마우스는 extract를 투여하지 않은 마우스에 비해서 luminescence의 양이 2% 이하로 나타났다 (도 3.). 한편, 경구 투여의 경우 HPV16 PVs 공격 접종에 대한 luminescence의 양은 음성 대조군 마우스와 비슷하거나 오히려 높은 수준이었다. 따라서, S11-MC와 S11-HP(70-95)는 외용 투여하였을 때에, HPV 바이러스를 죽이거나 감염을 억제 할수 있어 공격 접종에 대한 완벽한 방어 효과를 확인할 수 있었다. 즉, S11-MC와 S11-HP(70-95)의 외용 투여가 HPV를 inhibition 할 수 있는 가장 효과적인 방법이라고 사료된다.
As a result of quantifying luciferase-induced luminescence, mice treated with S11-MC and S11-HP (70-95) externally showed less than 2% of luminescence compared to mice without extract (Fig. . In the case of oral administration, the amount of luminescence to
하기에 본 발명의 추출물 및 정제물을 함유하는 약학조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.
Hereinafter, formulation examples of the pharmaceutical composition containing the extract and purified product of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.
제제예Formulation example 1. One. 산제의Sanje 제조 Produce
S11-B 300 mgS11-B 300 mg
유당 100 mg
탈크 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.
The above components are mixed and filled in airtight bags to prepare powders.
제제예Formulation example 2. 정제의 제조 2. Preparation of tablets
S11-HP70 300 mgS11-HP70 300 mg
옥수수전분 100 mg
유당 100 mg
스테아린산 마그네슘 2 mgMagnesium stearate 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
제제예Formulation example 3. 캅셀제의 제조 3. Preparation of capsules
S11-MC 300 mgS11-MC 300 mg
결정성 셀룰로오스 3 mgCrystalline cellulose 3 mg
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.
The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
제제예Formulation example 4. 주사제의 제조 4. Preparation of injections
S11-H 300 mgS11-H 300 mg
만니톨 180 mg180 mg mannitol
주사용 멸균 증류수 2974 mgSterile sterilized water for injection 2974 mg
Na2HPO412H2O 26 mgNa 2 HPO 4 12 H 2 O 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당(2㎖) 상기의 성분 함량으로 제조한다.
(2 ml) per 1 ampoule according to the usual injection preparation method.
제제예Formulation example 5. 5. 액제의Liquid 제조 Produce
S11-T 300 mgS11-T 300 mg
이성화당 10 g10 g per isomer
만니톨 5 g5 g mannitol
정제수 적량Purified water quantity
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ㎖로 조절한 후 갈색 병에 충진하여 멸균시켜 액제를 제조한다.
Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.
제제예Formulation example 6. 건강 식품의 제조 6. Manufacture of health food
S11-HP70 1000 ㎎S11-HP70 1000 mg
비타민 혼합물 적량Vitamin mixture quantity
비타민 A 아세테이트 70 ㎍70 [mu] g of vitamin A acetate
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B1 0.13 ㎎0.13 mg of vitamin B 1
비타민 B2 0.15 ㎎0.15 mg of vitamin B 2
비타민 B6 0.5 ㎎0.5 mg of vitamin B 6
비타민 B12 0.2 ㎍Vitamin B 12 0.2 g
비타민 C 10 ㎎10 mg vitamin C
비오틴 10 ㎍Biotin 10 μg
니코틴산아미드 1.7 ㎎Nicotinic acid amide 1.7 mg
엽산 50 ㎍50 ㎍ of folic acid
판토텐산 칼슘 0.5 ㎎Calcium pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture quantity
황산제1철 1.75 ㎎1.75 mg of ferrous sulfate
산화아연 0.82 ㎎0.82 mg of zinc oxide
탄산마그네슘 25.3 ㎎Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎15 mg of potassium phosphate monobasic
제2인산칼슘 55 ㎎Secondary calcium phosphate 55 mg
구연산칼륨 90 ㎎
탄산칼슘 100 ㎎100 mg of calcium carbonate
염화마그네슘 24.8 ㎎24.8 mg of magnesium chloride
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
제제예Formulation example 7. 건강 음료의 제조 7. Manufacture of health drinks
S11-MC 1000 ㎎S11-MC 1000 mg
구연산 1000 ㎎Citric acid 1000 mg
올리고당 100 g100 g of oligosaccharide
매실농축액 2 gPlum concentrate 2 g
타우린 1 gTaurine 1 g
정제수를 가하여 전체 900 ㎖Purified water was added to a total of 900 ml
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 ℓ 용기에 취득하여 밀봉, 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. 상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.The above ingredients were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the resulting solution was filtered and obtained in a sterilized 2 liter container, sealed, sterilized and refrigerated It is used in the production of the health beverage composition of the present invention. Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
Claims (18)
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KR1020120058332A KR101477966B1 (en) | 2012-05-31 | 2012-05-31 | A composition comprising the extract of pine tree leaf for the prevention and treatment of cervical cancer |
EP13796984.6A EP2788311A4 (en) | 2012-05-31 | 2013-05-29 | A composition comprising the extract of pine tree leaf or the compounds isolated therefrom for the prevention and treatment of cancer disease by inhibiting hpv virus and the uses thereby |
US14/366,093 US20140363530A1 (en) | 2012-05-31 | 2013-05-29 | Composition comprising the extract of pine tree leaf or the compounds isolated therefrom for the prevention and treatment of cancer disease by inhibiting hpv virus and the uses thereby |
PCT/KR2013/004698 WO2013180462A1 (en) | 2012-05-31 | 2013-05-29 | A composition comprising the extract of pine tree leaf or the compounds isolated therefrom for the prevention and treatment of cancer disease by inhibiting hpv virus and the uses thereby |
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Non-Patent Citations (4)
Title |
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Journal of Pathology. Vol.189(1), pp.12-19 * |
Journal of Pathology. Vol.189(1), pp.12-19* |
한국식품저장유통학회지. Vol.12(2), pp.179-183 * |
한국식품저장유통학회지. Vol.12(2), pp.179-183* |
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