KR100508403B1 - An Extract having antiaging activity - Google Patents
An Extract having antiaging activity Download PDFInfo
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- KR100508403B1 KR100508403B1 KR10-2005-0034632A KR20050034632A KR100508403B1 KR 100508403 B1 KR100508403 B1 KR 100508403B1 KR 20050034632 A KR20050034632 A KR 20050034632A KR 100508403 B1 KR100508403 B1 KR 100508403B1
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Abstract
본 발명은 한약재 추출물에 관한 것으로 보다 상세히는 와송(바위솔; Orostachys japonicus), 지유(오이풀; Sanguisorba officinalis), 금은화(Lonicera japonica), 귀전우(화살나무; Winged spindle), 삼칠근(Notoginserg Radix), 백지(구릿대; Angelica dahurica), 마치현(쇠비름; Portulaca oleracea), 우피두견(만병초; Rhododendron brachycarpum), 지구목(헛개나무; Hovenia dulcis), 유근피(드릅나무; Ulmus Macrocarpa Hance)로 구성된 한약재를 열수추출한 항종양활성, 간질환억제활성, 노화방지 및 면역증강활성을 갖는 한약재 추출물에 관한 것이다.The present invention relates to the herbal extracts, and more specifically, hawthorn (rocksol; Orostachys japonicus), fat milk (cucumber; Sanguisorba officinalis), Lonicera japonica, Winged spindle, Winged spindle, Notoginserg Radix, Extracted Chinese herbal medicine consisting of white paper (holders; Angelica dahurica), portulaca (Portulaca oleracea), cow skin dogs (Ranoddron); The present invention relates to an herbal extract having antitumor activity, liver disease inhibitory activity, anti-aging and immune enhancing activity.
Description
본 발명은 한약재 추출물에 관한 것으로 보다 상세히는 와송(바위솔; Orostachys japonicus), 지유(오이풀; Sanguisorba officinalis), 금은화(Lonicera japonica), 귀전우(화살나무; Winged spindle), 삼칠근(Notoginserg Radix), 백지(구릿대; Angelica dahurica), 마치현(쇠비름; Portulaca oleracea), 우피두견(만병초; Rhododendron brachycarpum), 지구목(헛개나무; Hovenia dulcis), 유근피(드릅나무; Ulmus Macrocarpa Hance)로 구성된 한약재인 코리원(상표)을 열수추출한 항종양활성, 간질환억제활성, 항산화성 및 면역증강활성을 갖는 한약재 추출물에 관한 것이다.The present invention relates to the herbal extracts, and more specifically, hawthorn (rocksol; Orostachys japonicus), fat milk (cucumber; Sanguisorba officinalis), Lonicera japonica, Winged spindle, Winged spindle, Notoginserg Radix, Corriwon, a herb that consists of white paper (holders; Angelica dahurica), machi (purslane; Portulaca oleracea), cow skin dogs (Rhododendron brachycarpum), terrestrial tree (Holberry; Hovenia dulcis), and root roots (Hulm Macrocarpa Hance) It relates to herbal extracts having anti-tumor activity, hepatic disease inhibitory activity, antioxidant activity and immuno-enhancing activity from hot water extracted.
암의 전이(metastasis)는 초기종양에서 암세포가 신체의 다른 부위로 전파되는 과정으로 악성 종양의 가장 큰 특징이라 할 수 있다. 최근 암 연구보고에서 암전이로 인한 사망은 암 관련환자 사망의 약 90%를 차지하며, 바로 암의 전이가 암환자 사망의 중요한 원인임을 확인하였다. 암의 전이는 암세포와 암세포 주위와의 복잡한 상호작용으로 발생한다. 암세포 주위는 암세포가 정상적으로는 통과할 수 없는 지지구조체인 세포외 기질(extracellular matrix, ECM)과 기저막(basement membrane)으로 되어 있다. 암세포는 이러한 세포외 장벽을 극복하기 위하여 ECM과 기저막을 분해할 수 있는 다양한 종류의 단백질 분해효소(proteinase)를 생산ㆍ분비하는데 그 중에서도 MMP(matrix metalloproteinase)가 중요한 역할을 담당한다. 정상적인 생리환경에서 MMP는 TIMP(tissue inhibitors of metalloproteinases)라는 내생 MMP 저해제에 의하여 엄격하게 조절된다. 그러나 암의 증식, 침윤 및 전이과정 중에는 MMP와 TIMP 간의 평형이 무너지고 과다한 MMP가 분비된다. Metastasis of cancer is the most characteristic of malignant tumors in the process of spreading cancer cells to other parts of the body in early tumors. Recent cancer research reports indicate that cancer-related deaths account for about 90% of cancer-related deaths, and cancer metastasis is an important cause of cancer deaths. Cancer metastasis results from complex interactions between cancer cells and their surroundings. Around the cancer cells is an extracellular matrix (ECM) and basement membrane, which are support structures that cancer cells cannot normally pass through. In order to overcome these extracellular barriers, cancer cells produce and secrete various kinds of proteinases capable of decomposing ECM and the basement membrane. Among them, matrix metalloproteinase (MMP) plays an important role. Under normal physiological conditions, MMPs are tightly regulated by endogenous MMP inhibitors called tissue inhibitors of metalloproteinases (TIMPs). However, during cancer proliferation, infiltration and metastasis, the balance between MMP and TIMP is broken and excess MMP is secreted.
지금까지 보고된 바에 의하면 타입 IV 콜라게나제 또는 젤라티나제 (collagenase or gelatinaes)인 MMP-2와 MMP-9가 암의 전이와 밀접한 관련이 있다고 알여졌다. 암 전이에 가장 큰 장벽인 ECM과 기저막의 주된 성분은 타입 IV 콜라겐인데, MMP-2와 MMP-9는 촉매적 도메인(catalytic domain) 내에 젤라틴과 콜라겐에 대해 친화성이 있는 유사 피므로넥틴 도메인(fibronectin-like domain)을 포함하고 있으며, 타입 IV 콜라겐에 대하여 기질 특이성을 갖고 있다. 뿐만 아니라 인간 암세포주 배양과 실험 동물모델에서 MMP-2와 MMP-9가 암의 전이와 연관되었다고 입증되었음으로, 이를 분비하는 세포주를 이용하여 MMP 저해제를 탐색하는 전략이 효과적이다.It has been reported that MMP-2 and MMP-9, type IV collagenase or gelatinaes, are closely related to cancer metastasis. The major components of ECM and basement membranes, which are the biggest barriers to cancer metastasis, are type IV collagen, and MMP-2 and MMP-9 are similar pyrinenectin domains with affinity for gelatin and collagen in the catalytic domain. fibronectin-like domain) and has substrate specificity for type IV collagen. In addition, MMP-2 and MMP-9 have been shown to be involved in metastasis of cancer in human cancer cell line cultures and experimental animal models.
타입 IV 콜라게나제를 분비하는 세포주 탐색을 위해 HT1080, C32TG, HepG2 T98G, MIA-PaCa2, UAcc62, U937, G361 등 다양한 종양 세포주를 이용하여 무 혈청 배지에 TPA와 TGF- 1등의 첨가에 따른 타입 IV 콜라게나제의 유도정도를 확인한다. 젤라티나제의 효소 활성측량법으로는 방사능 동위원소가 표지된 젤라틴(3H-acetylated gelatin)이나 광합성 펩타이드(fluorogenic MMP-2/MMP-9 sustrate, DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg-OH)를 이용하여 분해산물의 방사능량이나 형광량을 측량하는 방법과, 활성 단백질 전기영동법을 이용한 젤라틴 자이모그라피 (gelatin zymography)가 있다. 이들 중 젤라틴 자이모그라피가 비교적 간편하고, 적은 시료량으로 효소활성을 측량이 가능하다. 또한 대략적인 분자량을 알 수 있기 때문에 분비되는 MMP를 동정할 수 있음으로 편리하다.In order to detect cell lines secreting type IV collagenase, various tumor cell lines such as HT1080, C32TG, HepG2 T98G, MIA-PaCa2, UAcc62, U937, G361 were used. Check the degree of induction of IV collagenase. Enzymatic activity measurement of gelatinases involves radiolabeled gelatin (3H-acetylated gelatin) or photosynthetic peptides (fluorogenic MMP-2 / MMP-9 sustrate, DNP-Pro-Leu-Gly-Met-Trp-Ser-). Arg-OH) is used to measure the radioactivity or fluorescence of the degradation products, and gelatin zymography using active protein electrophoresis. Among them, gelatin zymography is relatively simple, and enzyme activity can be measured with a small sample amount. In addition, since the approximate molecular weight is known, it is convenient to identify the secreted MMP.
따라서, MMP 저해제는 암의 침윤과 전이를 효과적으로 막을 수 있는 새로운 표적으로 기대되어 많은 암 연구자들이 새로운 개념의 항암 치료제로서 MMP 저해제 개발에 연구를 집중하고 있다. 현재 MMP에 대한 저분자의 선택적 저해제 탐색 및 합성 디자인의 연구가 활발히 진행되고 있으며, 화학적으로 합성된 저해제와 천연물로부터 분리된 저해제의 전임상 또는 임상실험의 결과는 MMP 저해제가 암의 치료에 있어서 새롭고 유망한 치료제가 될 수 있음을 보여주고 있다. Therefore, MMP inhibitors are expected to be new targets to effectively prevent cancer invasion and metastasis, and many cancer researchers are focusing on developing MMP inhibitors as a new anticancer drug. There is an active search for a low molecular weight selective inhibitor for MMP and the study of synthetic design. The results of preclinical or clinical trials of chemically synthesized inhibitors and inhibitors isolated from natural products show that MMP inhibitors are new and promising therapeutic agents in the treatment of cancer. It can be shown.
그러나, 기존의 펩타이드 구조의 부분적인 변형에 의한 합성 저해제는 제한된 생체이용성(bioavailabilty), 짧은 반감기(half-life), 효소 선택성의 결여 및 수용액에서 낮은 용해도 등의 적지 않은 문제점들이 있다. MMP의 중요한 생리적 활성에 비추어 볼 때 특정 MMP에 대해 선택성이 떨어지는 강력한 저해제의 지속적인 투여는 심각한 부작용을 야기할 것으로 예상할 수 있다. 식물이나 미생물 등의 천연물로부터 몇 종류의 MMP 저해제가 탐색되어 개발과정 중에 있지만 효소 저해활성의 빈약함과, 강한 세포독성 때문에 치료약으로 개발되기까지는 많은 문제점이 있다.However, synthetic inhibitors by partial modification of existing peptide structures have a number of problems such as limited bioavailabilty, short half-life, lack of enzyme selectivity and low solubility in aqueous solutions. In view of the important physiological activity of MMPs, continuous administration of potent inhibitors that are less selective for certain MMPs can be expected to cause serious side effects. Several kinds of MMP inhibitors have been discovered from natural products such as plants and microorganisms, and there are many problems until the development of therapeutic drugs due to the poor enzyme inhibitory activity and strong cytotoxicity.
따라서, 기존의 MMP 저해제들의 이러한 문제점들을 극복하기 위한 방법으로 민간에서 임상효능이 입증된 한약재로부터 MMP 저해제를 탐색하여 치료약으로 개발하는 방법을 들수 있다. 한방에서 오랜 경험에 의하여 암 치료에 사용되어온 많은 약재들을 몇몇 암 연구가들이 항암제 개발에 사용하여 상당한 진전을 보였다. 지금까지 개발된 항암제 중에서 가장 우수한 것으로 평가받고 있으며 난소암에 특히 효능이 높은 탁솔(taxol)은 실제로 중국에서 많이 사용하고 있는 항암성 약재인 주목(Taxus류)으로부터 분리되며, DNA 토포이소머라제(topoisomerase)-I 저해제인 캄프토세신(camptothecin)류는 희수(Camptotheca acuminata)로부터 그리고, 빈크리스틴(vincristine), 빈블라스틴(vinblastine) 등은 장춘화(Catharanthus roseus)에서 분리되어 사용하고 있다. 그러나, 이들은 암세포의 분화와 증식억제라는 측면에서 탐색, 개발되었기 때문에 강한 임상독성을 갖고 있어 치료 중 상당한 부작용을 낳아 장기적인 암치료를 요하는 경우에는 적용이 불가능하다.Therefore, as a method for overcoming these problems of the existing MMP inhibitors, there is a method of searching for MMP inhibitors and developing them as therapeutic drugs from herbal medicines that have been clinically proven in the private sector. Many of the medicines that have been used to treat cancer with long experience in oriental medicine have been used by some cancer researchers to develop anticancer drugs. Taxol, which has been evaluated as the best among anticancer drugs developed so far and is particularly effective in ovarian cancer, is separated from taxus , an anticancer drug that is widely used in China, and DNA topoisomerase ( Camptothecins, which are topoisomerase-I inhibitors, are isolated from Camptotheca acuminata and vincristine and vinblastine from Catharanthus roseus . However, since they have been explored and developed in terms of differentiation and proliferation of cancer cells, they have strong clinical toxicity and are not applicable to long-term cancer treatment due to significant side effects during treatment.
지금까지 한방 생약재에서 암의 침윤, 전이, 암세포의 혈관신생 등을 방지하여 세포증식을 억제하는 세포독성 요법(cytostatic therapy) 개념의 치료제 개발을 위한 연구는 거의 없다. 이러한 맥락에서 앞으로 한약재로부터 생화학적 접근에 의한 MMP 저해제의 개발은 새로운 항암치료제 개발의 본보기가 될 수 있다.Until now, there are few studies for developing a therapeutic agent of cytostatic therapy that inhibits cell proliferation by preventing cancer invasion, metastasis and neovascularization of cancer cells in herbal medicines. In this context, the development of MMP inhibitors by a biochemical approach from herbal medicines could be an example of the development of new anticancer drugs.
일반적으로 대부분의 한방 생약재들은 건조된 상태로 보관 유통되고 있음으로 저장기간 중에 바이러스, 세균 곰팡이 등에 감염되었는지 유의하여야한다. 또한 다른 재료들이 유입되지 않았는지 유의하여야 하며, 특별히 고려해야할 점은 한약재료의 정확한 감정을 필요로 한다. 건조된 식물재료를 추출할 때는 적당한 크기로 자른 다음, 막자사발, 분쇄기 등을 이용하여 완전히 분말로 하여 유기용매와 열수추출을 한다. 건조된 재료는 먼저 메탄올이나 에탄올로 추출하는 것이 좋다. 알코올은 세포막을 파괴함으로 세포내의 성분을 추출할 수 있으나 에테르 또는 삼염화탄소와 같은 비극성 용매로는 세포내 성분을 용출시킬 수 없다. In general, most herbal medicines are stored and distributed in a dry state, so it should be noted that they are infected with viruses, bacterial fungi, etc. during the storage period. It is also important to note that no other ingredients have been introduced, and special considerations require accurate appraisal of herbal ingredients. When extracting the dried plant material, cut it to a suitable size, and then, using a mortar and pestle, grinder, etc. to completely powder the organic solvent and hot water extraction. The dried material is first extracted with methanol or ethanol. Alcohol can extract the components in the cell by breaking the cell membrane, but the intracellular components cannot be eluted with a nonpolar solvent such as ether or carbon trichloride.
최근, 대나무잎에서 추출한 다당류는 사르코마-180 (Sarcoma-180)이 이식된 생쥐의 복강암에 효과적인데 [Ikekawa, T.등 (1968) Gann 59: 155-157], 이러한 종양억제 약리활성은 간접적인 숙주를 매개로 한 면역증강효과에 기인하며, 특히 담자균류 세포벽에 존재하는 다당류의 활성이 중요한 것으로 알려져 있다 [Tanaka, T. 등 (1965) Gann, 56: 529-536; Hamuro, J. 등 (1978) Cancer Res., 38: 3080-3085; Maeda, Y. 및 Chihara, G., (1971) Nature, 229: 634-635; Komatsu, N. 등 (1969) Gann. 60: 137-144; Oh, G. T. 등 (1992) Arch. Pharm. Res., 15: 379-381].Recently, polysaccharides extracted from bamboo leaves are effective for celiac cancer in mice transplanted with Sarcoma-180 [Ikekawa, T. et al. (1968) Gann 59: 155-157]. Indirect host-mediated immunity-enhancing effects, in particular the activity of polysaccharides present in basidiomycete cell walls are known to be important [Tanaka, T. et al. (1965) Gann, 56: 529-536; Hamuro, J. et al. (1978) Cancer Res., 38: 3080-3085; Maeda, Y. and Chihara, G., (1971) Nature, 229: 634-635; Komatsu, N. et al. (1969) Gann. 60: 137-144; Oh, G. T. et al. (1992) Arch. Pharm. Res., 15: 379-381.
한방약제의 다른 약리활성에는 면역증강, 항응고활성 및 소화기계활성이 알려져 있다 [Wagner, H. (1989) Planta Medica 55: 235-241; Wagner, H. (1990) Pure Appl. Chem. 62: 1217-1222.; Hodges, L.C. 등 (985) Carbohyd. Polym. 5: 141-154; Kiyohara, H. 등 (1996) Planta Medica 62: 14-19]. 또한, Sargassum f㎕vellum의 알긴산, Sargassum kjellmanianum의 황화 갈락토퓨칸 (s㎕fated galactofucan) 등은 숙주를 매개로 한 항종양활성을 가지고 있으며 [Yamada, H. 등 (1987) Agric. Biol. Chem. 51, 785-790; Shimizu, S. 등 (1988) Biochem. Biophys. Res. Comm. 150, 335-341], Porphyra yezoensis의 다당류는 시험관내 및 생체내 탐식세포의 활성화 활성을 가지며 [Shimizu, S. 등 (1989) J. Am. Oil. Chem. Soc., 66, 237-241], Gracilaria verrucosa 다당류는 생쥐의 탐식활성을 증대시킨다 [Shinmen, Y. 등, (1989) Appl. Microbiol. Biotechnol. 31, 11-16].Other pharmacological activities of herbal medicines are known to be immune enhancing, anticoagulant and digestive system [Wagner, H. (1989) Planta Medica 55: 235-241; Wagner, H. (1990) Pure Appl. Chem. 62: 1217-1222; Hodges, L.C. Et al. (985) Carbohyd. Polym. 5: 141-154; Kiyohara, H. et al. (1996) Planta Medica 62: 14-19]. In addition, alginic acid of Sargassum fulvellum and sulfulated galactofucan of Sargassum kjellmanianum have anti-tumor activity through the host [Yamada, H. et al. (1987) Agric. Biol. Chem. 51, 785-790; Shimizu, S. et al. (1988) Biochem. Biophys. Res. Comm. 150, 335-341], Porphyra yezoensis polysaccharides have activating activity of phagocytic cells in vitro and in vivo [Shimizu, S. et al. (1989) J. Am. Oil. Chem. Soc., 66, 237-241], Gracilaria verrucosa polysaccharides enhance the phagocytic activity of mice [Shinmen, Y. et al., (1989) Appl. Microbiol. Biotechnol. 31, 11-16.
또한, 약리활성을 나타내는 식물계 황화다당류의 혈관내의 지방 제거활성과 항응고(anticoagulant) 활성은 이미 알려진 바 있으며 [Nakagawa, K. 등 (1972) Carbohyd. Res., 21: 420-426; Hatanaka, K. 등 (1987) J. Med. Chem. 30, 810-814], 다당류 중 덱스트란 설페이트(dextran sulfate), 펜토산 (pentosan), 폴리설페이트(polysulfate) [Erik De Clercq. (1989) J. Antimicrob. Chemother., 23 :35-46.22], 레티난 설페이트 (lentinan ulfate) [Hatanaka, K. 등 (1989) Jpn. J. Cancer Res. 80: 95-98.23], 리보퓨라난 설페이트 (ribofuranan sulfate) [Hatanaka, K. 등 (1991) J. Carbohy. Chem., 10: 681-690.24], 퓨칸 설페이트 (fucan sulfate) [Nishino, T. 등, (1991) Agric. Biol. Chem. 55: 791-796], 및 갈락탄 설페이트 (galactan sulfate) [Nagumo, T. 등 (1988) Kitasato Arch. of Exp. Med., 61: 59-67]는 인체 면역결핍 바이러스 (human immunodeficiency virus)에 의해 유발된 세포독성에 효과를 나타내며, 특히 리보퓨라난 설페이트 (ribofuranan sulfate)의 활성은 황화 (sulfation) 정도에 의존한다. In addition, the fat removal activity and anticoagulant activity in blood vessels of physiologically sulphide polysaccharides have been known [Nakagawa, K. et al. (1972) Carbohyd. Res., 21: 420-426; Hatanaka, K. et al. (1987) J. Med. Chem. 30, 810-814], dextran sulfate, pentosan, polysulfate in polysaccharides [Erik De Clercq. (1989) J. Antimicrob. Chemother., 23: 35-46.22], lentinan ulfate [Hatanaka, K. et al. (1989) Jpn. J. Cancer Res. 80: 95-98.23], ribofuranan sulfate (Hatanaka, K. et al. (1991) J. Carbohy. Chem., 10: 681-690.24], fucan sulfate (Nishino, T. et al., (1991) Agric. Biol. Chem. 55: 791-796, and galactan sulfate (Nagumo, T. et al. (1988) Kitasato Arch. of Exp. Med., 61: 59-67] have an effect on cytotoxicity induced by human immunodeficiency virus, in particular the activity of ribofuranan sulfate depends on the degree of sulfation. .
이러한 약리활성을 가지는 식물은 숙주방어활성(host-defense activity)과 탐식세포(macrophage)활성을 촉진하므로써 면역증강(immuno- potentiating)과 항종양활성(actitumoral activity)을 나타내는 것으로 평가된다. 또한, 면역계 활성화와 항종양에 대하여 임상적으로 효과가 인정되는 약물은 소수에 불과하므로 현재까지 항종양에 유효한 약물탐색이 많은 연구자에 의해 이루어지고 있다.Plants with such pharmacological activity are evaluated to exhibit immunopotentiating and antitumoral activity by promoting host-defense activity and macrophage activity. In addition, since only a few drugs have been clinically recognized as effective against immune system activation and anti-tumor, many researchers have been searching for effective drugs for anti-tumor.
한편, 예정된 세포사(apoptosis)는 간염의 주요 원인으로 Fas-FasL결합에 의한 간세포(hepatocyte)파괴로 급성간염과 만성간염을 야기한다[Feldmann, G. (1997) J. Hepatol. 36: 1-11]. 즉, 예정된 세포사멸은 종양괴사인자 수용체계 단백질인 Fas(또는 CD95, APO-1)가 그의 리간드(ligand)인 FasL과 결합함으로써 전개되며[Nagata,S. 및 Golstein, P. (1995) Science 267: 1449-1456], 이로 인해 간염 또는 당뇨병이 생성된다[Chervonsky, A. V. 등 Cell 89: 17-24; Griffith, T. S. 등 (1995)Science 270: 1189-1192; Bellgrau, D. (1995) Nature 377: 630-632]. 따라서, Fas-FasL 매개에 의한 세포사 억제활성을 나타내는 물질은 간염치료제로 유용하게 사용될 수 있다.On the other hand, scheduled apoptosis is a major cause of hepatitis and causes acute hepatitis and chronic hepatitis due to hepatocyte destruction by Fas-FasL binding [Feldmann, G. (1997) J. Hepatol. 36: 1-11. In other words, predetermined apoptosis is developed by binding Fas (or CD95, APO-1), a tumor necrosis factor receptor protein, with FasL, its ligand [Nagata, S. And Golstein, P. (1995) Science 267: 1449-1456, resulting in hepatitis or diabetes [Chervonsky, A. V. et al. Cell 89: 17-24; Griffith, T. S. et al. (1995) Science 270: 1189-1192; Bellgrau, D. (1995) Nature 377: 630-632. Therefore, the material showing cell death inhibitory activity by Fas-FasL media can be usefully used as a therapeutic agent for hepatitis.
따라서, 본 발명의 목적은 와송, 지유, 금은화, 귀전우, 삼칠근, 백지, 마치현, 우피두견, 지구목, 유근피로 구성된 한약재로부터 추출한 항종양, 감염억제, 항산화성 및 면역증강활성을 갖는 추출물을 제공하는 것이다.Therefore, an object of the present invention is an anti-tumor, infection inhibitory, antioxidant and immuno-enhancing activity extracted from Chinese herbal medicine consisting of Whasong, fat milk, gilding silver, ear cow, samchiol, white paper, machi, bovine dog, earth tree, root To provide.
또한, 본 발명의 목적은 상기 한약재 추출물을 유효성분으로 하는 건강보조식품을 제공하는 것이다.In addition, an object of the present invention is to provide a dietary supplement with the herbal extract as an active ingredient.
본 발명자들은 천연생약인 코리원(상표) 한약재 추출물의 활성을 연구하여 오던 중, 추출물에 대한 B-임파구(lymphocyte) 자극활성과 탐식세포 자극활성과 같은 면역증강활성 및 항종양활성을 확인하였으며, 본 발명의 추출다당류가 간세포 세포사 (apoptosis)에 있어서 간세포 염증의 원인인 Fas-FasL의 결합을 저해하고 FasL의 발현을 억제하는 효과를 가지고 있음을 알아내어 본 발명을 완성하였다.The inventors of the present invention, while studying the activity of the extract of the natural herbal Coryone (medicinal herb), confirmed the immuno-enhancing and anti-tumor activity such as B-lymphocyte stimulating activity and phagocytic stimulating activity against the extract, The extract polysaccharide of the present invention was found to have the effect of inhibiting the binding of Fas-FasL, which is the cause of hepatocellular inflammation, and inhibiting the expression of FasL in hepatocyte cell death (apoptosis).
상기 목적을 달성하기 위하여, 본 발명은 와송, 지유, 금은화, 귀전우, 삼칠근, 백지, 마치현, 우피두견, 지구목, 유근피로 구성되는 한약재로부터 추출되는 항종양활성, 간질환억제활성, 항산화성 및 면역증강활성을 갖는 한약재 추출물에 관한 것이다.In order to achieve the above object, the present invention is an anti-tumor activity, liver disease suppression activity, antioxidants extracted from Chinese herbal medicine consisting of Wasong, fat milk, gilding silver, jeonjeonwoo, samchieun, white paper, machi, cow skin It relates to herbal extracts having sex and immune enhancing activity.
또한, 본 발명은 상기 한약재를 각각 동량으로 사용하여 제조하는 한약재 추출물에 관한 것이다.In addition, the present invention relates to an herbal medicine extract prepared by using the same amount of each herbal medicine.
또한, 본 발명은 각각의 한약재를 세절하여 물, 수용액 또는 완충용액에서 분쇄한 뒤 원심분리 후의 침전물을 열수추출하여 제조되는 항종양활성, 간질환억제활성, 항산화성 및 면역증강활성을 갖는 한약재 추출물에 관한 것이다.In addition, the present invention is a herb extract having antitumor activity, liver disease inhibitory activity, antioxidant activity and immune enhancing activity prepared by severing each herbal medicine and grinding it in water, aqueous solution or buffer solution, and then extracting the precipitate after centrifugation. It is about.
또한, 본 발명은 상기 한약재 추출물을 유효성분으로 함유하는 항종양제, 간기능관련질환 치료제, 항산화제(노화방지제) 및 면역증강제용 약학적 조성물에 관한 것이다. The present invention also relates to an anti-tumor agent, a liver function-related disease treatment agent, an antioxidant (anti-aging agent) and an immuno-enhancer containing the herbal extract as an active ingredient.
또한, 본 발명은 Fas-FasL 상호작용을 매개로 하는 간세포사에 의한 염증, 간기능 장해, 알콜성 간염, 환경성 간염, 바이러스성 간염, 스트레스성 간염, 지방간 질환의 간기능관련질환에 대해 항종양제, 간기능관련질환 치료제, 항산화제(노화방지제) 및 면역증강제용 약학적 조성물을 제공할 수 있다.In addition, the present invention is anti-tumor against liver function-related diseases of inflammation, liver failure, alcoholic hepatitis, environmental hepatitis, viral hepatitis, stress hepatitis, fatty liver disease caused by hepatocellular death mediated through Fas-FasL interaction The pharmaceutical composition for the treatment of liver function related diseases, antioxidants (anti-aging agents) and immunostimulants can be provided.
약학적 조성물은 임상투여시에 경구 또는 비경구 투여가 가능하며 일반적인 의약품제제의 형태로 사용될 수 있다.The pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.
즉, 본 발명의 추출물은 실제 임상투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 생약추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘(calcium carbonate), 수크로스(sucrose) 또는 락토오스 (Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.That is, the extract of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in one or more herbal extracts. (sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
추출물의 유효용량은 200∼600mg/kg이고, 바람직하기로는 200∼500mg/kg 이며, 하루 1~3회 투여될 수 있다. 용량이 200mg/kg 미만이면 효과가 미미하며, 600mg/kg을 초과하면 투여량에 대한 기대효과의 증가치가 적어 경제적이지 못하다.The effective dose of the extract is 200-600 mg / kg, preferably 200-500 mg / kg, and may be administered 1-3 times a day. If the dose is less than 200mg / kg the effect is insignificant, if it exceeds 600mg / kg the increase in the expected effect on the dosage is not economical.
뿐만 아니라, 본 발명은 상기 한약재 추출물을 유효성분으로 하는 건강보조식품을 제공할 수 있다.In addition, the present invention can provide a dietary supplement comprising the herbal extract as an active ingredient.
본 명세서에는 "조추출물", "열수추출물", "코리원(상표) 한약재 추출물", "추출물" 및 "한약재 추출물"을 혼용하였다.In the present specification, "crude extract", "hot water extract", "Koriwon (trademark) herbal medicine extract", "extract" and "herbal medicine extract" were mixed.
이하 실시예에 의하여 본 발명을 상세하게 설명한다.The present invention will be described in detail by the following examples.
하기 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.
<실시예 1: 생약추출>Example 1: Herbal Medicine Extraction
*건조 한약재 500g을 세절하여, 50mM PBS(pH 7.2)용액에서 분쇄한 뒤, 4℃, 15,000 X g에서 20분간 원심분리하였다. 그 후 샘플 5g을 취하여 10배량의 메탄올을 가하여 70℃에서 3시간동안 가온 침출하여 여과(동양여지 No. 1)하고 여액을 모아 감압농축하여 추출물을 수득하였다.* 500 g of the dried herbal medicine was chopped, ground in a 50 mM PBS (pH 7.2) solution, and then centrifuged at 4 ° C. and 15,000 × g for 20 minutes. Thereafter, 5 g of the sample was taken, 10 times the amount of methanol was added thereto, and the mixture was leached by heating at 70 ° C. for 3 hours, filtered (oriented filter No. 1), and the filtrates were concentrated under reduced pressure to obtain an extract.
<실시예 2: 생약추출물의 MMP-9 저해활성>Example 2: MMP-9 Inhibitory Activity of Herbal Extracts
MMP-9를 생산하는 Hep3B 세포주를 이용하여 실시예1의 추출물에 대한 MMP-9 저해활성을 실험하고 그 결과를 도 1에 나타내었다. 도 1에서 보는 바와 같이 본 발명의 한약재 대부분 성분들이 MMP-9 저해활성을 갖는 것을 확인하였다.MMP-9 inhibitory activity against the extract of Example 1 was tested using the Hep3B cell line producing MMP-9 and the results are shown in FIG. 1. As shown in Figure 1 it was confirmed that most of the components of the herbal medicine of the present invention has MMP-9 inhibitory activity.
<실시예 3: 생약추출물의 사람간암세포(Hep 3B)에 대한 세포독성>Example 3: Cytotoxicity of the Herbal Extract of Human Liver Cancer Cells (Hep 3B)>
사람의 간암세포(Hep3B, 5×103)를 각각 96웰 마이크로플레이트에 웰당 198㎕씩 분주한 후 MTT 어세이를 실시하여 세포독성을 검사한 결과, 간암세포에 선택적인 독성을 나타냈다. 도 2에 세포독성의 결과를 나타냈으며, 도2의 B)에 나타난 화살표는 세포사(apoptosis)유발 세포독성을 나타낸 것이다.Human liver cancer cells (Hep3B, 5 × 10 3 ) were each dispensed in 96-well microplates at 198 μl per well, followed by MTT assay and cytotoxicity. The results of cytotoxicity are shown in FIG. 2, and the arrows shown in B) of FIG. 2 indicate apoptosis-induced cytotoxicity.
<실시예 4: Fas-FasL 매개에 의한 간세포의 세포사에 대한 추출물의 억제효과> Example 4 Inhibitory Effect of Extracts on Cell Death of Hepatocytes by Fas-FasL Mediation
코라원 한약재 추출물의 Fas-FasL 매개에 의한 세포사 억제 및 간염의 치료효과를 확인하기 위하여, 생쥐의 초대 간세포(primary hepatocyte)를 분리 후 배양하였다(cm5당 2x105). 또한 FasL을 발현하거나 발현하지 않는 NIH3T3 섬유아세포 (cm5당 5x105)를 합하여 공배양하였다. 이때, 대조군으로는 Fas-FasL 결합에 의한 세포사의 핵심효소이자 카스파제(caspase) 저해제인 zVAD.fmk를, 카스파제-/-와 카스파제-3-는 대조생쥐군으로 사용하였다 [Hughes, D. P 및 Crispe, I. N. (1995) J. Exp. Med. 182: 1395-1401]. 간세포의 생존성은 3uM의 프로피디움 아이오다이드(PI)로 10분간 염색하여 현미경으로 관찰하였으며, 이때 정상적인 간세포의 형태는 PI-음성 생존세포로 구분하였다.In order to confirm the therapeutic effect of inhibiting cell death and hepatitis caused by Fas-FasL mediated collaboration of the original Chinese medicine extracts, the invitation liver (primary hepatocyte) of the mice were cultured after separation (2x10 5 per 5 cm). In addition, NIH3T3 fibroblasts ( 5 × 10 5 per cm 5 ) expressing or not expressing FasL were combined and co-cultured. In this case, zVAD.fmk, a key enzyme and caspase inhibitor of cell death by Fas-FasL binding, and caspase -/- and caspase- 3- were used as control mice. [Hughes, D P and Crispe, IN (1995) J. Exp. Med. 182: 1395-1401. The viability of hepatocytes was observed under a microscope after staining with 3 uM of propidium iodide (PI) for 10 minutes, and the normal hepatocytes were classified as PI-negative viable cells.
상기의 방법대로 간세포를 FasL을 발현하는 섬유아세포와 공배양하였을 때 생쥐의 간세포가 24시간 내에 사멸하였으나 대조군의 섬유아세포와 같이 배양된 간세포는 대부분이 생존하였다. 더욱이 간세포를 FasL 발현하는 섬유아세포와 동시에 배양하였을 때 일시적인 블렙(bleb) 형성이 시작되었으며 DNA절단이 확인되었으나, 카스파제 저해제인 zVAD.fmk를 첨가하였을 때는 간세포의 사멸이 일어나지 않았다(표 1). 이로부터 FasL이 간세포의 세포사의 원인이며, zVAD.fmk에 의해서 저해되는 카스파제가 세포사에 관여하고 있음을 알 수 있다. When hepatocytes were co-cultured with FasL-expressing fibroblasts as described above, mouse hepatocytes died within 24 hours, but most of the hepatocytes cultured with the control fibroblasts survived. Furthermore, when the hepatocytes were simultaneously cultured with FasL-expressing fibroblasts, transient bleb formation began and DNA cleavage was confirmed. However, the addition of the caspase inhibitor zVAD.fmk did not cause the death of hepatocytes (Table 1). This shows that FasL is the cause of cell death of hepatocytes, and that caspase inhibited by zVAD.fmk is involved in cell death.
한편, 열수조추출물을 50mg/15ml 농도로 첨가시, FasL발현-NIH3T3에 의한 세포사를 약 40%이상 감소시켜 간세포 생존능력을 향상시켰다. 이러한 결과는 섬유아세포가 생성하는 FasL에 의한 간세포의 Fas 수용체결합을 추출물이 방해 내지 저해함으로서 Fas 수용체에 세포사의 신호가 전달되는 것이 차단되기 때문으로 보인다(표 1). 또한 추출물의 보호효과는 야생형 간세포보다는 카스파제-3-이 결손된 생쥐에서 분리된 간세포에서 보다 효과적임이 확인되어 Fas-FasL에 의한 세포사는 주로 카스파제-3-를 매개하고 있음을 확인하였다(표 2).On the other hand, when hot water bath extract was added at a concentration of 50 mg / 15ml, cell death by FasL expression-NIH3T3 was reduced by about 40% or more to improve hepatocyte viability. This result seems to be due to the blockage or inhibition of Fas receptor binding of hepatocytes by FasL produced by the fibroblasts, the transmission of cell death signal to Fas receptor (Table 1). In addition, the protective effect of the extract was found to be more effective in hepatocytes isolated from caspase- 3 -deficient mice than wild-type hepatocytes, indicating that apoptosis by Fas-FasL mediates caspase- 3- (Table) 2).
한편, 추출물의 각종 분획들이 섬유아세포와 24시간 공배양시 간세포의 세포사에 대한 억제효과(PI-음성 세포)를 검정한 결과, 조추출물에서 강한 억제효과를 보였다(표 3).On the other hand, the various fractions of the extract showed a strong inhibitory effect in the crude extract as a result of assaying the inhibitory effect (PI-negative cells) on the cell death of hepatocytes during coculture with fibroblasts for 24 hours (Table 3).
<실시예 5: 추출물의 화학적 조성>Example 5: Chemical Composition of Extracts
한약재 추출물의 화학적 특성을 조사하기 위하여 다음과 같이 실시하였다. 총당(total sugar)의 정량은 페놀-황산법[Dubois, M. 등 (1956) Anal. Chem. 28: 350-354]을 사용하였으며, 환원당은 넬슨-쏘모지(Nelson-Somogyi)방법[Nelson, N. J. 및 Somogyi, M. (1952) J. Biol. CHem. 195: 19-23]을, 표준물질로는 포도당을 사용하였다. 단백질 정량은 소혈청알부민(BSA)을 표준으로 하여 로우리(Lowry)법 [Lowry, H.O.,등 (1951) J. Biol. Chem., 193: 265-275]으로 하였으며, 다당류의 가수분해는 2M 황산으로 100℃에서 6시간동안 스크루캡 바이알에서 수행하였다. 가수분해 후 탄산바륨(BaCO3)으로 중화시키고 엠버라이트(Amberite) IR-120(H+ form)컬럼에서 분리하였다. 갈락토퓨라노실(galactofuranosyl) 잔기를 제거하기 위한 다당류의 부분가수분해는 25mM 황산(10ml)으로 100℃에서 4시간동안 수행하였으며, 이때 0.5M 수산회나트륨으로 중화하고 증류수로 투석하였다.In order to investigate the chemical properties of the herbal extracts were carried out as follows. Quantification of total sugars was carried out using the phenol-sulfuric acid method [Dubois, M. et al. (1956) Anal. Chem. 28: 350-354, and the reducing sugars were used in the Nelson-Somogyi method [Nelson, NJ and Somogyi, M. (1952) J. Biol. CHem. 195: 19-23] and glucose was used as a standard. Protein quantitation is based on Low Serum Albumin (BSA), Lowry method [Lowry, HO, et al. (1951) J. Biol. Chem., 193: 265-275], and the hydrolysis of the polysaccharides was carried out in a screwcap vial for 6 hours at 100 ℃ with 2M sulfuric acid. After hydrolysis it was neutralized with barium carbonate (BaCO 3 ) and separated in Amberlite IR-120 (H + form) column. Partial hydrolysis of polysaccharides to remove galactofuranosyl residues was performed with 25 mM sulfuric acid (10 ml) at 100 ° C. for 4 hours, neutralized with 0.5 M sodium hydroxide and dialyzed with distilled water.
표 4a로부터 조추출물의 구성성분 중 대부분이 당류(96.1%)이며, 미량의 단백질과 황이 포함되어 있음을 확인했다. 따라서, 본 발명의 추출물에 존재하는 황에 의한 항바이러스제로서의 기능도 예상된다. 또한 조추출물의 중성다당성분을 분석한 결과를 도4b에 나타내었다.It was confirmed from Table 4a that most of the components of the crude extract were saccharides (96.1%) and contained trace amounts of protein and sulfur. Therefore, the function as an antiviral agent by sulfur present in the extract of the present invention is also expected. In addition, the result of analyzing the neutral polysaccharide component of the crude extract is shown in Figure 4b.
<실시예 5: 추출물의 B-임파구 자극활성>Example 5: B-lymphocyte stimulating activity of the extract
본 발명의 한약재 조추출물에 대한 B-임파구 자극활성을 검토하기 위해 실험동물은 암컷의 생쥐(C57BL/6XC3H) F1(B6C3F1)을 생명공학연구소로부터 구입하여 17~22g 중량이 될 때 비장적출실험에 이용하였다. 양의 적혈구(sRBC)는 한국배지주식회사(서울)에서 구입하였고 기니아피그 보체와 RPMI 1640배지는 Gibco BRL(Grand Island, NY, USA)사 제품을, 리포폴리사카라이드(Lipopolysaccharide, LPS)는 시그마사 제품을 사용하였다.In order to examine the B-lymphocyte stimulating activity of the crude medicinal herb extract of the present invention, the experimental animal was purchased from the biotechnology research institute for the mouse (C57BL / 6XC3H) F1 (B6C3F1) in the spleen extraction experiment. Was used. Sheep red blood cells (sRBC) were purchased from Korea Medium Company (Seoul), guinea pig complement and RPMI 1640 medium from Gibco BRL (Grand Island, NY, USA), and lipopolysaccharide (LPS) from Sigma The product was used.
임파구의 시험관 내 활성화 및 항체생성세포수(AFC) 검정은 다음과 같다. 비장세포를 RPMI 1640배지(10% 소태아혈청함유)에서 배양하여 세포수를 5x106 cells/ml로 조정한 후, 웰당 0.5 ml씩(처리군당 4개씩) 48-웰 플레이트(Costar사 제품)에 옮긴 후, 시료 또는 LPS (25 ㎍/ml, 시그마)를 첨가한다. 시험관 내 자극을 위하여 플레이트를 37℃의 Bellco(Bellco Biotech., Vineland, NJ, USA) 스테인레스스틸 조직배양기에서 흔들어 가면서 배양한다.In vitro activation and lymphocyte production (AFC) assay of lymphocytes is as follows. Splenocytes were cultured in RPMI 1640 medium (containing 10% fetal bovine serum) to adjust the cell number to 5x10 6 cells / ml, and then in a 48-well plate (Costar Co.) at 0.5 ml per well (4 per treatment group). After transfer, the sample or LPS (25 μg / ml, Sigma) is added. Plates are incubated with shaking in a Bellco (Bellco Biotech., Vineland, NJ, USA) stainless steel tissue incubator at 37 ° C for in vitro stimulation.
항체생성은 배양 2일 뒤에 측정하며 TNP(trinitrophenyl)-합텐 처리된 sRBC에 대한 AFC를 Jerne 플라크검정법으로 측정하고 세포수는 헤마시토미터 (hemacytometer)를 사용하였다. 이때 항체생성 세포수를 활성치로 나타내고 비활성은 다음과 같이 나타냈다. 즉, 일반적으로 항체생성능이 강한 대조군으로 사용되는 리포폴리사카라이드(lipopolysaccharide; LPS) 25㎍/㎖를 대조군으로 처리하였을 때를 기준했다. LPS의 활성이 거의 25 ㎍/㎖농도에서 포화되므로 이 양을 대조군으로 사용하였다.Antibody production was measured 2 days after the culture, AFC for TNP (trinitrophenyl) -hapten treated sRBC was measured by Jerne plaque assay and the number of cells using a hemacytometer. In this case, the antibody-producing cell number was expressed as an activity value, and the specific activity was expressed as follows. That is, it was based on the treatment of 25 μg / ml lipopolysaccharide (LPS), which is generally used as a control with high antibody production, as a control. The amount of LPS was used as a control since the activity of LPS was saturated at nearly 25 μg / ml.
조추출물 2mg를 B-임파구 자극활성을 C57BL/6XC3H 생쥐에서 다클론 항체생성으로 검정한 결과를 표 5에 나타냈다.Table 2 shows the results of assaying 2 mg of crude extract for B-lymphocyte stimulating activity by polyclonal antibody production in C57BL / 6XC3H mice.
<실시예 6: 추출물의 항종양활성의 검정>Example 6: Assay of Antitumor Activity of Extracts
추출물에 있는 다당류의 항암작용을 다음 방법으로 검정하였다. 생후 4주된 ICR계 생쥐에 사르코마(Sarcoma) 180세포를(0.1 ml, 7 x 106 세포) 쥐의 우측서혜부 피하에 이식하였다. 실험표본 물질은 PBS에 적당한 농도로 용해시켜 고압멸균한 뒤 내부복막 가까이에 종양이식한 후, 24시간 이후부터 10일동안 매일 주사(2 mg/100 g)하였다. 모든 쥐들을 5주간 관찰한 후 종양의 성장에 미치는 영향을 알아보기 위해, 쥐를 죽이고 종양을 절제하여 무게를 측정하므로써 다음의 식으로부터 종양의 성장억제비율을 구하였다.The anticancer activity of the polysaccharides in the extract was assayed by the following method. Sarcoma 180 cells (0.1 ml, 7 × 10 6 cells) were implanted subcutaneously in the right groin of rats at 4 weeks old ICR mice. Experimental samples were dissolved in PBS at an appropriate concentration, autoclaved, and transplanted near the inner peritoneum, followed by daily injection (2 mg / 100 g) for 10 days after 24 hours. After observing all the rats for 5 weeks, the tumor growth inhibition rate was calculated from the following equation by killing the rats, resecting the tumors, and weighing them.
억제율 (%) = {(A-B)/A} ×100Inhibition Rate (%) = {(A-B) / A} × 100
A는 대조군의 종양무게 평균값이며 B는 실험군의 종양무게 평균값이다. 완전한 퇴행은 실험쥐 숫자에 대한 완전한 퇴행을 보이는 쥐 숫자의 비율이다. A is the mean tumor weight of the control group and B is the mean tumor weight of the experimental group. Complete regression is the ratio of rat numbers showing complete regression to the number of mice.
그 결과 조추출물은 사르코마 180 고형종양을 이식한 쥐에서 항종양활성이 2 mg/100g 투여로 44.7%로 매우 높은 억제활성을 나타냈다(표 6).As a result, crude extract showed very high inhibitory activity of 44.7% with 2 mg / 100 g of antitumor activity in Sarcoma 180 solid tumors (Table 6).
<실시예 7: 추출물의 B-임파구 자극 활성 검정>Example 7: B-lymphocyte stimulating activity assay of extract
조추출물을 각각의 시료 0.1mg/ml에서 1.0 mg/ml에 대한 면역자극 검정을 한 결과, 대조군인 LPS군의 89% 활성을 나타내었다(표 7).The crude extract was immunostimulated for 1.0 mg / ml in 0.1 mg / ml of each sample, showing 89% activity of the control LPS group (Table 7).
<실시예 8: 추출물의 탐식세포 자극활성 (macrophage-stimulating activity)> Example 8: Macrophage-stimulating activity of extracts
열수추출물은 복강 및 구강식이를 위해 PBS에 녹인 뒤 투여하였다. 구강식이를 위해서 강제로 섭취하게 하였으며, PEC 분리와 비장의 탐식세포 활성을 위해 적출하였다. Hot water extract was administered after dissolving in PBS for abdominal cavity and oral diet. Forced intake for oral diet and extraction for PEC isolation and phagocytic activity of the spleen.
비장의 탐식세포(Splenic macrophage)분리는 조직을 부드럽게 부순 뒤 Tris완충액-염화암모늄용액으로 조직을 단일 세포화시키고, 세포 추출기 (Becton Dickinson Labware, Franklin Lakes, NJ, USA)를 통과시켜 수행하였다. 잔존세포는 HBSS(Hank의 균형용액)를 완벽하게 세척하고, RPMI배지(10% 가열불활성화 소태아혈청, 100U/ml 페니실린, 100g/ml 스트렙토마이신 함유, RPMI-FCS)로 재희석하였다. 희석된 세포는 플라스틱 용기에, 배양된 탐식세포는 RPMI-FCS-HEPES에 부착시키도록 만들었다.Splenic macrophage separation of the spleen was performed by gently crushing the tissue, single cellizing the tissue with Tris buffer-ammonium chloride solution, and passing through a cell extractor (Becton Dickinson Labware, Franklin Lakes, NJ, USA). The remaining cells were completely washed with HBSS (Hank's balance solution) and rediluted with RPMI medium (10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 g / ml streptomycin, RPMI-FCS). Diluted cells were made to adhere to plastic containers and cultured phagocytes to RPMI-FCS-HEPES.
PEC는 추출물을 복강 및 구강섭취 후에 5ml의 Hank의 균형용액(HBSS)으로 말초동공으로부터 분리하고, 원심분리하여 세포덩어리를 HBSS로 두 번 세척한 후 HBSS(25mM-HEPES(-2-hydroxyethyl-peperazine-N-2-ethane s㎕fonic acid 함유) 1 ml에 녹였다. 이렇게 얻은 PEC는 F-300 세포측정기(Medical Electronics, Kobe, Japan)를 사용하여 그 수를 측정하였다.PEC extract the extract from the peripheral cavity with 5 ml Hank's Balance Solution (HBSS) after intraperitoneal and oral ingestion, centrifuge and wash the cell mass twice with HBSS and then HBSS (25mM-HEPES (-2-hydroxyethyl-peperazine). It was dissolved in 1 ml of -N-2-ethane sulfonic acid) The number of PECs thus obtained was determined using an F-300 cytometer (Medical Electronics, Kobe, Japan).
PEC의 탐식활성의 측정은 형광 마이크로파티클(microparticle)에서 배양하여 측정한다. 즉, PEC(1 x 105)를 원심분리하고 100㎕의 HBSS-HEPES에 재희석 한 뒤, HBSS-HEPES로 100배 희석시킨 플루오레스브라이트 카복실레이트 마이크로스피어즈 (Fluoresbrite carboxylate microspheres, 2.0m; Polyscience, Warrington, PA, USA)를 20㎕를 첨가하여 37℃에서 1시간 배양하였다. 배양액에 2ml의 냉PBS(3 mM ethylenediaminetetraacetic acid disodium salt; EDTA-PBS함유)를 첨가하여 반응을 정지시킨 후 세포덩어리를 모아 EPICS-프로파일 II 플로우시토미터 (EPICS-Profile II flow-cytometer, Coulter, Hialeah, FL, USA)를 이용하여 탐식활성을 측정하였다.The phagocytic activity of PEC is measured by culturing in fluorescent microparticles. In other words, PEC (1 × 10 5 ) was centrifuged, rediluted in 100 μl of HBSS-HEPES, and then diluted 100-fold with HBSS-HEPES to Fluoresbrite carboxylate microspheres (2.0 m; Polyscience, Warrington, PA, USA) was incubated for 1 hour at 37 ℃ to 20μl. 2 ml of cold PBS (containing 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA-PBS)) was added to the culture to stop the reaction, and the cell masses were collected to collect the EPICS-Profile II flow-cytometer, Coulter, Hialeah , FL, USA) was used to measure phagocytic activity.
화학 루미네센스 활성측정은 F-300 마이크로셀(microcell) 측정기를 사용하여 PEC와 비장의 탐식세포 밀도가 4 x 105/ml이 되도록 트리판 블루(Trypan Blue) 염색으로 세포수를 세고 RPMI-FCS-HEPES로 조정한 뒤, 500㎕의 세포용액을 20㎕의 0.2% 루미놀(luminol, 5-amino-2,3-dihydro-1,4-phthalazine-dione, Sigma, Co., USA)이 함유된 폴리스티렌 큐벳에 옮긴다. 각각의 큐벳을 부드럽게 섞은 뒤 15분 후에 루미노미터(luminometer, Multi-biolumat LB9505; Berthold, Wildbad, Germany)를 사용하여 측정하였다. 활성은 15분동안의 총활성으로 나타냈다.For chemical luminescence activity measurement, the cells were counted by trypan blue staining using the F-300 microcell measuring instrument so that the phagocytic density of PEC and spleen was 4 x 10 5 / ml and RPMI- After adjusting with FCS-HEPES, 500 μl of cell solution contained 20 μl of 0.2% luminol (luminol, 5-amino-2,3-dihydro-1,4-phthalazine-dione, Sigma, Co., USA) Transfer to polystyrene cuvettes. After 15 minutes of mixing each cuvette gently, it was measured using a luminometer (luminometer, Multi-biolumat LB9505; Berthold, Wildbad, Germany). Activity was expressed as total activity for 15 minutes.
비장의 탐식세포 활성에 대한 효과를 측정하기 위하여, 추출물을 10마리의 생쥐 복강 내에 투여하여 이 중 2마리에서 비장의 탐식세포를 투여후 3, 18일째에 분리하여 일시적인 화학 루미네센스를 측정하였다(표8). 추출물 투여 생쥐에서의 화학 루미네센스가 대조군보다 증가하였다.In order to measure the effect on the phagocytic activity of the spleen, extracts were administered intraperitoneally in 10 mice, two of which were isolated from the spleen phagocytes 3 and 18 days after administration to measure the transient chemiluminescence. Table 8. The chemical luminescence in the mice receiving the extract was higher than that of the control group.
생쥐당 5mg씩 추출물을 투여한 생쥐의 PEC가 가장 높은 화학 루미네센스를 나타내었다. 이들 생쥐에서 비장의 탐식세포의 생성은 투여량 의존적임이 확인되었다. 추출물의 B세포자극활성을 다클론 항체생성 세포를 C57BL/6XC3H 생쥐에서 측정한 결과 5 mg 투여에서 가장 높았으며(표 9), B-세포자극활성을 나타내었다.PEC of the mice administered with the extract of 5 mg per mouse showed the highest chemical luminescence. The production of phagocytic phagocytes in these mice was found to be dose dependent. The B cell stimulatory activity of the extract was measured in polyclonal antibody-producing cells in C57BL / 6XC3H mice, which was the highest at 5 mg (Table 9), and showed B-cell stimulatory activity.
<실시예 9: 열수추출물의 독성 및 혈중누출효소에 대한 영향>Example 9: Toxicity of Hot Water Extract and Effect on Blood Leak Enzyme>
상기에서 추출된 열수추출물을 임상적으로 사용하려면 독성이 없어야 하므로, 독성검사와 간세포계 효소에 미치는 영향을 검토하였다. 무처리군의 정상랫트 (1군 5마리)에 대한 열수추출 다당류의 복강내 투여시의 치사량을 구하고자 하였으나, 5일동안 950mg/kg 이하 투여시에 사망을 일으키지 않았다. 또한 950 mg/kg을 복강내 투여시, 24시간 후의 S-GOT, S-GPT, S-Alp에 어떠한 영향도 미치지 않았다(표 10).In order to use the extracted hot water extract clinically, it should not be toxic, and the effects on toxicity and hepatocellular enzymes were examined. To evaluate the lethal dose of intraperitoneal administration of hot-water-extracted polysaccharides to normal rats (5 rats in group 1), no death occurred at 5 mg or less for 5 days. In addition, intraperitoneal administration of 950 mg / kg had no effect on S-GOT, S-GPT, S-Alp after 24 hours (Table 10).
<실시예 12. 열수추출물의 노화방지(항산화성)에 대한 효과><Example 12. Effect of anti-aging (antioxidative) of hot water extracts>
열수추출물에 대한 항산화성 유무를 확인하기 위해서 실험동물은 일본 에어로지수틱스(Air Logistics)사의 4주령 B6C3F1 숫컷 생쥐로 한국 바이오 제노믹스사를 통하여 구입하여 다섯 군(1군 10마리)으로 나누어 실험하였다. 제1군(이하, 1군)은 정상군으로 물과 사료만으로 사육하였다. 제2군(이하, 2군)은 생쥐당 17.5mg/㎏의 비율로 DEN(N,N-diethylnitrosoamine)을 주 2회씩 8주간 복강 주사하였고[Kolaja, K. L., Xu, Y., Walborg, E. F., Jr., Stevenson, D. E. and Klaunig, J. E. (1998) J. Toxicol. Environ. Health A 53: 479-492] 이후에 12주를 더 사육한 다음에 경추 탈구법으로 도살하여 생화학적 검사를 실시하는데 이용하였다. 제3군(이하, 3군)은 처음부터 열수추출물을 물에 희석하여(500 ppm) 사료와 함께 사육하였고, 제4군(이하, 4군)은 DEN을 8주간 복강 주사한 후에 9주부터 열수추출물을 물에 희석하여 사료와 함께 사육하였다. 제5군(이하, 5군)은 DEN을 복강 주사하면서 열수추출물과 사료로 사육하는 군으로 분류하여 실험하였다.In order to confirm the antioxidant activity of the hot water extract, the experimental animals were 4 weeks old B6C3F1 male mice of Japan's Air Logistics, purchased through Korea Biogenomes, and divided into five groups (10 groups). The first group (hereinafter referred to as group 1) was a normal group and reared only with water and feed. Group 2 (hereinafter, Group 2) was intraperitoneally injected with DEN (N, N-diethylnitrosoamine) twice a week for 8 weeks at a rate of 17.5 mg / kg per mouse [Kolaja, KL, Xu, Y., Walborg, EF, Jr., Stevenson, DE and Klaunig, JE (1998) J. Toxicol. Environ. Health A 53: 479-492], followed by a further 12 weeks and then slaughtered by cervical dislocation and used for biochemical testing. The third group (hereinafter referred to as group 3) was bred with water (500 ppm) after diluting the hot water extract from the beginning, and the fourth group (hereinafter referred to as group 4) was given 9 weeks after intraperitoneal injection of DEN for 8 weeks. Hot water extracts were diluted with water and bred with feed. Group 5 (hereinafter referred to as group 5) was classified into groups breeding hot water extract and feed while intraperitoneally injecting DEN.
글루타사이온 퍼옥사이다제(Glutathione peroxidase)는 생체 내에서 생성되는 과산화수소를 물로 바꿔주는 과정에 관여하는 효소이며, 글루타사이온 퍼옥사이다제의 활성도는 이 효소에 의해 생성된 산회글루타사이온(oxidized glutathione; GSSG)이 글루타사이온 환원제에 의해 글루타사이온(glutathione;GSH)으로 되어 일정수준의 GSH를 유지할 때, NADPH가 산화되는 것을 측정하는 방법으로 그 검정방법은 다음과 같다.Glutathione peroxidase is an enzyme involved in the process of converting hydrogen peroxide produced in vivo into water, and the activity of glutathione peroxidase is produced by When oxidized glutathione (GSSG) is glutathione-reducing agent to glutathione (GSH) to maintain a certain level of GSH, the assay method is as follows.
0.1mM EDTA를 함유하는 0.1mM 인산염 완충용액(pH 7.0) 0.5ml와 글루타사이온 환원제 0.1 ml(0.24 unit), 10mM GSH 0.1ml 그리고 시료 0.1ml를 혼합하였다. 이 혼합용액에 1.5mM NADPH 용액 0.1ml를 넣고 12mM 쿠메니 하이드로퍼옥사이드 (cumene hydroperoxide) 0.1ml를 가하여 340nm에서 5분간 흡광도의 감소를 측정하였다. 1분동안에 1uM의 NADPH를 산화시키는 효소의 양을 1unit로 하였다. 0.5 ml of 0.1 mM phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.1 ml (0.24 unit) of glutathione reducing agent, 0.1 ml of 10 mM GSH and 0.1 ml of sample were mixed. 0.1 ml of 1.5 mM NADPH solution was added to the mixed solution, and 0.1 ml of 12 mM cumene hydroperoxide was added thereto. The decrease in absorbance was measured at 340 nm for 5 minutes. The amount of enzyme that oxidizes 1 uM of NADPH in 1 minute was 1 unit.
표 11은 열수추출물의 과산화물분해효소에 대한 영향을 측정한 결과를 나타낸 것이다. 암 유발 물질을 투여하지 않은 군들(1 군과 2 군)간의 비교에서 열수추출물의 투여군에서 과산화물분해효소의 활성이 29 % 이상 증가함을 나타내었다, 또한 암 유발 물질의 투여군들간(2 군, 4군 그리고 5 군)의 비교에서도 열수추출물의 투여군(4 군과 5 군)에서 과산화물분해효소의 활성이 유의적으로 증가하였다.Table 11 shows the results of measuring the effect of hot water extract on the peroxidase. Comparison between the groups that did not receive cancer-causing agents (groups 1 and 2) showed an increase in activity of peroxide dehydrogenase by more than 29% in the group that received hot water extracts, and also between groups that received cancer-causing substances (groups 2, 4). In the comparison between group 5 and group 5, the activity of peroxidase was significantly increased in the hot water extract group (groups 4 and 5).
이상과 같은 본 발명에 의하면, 본 발명의 추출물은 항종양활성, 간염억제활성, 면역증강활성 및 노화방지(항산화성) 특성을 가지며, 이에 따라 항종양제, 간기능관련치료제, 면역활성화제용 치료제 및 항산화제(노화방지제)로 사용될 수 있다. According to the present invention as described above, the extract of the present invention has anti-tumor activity, hepatitis inhibitory activity, immuno-enhancing activity and anti-aging (antioxidative) properties, accordingly antitumor agent, liver function-related therapeutic agent, therapeutic agent for immunoactivator And antioxidants (antiaging agents).
또한, 본 발명의 추출물은 독성이 전혀 없으므로 건강보조식품으로도 널리 이용될 수 있다.In addition, since the extract of the present invention is not toxic at all, it can be widely used as a health supplement.
도 1은 MMP-9를 Hep3B sup. 세포주를 이용하여 본 발명의 한약재의 열수추출물을 이용하여 MMP 자이모그라피를 한 결과를 나타낸 것이다.1 supra MMP-9 Hep3B. It shows the results of MMP Zymograph using the hot water extract of the herbal medicine of the present invention using a cell line.
1: 대조군(Hep3B sup., MMP9) 2: 삼칠근1: control group (Hep3B sup., MMP9) 2: tricuspid muscle
3: 금은화 4: 백지3: gold and silver 4: white paper
5: 대조군(Hep3B sup., MMP9, 레인1의 1/5 양을 영동함.)5: Control (Hep3B sup., MMP9, run 1/5 of Lane 1).
6: 지구목 7: 유근피6: earth tree 7: roots
8: 귀전우8: Earnest
도 2는 간암세포주(Hep3B)에 대한 세포독성의 결과이다. 2 shows the results of cytotoxicity against hepatocellular carcinoma cell line (Hep3B).
A) 정상간세포주, B) 간암세포주 Hep3B.A) normal liver cell line, B) liver cancer cell line Hep3B.
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