KR20130040578A - Pharmaceutical composition for preventing or treating liver cancer comprising herbal extracts - Google Patents
Pharmaceutical composition for preventing or treating liver cancer comprising herbal extracts Download PDFInfo
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- KR20130040578A KR20130040578A KR1020110105433A KR20110105433A KR20130040578A KR 20130040578 A KR20130040578 A KR 20130040578A KR 1020110105433 A KR1020110105433 A KR 1020110105433A KR 20110105433 A KR20110105433 A KR 20110105433A KR 20130040578 A KR20130040578 A KR 20130040578A
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- liver cancer
- red ginseng
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Abstract
Description
본 발명은 생약 추출물을 포함하는 간암 예방 또는 치료용 약학적 조성물에 관한 것으로, 더욱 상세하게는 홍삼, 당귀, 오미자, 토사자 및 울금의 추출물을 유효성분으로 포함하여, 높은 항산화 활성을 가지며, 특히, 간암에 대한 항암 효과가 탁월한 간암 예방 또는 치료용 약학적 조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for preventing or treating liver cancer, including a herbal extract, and more particularly, including extracts of red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric as an active ingredient, having high antioxidant activity, in particular, It relates to a pharmaceutical composition for preventing or treating liver cancer having excellent anticancer effect on liver cancer.
간암은 세계적으로 가장 치명적인 암의 하나로, 우리나라를 비롯하여 아시아권 국가에서 특히 많이 발생하여 심각한 문제가 되고 있다. 간암의 90% 이상을 차지하는 고형 암종은 치료가 힘들고 예후도 매우 불량한데, 그 이유는 초기에는 별 증상이 없다가 발견 당시에는 치료가 불가능할 정도로 진행되었을 경우가 대부분이고, 또한 간암 환자의 80% 정도는 간경변에서 시작하여 그 합병증으로 사망하는 경우가 많기 때문이다. 이러한 간암의 치료에서 가장 중요한 방법은 수술로 제거하는 것이다. 수술로 암조직을 제거할 수 없는 경우 간이식, 간동맥 색전술, 경피적 에탄올 주입술, 면역화학요법 등을 사용한다. 간 절제술은 간암 환자 중에서 전이가 발생하지 않은 비교적 초기 환자의 치료법으로서, 현재 전체 간암 환자의 약 20% 내외가 간 절제술에 의한 치료를 받고 있으나, 간 절제술을 받은 간암환자의 경우에도 장기 생존율은 높지 않은 편이다. 특히, 수술 후 5년 이내 생존율이 30-40%정도로 사망하는 환자가 많은 실정이다. 또한, 수술요법은 출혈, 감염 등의 부작용이 발생될 수 있으며, 면역화학요법 시행시 면역억제제에 대한 과민반응, 오한 및 발열, 오심 및 구토, 전신 쇠약감, 식욕부진, 두통, 빈혈, 백혈구감소증 등이 발생할 수 있다.
Liver cancer is one of the most deadly cancers in the world, and it is a serious problem because it is particularly common in Asian countries. Solid carcinoma, which accounts for more than 90% of liver cancers, is difficult to treat and has a very poor prognosis. Most of the cases are due to the fact that there is no symptom at the beginning, and progressed beyond treatment at the time of discovery, and about 80% of patients with liver cancer. This is because most people start with cirrhosis and die from complications. The most important method in the treatment of liver cancer is surgical removal. If the cancer cannot be removed by surgery, liver transplantation, hepatic artery embolization, percutaneous ethanol injection, and immunochemotherapy are used. Liver resection is the treatment of relatively early patients without metastases among liver cancer patients. Currently, about 20% of all liver cancer patients are treated by liver resection, but long-term survival rate is high even in liver cancer patients who have undergone liver resection. I'm not. In particular, many patients die at 30-40% survival rate within 5 years after surgery. In addition, surgical therapy may cause side effects such as bleeding and infection, and hypersensitivity reactions to immunosuppressive agents, chills and fever, nausea and vomiting, general weakness, anorexia, headache, anemia, and leukopenia during immunochemotherapy This can happen.
따라서, 독성과 부작용이 적으면서 간암에 대한 항암효과를 나타내는 천연물을 찾아내기 위하여 많은 연구가 진행되고 있으며, 특히, 비교적 인체에 안전한 한약재를 이용한 항암 치료약 및 항암 치료보조제 개발 연구가 활발하게 진행되고 있다.
Therefore, many studies have been conducted to find natural products exhibiting anti-cancer effects against liver cancer with less toxicity and side effects, and in particular, researches on the development of anti-cancer drugs and anti-cancer treatment aids using herbal medicines that are relatively safe for human body are being actively conducted. .
한편, 실험적으로 산화스트레스를 유발하여 장기 손상을 일으키는 물질에는 carbontetrachoride(CCl4), chloroform, phophorus, DEN(diethylnitrosamine) 등이 있다. 특히 DEN(diethylnitrosamine)은 유전자 독성 발암물질(genotoxic carcinogen)로 간암, 대장암 및 신세포암 등을 유발할 수 있다고 알려져 있다.
On the other hand, there are carbontetrachoride (CCl4), chloroform, phophorus, DEN (diethylnitrosamine), etc., which cause oxidative stress and cause organ damage. In particular, DEN (diethylnitrosamine) is a genotoxic carcinogen known to cause liver cancer, colon cancer and renal cell carcinoma.
현재 빈번히 발생되는 암을 치료하기 위한 많은 연구가 진행되고 있으며 그 결과 새로운 치료약이 계속해서 개발되고 있다. 그러나 그 개발된 약물의 부작용 또한 매우 커서 부작용이 적으면서도 암을 치료할 수 있는 안전한 치료약을 개발하기가 많은 연구의 목적이 되고 있다. 이러한 목적에 도달하기에 천연물인 한약재는 과거 수 천년의 복용경험과 효능에 있어 우선적으로 인식되고 있는 개발원료가 되고 있다.Currently, a lot of research is being conducted to treat cancers that occur frequently. As a result, new therapeutic drugs are continuously being developed. However, the side effects of the developed drug is also very large, and it is the purpose of many studies to develop a safe therapeutic drug that can treat cancer with less side effects. To achieve this purpose, herbal medicine, a natural product, has become a developmental material that is primarily recognized in the past thousands of years of taking experience and efficacy.
한편"군신좌사론(君臣佐使論)"이란, 고대 군주제도의 통치형태를 본따서 성립된 방제구성의 기본원칙으로 여러 가지 약물을 배합하여 약물의 효능을 높이고 부작용을 줄이는 방법이라 하겠다. 한의학에서는 이러한 원리를 적용하여 각각의 성분이 특유의 역할을 하여 전체적인 조화와 균형의 치료를 목적으로 한다. 다양한 질병의 치료목적으로 복용되었던 한약재는 불순물을 걸려 정제하는 포제법이 그리 발달되지 않았으나 단순히 독삼탕과 같은 한 종류 약물의 복용에서부터 여러 종류의 약물 복용으로 발전되어 간 방제학의 크나큰 발전을 시사하는 바가 크다고 할 수 있다. 이는 인체를 조절하는 인자가 굉장히 다양하다는 것을 선인들이 인식하고 있었으며, 질병 내지는 통증을 제어하는 방법에 대하여 단순히 또는 중요한 한가지 인자를 조절하는 것보다 다양한 약물을 가지고 인체 내 다양한 인자를 조절하는 것이 더 유익하다는 것으로 선인들의 경험적 지식이 집약되고 있다는 것으로 귀결할 수 있기 때문이다.
On the other hand, the "military priesthood theory" (君臣 佐使 論) is the basic principle of the composition of the control, which is modeled after the rule of the ancient monarchy system. In oriental medicine, this principle is applied, and each ingredient plays a unique role to treat the whole harmony and balance. Herbal medicines, which were used for the treatment of various diseases, have not been developed as a method of refining them due to impurity, but they have been developed from simply taking one type of drug like Doksamtang to taking various types of drugs, suggesting a great development of liver control. can do. Predecessors have recognized that there are a great variety of factors that control the human body, and it is more beneficial to control various factors in the body with different drugs than simply or one important factor in how to control disease or pain. It can be concluded that the empirical knowledge of the predecessors is concentrated.
본 발명자들은 400여 가지 약재에 대한 실험과정 홍삼, 당귀, 오미자, 토사자, 울금의 조합 구성의 유효성을 관찰하고자 HepG2 간암 세포주를 배양하여 구성물을 단미(單味)와 복미(復味)로 나누어 각각의 세포증식에 미치는 효과를 비교하고 조합의 의의를 군신좌사론에 의거하여 살펴보고, 또한, 홍삼, 구기자, 산수유, 오미자, 복분자, 토사자, 울금 및 당귀의 생약 복합 추출물이 DEN의 지속적 경구투여에 의한 간암 유발 자극에 대해 간세포 보호효과가 있음을 발견함으로써 본 발명을 완성하였다.
The present inventors cultured HepG2 liver cancer cell line to observe the efficacy of the combination of red ginseng, Angelica, Schisandra chinensis, Tosa, and turmeric for the experimental process on about 400 kinds of medicinal herbs. To compare the effect on the cell proliferation and to examine the significance of the combination according to the history of military affairs, the herbal extracts of red ginseng, wolfberry, cornus, Schisandra chinensis, Bokbunja, Tosaja, turmeric and Angelica spermatozoa were used for continuous oral administration of DEN. The present invention was completed by discovering that hepatocellular protective effect is observed against liver cancer-induced stimulation.
본 발명의 목적은 홍삼, 당귀, 오미자, 토사자 및 울금의 추출물을 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물을 제공하는 것이다. 상기 조성을 통하여 약물의 효능을 높이고 부작용을 높이는 효과를 달성하려고 한다. An object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer comprising red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric extract as active ingredients. Through the composition is to achieve the effect of increasing the efficacy and side effects of the drug.
본 발명의 다른 목적은 상기 추출물을 유효성분으로 포함하는 간암 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.
Another object of the present invention to provide a health functional food composition for preventing or improving liver cancer comprising the extract as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 홍삼, 당귀, 오미자, 토사자 및 울금의 추출물을 유효성분으로 포함하는 간암암 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric extract as active ingredients.
또한, 상기 조성물에 추가적으로 구기자, 산수유 및 복분자 추출물을 더 포함하는 것을 특징으로 하는 간암 예방 또는 치료용 약학적 조성물을 제공한다. In addition, it provides a pharmaceutical composition for preventing or treating liver cancer, characterized in that it further comprises a gojija, cornus and bokbunja extract in addition to the composition.
또한, 상기 추출물을 유효성분으로 포함하는 간암 예방 또는 개선용 건강기능식품용 조성물.
In addition, liver cancer prevention or improvement for health functional food composition comprising the extract as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 홍삼, 당귀, 오미자, 토사자 및 울금의 추출물을 유효성분으로 포함하는 간암 예방 또는 치료용 약학적 조성물을 제공한다. 상기 조성물은 당귀 100 중량부를 기준으로 각각 홍삼 100~500 중량부, 오미자 50~200 중량부, 토사자 20~200 중량부, 울금 20~200 중량부를 포함하는 것을 특징으로 하는 간암 예방 또는 치료용 약학적 조성물인 것이 바람직하다. The present invention provides a pharmaceutical composition for preventing or treating liver cancer, comprising extracts of red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric as active ingredients. The composition is based on 100 parts by weight of red ginseng based on 100 to 500 parts by weight, Schisandra chinensis 50 to 200 parts by weight, earth and
본 발명은 홍삼, 당귀, 오미자, 토사자, 울금 구기자, 산수유 및 복분자 추출물을 포함하는 것을 특징으로 하는 간암 예방 또는 치료용 약학적 조성물을 제공한다. 상기 조성물은 당귀 100 중량부를 기준으로 각각 홍삼 100~500 중량부, 오미자 50~200 중량부, 토사자 20~200 중량부, 울금 20~200 중량부, 구기자 100~500중량부, 산수유 50~200중량부, 복분자 50~200 중량부를 포함하는 것을 특징으로 하는 간암 예방 또는 치료용 약학적 조성물인 것이 바람직하다. The present invention provides a pharmaceutical composition for preventing or treating liver cancer, comprising red ginseng, Angelica, Schisandra chinensis, Tosa, turmeric wolfberry, Cornus and Bokbunja extracts. The composition is 100 to 500 parts by weight of red ginseng, 50 to 200 parts by weight of Schisandra chinensis, 20 to 200 parts by weight of earthenware, 20 to 200 parts by weight of turmeric, 100 to 500 parts of wolfberry, 50 to 200 parts by weight of cornus oil It is preferred that the pharmaceutical composition for preventing or treating liver cancer, characterized in that it comprises 50 to 200 parts by weight of bokbunja.
또한, 상기 조성물은 항산화 활성을 갖는 것이 바람직하고, 상기 추출물은 물, 탄소수 1-4개의 저급 알코올, 아세톤, 에테르, 에틸아세테이트, 부틸아세테이트, 클로로포름, 메틸렌클로라이드, 헥산, 1,3 부틸렌 글리콜 및 이들의 혼합용매로 이루어진 군중에서 선택된 용매로 추출된 것이 바람직하다. In addition, the composition preferably has an antioxidant activity, the extract is water, lower alcohol having 1-4 carbon atoms, acetone, ether, ethyl acetate, butyl acetate, chloroform, methylene chloride, hexane, 1,3 butylene glycol and It is preferable to extract with a solvent selected from the group consisting of these mixed solvents.
그리고, 상기 조성물은 고본, 천마, 시호, 도인, 계지, 대황, 감초, 천궁, 진피, 택사, 황련, 황금, 복령, 작약, 백출, 황백, 치자, 반하, 조구등, 지실, 인삼, 맥문동, 원지, 석창포, 창출, 감국, 방풍, 생강, 망초, 대조, 단삼, 목단피, 지황, 박하, 산약, 저령, 하수오, 구자, 결명자, 독활, 두충, 백화사설초, 삼백초, 인진, 지모, 홍화, 황기, 석송자, 은행잎, 황정, 연자육, 용골, 지골피, 우슬, 숙지황, 흑임자, 백자인, 맥아, 파극천 및 해송으로 구성된 군에서 선택되는 하나 이상의 생약 추출물을 추가로 포함할 수 있다. In addition, the composition is Gobon, Cheonma, Shiho, Doin, Gyeji, Rhubarb, Licorice, Cheongung, Dermis, Taxa, Huangyan, Golden, Fuling, Peony, Baekchul, Hwangbaek, Gardenia, Banja, Jaguk, Jisil, Ginseng, Macmundong, Wonji , Seokchangpo, Creation, Gamkook, Windproof, Ginger, Forget-me-not, Contrast, Dansam, Barberry, Chihwang, Mint, Potion, Spirit, Shouo, Guja, Absolute, Poisonous life, Tochung, Baekhwasaeng, Three hundred seconds, Injin, Jimo, Safflower, Astragalus, It may further include one or more herbal extracts selected from the group consisting of calendula, ginkgo biloba, yellow flower, lotus root, keel, phalanges, dew, sukji sulfur, black sesame seed, white porcelain, malt, pagokcheon and sea song.
그리고, 상기 조성물은 항암 공진단 제조를 위한 것일 수 있다.
And, the composition may be for the production of anticancer resonance stage.
한의학에서는 질병을 치료하기 위하여 본초의 약성을 강화하고 부작용을 줄이는 방법이 다양하게 존재하였다. 포제법과 약물의 사기와 오미, 그리고 귀경론, 승강부침을 이용한 약물의 보사법이 한 가지 약물로 질병을 치료하고자 할 시 약물의 효능을 높이고 부작용을 줄이는 방법이라고 한다면 앞서 언급한 군신좌사론은 여러 가지 약물을 배합하여 약물의 효능을 높이고 부작용을 줄이는 방법이라 하겠다. 군신좌사론은 복잡다양한 병증을 일정한 입방법도에 의하여 보다 훌륭하게 약물의 치료효과를 발휘하도록 하며 혹은 그 편승의 작용을 제지하려는 방침으로 다음과 같이 설명될 수 있다. 군약(君藥)이라 함은 병인이나 주병증에 대하여 중요하게 치료작용을 증강시킬 수 있는 약물을 말하며 신약(臣藥)이란 군약을 협조하여 그 치료작용을 증강시키는 약물을 말한다. 좌약(佐藥)이란 세가지 의미로 설명되는데 첫째, 겸증이나 차증후를 치료하는 약물을 말하며, 둘째로는 군약(君藥)이 유독하거나 약성일 준렬한 것을 제약 시키려는데 있고, 셋째로는 반좌작용(反佐作用)으로 즉, 주치약의 약성과 상반되는 약물을 써서 보좌를 유도하는 것을 말한다. 예를 들면 온열제 중에 소량의 한량성약물을 배합하거나 한량제 중에 비교적 소량의 온열성약물을 배합하여 응용하므로써 한열이 서로 저항함이 없이 약물을 잘 받아들이게 되기 때문에 치료의 목적에 이르게 되는 것이다. 사약(使藥)이란 즉 인경약 혹은 제약성을 조화시키는 약물로써 흔히 용량을 최소량으로 배합하는 것을 말한다.In oriental medicine, various methods of strengthening herbal medicine and reducing side effects exist to treat diseases. If the method of scavenging, cheating and chewing of drugs, and the method of compensating medicine using ear pain theory and elevation up and down, is a way to improve the efficacy of the drug and reduce side effects when treating a disease with one drug, Formulated with a variety of drugs to increase the efficacy and reduce side effects. The history of military affairs can be explained as follows in order to prevent the various effects of the drug more effectively by a certain method of legislation or to restrain the effects of piggybacking. Gun drug (君 藥) refers to a drug that can significantly enhance the therapeutic action for the etiology or main disease, and new drug (臣 藥) refers to a drug that enhances the therapeutic action in cooperation with military drugs. Suppository (佐 藥) is described in three meanings, first, drugs to treat humility or secondary symptoms, and second, to limit the toxic or weak order of military drugs (君 藥), and third, anti-left action (反 佐 作用), that is, to induce the throne by using drugs contrary to the weakness of the main medicine. For example, by applying a small amount of a thermotropic drug in a warming agent or by applying a relatively small amount of a thermosetting drug in a thermosetting agent, it is possible to reach the purpose of treatment because the heat of the heat is well received without resistance to each other. Drugs (使 藥), that is, a combination of drugs or pharmaceutical drugs, often means a minimum amount of combination.
또한 약물의 배합론에 있어서도 두 가지의 효능이 유사한 약물을 배합하여 원래 가지고 있는 치료효능을 현저하게 증강시키는 것을 이름하여 상수(相須)라 하며, 두 가지의 효능이 서로 다른 약물을 주약과 보약으로 구분하여 배합함으로써 주약의 치료효능을 증가시키는 상사(相使), 두 가지의 서로 다른 약물을 배합했을 때 한 가지 약물의 독성반응이나 혹은 부작용이, 또 다른 약물에 의해 억제되어 그 독성이나 약효가 감소되거나 제거되는 상외(相畏), 두 가지의 서로 다른 약물을 배합했을 때 한 가지 약물이 다른 한 가지 약물의 독성 혹은 부작용을 감소시키거나 제거하는 상살(相殺), 두 가지 약물을 배합했을 때 한 가지 약물이 다른 한가지 약물과 상호 견제하면서 원래의 성질과 효능을 감약시키거나 심할 경우에는 파괴하는 것을 상악(相惡), 두 가지 약물을 배합했을 때 상호작용으로 독성반응이나 혹은 극렬한 부작용을 일을 것을 상반(相反)이라 하여 질병의 치료목적에 응용하였다.
In addition, in the formulation of the drug, two drugs having similar efficacy are combined to reinforce the original therapeutic effect. The name is called constant, and the two drugs are different from each other. When the two different drugs are combined, the toxic reaction or side effect of one drug is suppressed by another drug, and the toxicity or efficacy When two different drugs are combined, one drug may combine two drugs, one that reduces or eliminates the toxicity or side effects of the other. When one drug intervenes with another drug, weakens or destroys the original properties and efficacy, or destroys it in the worst case. Of the formulation when the drug toxicity reactions or interactions or a fierce side effects as opposed to (相反) that the work was applied for the treatment of disease.
이에, 홍삼, 당귀, 오미자, 토사자, 울금의 약물학적 조합이 간암세포주의 세포생존율에 미치는 영향을 관찰한 결과의 의의는 첫째, 군신좌사론에 입각하여 이 한약재의 조합 각각이 어떠한 역할을 담당하고 있는 지와 간암에 치료 또는 보조적으로 상복할 수 있는 안전한 약물조합이 될 수 있음을 확인하였다. 그 결과, 본 발명은 상기 군신좌사론(君臣佐使論; oriental medicine formula)의 기본적 원리에 따라서 HepG2의 세포 활성을 관찰하는 방법으로 당귀(angelica gigantis radix), 홍삼(red ginseng), 울금(turmeric (curcuma longa. L.)), 토사자(cuscuta japonica) 및 오미자(schisandra chinesis)로 구성된 제형을 연구함에 의해 이루어졌다. 그 결과, 홍삼과 오미자는 유효하게 간 세포 보호를 갖고 있고, 당귀는 유효하게 HepG2의 세포의 활성을 감소시키며, 토사자와 울금은 HepG2의 세포 활성에 영향을 미치지 못한다. 이러한 결과를 토대로, 군신좌사론에 따르면 홍삼은 세포 보호효과를 갖는 군(君) 약물에 해당하고, 당귀는 세포 살상효과를 갖는 신(臣) 약물에 해당하며, 오미자는 세포보호 효과를 갖는 홍삼을 돕는 좌(佐) 약물에 해당한다. 토사자와 울금은 사(使) 약물에 해당한다. 고정적으로 정해져 있는 것은 아닌 것으로 보이며, 조합과 작용 대상에 따라 상호작용을 한다. Therefore, the results of observing the effect of pharmacological combinations of red ginseng, donkey, Schisandra chinensis, Tosa and turmeric on the cell survival rate of hepatocarcinoma cell lines were as follows. First, based on the history of military affairs, each combination of these herbs plays a role. It was confirmed that it could be a safe drug combination that can be treated or assisted with liver cancer. As a result, the present invention is a method for observing the cellular activity of HepG2 according to the basic principle of the oriental medicine formula (상기 臣 使 使; oriental medicine formula), Angelica gigantis radix, red ginseng, turmeric (curcuma) longa.L.)), cuscuta japonica and schizandra chinesis. As a result, red ginseng and Schisandra chinensis had effective liver cell protection, Angelica effectively reduced HepG2 cell activity, and earth and sand did not affect HepG2 cell activity. Based on these results, according to the military affairs theory, red ginseng corresponds to a group drug having a cell protective effect, a donkey corresponds to a renal drug having a cell killing effect, and Schisandra chinensis has a cell protective effect. Corresponds to the left drug to help. Tosa and turmeric are four drugs. It does not appear to be fixed, but interacts with combinations and objects of action.
구체적으로, 실험결과 홍삼과 오미자, 토사자, 울금은 간암세포주의 세포생존율에 그다지 큰 영향을 미치고 있지 않으나, 당귀는 상당히 많은 영향을 미치고 있다(도 1). 홍삼과 당귀, 오미자, 토사자, 울금의 혼합투여는 홍삼, 오미자, 토사자, 울금 보다는 유의성 있는 세포생존율의 감소가 관찰되었고 당귀 단독 처리군보다는 유의성 없는 감소경향이 관찰되었다. 또한 재미있는 관찰결과는 홍삼, 당귀, 토사자, 울금군에서의 세포생존율이 홍삼, 당귀, 토사자, 오미자, 울금군에서의 세포생존율보다 낮아(p<0.01), 이 처방에서의 오미자는 세포를 보호하는 결과를 가져온다고 사료되었다. 홍삼 단독처리군(약 94.8%)과 당귀 단독처리군(약 15.6%)의 실험결과와 홍삼과 당귀 혼합처리군(약 88.8%)에서의 세포생존율이 그 중간에 해당하므로 홍삼은 세포를 보호한다는 결과라고 사료되었다.Specifically, the results of the red ginseng, Schisandra chinensis, Tosa and turmeric are not significantly affecting the cell survival rate of liver cancer cell lines, but the donkey has a considerable effect (Fig. 1). In the combination of red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric, there was a significant decrease in cell viability than that of red ginseng, Schisandra chinensis, Tosa and turmeric. Also, interesting observations showed that the cell viability of red ginseng, donkey, sediment, and turmeric group was lower than that of red ginseng, donkey, sediment, schizandra, and turmeric group (p <0.01). It was thought to bring about the result. Red ginseng protects cells because the results of experiments of red ginseng alone (about 94.8%) and donkeys alone (about 15.6%) and cell survival rate of red ginseng and donkeys mixed (about 88.8%) are in between. It was considered a result.
따라서, 단순히 간암세포주에 대한 세포생존율을 대상으로 한 본 실험연구를 바탕으로 고려한다면 방제학상 군약에 해당하는 홍삼은 세포를 보호하며, 신약에 해당하는 당귀가 간암세포를 살상하고 오미자, 토사자, 울금이 군약의 역할에 도움을 주는 좌약에 해당한다고 할 수 있다. 이러한 역할적 해석은 세포의 살상보다는 부작용 감소를 우선으로 한 해석이다. 이는 최근에 시술되고 암치료법이 수술요법, 방사선 요법, 화학요법 등으로 부작용이 발생되고 있는 상황에서 이를 줄이기 위한 노력으로 면역요법 등을 고려한다면 적절한 구성이라고 사료되나 더욱 다양한 각도에서의 연구가 진행되어야 할 것으로 사료된다.
Therefore, based on this experimental study aimed at the cell survival rate of liver cancer cell line, red ginseng, which is a control drug, protects the cells, and the donkey, which is a new drug, kills liver cancer cells. This can be said to be a suppository to help the role of the military. This role-based interpretation focuses on reducing side effects rather than killing cells. This is an appropriate configuration considering the immunotherapy as an effort to reduce the situation in recent times and the cancer treatment is occurring side effects due to surgery, radiation therapy, chemotherapy, etc., but studies from various angles should be conducted. It is believed to be.
최종적으로, 홍삼, 당귀, 오미자, 토사자, 울금의 조합이 간암세포주의 세포생존율에 미치는 영향을 관찰하여 한의학에서 중요시 되고 있는 군신좌사론의 방제학적 구성에 어떻게 해당되는 지를 관찰한 본 실험결과에서 다음과 같은 결론을 얻었다. 홍삼과 오미자는 간암세포주의 세포 보호효과가 있었다. 당귀는 간암세포주의 세포생존율을 유의하게 감소시켰다. 토사자, 울금은 간암세포주의 세포생존율에 영향을 미치지 않았다.Finally, the effect of the combination of red ginseng, donkey, Schisandra chinensis, Tosa and turmeric on the cell survival rate of liver cancer cell line was examined. The same conclusion was obtained. Red ginseng and Schisandra chinensis were effective against hepatocellular carcinoma cells. Angelica significantly reduced the cell survival rate of liver cancer cell lines. Tosa and turmeric did not affect the cell survival rate of liver cancer cell lines.
상기 연구결과로 군신좌사론에 의한다면 군약에 해당하는 홍삼은 세포를 보호하며, 신약에 해당하는 당귀가 간암세포를 살상하고 오미자가 군약의 보좌하는 좌약이라 할 수 있으며, 토사자, 울금이 사약에 해당한다고 볼 수 있을 것으로 사료된다.
According to the results of the military study, the red ginseng corresponding to the military drug protects the cells, and the donkey corresponding to the new drug kills the liver cancer cells, and Somi is the suppository of the military medicine. It seems to be applicable.
본 발명의 조성물에 포함되는 홍삼은 오갈피나무과(Araliaceae) 인삼 속 (Panax)에 속하는 다년생 초본류인 인삼(Yang JP, Yeo IS: [A study on the origins of 'Korean ginseng']. Uisahak.2004;13:1-19)을 장기간 저장할 목적으로 수증기로 2-3시간 정도 찐 다음 말리는 과정, 즉 수삼(水蔘)의 전분이 호환되는 수치 과정을 거치면서 수삼 표피의 색이 붉게 되기 때문에 홍삼이라 불리고 있다. 최근에 보고된 홍삼의 약리효과는 항당뇨 효과(Lee HJ, Lee YH, Park SK, et al.: Korean red ginseng (Panax ginseng) improves insulin sensitivity and attenuates the development of diabetes in Otsuka Long-Evans Tokushima fatty rats. Metabolism. 2009;58(8):1170-1177 ; Vuksan V, Sung MK, Sievenpiper JL, et al.: Korean red ginseng (Panax ginseng) improves glucose and insulin regulation in well-controlled, type 2 diabetes: results of a randomized, double-blind, placebo-controlled study of efficacy and safety. Nutr Metab Cardiovasc Dis. 2008;18(1):46-56), 항산화효과(Hong CE, Lyu SY: Anti-inflammatory and Anti-oxidative Effects of Korean Red Ginseng Extract in Human Keratinocytes. Immune Netw. 2011;11(1):42-49), 피부보습효과(Kim H, Oh I, Park KH, Kim NM, Do JH, Cho Y: Stimulatory effect of dietary red ginseng on epidermal hydration and ceramide levels in ultraviolet-irradiated hairless mice. J Med Food. 2009;12(4):746-754), 항고지혈증효과(Hwang SY, Son DJ, Kim IW, et al.: Korean red ginseng attenuates hypercholesterolemia-enhanced platelet aggregation through suppression of diacylglycerol liberation in high-cholesterol-diet-fed rabbits. Phytother Res. 2008;22(6):778-783) 등으로 알려져 있다. 성미는 달고 조금 쓰며 성질은 평 (平)하며 비 (脾)와 폐 (肺)로 들어가 작용하며 한의학에서 주로 이용되며 중추신경에 대한 진정작용과 흥분작용이 있고 순환계에 작용하여 고혈압이나 동맥경화 예방 효과가 있어 강심작용, 항산화작용, 항피로작용, 항방사능작용과 혈당강하작용 기능을 한다. 주요성분으로는 사포닌(saponin: ginsenosides), 파낙시놀(panaxynol), 베타-엘레멘(β-elemene), 말톨(maltol) 등이 있다.Red ginseng included in the composition of the present invention is a perennial herbaceous ginseng (Yang JP, Yeo IS: A study on the origins of 'Korean ginseng') belonging to the genus Araliaceae Panax ginseng . : 1-19) It is called red ginseng because the skin of red ginseng becomes red during the process of steaming 2-3 hours with water vapor for the purpose of long-term storage. . Recently reported pharmacological effects of red ginseng are Lee HJ, Lee YH, Park SK, et al .: Korean red ginseng (Panax ginseng) improves insulin sensitivity and attenuates the development of diabetes in Otsuka Long-Evans Tokushima fatty rats Metabolism. 2009; 58 (8): 1170-1177; Vuksan V, Sung MK, Sievenpiper JL, et al .: Korean red ginseng (Panax ginseng) improves glucose and insulin regulation in well-controlled,
본 발명의 조성물에 포함되는 당귀는 미나리과(Umbelliferae)에 속한 다년생 초본인 당귀 참당귀 Angelica gigas Nakai의 뿌리를 건조시킨 것이다. 방향성분인 정유 및 데쿠신, 진정작용하는 베타시토스테롤, 자당, 니코틴산, 엽산 등의 성분으로 구성되어 있다. 각종 여성 질환, 생리불순, 생리통, 빈혈, 자궁 발육 부진, 산후 혈액 부족, 갱년기 장애 등에 두루 효과가 있고 여성 호르몬 분비를 원활하게 도와주는 작용이 있다. 최근의 연구결과로는 당귀가 세포성 면역반응에 우수한 효과가 있는 것으로 알려져 있다. 한의학적으로는 성질이 따뜻하고 맛은 달고 매우며 심장, 간, 비장에 들어가 작용한다. 혈을 보충하고 조화롭게 하며 생리(월경)를 조절하고 변을 잘 통하게 한다. The Angelica gigas Angelica gigas Nakai, a perennial herb belonging to the genus Arum ( Umbelliferae ), dried in the composition of the present invention is dried. Consists of essential oils and decousins, sedating beta sitosterol, sucrose, nicotinic acid and folic acid. Various women's diseases, menstrual disorders, menstrual cramps, anemia, poor uterine development, postpartum blood deficiency, menopausal disorders, etc. are effective throughout the female hormone secretion is effective. Recent studies have shown that donkeys have an excellent effect on cellular immune responses. In oriental medicine, it is warm in nature, sweet in taste, and works in the heart, liver and spleen. It replenishes and harmonizes the blood, regulates menstruation (menstruation), and helps stool well.
본 발명의 조성물에 포함되는 오미자는 오미자과(Schisandraceae)에 속한 오미자(북오미자) Schisandra chinensis Baill. 또는 화중오미자(남오미자) S. sphenanthera Rehd. et Wils.의 완숙한 과실을 건조한 것으로, 정유 약 3%가 함유되어 있으며, 정유 속에 다양의 sesquicarene, β2-bisabclene, β-chamigrene, α-ylangene 등이 함유되어 있다. 건조된 과실은 citral 12%, 사과산 10% 및 소량의 주석산을 함유하고 그 밖에 과당, 수지 등이 함유되어 있다. 성질은 따뜻하고 맛을 시고 달다. 폐, 심, 신장으로 들어가 수렴하고 거두어 들이며 기를 보충하고 진액을 생산하며 신장의 기운을 보충하고 진액을 생성하게 한다. Schisandra chinensis is included in the composition of the present invention belong to the Schisandra schisandraceae (Schisandraceae) (North Schisandra) Schisandra chinensis Baill. Or Schisandra chinensis ( South Schisandra chinensis) S. sphenanthera Rehd . et Wils.'s ripe fruits are dried, containing about 3% essential oils, and various sesquicarene, β2-bisabclene, β-chamigrene, and α-ylangene. The dried fruit contains 12% citral, 10% malic acid, and a small amount of tartaric acid, in addition to fructose, resin and the like. The nature is warm, flavored and sweet. It enters the lungs, heart and kidneys, converges and recollects, replenishes and produces essences, replenishes the energy of the kidneys and produces essences.
토사자는 선화과(메꽃과 ; Convolvulaceae)에 속한 일년생 기생초본인 갯실사삼(Cuscuta chinensis LAM.), 새삼(C. japonica CHOISY), 실새삼(C. australis R. BROWN)의 동속 근연식물의 성숙한 종자를 건조한 것(전국한의과대학, ed 본초학. 서울: 영림사; 1994)으로 간장기능 장애회복 작용(이 명렬, 전 성주: 토사자 추출물이 실험적으로 유발된 가토의 간장기능장애에 미치는 영향. J. Korean Soc. Food Nutr. 1989;18(3):333-337), 항암작용(이 재복, 이 병렬: 토사자 약침의 항암작용 및 면역효과에 대한 실험적 연구. 대한침구학회지. 2001;18(3)), 항산화작용( Bao X, Wang Z, Fang J, Li X: Structural Features of an immunostimulating and Antioxidant Acidic Polysaccharide from the Seeds of Cuscuta Chinensis. Planta Med. 2002;68(3):237-243), 혈관이완 효과(Jin SS, Whang EH, Kim HC, Kim CS, Oh KS, Han JH: Study on the Effect of Semen Cuscute. TheJournal of Traditional Korean Medicine. 2002;12(1):167-177) 등이 알려져 있다. 토사자는 강정, 강장제로서의 효능이 알려져 있으며, 수지 배당체, 비타민 A을 비롯하여 β-카로틴-5(β-carotene-5), 6-에폭시드(6-epoxide), 타라산틴(taraxanthin) 및 루틴(lutein) 등을 함유하는 것으로 알려져 있다.Earthworms are mature seeds of the same annual plant of Cuscuta chinensis LAM., C. japonica CHOISY, and C. australis R. BROWN. to dry (national college of Oriental Medicine, ed herbalism Seoul: Younglim four; 1994), a liver dysfunction recovery action (the myeongryeol, former lord: effects on liver function disorders of the tosaja extract experimentally induced rabbits J. Korean Soc. Food Nutr. 1989; 18 (3): 333-337), Anticancer Activity (Lee Bok Lee, Parallel: An Experimental Study on the Anticancer Activity and Immune Effect of Tosa Herbal Acupuncture. Journal of The Korean Acupuncture Society. 2001; 18 (3)), Antioxidant activity (Bao X, Wang Z, Fang J, Li X: Structural Features of an immunostimulating and Antioxidant Acidic Polysaccharide from the Seeds of Cuscuta Chinensis.Planta Med. 2002; 68 (3): 237-243) Jin SS, Whang EH, Kim HC, Kim CS, Oh KS, Han JH: Study on the Effect of Semen Cuscute.The Journal of Traditional Korean Medicine. 2002; 12 (1): 167-177). Tosa is known for its efficacy as a hardening and tonic agent, including resin glycosides, vitamin A, and β-carotene-5, 6-epoxide, taraxanthin and lutein. And the like.
본 발명의 조성물에 포함되는 울금(鬱金, Curcuma aromatica Salisb.)은 다년생 숙근성 초본식물로서 생강과 식물인 강황, 울금, 아출의 덩이뿌리(塊根)이다. 항약(생약)대사전에 의하면 지질대사작용(동물실험)에 영향을 주며, 주동맥 및 관상동맥의 내막 반괴의 형성, 지질의 침적을 감소시킬 수 있고 함유된 정유는 담즙의 분비와 배설을 촉진하는 작용이 있어 담낭을 수축시킴으로써 이담작용을 가지며 사상결석을 용해하므로 담석증에 쓰인다고 알려져 있다. 또한 중약대사전에 의하면 맛은 맵고 쓰며 성질은 한하고 독이 없으며 기의 순환을 촉진하고 울결된 것을 풀며 혈을 량하게 하고 어혈을 없애는 효능이 있다. 흉복협륵의 제통, 실심전광, 열병신혼, 토혈, 비출혈, 혈뇨, 혈림, 여자의 대상월경, 황달을 치료한다고 알려져 있다.
Curcuma ( Curcuma aromatica Salisb. ) Included in the composition of the present invention is a perennial ripening herbaceous plant, which is a turmeric root, turmeric, turmeric, and buds. Antibiotic metabolism affects lipid metabolism (animal testing), which can reduce the formation of endothelial masses in the main and coronary arteries, reduce the deposition of lipids, and contain essential oils that promote bile secretion and excretion. It is known to be used for cholelithiasis because it has a dipharytic effect by contracting the gallbladder and dissolves filamentous stones. In addition, according to the Chinese herbal medicine dictionary, the taste is spicy and bitter, and the nature is limited and nontoxic, and it promotes the circulation of the qi, loosening the loose, bleeding blood and removing the blood. It is known to treat thorax abdominal pain, vaginal embolism, fever newlyweds, hemorrhage, nasal bleeding, hematuria, hemorrhage, menstruation of men and jaundice.
한편, 구기자, 산수유, 복분자는 면역증강적 측면에서 추가하여 사용될 수 있으며, 홍삼, 당귀, 오미자, 토사자, 울금 구기자, 산수유 및 복분자 추출물의 조합이 군신좌사론과 아울러 전체적 면역증강적 측면에서 효과가 있다. On the other hand, goji berry, cornus and bokbunja can be used in addition to the immune-enhancing aspects, and the combination of red ginseng, donkey, schisandra chinensis, earth and sand, turmeric wolfberry, cornus and bokbunja extract is effective in terms of overall immunity in addition to the military affairs theory have.
본 발명의 조성물에 포함되는 상기 구기자는 가지과(茄科:Solanaceae)에 속한 구기자나무 Lycium chinense Mill. 또는 기타 동속식물의 성숙한 과실로, Carotene, vitamin B1, vitamin B2, nicotin acid, vitamin C을 주로 함유하고, β-sitosterol, linoleic acid 등이 함유되어 있다. 한의학적으로 성질은 평하고 맛은 달며 간장과 신장에 들어가 기능을 보강하고 정을 보충시키며 눈을 맑게 한다고 하였다. 이것을 지속적으로 복용하면 콜레스테롤을 줄이는 동시에 동맥경화증 예방 치료에 효과적이다. 이것의 뿌리 또한 약으로 사용할 수 있는데 뿌리껍질의 약리 실험에 의하면 혈압강하, 혈당량 저하, 살균작용이 있는 것으로 나타나 있다.The Gugija included in the compositions of the invention Solanaceae: Gugija tree belonging to the (茄科Solanaceae) Lycium chinense Mill. Or other mature fruit of the same plant, mainly contains carotene, vitamin B1, vitamin B2, nicotin acid, vitamin C, β-sitosterol, linoleic acid and the like. According to the oriental medicine, the properties are flat, the taste is sweet, and it enters the liver and kidney to strengthen the function, supplement the tablets, and clear the eyes. Consistently taking it will reduce cholesterol and help prevent atherosclerosis. Its roots can also be used as a medicine, and pharmacological experiments of the root bark have shown that there is a drop in blood pressure, lowering blood sugar, and bactericidal action.
본 발명의 조성물에 포함되는 상기 산수유는 층층나무과(山茶萸科:Cornaceae)에 속한 낙엽소교목인 산수유나무의 성숙한 열매를 건조한 것으로, 과육에는 cornin, 즉 verbenalin, saponin, tannin, ursolic acid 및 vitamin A가 함유되어 있고, 종자의 지방유에는 palmitin acid, olein acid, linol acid 등이 함유되어 있다. 위 성분 중 코르닌은 신진 대사에 활력을 향상시켜주며, 항당뇨 효과가 추정되는 물질도 포함하고 있다. 최근 연구결과로는 산수유가 면역세포의 활성을 항진시켜 면역증강능력을 하고 또한 일정 정도의 항암효과가 있다는 보고가 있다. 한의학적으로 성질은 따뜻하고 맛은 시거나 떫고 간장과 신장에 작용한다. 간장, 신장의 기능을 보충하고 정액이 흘러나가는 것을 붙잡아 준다.The cornus in the composition of the present invention is dried mature fruit of the cornus tree, which is a deciduous arborescent belonging to the dogwood (山茶 : 科 : Cornaceae ), the flesh of cornin, that is, verbenalin, saponin, tannin, ursolic acid and vitamin A The seed oil contains palmitin acid, olein acid and linol acid. Cornine, a potent ingredient in the stomach, boosts metabolism and contains anti-diabetic substances. Recent studies have reported that cornus milk boosts the activity of immune cells, boosts immune function, and has some degree of anti-cancer effect. In oriental medicine, the properties are warm, the taste is sour or steamed, and it acts on the liver and kidneys. Replenishes the function of the liver, kidneys and catches semen outflow.
본 발명의 조성물에 포함되는 복분자는 장미과의 낙엽관목인 루부스 코리아넘(Rubus coreanus)의 덜 익은 열매, 즉 미성숙과실을 일컫는다. 복분자는 예로부터 식용으로 이용되고 있을 뿐만 아니라 한방이나 민간에서 신장기능, 불임증, 음위증, 유정몽설 및 강정제에 치료 목적의 약재로 사용되고 있다. 복분자는 보간신, 명목 및 이뇨 효과가 있는 것으로 알려져 있다. 루부스 코리아넘의 줄기에서 탄닌 성분 (-)-에피카테킨, (+)-카테킨 및 프로안토시아니딘이 함유되어 있음이 확인되었고, 다양한 플라보노이드 화합물이 잎에 함유되어 있음이 확인되었다.
Bokbunja contained in the composition of the present invention refers to less ripe fruit, that is, immature fruit of Rubus coreanus, a deciduous shrub of the Rosaceae family. Bokbunja has not only been used for food, but also as a medicinal herb for renal function, infertility, erosion, well-ventilation and jeongjeongje in oriental medicine and folk medicine. Bokbunja are known to have interpolation, nominal and diuretic effects. It was confirmed that the stem of Rubus Koreanum contained tannin components (-)-epicatechin, (+)-catechin and proanthocyanidins, and various flavonoid compounds were contained in the leaves.
본 발명의 조성물에 포함되는 상기 각 생약 재료들은 재배한 것 또는 시판되는 것 등을 제한 없이 사용할 수 있으며, 건조상태 또는 자연 그대로 추출하여 이용할 수 있다.Each herbal material included in the composition of the present invention may be used without limitation, such as cultivated or commercially available, and can be extracted or used as it is in a dry or natural state.
본 발명의 생약 추출물은 당업계에 공지된 통상의 방법, 즉, 통상적인 온도와 압력의 조건하에서, 통상적인 용매를 사용하여 제조될 수 있다. 본 발명의 생약 추출물 제조에 사용될 수 있는 추출용매로는 예를 들면, 물, 탄소수 1-4개의 저급 알코올, 아세톤, 에테르, 에틸아세테이트, 부틸아세테이트, 클로로포름, 메틸렌클로라이드, 헥산 및 1,3 부틸렌 글리콜 등의 추출용매를 단독으로 또는 혼합하여 사용 가능하며, 바람직하게는 메탄올 또는 에탄올이지만, 이에 제한되는 것은 아니다. 상기 추출 용매의 적합한 양은 생약 건조 중량의 1 내지 20배 정도이며, 바람직하게는 2 내지 10배이다. 또한, 추출방법으로는 열 추출, 냉침 추출, 환류 냉각 추출 및 초음파 추출 등을 사용할 수 있으며, 1회 또는 다수회 반복하여 추출시켜 사용할 수 있다. 또한, 추출온도 및 추출시간은 각 생약의 유용성분의 유효활성이 제거되지 않을 정도의 온도 및 시간이면, 특별히 제한되지 않는다.The herbal extracts of the present invention can be prepared using conventional methods known in the art, i.e., under conventional conditions of temperature and pressure, using conventional solvents. Extractive solvents that may be used to prepare the herbal extracts of the present invention include, for example, water, lower alcohols having 1 to 4 carbon atoms, acetone, ether, ethyl acetate, butyl acetate, chloroform, methylene chloride, hexane and 1,3 butylene Extraction solvents, such as glycol, may be used alone or in combination, preferably methanol or ethanol, but is not limited thereto. A suitable amount of the extraction solvent is about 1 to 20 times the dry weight of the herbal medicine, preferably 2 to 10 times. As the extraction method, it is possible to use heat extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and the like, and they can be extracted once or repeatedly. In addition, the extraction temperature and extraction time is not particularly limited as long as the temperature and time such that the effective activity of the useful ingredient of each herbal medicine is not removed.
또한, 본 발명의 생약 추출물은 통상의 정제과정을 통해 정제시켜 이용할 수 있다. 예를 들면, 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획을 이용할 수 있다. 또한, 본 발명의 생약 추출물은 감압 증류, 동결 건조 및 분무 건조 등과 같은 건조 과정을 통해 분말 상태로 제조하여 사용할 수 있다.
In addition, the herbal extract of the present invention can be used by purification through a conventional purification process. For example, various purification methods additionally performed, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through can be used. In addition, the herbal extract of the present invention can be prepared and used in a powder state through a drying process such as distillation under reduced pressure, freeze drying and spray drying.
본 발명의 상기 생약 추출물을 포함하는 약학적 조성물은 항산화 활성을 가지며, 간암에 대한 항암 효과를 나타낸다. 상기 조성물은 페놀성 화합물을 다량 함유함을 특징으로 한다. 페놀성 화합물은 식물계에 널리 분포되어 있는 2차 대사산물의 하나로서 다양한 구조와 분자량을 가지며 플라보노이드와 탄닌이 주성분이다. 플라보노이드(flavonoid)는 방향족 폴리페놀로서 크게 플라보놀(flavonol), 플라본(flavone), 플라바논(flavanone), 플라바놀(flavanol), 안토시아니딘(anthocyanidin), 이소플라본(isoflavone)등으로 분류된다. 이들은 phenol hydroxyl(OH)기를 가지기 때문에 단백질 및 기타 거대분자들과 쉽게 결합하며 항산화, 항암 등의 다양한 생리활성을 가진다. 따라서 페놀성 화합물의 함량이 증가할 수록 항산화 활성이 증진된다.The pharmaceutical composition comprising the herbal extract of the present invention has antioxidant activity and exhibits anticancer effect on liver cancer. The composition is characterized by containing a large amount of phenolic compound. Phenolic compounds are one of the secondary metabolites widely distributed in the plant system and have various structures and molecular weights, and flavonoids and tannins are the main components. Flavonoids are aromatic polyphenols and are broadly classified into flavonols, flavones, flavanones, flavanones, flavanols, anthocyanidins and isoflavones. . Because they have phenol hydroxyl (OH) groups, they easily bind to proteins and other macromolecules and have various physiological activities such as antioxidants and anticancer. Therefore, as the content of phenolic compound increases, antioxidant activity is enhanced.
또한, 본 발명의 상기 생약 추출물은 산화적 스트레스에 의한 간세포 손상을 억제함으로써 간암에 대한 항암 활성을 나타낸다. 본 발명의 구체적 일실시예에서 본 발명의 약학적 조성물은 산화적 스트레스로 장기 손상을 일으키는 발암물질로 알려진 DEN(diethylnitrosamine)에 의한 간세포 손상을 억제하는 것을 확인할 수 있다.
In addition, the herbal extract of the present invention exhibits anticancer activity against liver cancer by inhibiting hepatocyte damage caused by oxidative stress. In a specific embodiment of the present invention, the pharmaceutical composition of the present invention can be confirmed to inhibit the hepatocyte damage by DEN (diethylnitrosamine) known as a carcinogen causing long-term damage by oxidative stress.
본 발명의 조성물은 유효 성분으로서 상기 홍삼, 구기자, 산수유, 오미자, 복분자, 토사자, 울금 및 당귀의 생약 추출물 외에 상기 생약 추출물의 약리적 항암 효과 증진을 위한 다양한 다른 종류의 생약 추출물을 추가로 포함할 수 있다. 본 발명의 조성물에 추가로 포함될 수 있는 생약 추출물로는 약학적으로 허용가능한 생약으로서, 고본, 천마, 시호, 도인, 계지, 대황, 감초, 천궁, 진피, 택사, 황련, 황금, 복령, 작약, 백출, 황백, 치자, 반하, 조구등, 지실, 인삼, 맥문동, 원지, 석창포, 창출, 감국, 방풍, 생강, 망초, 대조, 단삼, 목단피, 지황, 박하, 산약, 저령, 하수오, 구자, 결명자, 독활, 두충, 백화사설초, 삼백초, 인진, 지모, 홍화, 황기, 석송자, 은행잎, 황정, 연자육, 용골, 지골피, 우슬, 숙지황, 흑임자, 백자인, 맥아, 파극천 및 해송 등이 가능하며, 각 성분의 생약 추출물을 단독으로 또는 혼합하여 이용할 수 있다. 이때, 상기 '약학적으로 허용가능한'이란 생리학적으로 허용되고 생물체에게 투여될 때, 통상적으로 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 것을 말한다.
The composition of the present invention may further include various other types of herbal extracts for enhancing the pharmacological anticancer effect of the herbal extracts, in addition to the herbal extracts of the red ginseng, goji berry, cornus oil, Schisandra chinensis, bokbunja, earth and sand, turmeric, and Angelica as active ingredients. have. Herbal extracts that may be additionally included in the composition of the present invention include pharmaceutically acceptable herbal medicines, including Gobon, Cheonma, Shiho, Doin, Gyeji, Rhubarb, Licorice, Cheongung, Dermis, Taxa, Hulk, Golden, Bokyeong, Peony, Baekchul, Hwangbaek, Gardenia, Banja, Morning Light, Jisil, Ginseng, Macmun-dong, Wonji, Seokchangpo, Creation, Gamkook, Windproof, Ginger, Forget-me-not, Contrast, Sweet Ginseng, Barberry, Chihwang, Mint, Mountain Fertilizer, Spirit, Shou, Guja, Absolute, Poisonous life, tofu, baekryeoksaengcho, three hundred seconds, jinjin, jimo, safflower, Astragalus, calendula, ginkgo biloba, hwangjeong, lotus root, keel, phalanges, dew, sagejiku, black sesame, white porcelain, malt, pageokcheon and seaweed are available. The herbal extracts may be used alone or in combination. In this case, the 'pharmaceutically acceptable' refers to a physiologically acceptable and, when administered to an organism, usually does not cause an allergic reaction or the like.
본 발명의 약학적 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있으며, 담체와 함께 제제화될 수 있다. 이때, '약학적으로 허용가능한 담체'란 생물체를 자극하지 않고 투여 물질의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier. In this case, the 'pharmaceutically acceptable carrier' refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered substance.
상기 조성물은 생약 추출물을 캡슐에 부형제 없이 충전하거나 미립화된 고체 담체, 액체 담체 또는 그 양자와 균일하게 충분히 접촉시켜 제조될 수 있으며, 필요한 경우 생성물을 바람직한 제제로 성형하여 사용할 수 있다. 적합한 담체 부형제의 예로서 전분, 물, 식염수, 에탄올, 글리세롤, 링거액 및 덱스트로스 용액 등을 들 수 있으며, 문헌(Remington`s Pharmaceutical Science, 19thEd., 1995, Mack Publishing Company, Easton PA)등에 개시되어 있는 바에 따라 당해 기술 분야에 알려진 적합한 제제로 제제화할 수 있다.The composition may be prepared by filling the herbal extract with no excipient in the capsule or uniformly and sufficiently in contact with the atomized solid carrier, liquid carrier or both, and, if necessary, the product may be molded into a desired formulation. Examples of suitable carrier excipients include starch, water, saline, ethanol, glycerol, Ringer's solution and dextrose solution, and the like (Remington's Pharmaceutical Science, 19 th Ed., 1995, Mack Publishing Company, Easton PA) and the like. As disclosed, it may be formulated into a suitable formulation known in the art.
또한, 본 발명의 약학적 조성물은 이를 유효성분으로 포함하는 어떠한 제형으로도 적용가능하며, 경구용 또는 비경구용 제형으로 제조할 수 있다. 본 발명의 약학적 제형은 구강(oral), 직장(rectal), 비강(nasal), 국소(topical; 볼 및 혀 밑을 포함), 피하, 질(vaginal) 또는 비경구(parenteral; 근육내, 피하 및 정맥내를 포함) 투여에 적당한 것 또는 흡입(inhalation) 또는 주입(insufflation)에 의한 투여에 적당한 형태를 포함한다. In addition, the pharmaceutical composition of the present invention can be applied to any formulation containing it as an active ingredient, it can be prepared in oral or parenteral formulations. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.
본 발명의 조성물을 유효성분으로 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제로 제제화할 수 있다. 정제 및 캡슐 등의 제형으로 제제화하기 위해, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마스네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다.Examples of formulations for oral administration comprising the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
본 발명의 조성물을 유효성분으로 포함하는 비경구 투여용 제형으로는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 구체적으로, 피하주사, 정맥주사 또는 근육내 주사 등의 주사용 형태, 좌제 주입방식 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화할 수 있다. 주사용 제형으로 제제화하기 위해서는 본 발명의 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제화할 수 있다. 좌제로 주입하기 위해서는, 코코아버터 또는 다른 글리세라이드 등 통상의 좌약 베이스를 포함하는 좌약 또는 체료 관장제와 같은 직장투여용 조성물로 제제화할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.
Formulations for parenteral administration comprising the composition of the present invention as an active ingredient include injectables, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants in the form known in the art. Can be mad. Specifically, it may be formulated for injection such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method or aerosol for inhalation through respiratory system. For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials. For injection into suppositories, it may be formulated into rectal compositions such as suppositories or enema preservatives, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
본 발명의 상기 약학적 조성물은 대표적인 한의학 처방약인 공진단의 주요구성성분으로 알려진 산수유 및 당귀를 포함하고, 다른 생약들, 즉, 홍삼, 구기자, 오미자, 복분자, 토사자 및 울금을 적정 함량으로 포함함으로써, 항암 작용을 나타내므로, 간암 예방 또는 치료를 위한 항암 공진단 제조에 이용될 수 있다. 따라서, 본 발명의 상기 조성물은 특히 환제 형태로 가공되는 것이 바람직하다. 이러한 환제 형태는 생약을 추출하여 농축한 후 각 약재를 분쇄기를 이용하여 극세말하여 추출물과 가루를 혼합하고, 약액과 혼합한 가루는 다시 분쇄기에 넣어 극세말을 하여 300 내지 500 mesh의 체에 걸러내 미세한 입자로 만들어 꿀과 함께 환(丸) 또는 단(丹), 원(元) 등으로 제조할 수 있다. 환ㆍ단ㆍ원으로 만든 약재는 다시 식용 금박(또는 은박)을 사용하여 포장을 할 수 있다. 금 또는 은은 예로부터 환약을 만들 때 사용되어 마음을 안정시키고 방충 방부의 효과로 공진단이나 우황청심원 같은 처방에 보조재료로 이용하여 왔다. 또한, 다른 형태로는 환을 만드는 과정은 동일하게 하고 자초나 단삼 같은 한약재 중에서 과거부터 염료로 사용해왔고 방충 방부의 효과가 있는 약재를 추출하여 표면에 코팅하여 사용할 수 있다.
The pharmaceutical composition of the present invention comprises arithmetic and Angelica known as the main constituents of the resonant stage, which is a representative herbal medicine, and contains other herbal medicines, that is, red ginseng, wolfberry, Schisandra chinensis, bokbunja, earthenware and turmeric in an appropriate amount, Since it exhibits anti-cancer activity, it can be used to manufacture anti-cancer resonators for preventing or treating liver cancer. Therefore, the composition of the present invention is particularly preferably processed in the form of pills. The pill form is extracted and concentrated by using a pulverizer, each herbal extract is finely pulverized by mixing the extract and powder, and the powder mixed with the chemical solution is put into a pulverizer finely filtered through a sieve of 300 to 500 mesh to fine It can be made into particles and honey together with honey (환) or dan (원), circle (元). Herbs made of round, sweet and round can be packaged again using edible gold leaf (or silver leaf). Gold or silver has long been used in making pills to stabilize the mind and have been used as an auxiliary material in prescriptions such as resonant stages and cow sulfur cheongsimwon with the effect of insect repellent protection. In addition, the process of making the ring in the other form is the same, and has been used as a dye from herbal medicines such as porcelain and salvia and has been used as a dye from the past, and can be used by coating the surface to extract the effective insect repellent.
본 발명의 상기 간암 예방 또는 치료방법은 상기 약학적 조성물을 투여하는 것을 포함함을 특징으로 한다.The method for preventing or treating liver cancer of the present invention is characterized by comprising administering the pharmaceutical composition.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있으며, 구체적으로, 구강, 직장, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비측내, 흡입, 안구 내 또는 피내경로를 통해 통상적인 방식으로 투여될 수 있다. 바람직하게는 경구 투여한다. As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be various oral or parenteral routes as long as it can reach the desired tissue. It may be administered through, or specifically, can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal route. . Preferably oral administration.
본 발명의 치료방법은 상기 생약 추출물을 포함한 조성물을 약학적 유효량으로 투여하는 것을 포함한다. 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이며, 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다. 따라서 본 발명의 목적에 적합한 약학적 조성물의 유효량은 전술한 사항을 고려하여 결정하는 것이 바람직하다. 그러나, 바람직한 효과를 위해서, 본 발명의 화합물은 0.0001 내지 100 ㎎/㎏, 바람직하게는 0.01~10 ㎎/㎏의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다.The treatment method of the present invention comprises administering a composition containing the herbal extract in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment and may be administered to the patient in a single dose and in multiple doses. Administration may be by long-term, fractionated treatment protocols. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of a pharmaceutical composition suitable for the purpose of the present invention is preferably determined in consideration of the above matters. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.01 to 10 mg / kg, divided once to several times daily.
또한, 본 발명의 간암 예방 또는 치료하는 방법은 간암이 발생할 수 있는 임의의 동물에 적용가능하며, 동물은 인간 및 영장류뿐만 아니라, 소, 돼지, 양, 말, 개 및 고양이 등의 가축을 포함한다.In addition, the method for preventing or treating liver cancer of the present invention is applicable to any animal in which liver cancer may occur, and animals include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, and cats. .
또한, 본 발명의 조성물은 간암 치료를 위하여 단독으로 사용하거나, 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.
In addition, the composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the treatment of liver cancer.
또한, 본 발명은 당귀의 추출물을 유효성분으로 포함하는 간암 예방 또는 개선용 건강기능식품용 조성물을 제공한다.In another aspect, the present invention provides a composition for preventing or improving liver cancer comprising the extract of Angelica tang as an active ingredient.
본 발명의 간암 예방 또는 개선용 건강기능식품용 조성물에 있어서, 상기 조성물은 홍삼, 구기자, 산수유, 오미자, 복분자, 토사자, 울금 및 당귀의 추출물을 유효성분으로 포함하는 것이 바람직하다.In the composition for preventing or improving liver cancer of the present invention, the composition preferably comprises extracts of red ginseng, goji berry, cornus oil, Schisandra chinensis, bokbunja, earth and sand, turmeric, and Angelica as active ingredients.
본 발명의 상기 조성물은 간암 예방 또는 개선을 위한 목적으로 건강기능식품에 첨가될 수 있다. 이때, 조성물에 포함되는 각 생약 재료들은 추출물 형태 외에도 재료 자체를 분말화하여 추가로 첨가하는 방식으로도 이용될 수 있다.The composition of the present invention may be added to a dietary supplement for the purpose of preventing or improving liver cancer. In this case, each herbal material included in the composition may be used in a manner of additionally adding the powder itself in addition to the extract form.
상기 건강기능식품은 최종 제품의 상품성과 소비자 기호도에 따라 다른 식품소재를 첨가하여 제조될 수 있다. 상기 조성물의 유효한 첨가량은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있으며, 바람직하게는 총 원료에 대하여 생약 건조 중량을 기준으로 0.001 내지 90중량%, 더욱 바람직하게는 5 내지 30중량%로 첨가될 수 있다.The health functional food may be prepared by adding other food materials according to the commerciality and consumer preference of the final product. The effective amount of the composition can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), preferably 0.001 to 90% by weight, more preferably 5 to 5, based on the dry weight of the herbal medicine relative to the total raw material. It can be added at 30% by weight.
본 발명의 약학적 조성물은 식품학적으로 허용가능한 식품첨가제를 추가로 포함하여 제제화함으로써 제제 형태의 건강기능식품으로 제조할 수 있으며, 제제의 형태로는 정제, 캡슐제, 환제, 액제 등이 가능하다. The pharmaceutical composition of the present invention can be prepared as a health functional food in the form of a preparation by further comprising a food additive acceptable food formulation, the form of the preparation can be tablets, capsules, pills, liquids and the like. .
또한, 상기 조성물을 첨가할 수 있는 식품의 종류에는 특별한 제한은 없다. 예를 들면, 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예 : 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등이 가능하며, 통상적인 의미에서의 기능성 식품을 모두 포함한다. In addition, there is no particular limitation on the kind of food to which the composition can be added. For example, beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage cornebi, etc.) , Breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malts, dairy products (e.g. butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, retort food , Frozen foods, various seasonings (e.g., miso, soy sauce, sauce, etc.) are possible, and includes all functional foods in the usual sense.
또한, 본 발명의 건강기능식품은 다양한 향미제, 천연 탄수화물, 감미제, 비타민, 전해질, 착색제, 펙트산, 알긴산, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산화제 등을 추가로 함유할 수 있다. 특히, 상기 천연 탄수화물로는 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜을 사용할 수 있으며, 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 ㎖ 당 0.01~10 g 정도의 범위에서 선택될 수 있다.
In addition, the health functional food of the present invention is a variety of flavors, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonating agents And the like may be further contained. In particular, the natural carbohydrate may be a sugar such as glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. The ratio of such additives is not critical but may be selected in the range of 0.01 to 10 g per 100 ml of the composition of the present invention.
상기에서 살펴본 바와 같이, 본 발명의 간암 예방 또는 치료용 약학적 조성물은 항산화 활성을 가지며, 산화적 스트레스에 의한 폐세포 손상 억제를 통한 항암 효과를 나타내므로, 간암 치료 또는 예방을 위한 약학적 제제 및 건강기능식품 제조시 유용하게 이용될 수 있다. 또한, 상기 약학적 조성물은 환제 형태로 가공시 식감이 우수하여 간암 치료 또는 예방을 위한 항암공진단 제조에 이용될 수 있다. 더욱이 본 발명의 조합은 군신좌사론적 측면과 전체적 면역증강 측면에서 그 효과가 더욱 의미있다. As described above, the pharmaceutical composition for preventing or treating liver cancer of the present invention has an antioxidant activity and exhibits an anticancer effect through inhibiting lung cell damage caused by oxidative stress, and thus, a pharmaceutical agent for treating or preventing liver cancer and It can be usefully used in the manufacture of dietary supplements. In addition, the pharmaceutical composition is excellent in the texture when processed in the form of pills can be used for the production of anti-cancer diagnostics for the treatment or prevention of liver cancer. Moreover, the combination of the present invention is more significant in terms of military history and overall immunity.
도 1은 HepG2의 세포 활성에 약물을 구성하는 생약 각각의 단미 에탄올 추출물의 각각의 효과를 나타낸 그래프이고(A : 인삼, B : 당귀, C : 외미자, D : 토사자, E : 울금), 이때 데이터는 세 번의 반복 실험결과의 평균 ± 표준편차를 나타낸다.
도 2는 HepG2의 세포 활성에 약물을 구성하는 생약의 복미 에탄올 추출물의 각각의 효과를 나타낸 그래프이고(A : 홍삼 + 당귀, B : 홍삼 + 오미자, C : 당귀 + 울금, D : 홍삼 + 당귀 + 울금, E : 당귀 + 토사자 + 울금), 이때 데이터는 세 번의 반복 실험결과의 평균 ± 표준편차를 나타낸다.
도 3은 HepG2의 세포 활성에 약물을 구성하는 생약의 단미 및 복미 에탄올 추출물의 각각의 효과를 비교한 그래프이고(이때, 실험에 사용한 각 약재의 농도는 다음과 같다: 홍삼 40.6 ㎎/㎖, 당귀 15.6 ㎎/㎖, 오미자 15.6 ㎎/㎖, 토사자 6.3 ㎎/㎖, 울금 6.3 ㎎/㎖이고, a ; 홍삼, b : 당귀, c : 오미자, d : 토사자, e : 울금, f : 홍삼 + 당귀, g : 홍삼 + 오미자, h : 당귀 + 울금, i : 홍삼 + 당귀 + 울금, j : 당귀 + 토사자 + 울금, k : 홍삼 + 당귀 + 토사자 + 울금, l : 홍삼 + 당귀 + 오미자 + 토사자 + 울금, t-test by origin 6.0, * ; p<0.05, ** ; p<0.01, *** ; p<0.001, vs l group. + ; p<0.01, vs b group, 이때 데이터는 세 번의 반복 실험결과의 평균 ± 표준편차를 나타낸다.
도 4는 상기 표 1에 기재된 데이터를 그래프로 나타낸 것이다.
도 5는 본 발명에 따른 생약 추출물의 메탄올, 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 수층 분획을 얻는 일련의 과정을 도시한 것이다.
도 6은 본 발명에 따른 생약 추출물의 ABST radical 소거능을 측정한 그래프이다.
도 7은 본 발명에 따른 생약 추출물의 SOD(Superoxide dismutase) 유사 활성 측정을 측정한 그래프이다.
도 8은 본 발명에 따른 생약 추출물을 간암 유도된 랫트에 투여한 후 간조직의 형태를 조사한 결과이다.
도 9는 본 발명에 따른 생약 추출물이 간암 유도된 랫트의 LDH 활성에 미치는 영향을 평가한 그래프이다.
도 10은 본 발명에 따른 생약 추출물이 간암 유도된 랫트의 AST 활성에 미치는 영향을 평가한 그래프이다.
도 11은 본 발명에 따른 생약 추출물이 간암 유도된 랫트의 ALT 활성에 미치는 영향을 평가한 그래프이다.
도 12는 본 발명에 따른 생약 추출물이 간암 유도된 랫트의 ALP 활성에 미치는 영향을 평가한 그래프이다. 1 is a graph showing the effects of the ethanol extract of each of the herbal medicines constituting the drug on the cellular activity of HepG2 (A: ginseng, B: Angelica, C: foreigner, D: earthworm, E: turmeric) The data represent the mean ± standard deviation of the results of three replicates.
Figure 2 is a graph showing the effects of the ethanol extract of the herbal medicines of the herbal medicine constituting the drug on the cell activity of HepG2 (A: red ginseng + Angelica, B: red ginseng + Schisandra chinensis, C: Angelica + turmeric, D: red ginseng + Angelica + Turmeric, E: Angelica + sediment + turmeric), where data represent mean ± standard deviation of the results of three replicates.
3 is a graph comparing the effects of the sweet and ethanol extracts of the herbal medicines constituting the drug on the cell activity of HepG2 (at this time, the concentration of each medicinal herb used in the experiment is as follows: red ginseng 40.6 mg / ㎖, Angelica gigas 15.6 mg / ml, Schisandra chinensis 15.6 mg / ml, Tosa 6.3 mg / ml, turmeric 6.3 mg / ml, a; Red ginseng, b: Angelica, c: Schisandra chinensis, d: Tosa, e: Turmeric, f: Red ginseng + Angelica, g: red ginseng + Schisandra chinensis, h: donkey + turmeric, i: red ginseng + donna + turmeric, j: donkey + sesame + turmeric, k: red ginseng + donkey + Tosa + turmeric, l: red ginseng + S. donna + Schisandra + turmeric, t-test by origin 6.0, *; p <0.05, **; p <0.01, ***; p <0.001, vs l group. +; p <0.01, vs b group, where data is the result of three replicates Mean ± standard deviation of.
4 is a graph showing the data described in Table 1 above.
Figure 5 shows a series of procedures for obtaining methanol, hexane, chloroform, ethyl acetate, butanol and aqueous layer fraction of the herbal extract according to the present invention.
Figure 6 is a graph measuring the ABST radical scavenging ability of the herbal extracts according to the present invention.
Figure 7 is a graph measuring the measurement of SOD (Superoxide dismutase) -like activity of the herbal extract according to the present invention.
8 is a result of examining the form of liver tissue after administration of the herbal extract according to the present invention to the rat liver induced rats.
9 is a graph evaluating the effect of the herbal extracts according to the present invention on LDH activity of rats induced liver cancer.
10 is a graph evaluating the effect of the herbal extract according to the present invention on the AST activity of rats induced liver cancer.
11 is a graph evaluating the effect of the herbal extract according to the present invention on ALT activity of liver cancer induced rats.
12 is a graph evaluating the effect of the herbal extract according to the present invention on ALP activity of liver cancer induced rats.
이하, 본 발명을 하기 실시예에 의거하여 보다 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명은 하기 실시예에 의해 한정되는 것이 아니고, 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 치환 및 균등한 타 실시예로 변경할 수 있음은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 명백할 것이다.
Hereinafter, the present invention will be described in more detail based on the following examples. It should be noted, however, that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The present invention is not limited to the following examples. Will be apparent to those skilled in the art to which the present invention pertains.
<실시예 1> 재료 준비Example 1 Material Preparation
<1-1> 약물 제조 및 추출방법<1-1> Drug Preparation and Extraction Method
본 발명자들은 우석대학교 본초학교실에서 검증 받은 홍삼(강화도 산지, 6년근), 당귀, 오미자, 토사자, 울금(이하, ㈜옴니허브, 한국)을 분말을 만든 후 각각 에탄올 100%에 3일간 냉침하여 용출시킨 추출물을 감압 농축 후 동결 건조하여 사용하였다. 이들을 각각 DMSO에 녹인 후 사용전까지 냉동보관하였다.
The present inventors made powders of red ginseng (Ganghwado, 6 years old), donkey, schisandra chinensis, earth and sand, Ulgeum (hereinafter, Omni Herb, Korea), which were verified in the main school of Woosuk University, and then eluted by cooling them in 100% ethanol for 3 days. The extract was concentrated under reduced pressure and freeze dried. These were each dissolved in DMSO and stored frozen until use.
<1-2> 암세포주 배양<1-2> Cancer Cell Line Culture
HepG2 간암 세포주는 국립암센터에서 분양 받아 사용하였다. 배양액은 Lonza 사의 MEGM BulletKit을 하용하였고 간암세포주를 BulletKit에 포함된 BPE (Bovine Pituitary Extract), hEGF (human epidermal growth factor), hydrocortisone, isulin과 추가로 100 ng/㎖ cholera toxin, penicillin, streptomycin이 첨가된 MEGM medium에서 며칠간 배양하였다.HepG2 liver cancer cell line was used by the National Cancer Center. The culture medium used MEGM BulletKit from Lonza, and liver cancer cell lines were supplemented with BPE (Bovine Pituitary Extract), hEGF (human epidermal growth factor), hydrocortisone, isulin and 100 ng / ml cholera toxin, penicillin, and streptomycin added to BulletKit. Incubated for several days in MEGM medium.
<실시예 2> 세포 생존율 측정Example 2 Measurement of Cell Viability
배양 중이던 세포를 trypsin/EDTA 용액으로 처리한 후, 96 well plate에 well당 각각 HepG2 간암 세포주(10 x 103 cells)로 분주하고 배양하였다. 24시간이 지난 후에 배양 중이던 medium을 제거한 후, supplements를 제외하고 penicillin과 streptomycin만이 첨가된 MEGM medium에 각각의 추출물을 지정된 농도로 희석하여 각각의 well에 주입하였으며 48시간을 추가로 배양한 후 세포의 생존율을 PrestoBlue (Invitrogen) 시약으로 확인하였다.
Cells were incubated with trypsin / EDTA solution, and then dispensed into 96 well plates with HepG2 liver cancer cell lines (10 × 10 3 cells) per well and incubated. After 24 hours, the medium that was incubated was removed, and each extract was diluted to the specified concentration in MEGM medium containing only penicillin and streptomycin except supplements, and injected into each well. Survival was confirmed with PrestoBlue (Invitrogen) reagent.
통계처리는 모든 실험은 3회 반복으로 수행하여 평균값과 표준편차로 나타내었다. 자료의 통계적 처리와 상관계수 값은 Microsoft Office Excel 2007과 origin 6.0 소프트웨어를 사용하였다.
Statistical processing was performed in three replicates for all experiments, expressed as mean value and standard deviation. Statistical processing and correlation coefficient values were used for Microsoft Office Excel 2007 and origin 6.0 software.
<실시예 3> 실험결과Example 3 Experimental Results
<3-1> 간암세포주(HepG2)에 대한 구성약물 중 단미가 세포생존율에 미치는 영향<3-1> Effect of Danmi in Cell Drugs on Hepatocellular Carcinoma Cell Line (HepG2)
강화도 산지의 홍삼 6년근 에탄올 추출물이 간암세포주(HepG2)의 세포 생존율에 미치는 영향을 다양한 농도에서 살펴본 결과, 홍삼 에탄올 추출물은 간암세포의 생존율에 직접적인 영향을 미치지 않는 것으로 나타났다. 실험에 관찰된 홍삼 에탄올 추출물의 농도는 약 160 ㎎/㎖에서 5 ㎎/㎖까지 관찰하였으며 160 ㎎/㎖의 농도에 이르러서야 71.5%의 세포 생존율이 관찰되어 크게 의미를 부여하지 못하였다(도 1A). The effects of red ginseng 6-year-old ethanol extract from Ganghwa Island on the cell viability of hepatocellular carcinoma cell line (HepG2) at various concentrations showed that red ginseng ethanol extract did not directly affect the survival rate of liver cancer cells. The concentration of red ginseng ethanol extract observed in the experiment was observed from about 160 mg / ml to 5 mg / ml, and the cell survival rate of 71.5% was not reached until the concentration of 160 mg / ml (Fig. 1A). ).
㈜옴니허브에서 구입한 당귀의 에탄올 추출물이 간암세포주(HepG2)의 세포 생존율에 미치는 영향을 여러 농도에서 살펴본 결과, 당귀 에탄올 추출물은 비교적 낮은 농도에서 간암세포주의 세포 생존율에 영향을 미치고 있었다. 당귀 에탄올 추출물 15.6 ㎎/㎖에서 약 82.9%의 세포생존율이 관찰되었으며 31.3 ㎎/㎖에서는 약 39.4%의 세포생존율로 당귀 에탄올 추출물은 간암세포주의 생존율을 억제하고 있음이 관찰되었다(도 1B).The effect of ethanol extract of Angelica gigas from Omnihub Co., Ltd. on cell viability of hepatocellular carcinoma cell line (HepG2) was examined at various concentrations. The cell survival rate of about 82.9% was observed at 15.6 mg / ml of the Angelica gigas ethanol extract, and the cell survival rate of about 39.4% at 31.3 mg / ml was observed to inhibit the survival of hepatocellular carcinoma cell lines (FIG. 1B).
오미자의 에탄올 추출물이 간암세포주(HepG2)의 세포 생존율에 미치는 영향은 7.8 ㎎/㎖의 농도에서는 약 110.0%로 다소 증가시키나 농도가 높아져 62.5 ㎎/㎖에서는 78.2%를 나타내어 간암세포주의 세포생존율에는 영향을 약간 미치는 것으로 판단되었다(도 1C).The effect of ethanol extract of Schisandra chinensis on the cell viability of hepatocellular carcinoma cell line (HepG2) was slightly increased to 110.0% at the concentration of 7.8 mg / ml, but increased to 78.2% at 62.5 mg / ml, affecting the cell survival rate of hepatocarcinoma cell line. Was judged to exert slightly (Figure 1C).
토사자 에탄올 추출물이 간암세포주의 세포 생존율에 미치는 영향은 비교적 낮은 0.8 ~ 25.0 ㎎/㎖의 농도에서 홍삼에탄올 추출물의 결과와 유사한 결과는 나타내어 영향을 미치지 않는 것으로 관찰되었다(도 1D).The effect of Tosa ethanol extract on the cell viability of hepatocellular carcinoma cell line was observed to be similar to that of the red ginseng ethanol extract at a relatively low concentration of 0.8-25.0 mg / ml (FIG. 1D).
울금 에탄올 추출물이 간암세포주의 세포 생존율에 미치는 영향은 오미자 에탄올 추출물의 관찰결과와 유사하며 낮은 농도인 0.8 ~ 12.5 ㎎/㎖에서는 간암세포주의 세포생존율을 다소 증가시키지만 25 ㎎/㎖에서는 83.7%의 세포생존율이 관찰되었다(도 1E).
The effect of turmeric ethanol extract on the cell viability of hepatic cancer cell line was similar to that of Schizandra ethanol extract, and the low concentration of 0.8 ~ 12.5 ㎎ / mL increased the survival rate of hepatocarcinoma cell line slightly, but it was 83.7% at 25 ㎎ / mL. Survival was observed (FIG. 1E).
<3-2> 간암세포주(HepG2)에 대한 구성약물 중 복미가 세포생존율에 미치는 영향 <3-2> Effect of Cicada on Cell Viability in Constituent Drugs for Hepatocellular Carcinoma Cell Line (HepG2)
다음은 구성약물 별 상승효과(synergy effect)를 관찰하기 위하여 홍삼과 당귀의 상승효과(A), 홍삼과 울금의 상승효과(B), 당귀와 울금의 상승효과(C), 홍삼, 당귀와 울금의 상승효과(D), 당귀 그리고 토사자, 울금의 상승효과(E)를 관찰하였다. 각각의 농도는 도 1에서 실시한 농도를 각각 혼합하여 M1 (Mixture Level 1), M2, M3, M4, M5, M6, M7로 표기하였다. Next, to observe the synergy effect of each constituent drug, the synergistic effect of red ginseng and donkey (A), synergistic effect of red ginseng and turmeric (B), synergistic effect of donkey and turmeric (C), red ginseng, donkey and turmeric The synergistic effect of (D), Angelica and sediment and sediment were observed (E). Each concentration was expressed as M1 (Mixture Level 1), M2, M3, M4, M5, M6, and M7 by mixing the concentrations performed in FIG. 1.
홍삼과 당귀의 조합에서는 M5 농도에서 다소 세포생존율이 떨어지기 시작하여 약 88.8%로 관찰되었고 이후 M6 농도에서 37.6%, M7 농도에서 17.9%로 관찰되었다(도 2A).In the combination of red ginseng and Angelica, cell viability began to drop slightly at the M5 concentration, which was observed to be about 88.8%, followed by 37.6% at the M6 concentration and 17.9% at the M7 concentration (FIG. 2A).
홍삼과 울금의 조합에서 M2 농도에서는 약 106.9%의 세포생존율이, M3 농도에서는 약 108.1%의 세포생존율이, M4 농도에서는 약 106.2%가, M5 농도에서는 약 109.1%의 세포생존율이, M6 농도에서는 약 99.7%의 세포생존율이, M7 농도에서는 약 76.7%의 세포생존율이 관찰되었다(도 2B).In the combination of red ginseng and turmeric, the cell survival rate was about 106.9% at M2 concentration, about 108.1% at M3 concentration, about 106.2% at M4 concentration, about 109.1% at M5 concentration, and at M6 concentration. A cell viability of about 99.7% was observed and a cell viability of about 76.7% was observed at the M7 concentration (FIG. 2B).
당귀와 울금의 농도별 조합에서의 관찰된 간암세포주의 세포생존율은 다음과 같았다. 무처리군인 M1 농도에서의 세포생존율을 100.0%라 환산하였을 경우, M2 농도에서의 세포생존율은 약 96.3%, M3 농도에서의 세포생존율은 약 101.0%, M4 농도에서의 세포생존율은 약 94.9%, M5 농도에서의 세포생존율은 약 80.0%, M6 농도에서의 세포생존율은 약 41.7%, M7 농도에서의 세포생존율은 약 18.3%로 관찰되었다(도 2C).The cell survival rates of the hepatocellular carcinoma cell lines were as follows. When the cell viability at the M1 concentration of the untreated group was converted to 100.0%, the cell viability at the M2 concentration was about 96.3%, the cell viability at the M3 concentration was about 101.0%, and the cell viability at the M4 concentration was about 94.9%, Cell viability at M5 concentration was about 80.0%, cell viability at M6 concentration was about 41.7%, and cell viability at M7 concentration was about 18.3% (FIG. 2C).
홍삼 그리고 당귀와 울금의 농도별 조합으로 간암세포주의 세포생존율에 미치는 영향은 다음과 같았다. M2 농도에서의 세포생존율은 약 105.5%, M3 농도에서의 세포생존율은 약 100.5%, M4 농도에서의 세포생존율은 98.0%, M5 농도에서의 세포생존율은 약 79.8%, M6 농도에서의 세포생존율은 약 37.4%, M7 농도에서의 세포생존율은 약 16.7%로 관찰되었다(도 2D).The effects of the combination of red ginseng and donkey and turmeric on the cell survival rate of liver cancer cell lines were as follows. Cell viability at M2 concentration was about 105.5%, cell viability at M3 concentration was about 100.5%, cell viability at M4 concentration was 98.0%, cell viability at M5 concentration was about 79.8%, and cell viability at M6 concentration was Cell viability at about 37.4% and M7 concentration was observed to be about 16.7% (FIG. 2D).
당귀와 토사자, 울금의 농도별 조합으로 간암세포주의 세포생존율에 미치는 영향은 다음과 같았다. 무처리군인 M1 농도에서의 세포생존율을 100.0%라 환산하였을 경우, M2 농도에서의 세포생존율은 약 99.3%, M3 농도에서의 세포생존율은 약 99.7%, M4 농도에서의 세포생존율은 약 92.3%, M5 농도에서의 세포생존율은 약 68.2%, M6 농도에서의 세포생존율은 약 38.7%, M7 농도에서의 세포생존율은 약 17.3%로 관찰되었다(도 2E).
The effects of the combination of donkey, earth and sand and turmeric on the cell survival rate of liver cancer cell lines were as follows. When the cell viability at the M1 concentration of the untreated group was converted to 100.0%, the cell viability at the M2 concentration was about 99.3%, the cell viability at the M3 concentration was about 99.7%, the cell viability at the M4 concentration was about 92.3%, The cell viability at M5 concentration was about 68.2%, the cell viability at M6 concentration was about 38.7%, and the cell viability at M7 concentration was about 17.3% (FIG. 2E).
<3-3> 간암세포주(HepG2)에 대한 구성약물 중 단미와 복미가 세포생존율에 미치는 영향비교<3-3> Comparison of the Effect of Sweet Rice and Red Rice Fructus on the Cell Viability of Hepatic Cancer Cell Line (HepG2)
다음은 단미와 복미의 효율성을 비교하기 위하여 특정 농도를 정하여 단미와 복미가 간암세포주의 세포생존율에 미치는 영향을 관찰 비교하였다. Next, to compare the efficiency of sweet and scented rice, we determined the specific concentrations and compared the effects of scavengers on the cell survival rate of liver cancer cell lines.
홍삼군은 농도 40.6 ㎎/㎖에서 간암세포주의 세포생존율은 약 94.8%이며, 당귀군은 농도 15.6 ㎎/㎖에서 약 82.9%의 세포생존율이 관찰되었다. 오미자군은 농도 15.6 ㎎/㎖에서 약 104.9%의 세포생존율이 관찰되었으며, 토사자군은 농도 6.3 ㎎/㎖에서 약 102.0%의 농도가 관찰되었고, 울금군은 농도 6.3 ㎎/㎖에서 약 113.7%의 세포생존율이 관찰되었다.In the red ginseng group, the cell survival rate of the liver cancer cell line was about 94.8% at the concentration of 40.6 mg / ml, and the cell survival rate of about 82.9% was observed at the concentration of 15.6 mg / ml in the Angelica ginseng group. The cell viability of Schisandra chinensis was observed at 15.6 ㎎ / ml at about 105.6%, while the Tosa sperm at about 6.3 ㎎ / ml at 106.3%. Cell viability was observed.
홍삼과 당귀군(f) 각각의 농도에서 세포생존율은 약 88.8%, 홍삼과 울금군(g) 각각의 농도에서 세포생존율은 약 109.1%가 관찰되었다. 당귀와 울금군(h) 각각의 농도에서 세포생존율은 약 80.0%가 관찰되었으며, 홍삼, 당귀와 울금군(i) 각각의 농도에서는 약 79.8%의 세포생존율이 관찰되었다. 당귀, 토사자, 울금군(j) 각각의 농도에서는 약 68.2%의 세포생존율이 관찰되었고, 홍삼, 당귀, 토사자, 울금군(k) 각각의 농도에서는 약 67.8%의 세포생존율이 관찰되었으며, 홍삼, 당귀, 오미자, 토사자, 울금군(l) 모두 포함한 실험군에서는 약 79.5%의 세포생존율이 관찰되었다(도 3).
The cell viability was about 88.8% at the concentrations of red ginseng and donkey group (f) and about 109.1% at the concentrations of red ginseng and turmeric group (g). The cell viability was about 80.0% at the concentrations of the Angelica and Ulgum group (h), and the cell viability was about 79.8% at the concentrations of the red ginseng, Angelica and Ulgum group (i). Cell concentrations of about 68.2% were observed at the concentrations of Angelica, Seongsa, and Ulgeum (j), and 67.8% at the concentrations of red ginseng, Angelica, Tosa, and Ulgeum (k). About 79.5% of cell viability was observed in the experimental group including all of Angelica, Schisandra chinensis, Tosa, and turmeric group (l).
상기 실시예 3-1 내지 3-3의 데이터의 평균값과 표준편차의 수치를 하기 표 1 및 도 4에 기재하였다.
The average values and standard deviation values of the data of Examples 3-1 to 3-3 are shown in Table 1 and FIG. 4.
Red ginseng
Angelica
Schisandra
Tosa
Turmeric
Red Ginseng + Angelica
Red ginseng + turmeric
Donkey + turmeric
Red Ginseng + Donkey + Turmeric
Donkey + Earthworker + Turtle
Red ginseng + Angelica + earthenware + turmeric
평균
Average
Standard Deviation
<실시예 4> 재료 준비Example 4 Material Preparation
<4-1> 생약 추출물 제조<4-1> herbal extract preparation
본 발명의 조성물에 포함되는 재료로서, 홍삼, 구기자, 산수유, 오미자, 복분자, 토사자, 울금 및 당귀를 약재상에서 구입한 후 분쇄하였다. 분말화된 생약재들을 표 2의 조성으로 총 약재 중량이 1 ㎏이 되도록 한 후, 이를 추출기를 이용하여 100% 메탄올 4 L로 70℃에서 3시간 동안 추출하여 생약 추출물을 제조하였다.
As a material contained in the composition of the present invention, red ginseng, wolfberry, cornus oil, Schisandra chinensis, bokbunja, earth and sand, turmeric and Angelica were purchased and then ground. The powdered herbal medicines were made to have a total herbal weight of 1 kg in the composition of Table 2, and then extracted with 4 L of 100% methanol at 70 ° C. for 3 hours using an extractor to prepare a herbal extract.
<4-2> 생약 추출물 분획 제조<4-2> Preparation of herbal extract fraction
상기 <4-1> 과정에서 제조된 100% 메탄올 추출물은 도 5와 같은 과정으로 용매의 극성차를 이용하여 비극성 용매로부터 극성 용매 순으로 분리하여 5가지 분획으로 준비하였다. 즉 메탄올 추출물을 감압농축하여 n-hexane과 물을 혼합하여 용해시킨 후 분액깔대기에 넣고 n-hexane 분획층을 얻었다. 그 후 수층을 분액깔대기에 넣고 같은 방법으로 클로로포름, 에틸아세테이트, n-부탄올 및 수층(aqueous) 분획을 차례로 얻었다. 상기 메탄올 추출물, 헥산 분획, 클로로포름 분획, 에틸아세테이트 분획, 부탄올 분획 및 수층 분획의 추출물은 후 감압 농축하고 동결건조한 후 중량법으로 각 분획의 수율을 구하였다. 각 분획 추출물은 -70℃에 냉동 보관하여 실험에 사용하였다. 이렇게 수득한 생약 추출물을 '항암공진단 추출물'로 명명하였다.
The 100% methanol extract prepared in the above <4-1> process was prepared in five fractions by separating the polar solvent from the nonpolar solvent in the order of polar solvent using the polarity difference of the solvent in the same manner as in FIG. 5. In other words, the methanol extract was concentrated under reduced pressure, n-hexane and water were mixed and dissolved in the separatory funnel to obtain an n-hexane fraction layer. Thereafter, the aqueous layer was placed in a separatory funnel, and chloroform, ethyl acetate, n-butanol and aqueous fractions were sequentially obtained in the same manner. The methanol extract, the hexane fraction, the chloroform fraction, the ethyl acetate fraction, the butanol fraction, and the extract of the aqueous layer were then concentrated under reduced pressure, lyophilized, and the yield of each fraction was determined by gravimetric method. Each fraction extract was stored frozen at -70 ℃ was used in the experiment. The herbal extract thus obtained was named 'anticancer diagnostic diagnosis extract'.
<4-3> 실험동물의 준비<4-3> Preparation of Experiment Animal
체중 180± 10 g 내외의 주령 흰쥐(Rat, Sprague Dawley)를 샘타코(오산, 한국)로부터 구입하였다. 실험동물은 원광대학교 한의과대학 동물사육실에서 일정한 조건 (온도 : 21± 2℃, 습도 : 50-60%, 12시간 명/암 주기)하에서 일반 고형사료(샘타코, 흰쥐 용)와 물을 충분히 공급하면서 1주 동안 적응시킨 후 실험에 사용하였다. Age rats (Rat, Sprague Dawley) of about 180 ± 10 g body weight were purchased from Samtaco (Osan, Korea). The experimental animals were supplied with sufficient solid food (for samtaco, rats) and water under certain conditions (temperature: 21 ± 2 ℃, humidity: 50-60%, 12 hours light / dark cycle) in the animal breeding room of Wonkwang University. Adapted for 1 week while being used in the experiment.
모든 동물 실험은 원광대학교 동물 관리 위원회에 의해 승인된 프로토콜에 따라 수행되었다.
All animal experiments were performed according to a protocol approved by Wonkwang University Animal Care Committee.
<< 실시예Example 5> 생약 추출물의 항산화 활성 검사 5> Antioxidant activity test of herbal extract
산화적 스트레스에 의한 암의 형성과 발전과정은 익히 알려진 사실이다. 또한 발암물질인 DEN 자극의 주된 기전은 산화적 스트레스를 유발함에 있다. 따라서, 본 발명자들은 상기 생약 추출물이 산화적 스트레스를 억제할 수 있는지를 확인하기 위해 산화적 스트레스 억제 정도를 확인하였다.
The formation and development of cancer by oxidative stress is well known. In addition, the main mechanism of DEN stimulation, a carcinogen, is in inducing oxidative stress. Therefore, the present inventors confirmed the degree of oxidative stress inhibition to determine whether the herbal extracts can inhibit oxidative stress.
<5-1> ABST radical 소거능 측정<5-1> ABST radical scavenging ability measurement
ABST법은 항산화능 측정 방법 중 하나인 간접적인 방법 중에서 가장 많이 사용되는 방법이다. ABTS radical을 이용한 항산화능의 측정은 potassium persulfate와의 반응에 의해 생성되는 ABTS free radical이 샘플 내의 항산화 물질에 의해 제거되어 radical 특유의 색인 청록색이 탈색되는 것을 이용한 방법으로 Van den Berg 등의 방법을 변형하여 측정하였다. 구체적으로 7 mM 2.2-azino-bis(3-rthylbenzthiazoline-6-sulfonic acid)(ABTS)와 2.45 mM potassium persulfate를 최종농도로 혼합하여 실온인 암소에서 24시간 동안 방치하여 ABTS+radical을 형성시킨 후 732 ㎚에서 흡광도 값이 0.9(± 0.1)가 되게 증류수로 희석하였다. 희석된 ABTS radical 용액 950 ㎕에 각 분획의 생약 추출물(메탄올 추출물, 클로로포름 분획, 에탄올 분획 및 헥산 분획) 50 ㎕(10 ㎎/㎖ 농도)를 가하여 혼합 후 흡광도의 변화를 위해 10분 후에 측정하였으며 표준물질로서 Trolox를 동량 첨가하였다. ABST 소거능은 하기 수학식 1의 방법으로 계산하였으며, 그 결과를 도 6에 나타냈다.ABST method is the most frequently used method of indirect one of the antioxidant capacity measurement method. Antioxidant activity was measured using ABTS radicals by removing the ABTS free radicals produced by the reaction with potassium persulfate by the antioxidants in the sample and decolorizing the cyan color which is specific to radicals. Measured. Specifically, 7 mM 2.2-azino-bis (3-rthylbenzthiazoline-6-sulfonic acid) (ABTS) and 2.45 mM potassium persulfate were mixed at the final concentration for 24 hours in a cow at room temperature to form ABTS + radical. Diluted with distilled water at 0.9 nm so that the absorbance value is 0.9 (± 0.1). 50 µl (10 mg / ml concentration) of crude extracts (methanol extract, chloroform fraction, ethanol fraction and hexane fraction) of each fraction was added to 950 µl of diluted ABTS radical solution and measured after 10 minutes for change of absorbance after mixing. Equivalent amount of Trolox was added as material. ABST scavenging ability was calculated by the method of
[수학식 1][Equation 1]
ABTS scavenging ability(%) = 1-(시료 처리구 흡광도/무처리구 흡광도)x100
ABTS scavenging ability (%) = 1- (Sample absorbance / untreated absorbance) x100
도 6에서 보듯이, 메탄올 추출물의 ABST 항산화능은 약 81.17 ± 0.36%로 관찰되었고, chloroform 분획물은 72.40 ± 0.24%의 항산화능을 나타내었다. Ethyl acetate 분획물은 약 65.13 ± 0.14%로 가장 낮은 ABST 항산화능을 나타내었으며, n-hexan 분획물은 92.68 ± 0.24%로 가장 높은 ABST 항산화능을 나타내었다. 이러한 결과는 본 발명의 생약 추출물(항암공진단 추출물)에 천연 수용성 페놀성 물질이 다량 함유하고 있음을 추정할 수 있으며 특히, n-hexan 분획층에 가장 많은 천연 수용성 페놀성 물질이 함유되어 있음을 추정할 수 있다.
As shown in Figure 6, the ABST antioxidant activity of the methanol extract was observed to be about 81.17 ± 0.36%, the chloroform fraction showed an antioxidant capacity of 72.40 ± 0.24%. Ethyl acetate fraction showed the lowest ABST antioxidant activity of about 65.13 ± 0.14%, and n-hexan fraction showed the highest ABST antioxidant activity of 92.68 ± 0.24%. These results suggest that the herbal extract (anticancer diagnostic diagnostic extract) of the present invention contains a large amount of a natural water-soluble phenolic substance, and in particular, it is estimated that the most natural water-soluble phenolic substance is contained in the n-hexan fraction layer. can do.
<5-2> 총 페놀 함량 측정<5-2> Total Phenolic Content Measurement
상기 생약 추출물의 총 폴리페놀 함량은 Dewanto10)의 방법에 따라 Folin-ciacalteu rearent가 항암공진단의 폴리페놀 화합물에 의해 환원된 결과 몰리브덴 청색으로 발색하는 것을 원리로 분석하였다. 각 분획의 생약 추출물(메탄올 추출물, 클로로포름 분획, 에탄올 분획 및 헥산 분획) 100 ㎕에 Folin-Ciocalteau's phenol regent 100 ㎕을 넣고 혼합하여 실온에서 3분간 정치한 뒤 2% Na2CO3 용액 100 ㎕을 가하여 혼합 한 후 실온에서 1시간 방치시키고 750 ㎚에서 흡광도를 측정하였다. 용매에 따른 추출 효율을 비교하기 위해 상기 실시예 <4-1>에서 메탄올 대신 증류수로 생약을 추출한 추출물을 함께 실험하였다. 총 페놀함량을 정량분석하기 위해 표준물질인 gallic acid를 증류수에 녹여 일정한 농도별로 조제하고 시료와 동일한 방법으로 실험하여 검량선을 작성하고 시료의 총 페놀 함량을 측정하였으며, 그 결과를 하기 표 3에 나타내었다. The total polyphenol content of the herbal extracts was analyzed based on the principle that the Folin-ciacalteu rearent was colored by molybdenum blue as a result of reduction by the polyphenol compound of the anticancer resonance group according to the method of Dewanto10). To 100 μl of herbal extracts (methanol extract, chloroform fraction, ethanol fraction and hexane fraction) of each fraction, add 100 μl of Folin-Ciocalteau's phenol regent, mix, hold for 3 minutes at room temperature, and add 100 μl of 2% Na 2 CO 3 solution. After mixing, the mixture was left at room temperature for 1 hour and the absorbance was measured at 750 nm. In order to compare the extraction efficiency according to the solvent, the extract extracting the herbal medicine with distilled water instead of methanol in Example <4-1> was experimented together. In order to quantify the total phenol content, gallic acid, a standard substance, was dissolved in distilled water, and prepared according to a certain concentration, and the experiment was performed in the same manner as the sample to prepare a calibration curve and to measure the total phenol content of the sample. The results are shown in Table 3 below. It was.
± 0.031.19
± 0.03
± 0.011.19
± 0.01
± 0.0081.05
± 0.008
± 0.0041.17
± 0.004
± 0.011.32
± 0.01
± 1.2327.87
± 1.23
± 1.0732.80
± 1.07
± 0.7230.69
± 0.72
± 0.5414.09
± 0.54
± 0.59121.62
± 0.591
표 3에서 보듯이, 생약 추출물의 총페놀 함량은 1.19 ± 0.03 ㎎/g이였으며, 메탄올 추출물의 총페놀 함량은 약 1.19 ± 0.01 ㎎/g로 관찰되었다. Chloroform 분획물은 1.05 ± 0.01 ㎎/g의 총페놀 함량을 나타내었으며, ethyl acetate 분획물은 약 1.17 ± 0.01 ㎎/g의 총페놀 함량을 나타내었으며, n-hexan 분획물은 1.32 ± 0.01 ㎎/g로 가장 높은 총페놀 함량을 나타내었다. 생약 추출물의 총플라보노이드 함량은 27.87 ± 1.23 ㎎/g이였으며, 메탄올 추출물의 총플라보노이드 함량은 약 32.80 ± 1.07 ㎎/g로 관찰되었다. Chloroform 분획물은 30.69 ± 0.72 ㎎/g의 총페놀 함량을 나타내었으며, ethyl acetate 분획물은 약 14.09 ± 0.54 ㎎/g의 총플라보노이드 함량을 나타내었으며, n-hexan 분획물은 21.62 ± 0.59 ㎎/g의 총플라보노이드 함량을 나타내었다.
As shown in Table 3, the total phenol content of the herbal extract was 1.19 ± 0.03 mg / g, and the total phenol content of the methanol extract was about 1.19 ± 0.01 mg / g. The chloroform fraction showed a total phenolic content of 1.05 ± 0.01 mg / g, the ethyl acetate fraction had a total phenolic content of about 1.17 ± 0.01 mg / g, and the n-hexan fraction had the highest total content of 1.32 ± 0.01 mg / g. Total phenolic content is shown. The total flavonoid content of the herbal extract was 27.87 ± 1.23 mg / g, and the total flavonoid content of the methanol extract was about 32.80 ± 1.07 mg / g. The chloroform fraction showed a total phenolic content of 30.69 ± 0.72 mg / g, the ethyl acetate fraction had a total flavonoid content of about 14.09 ± 0.54 mg / g, and the n-hexan fraction had a total flavonoid of 21.62 ± 0.59 mg / g. Content is indicated.
<5-3> Superoxide dismutase(SOD) 유사 활성 측정<5-3> Superoxide dismutase (SOD) -like activity measurement
SOD는 superoxide를 정상 상태의 산소로 환원시킴으로서 superoxide가 관여하는 암 등의 각종 질병이나 노화를 억제할 수 있는 효소로 알려져 있다. SOD 유사활성 반응에서 pyrogallol은 물에 존재하는 superoxide radical에 의해 자동 산화가 일어나 갈색 물질을 형성하여 이를 분광광도계로 분석하고, superoxide 포착활성이 있는 물질이 존재시 pyrogallol의 산화속도가 낮아지는 원리를 이용하여 superoxide 포착활성을 간접적으로 측정할 수 있다. SOD 유사활성은 Markund 등의 방법에 따라 측정하였다. 각 분획의 생약 추출물(메탄올 추출물, 클로로포름 분획, 에탄올 분획 및 헥산 분획) 0.2 ㎖(1 ㎎/㎖ 농도)에 pH 8.5로 보정한 Tris-HCl buffer(50 mM tris amino-methane과 10 mM EDTA) 2.6 ㎖과 7.2 mM pyrogallol 0.2 ㎖을 첨가하여 25℃에서 20분간 반응시킨 후 1 N HCl을 0.1 ㎖ 가하여 반응을 정지시켰다. 반응액 중 산화된 pyrogallol의 양은 420 ㎚에서 흡광도를 측정하였다. SOD 유사활성 측정은 하기 수학식 2로 계산하였으며, 그 결과를 도 7에 나타냈다.SOD is known as an enzyme that can suppress aging by reducing superoxide to normal oxygen and various diseases such as cancer involving superoxide. In the SOD-like activity, pyrogallol is automatically oxidized by superoxide radicals in water to form a brown substance, which is analyzed by spectrophotometer. Superoxide capture activity can be measured indirectly. SOD-like activity was measured according to Markund et al. Tris-HCl buffer (50 mM tris amino-methane and 10 mM EDTA) adjusted to pH 8.5 in 0.2 ml (1 mg / ml concentration) of herbal extracts (methanol extract, chloroform fraction, ethanol fraction and hexane fraction) of each fraction 2.6 After the reaction was carried out at 25 ° C. for 20 minutes by adding ㎖ and 0.2 ml of 7.2 mM pyrogallol, 0.1 ml of 1 N HCl was added to stop the reaction. The amount of oxidized pyrogallol in the reaction solution was measured for absorbance at 420 nm. SOD-like activity measurement was calculated by the
[수학식 2]&Quot; (2) "
SOD-like activity (%) = {1-(시료 처리구 흡광도 / 무처리구 흡광도)} x 100
SOD-like activity (%) = {1- (Sample Absorbance / Untreated Absorbance)} x 100
도 7에서 보듯이, 메탄올 추출물의 SOD 유사활성은 약 20.93 ± 0.91%로 관찰되었고, chloroform 분획물은 7.67 ± 0.31%의 SOD 유사활성을 나타내었다. Ethyl acetate 분획물은 약 26.52 ± 0.61%로 SOD 유사활성을 나타내었으며, n-hexan 분획물은 26.52 ± 3.15%로 SOD 유사활성을 나타내었다. As shown in Figure 7, the SOD-like activity of the methanol extract was observed to be about 20.93 ± 0.91%, the chloroform fraction showed a SOD-like activity of 7.67 ± 0.31%. Ethyl acetate fraction showed SOD-like activity at 26.52 ± 0.61% and n-hexan fraction showed SOD-like activity at 26.52 ± 3.15%.
이러한 결과는 생약 추출물의 SOD 유사활성이 비교적 높다고 할 수 있으며, 특히 ethyl acetate 층과 n-hexan 층의 SOD 유사활성이 높은 것으로 나타났다. 그러나, chloroform 분획층은 SOD 유사활성이 다소 떨어짐을 알 수 있었다.
These results indicate that the SOD-like activity of the herbal extracts is relatively high, especially the SOD-like activity of the ethyl acetate layer and the n-hexan layer is high. However, the chloroform fractionation layer was found to be slightly degraded SOD-like activity.
<실시예 6> 생약 추출물이 간암 유도된 랫트의 간조직에 미치는 영향 평가Example 6 Evaluation of Effects of Herbal Extracts on Liver Tissues of Rats Induced by Liver Cancer
상기 생약 추출물이 상기 실시예 <4-3>에서 준비된 랫트를 DEN으로 자극하여 간암을 유발시킨 후, 간조직에 미치는 영향을 알아보았다.The herbal extract was stimulated with DEN in the rat prepared in Example <4-3> to induce liver cancer, and then examined the effect on liver tissue.
구체적으로 간암 유도는 다음과 같이 수행하였다. 실험군은 난괴법에 의해 모두 5마리씩 3군으로 정상군(group Ⅰ), 대조군(DEN 주입, group Ⅱ), 식이섭취 실험군(DEN+항암공진단 메탄올 추출물, group Ⅲ)으로 나누어 실험하였다. 1주일간 예비사육한 후 대조군에 12주 동안 DEN 150ppm을 음수 형태로 투여하였다. 실험군(group Ⅲ)은 통상적인 사료와 DEN 150ppm + 100 ㎎/㎏의 항암공진단 메탄올 추출물을 용해하여 음수로 공급하였다.Specifically, induction of liver cancer was performed as follows. The experimental group was divided into three groups of five by the ingot method, divided into normal group (group I), control group (DEN injection, group II), and dietary intake experimental group (DEN + anticancer diagnostic methanol extract, group III). After preliminary breeding for 1 week, the control group was administered with 150 ppm of DEN in negative form for 12 weeks. The experimental group (group III) dissolved the conventional feed and ethanol extract of anticancer resonance diagnostic drug (150 ppm + 100 mg / kg) and supplied it as a negative water.
상기 각 군의 5마리 랫트로부터 전체 간을 얻은 후, 문헌[Histology a text and Atlas. Michael H. Ross, Wojciech Pawlina. 김진정 외 5인. 원색 조직학도보. 고려의학]에 기재된 방법을 참고로 하여 간조직의 형태를 조사하였다. 구체적으로, 수득한 간 조직을 NBF 용액으로 고정한 후 파라핀 포매 과정을 거쳐 마이크로톰으로 5㎛ 두께로 조직 절편을 제작한 다음, 헤마톡실린&에오신(H-E) 염색을 수행하였다. 100 필드(field) 이상을 갖는 표본을 이용하여 간 조직의 세포 형태를 현미경으로 관찰한 후 그 결과를 도 8에 나타내었다.
After obtaining whole liver from the five rats of each group, Histology a text and Atlas. Michael H. Ross, Wojciech Pawlina. Kim Jin-jung and five others. Color histology walk. The shape of liver tissue was examined by referring to the method described in [Korea Medicine]. Specifically, the obtained liver tissue was fixed with NBF solution, and then, paraffin embedding was performed to prepare tissue sections of 5 μm thickness using a microtome, and then hematoxylin & eosin (HE) staining was performed. After observing the cell morphology of liver tissue under a microscope using a specimen having more than 100 fields (micrometers), the results are shown in FIG.
도 8에서 보듯이, 적출한 간 조직을 광학현미경으로 관찰한 결과 정상군(Group Ⅰ)의 간 조직은 뚜렷한 둥근 핵을 갖고 있었고 세포질은 일정하게 eosin으로 염색되었으며, 간소엽의 구조도 잘 배열되어 있다.As shown in FIG. 8, the extracted liver tissues were observed under an optical microscope, and the liver tissues of the normal group (Group I) had distinct round nuclei, and the cytoplasm was uniformly stained with eosin. have.
NDEN으로 간암을 유발한 대조군(Group Ⅱ)의 간 조직은 정상군(Group Ⅰ)과 실험군(Group Ⅲ)에 비하여 간소엽의 구조가 일정하지 못하며 세포질의 공포화, 공포의 형성이 정상군(Group Ⅰ)과 실험군(Group Ⅲ)에 비하여 많이 형성되어 있다. 참고로, 기존 연구인 Mourao 등, Yoon J.S. 등의 NDEN로 유도된 간암에 대한 실험에서 NDEN만을 투여한 군에서 간 손상의 가장 두드러진 특징은 정상대조군과 비교하여 간세포의 팽대성 증가와 세포질 공포화 그리고 공포 형성의 유도라고 하였는데, 이는 본 실험의 결과와 일치하였다. The liver tissue of the control group (Group II), which induced liver cancer with NDEN, has a less uniform structure of hepatic lobules than the normal group (Group I) and the experimental group (Group III). It is more formed than I) and experimental group (Group III). For reference, Mourao et al., Yoon J.S. In the study of NDEN-induced liver cancer, the most prominent feature of liver injury in the NDEN-only group was the increase in hepatocellular swelling, cytoplasmic fear, and fear formation compared to the normal control group. Consistent with the results.
400배로 찍은 대조군(Group Ⅱ)의 파란색 원안에 표시되어 있는 세포는 정상군(Group Ⅰ)과 실험군(Group Ⅲ)에서는 보이지 않는 특이형태의 세포가 발견되었다. 공진단을 섭취한 실험군(Group Ⅲ)에서는 간소엽의 구조가 비교적 잘 유지되고 있었고, 핵 형태 또한 비교적 뚜렷했다. 실험군(Group Ⅲ)에서 세포질의 공포화 현상은 거의 관찰되지 않았으며, 대조군(Group Ⅱ)에서와 같은 특이형태의 세포 또한 관찰되지 않았다.
The cells shown in the blue circle of the control group (Group II) taken at 400 times were found to have a specific type of cells not seen in the normal group (Group I) and the experimental group (Group III). In the experimental group fed the resonant stage (Group III), the structure of hepatic lobe was relatively well maintained, and the nucleus shape was relatively clear. Almost no cytophobia was observed in the experimental group (Group III), and no specific cell types were observed as in the control group (Group II).
<실시예 7> 생약 추출물이 간암 유도된 랫트의 혈청 내 LDH, AST, ALT, ALP 활성에 미치는 영향 평가Example 7 Evaluation of Effects of Herbal Extracts on LDH, AST, ALT, and ALP Activities in Serum of Liver Cancer Induced Rats
상기 생약 추출물이 상기 실시예 <4-3>에서 준비된 랫트를 DEN으로 자극하여 간암을 유발시킨 후, 혈청 내 LDH(lactate dehydrogenase), AST(Aspartate aminotransferase)와 ALT(alanine aminotransferase) 및 ALP(alkaline phosphatase) 활성을 평가하였다. The herbal extract stimulates rats prepared in Example <4-3> with DEN to cause liver cancer, and then lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in serum. ) Activity was evaluated.
LDH는 비특이적 마커로서 장기의 세포 파괴시 혈중 농도가 증가하므로 주로 악성 종양의 존재여부를 추정할 때 이용된다.LDH is a nonspecific marker, which is used mainly to estimate the presence of malignant tumors because the concentration of blood increases during cell destruction of organs.
AST 및 ALT는 심장근과 간세포 내에 있는 특정효소로서 조직세포의 손상에 의해 혈청 중의 활성도가 증가되며, 이러한 활성도의 상승은 간담도 질환, 심장질환에서 일어나고 특히 만성간염의 경우에는 현저하게 증가된다.AST and ALT are specific enzymes in cardiomyocytes and hepatocytes, and the activity in serum is increased by damage to tissue cells. This increase in activity occurs in hepatobiliary disease and heart disease, especially in chronic hepatitis.
ALP는 만성간염이나 간경변증에서도 그 수치가 올라갈 수 있으나, 만들어진 담즙이 간 세포에서 잘 배출되지 못하거나 담도가 막혔을 때에도 현저히 증가되는 것으로 알려져 있다.
ALP can be elevated in chronic hepatitis and cirrhosis, but it is known that bile produced is significantly increased when bile is not well excreted from liver cells or blocked.
먼저, 간암 유도는 구체적으로 상기 실시예 6의 방법과 같이 수행하였다. 실험군은 난괴법에 의해 모두 5마리씩 3군으로 정상군(group Ⅰ), 대조군(DEN 주입, group Ⅱ), 식이섭취 실험군(DEN+항암공진단 메탄올 추출물, group Ⅲ)으로 나누어 실험하였다. 1주일간 예비사육한 후 대조군에 12주 동안 DEN 150ppm을 음수형태로 투여하였다. 식이섭취군(group Ⅲ)은 통상적인 사료와 DEN 150ppm + 100 ㎎/㎏의 GJD을 용해하여 음수로 공급하였다.First, induction of liver cancer was specifically performed in the same manner as in Example 6. The experimental group was divided into three groups of five by the ingot method, divided into normal group (group I), control group (DEN injection, group II), and dietary intake experimental group (DEN + anticancer diagnostic methanol extract, group III). After preliminary breeding for 1 week, the control group was administered with 150 ppm of DEN in negative form for 12 weeks. The dietary group (Group III) dissolved conventional feed and DEN 150ppm + 100 mg / kg of GJD and supplied it with negative water.
약물 투여 12주 후 랫트를 urethane으로 마취한 후 다음 복부의 정중선을 따라 절개하고 상행정맥채혈을 시행하였다. 혈액은 시험관에 넣어 실온에서 30분간 방치하여 혈액을 응고시킨 다음, 3000 rpm에서 원심분리하고 상등액을 회수하여 -80℃ 초저온 냉동고에 보관하면서 실험에 사용하였다.
After 12 weeks of drug administration, rats were anesthetized with urethane, and then dissected along the midline of the abdomen and ascended intravenous bleeding. The blood was placed in a test tube and allowed to stand at room temperature for 30 minutes to coagulate the blood, and then centrifuged at 3000 rpm, and the supernatant was collected and stored in a -80 ° C. cryogenic freezer for use in experiments.
<7-1> LDH 활성 영향 측정<7-1> LDH activity influence measurement
상기에서 얻은 혈청을 대상으로 LDH 활성도를 측정하였으며, LDH 활성도는 자동분석기용 kit 시약(동성제약)을 사용하여 측정하였으며, 그 결과를 도 9에 나타내었다.The serum obtained from the above was measured for LDH activity, and LDH activity was measured using a kit reagent for autoanalyzer (Dong-Pharma), and the results are shown in FIG. 9.
그 결과, 도 9에서 보듯이, LDH의 활성은 정상군에 비하여 대조군에서 유의있는 증가가 관찰되었으며, 생약 추출물을 동시투여한 실험군에서는 대조군에 비하여 유의하게 감소하는 결과가 관찰되었다.
As a result, as shown in Fig. 9, the LDH activity was significantly increased in the control group compared to the normal group, and in the experimental group coadministered with the herbal extract was significantly reduced compared to the control group was observed.
<7-2> AST 및 ALT 활성 영향 측정<7-2> AST and ALT Activity Impact Measurement
상기에서 얻은 혈청을 대상으로 AST 및 ALT 활성도를 측정하였으며, AST 및 ALT 활성도는 자동분석기용 kit 시약(동성제약)을 사용하여 측정하였으며, 그 결과를 도 10 및 도 11에 나타내었다.AST and ALT activity was measured in the serum obtained above, AST and ALT activity was measured using a kit reagent (Dong-Pharma Pharmaceutical) for the automatic analyzer, the results are shown in Figures 10 and 11.
그 결과, 도 10 및 도 11에서 보듯이, AST 및 ALT 활성은 정상군에 비하여 대조군에서 유의하게 증가되는 것이 관찰되었으며, 생약 추출물을 동시투여한 실험군에서는 대조군에 비하여 유의하게 감소하는 결과가 관찰되었다.
As a result, as shown in Figures 10 and 11, it was observed that the AST and ALT activity was significantly increased in the control group compared to the normal group, and in the experimental group coadministered with the herbal extract was significantly reduced compared to the control group was observed. .
<7-3> ALP 활성 영향 측정<7-3> ALP activity influence measurement
상기에서 얻은 혈청을 대상으로 ALP 활성도를 측정하였으며, ALP 활성도는 자동분석기용 kit 시약(동성제약)을 사용하여 측정하였으며, 그 결과를 도 12에 나타내었다.Serum obtained above was measured for ALP activity, and ALP activity was measured using an automatic analyzer kit reagent (Dong-Pharma). The results are shown in FIG.
그 결과, 도 12에서 보듯이, ALP 활성은 정상군에 비하여 대조군에서 증가되는 것이 관찰되었으며, 생약 추출물을 동시투여한 실험군에서는 대조군에 비하여 유의하게 감소하는 결과가 관찰되었다.
As a result, as shown in Figure 12, the ALP activity was observed to be increased in the control group compared to the normal group, and in the experimental group coadministered with the herbal extract was observed to be significantly reduced compared to the control group.
따라서, 종합적으로 본 발명의 생약 추출물은 산화적 스트레스로 인한 간암 유도 모델에서 간세포 손상을 유의적으로 억제하는 것을 확인할 수 있다.Therefore, it is confirmed that the herbal extract of the present invention significantly inhibits hepatocyte damage in a model of inducing liver cancer due to oxidative stress.
Claims (9)
Pharmaceutical composition for liver cancer prevention or treatment comprising red ginseng, Angelica, Schisandra chinensis, Tosa and turmeric extract as active ingredients.
According to claim 1, wherein the composition is a liver cancer preventive or therapeutic pharmaceutical composition, characterized in that it further comprises gojija, cornus and bokbunja extract.
According to claim 1, wherein the composition is liver cancer, characterized in that it comprises 100 to 500 parts by weight of red ginseng, 50 to 200 parts by weight, Schisandra chinensis 20 to 200 parts by weight, turmeric 20 to 200 parts by weight based on 100 parts by weight of Angelica Prophylactic or therapeutic pharmaceutical compositions.
According to claim 2, wherein the composition is based on 100 parts by weight of red ginseng, 100 to 500 parts by weight, Schisandra chinensis 50 to 200 parts by weight, earth and sand 20 to 200 parts by weight, turmeric 20 to 200 parts by weight, wolfberry 100 to 500 parts by weight , Cornus oil 50 ~ 200 parts by weight, bokbunja 50 to 200 parts by weight of liver cancer prevention or treatment pharmaceutical composition comprising a.
The pharmaceutical composition for preventing or treating liver cancer according to any one of claims 1 to 4, wherein the composition has antioxidant activity.
The extract according to any one of claims 1 to 4, wherein the extract is water, lower alcohol having 1-4 carbon atoms, acetone, ether, ethyl acetate, butyl acetate, chloroform, methylene chloride, hexane, 1,3 butylene glycol And a pharmaceutical composition for preventing or treating liver cancer, characterized in that extracted with a solvent selected from the group consisting of a mixed solvent thereof.
The composition according to any one of claims 1 to 4, wherein the composition is hardwood, cheonma, shiho, indoin, gyeji, rhubarb, licorice, celestial organ, dermis, taxa, yellow lotus, golden, biryeong, peony, white peach, yellow white, gardenia , Half, light bulb, fruit room, ginseng, macmundong, original ground, Seokchangpo, creation, country, windproof, ginger, forget-me-not, contrast, sweet ginseng, bark skin, huangwang, mint, pesticides, spirits, sewao, guja, faulty, poisonous, tofu, baekhwa In addition to one or more herbal extracts selected from the group consisting of private grass, three hundred seconds, injin, jin, safflower, yellow, lime, ginkgo biloba, hwangjeong, lotus root, keel, phalanges, dew, sage, black sesame, white porcelain, malt, pagokcheon Pharmaceutical composition for preventing or treating liver cancer, characterized in that it comprises.
The pharmaceutical composition for preventing or treating liver cancer according to any one of claims 1 to 4, wherein the composition is for producing an anticancer resonance stage.
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