KR100607907B1 - Pharmaceutical composition containing Phellinus linteus extract for preventing cancer metastasis to liver and excellent in immunomodulation - Google Patents
Pharmaceutical composition containing Phellinus linteus extract for preventing cancer metastasis to liver and excellent in immunomodulation Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
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- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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Abstract
본 발명은 이미 항암효과가 검증된 상황버섯에 동충하초, 길경, 진피, 산약, 당귀, 황기, 대추, 하수오, 익지인, 백출, 백복령, 산수유, 홍삼 엑기스를 약제학적으로 혼합한 복합물로서, 특히 간전이 억제 효과와 면역조절 효과가 있는 약학적 조성물에 관한 것이다. The present invention is a complex of pharmacological fungi, Cordyceps sinensis, Gilkyung, dermis, powder, Angelica, Astragalus, Jujube, Shouo, Ikjiin, Baekchul, Baekbokryeong, Cornus, Red ginseng extract, as a pharmaceutical compound, It relates to a pharmaceutical composition having this inhibitory effect and immunomodulatory effect.
상황버섯, 항암, 간전이, Colon26-L5, 대식세포, 자연 살상세포, 면역세포Situation mushroom, anticancer, liver metastasis, Colon26-L5, macrophage, natural killer cell, immune cell
Description
도 1은 본 발명의 상황배합복합조성물의 세포독성을 나타낸 것이다. Figure 1 shows the cytotoxicity of the situation composite composition of the present invention.
도 2a, 2b는 쥐 대장암주인 Colon26-L5 암주를 BALB/c 생쥐에 이식 전 일주일간 농도별로 본 발명의 상황배합복합조성물을 경구 투여한 후 암세포를 이식하고, 이식 후 18일 후 희생시켜 간전이에 미치는 효과를 나타낸 것이다. Figures 2a, 2b is a colon colon cancer cell line cancer colon26-L5 cancer in the BALB / c mice for a week before transplantation oral administration of the situational composite composition of the present invention by the concentration for a week before transplanting cancer cells, 18 days after transplantation to sacrifice liver It shows the effect.
도 3a와 3b는 상황배합복합조성물을 암세포 이식 전 일주일간 BALB/c 생쥐에게 농도별로 경구 투여한 후 암세포 이식 1일전에 anti-asialo GM1 serum을 200 ㎍을 복강 주사하여 자연 살상세포를 선택적으로 제거 한 후 Colon26-L5 암세포를 이식하여 자연 살상세포가 없는 상황에서 초기 면역 반응에 관계하는 대식세포가 간전이에 미치는 효과를 나타낸 것이며, 도 3c, 3d는 상황배합복합조성물을 암세포 이식 전 일주일간 경구투여하고 암세포 이식 1일전에 2-클로로아데노신을 50 ㎕ 정맥주사하여 마크로파지를 선택적으로 제거한 후 암세포 이식을 시행하여 마크로파지가 없는 상황에서 초기 면역 반응에 관계하는 자연 살상세포가 간전이에 미치는 효과를 나타낸 것이다. Figures 3a and 3b is orally administered to the BALB / c mice for one week prior to cancer cell transplantation of the situational composite composition and 200 ㎍ of anti-asialo GM1 serum intraperitoneally 1 day before cancer cell transplantation to selectively remove natural killer cells After transplanting Colon26-L5 cancer cells, macrophages involved in the initial immune response in the absence of natural killer cells showed the effect on liver metastasis. FIGS. 3C and 3D show a situation-complexed composition orally for one week before cancer cell transplantation. 50 μl intravenous 2-chloroadenosine was injected 1 day prior to cancer cell transplantation to selectively remove macrophages, followed by cancer cell transplantation to show the effect of natural killer cells involved in the initial immune response on liver metastasis in the absence of macrophages. will be.
도 4a, 4b 와 4c는 상황배합복합조성물을 일주일간 BALB/c 생쥐에게 농도별 로 경구투여한 후 비장세포를 수집하여 면역형광염색법으로 염색한 후 면역세포들의 표면항원인 Mac-1, NK1.1,CD3,CD19,CD4,CD8을 측정한 것 이다.Figures 4a, 4b and 4c is a situational composite composition orally administered to BALB / c mice by concentration for one week after splenocytes collected and stained by immunofluorescence staining surface antigens of immune cells Mac-1, NK1. 1, CD3, CD19, CD4, and CD8 are measured.
도 5는 BALB/c의 비장세포를 시험관내에서 배양하여 상황배합복합조성물을 농도별로 처리하여 면역세포에 대한 분열유도반응을 측정한 것이다. FIG. 5 shows the induction of response to immune cells by culturing BALB / c splenocytes in vitro and treating the situational composite composition by concentration.
본 발명은 항암효과를 갖는 한약재인 상황버섯을 에탄올 추출하여 그 추출물을 여러 가지 한약재와 배합한 복합조성물을 제조하여 생체 실험에서의 암세포의 간전이 억제 실험과 면역조절 효과를 검정함으로써 이루어진 것이다. The present invention is made by extracting a ethanol extract of a medicinal herb, an herbal medicine having an anticancer effect, and preparing a complex composition combining the extract with various medicinal herbs to test the liver metastasis suppression test and immunomodulatory effect of cancer cells in a biological experiment.
암은 현재 한국에서 사망원인 1위를 차지하는 질병으로서 환경문제, 식이의 서구화 등으로 인해 암 환자율이 급증하고 있는 실정이다. 현재의 암의 치료법으로 외과적 수술, 방사선치료 및 약물요법의 3가지로 분류되고 있다. Cancer is currently the number one cause of death in Korea, and the rate of cancer patients is increasing rapidly due to environmental problems and westernization of diet. Current treatments for cancer are classified into three types: surgical surgery, radiation therapy and drug therapy.
초기 단계의 암들은 외과적 수술로 치료가 가능하나, 암이 많이 진전되었거나, 전이가 일어난 경우에는 외과적 수술만으로는 치료가 어렵고 다른 방법을 함께 사용해야 한다. 방사선 치료방법의 경우 방사선 조사로 인한 새로운 암의 발병 또는 전이의 확산 시에 억제의 어려움이 있어 약물치료와 병행하고 있는 실정이다. 약물요법의 경우는 몸의 어떤 부위에 생긴 암이라도 약물이 도달할 수 있다는 점에서 방사선 치료나 외과적 수술에 뛰어난 치료법이며, 전이된 암을 치료할 수 있다는 점에서 전이된 암 증상을 완화시켜 환자의 삶의 질을 개선하고 수명연장을 시켜 주는 중요한 역할을 한다. 그러나 현재 임상적으로 사용되고 있는 항암제는 화학요법제제로서 상당한 부작용이 있으며, 인체 면역기능을 저하시키는 단점이 있는 것으로 알려져 있다. Early stage cancers can be treated with surgical procedures, but if the cancer has advanced or metastasized, it is difficult to treat with surgery alone and other methods must be used together. In the case of radiation therapy, there is a difficulty in suppressing the onset or metastasis of new cancer due to irradiation, which is concurrent with drug therapy. In the case of pharmacotherapy, it is an excellent treatment for radiation therapy or surgical operation in that the drug can reach any cancer of the body, and it can cure the metastatic cancer symptoms by treating the metastatic cancer. It plays an important role in improving the quality of life and extending lifespan. However, currently used anti-cancer drugs clinically have a significant side effect as a chemotherapeutic agent, is known to have a disadvantage of lowering human immune function.
또한 상황버섯은 소나무비늘버섯과(pellinus linteus)의 진흙버섯(Linteus) 속으로 분류되는 종류로서 Ikegawa 등에 의해 항암 효과가 확인되었으며, 다른 담자균들보다도 더 높은 항암 활성을 가지고 있음이 알려져 있다.(Ikagewa T., Uchara N., Naeda Y., Nakarishi M. and Fukioka F. Cancer research 734, 29.1969)In addition, the situation mushroom is classified as the genus of the pine mushroom, pellinus linteus , and mud mushrooms ( Linteus ). The anticancer effect was confirmed by Ikegawa et al. T., Uchara N., Naeda Y., Nakarishi M. and Fukioka F. Cancer research 734, 29.1969)
또한 상황버섯의 항종양 활성성분은 대부분 다당체 또는 단백 다당체로 존재하며, 그 항암효과는 화학요법제제와 같이 암세포를 직접 파괴하는 세포독성의 효과가 아니라 정상세포의 면역증강 효과에 기인하여 항암효과를 나타낸다( Komatsu N., Okuta S., Likumoto S., Kimura K., Saito G and Sokaki S.,Gann, 137,60,1960). 또한, 한약처방은 독성이 없으면서 면역조절 작용이 크다고 알려져 있다. In addition, most of the anti-tumor active ingredients of S. mushrooms exist as polysaccharides or protein polysaccharides, and their anticancer effects are not due to cytotoxic effects of direct destruction of cancer cells like chemotherapy agents, but due to the immune-enhancing effect of normal cells. (Komatsu N., Okuta S., Likumoto S., Kimura K., Saito G and Sokaki S., Gann, 137, 60, 1960). In addition, Chinese medicine prescription is known to have a large immune regulation without toxicity.
또한 본 발명자들은 한약재를 단독 물질이 아닌 복합적으로 사용한 보정방감탕이라는 처방이 암세포의 간전이 억제와 면역조절작용에 효과가 있음을 검정한 바 있다(Lee SJ., Saiki I., Hayakwa Y., Nunome S., Yamada H., Kim SH. Int immunopharmacol., 147-157,3,2003). In addition, the present inventors have tested that the prescription of corrected gambanggamtang using herbal medicines in combination with not a single substance is effective in inhibiting liver metastasis and immunomodulatory activity of cancer cells (Lee SJ., Saiki I., Hayakwa Y., Nunome S., Yamada H., Kim SH. Int immunopharmacol., 147-157, 3,2003).
본 발명자들은 이와 같은 종래 기술에 비추어 상황버섯을 단독으로 사용한 것이 아니라, 상황버섯의 에탄올 추출물을 이용하여 여러 가지 유용한 한약재들과 배합하여 상황버섯을 함유하는 약학적 복합 조성물을 제조하였다. 이와 같이 복합 조성물로서 제조할 경우, 여러 가지 한약재와 융합하여 서로에 대한 상승작용을 나타내어 상황버섯을 단독으로 사용한 경우에 비하여 뛰어난 항암작용을 나타낼 수 있다는 장점이 있다.The present inventors did not use the situation mushroom alone in the light of the prior art, and prepared a pharmaceutical composite composition containing the situation mushroom by combining with various useful herbal medicines using the ethanol extract of the situation mushroom. When prepared as a composite composition, there is an advantage that can exhibit excellent anti-cancer activity compared to the case of using the situation mushroom alone by showing a synergistic effect on each other by fusion with various herbal medicines.
본 발명은 이미 알려진 상황버섯의 항암효과를 응용하여 상황버섯의 에탄올추출물을 이용하여 여러 가지 한약재들을 약제학적으로 배합한 항암효과와 면역효과를 갖는 복합 조성물에 관한 것이다. 특히 본 발명의 조성물은 항암효과 중에서 암세포의 간전이 억제효과 및 면역조절 작용을 겸하고 있어 항암치료제 뿐 아니라 건강 보조식품으로도 활용이 가능하다.The present invention relates to a composite composition having an anticancer effect and an immune effect by combining various herbal medicines by using ethanol extract of the situation mushroom by applying the known anticancer effect of the situation mushroom. In particular, the composition of the present invention has the anti-cancer effect of liver cancer metastasis inhibitory effect and immunomodulatory effect can be utilized as a health supplement as well as anti-cancer treatment.
본 발명에서는 상황버섯의 에탄올 추출물을 여러 가지 한약제와 배합하여 신규한 상황버섯 추출물을 함유하는 약학적 조성물을 제공하며, 이 상황버섯을 함유하는 약학적 조성물은 상황버섯을 단독으로 사용한 경우에 비하여 보다 상승적인 항암 활성과 면역조절 효과를 갖는다.The present invention provides a pharmaceutical composition containing a novel situation mushroom extract by combining the ethanol extract of the situation mushroom with various herbal medicines, and the pharmaceutical composition containing the situation mushroom is more than when using the situation mushroom alone Has synergistic anticancer activity and immunomodulatory effect.
또한 본 발명에서는 상기 상황버섯 추출물을 함유하는 약학적 조성물을 가루제제, 액제로 제제화하여 제공한다. 또한 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물의 제조 시에는 종래의 한약 처방과는 다른 방법으로 약재를 배합한다. 기존의 한약 처방에서는 처방에 따라 정해진 양의 약재를 물로 달이는 방법을 사용하였지만, 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물 의 제조 시에는 동충하초, 길경, 진피, 산약, 당귀, 황기, 대추, 하수오, 익지인, 백복령, 백출, 산수유의 12가지 약재를 먼저 물로 달인 후, 이 달인 물에 상황버섯의 에탄올 추출액과 홍삼 엑기스를 배합한 후 감압농축기를 이용하여 농축한 후 동결건조기를 이용하여 건조시킨 후 가루제제로 만들어 사용한다. In addition, the present invention provides a pharmaceutical composition containing the situation mushroom extract is formulated into a powder formulation, a liquid formulation. In addition, the preparation of the pharmaceutical composition containing the situation mushroom extract according to the present invention is formulated with a method different from the conventional herbal medicine formulation. Conventional Chinese medicine prescription used a method of decoction of a predetermined amount of the medicine according to the prescription, but when preparing a pharmaceutical composition containing the situation mushroom extract according to the present invention Cordyceps sinensis, Gilkyung, dermis, powdered herbs, Angelica, Astragalus, Jujube First, decoction of 12 kinds of herbs of sewage, ripen, baekbokyeong, Baekchul, and cornus milk with water, and then combine ethanol extract and red ginseng extract of situation mushrooms in this month's water, concentrate using a reduced pressure concentrator, and then use lyophilizer. It is dried and used as a powder.
또한 상기 상황버섯 추출물을 함유하는 약학적 조성물은 암세포의 간으로의 전이 억제, 면역세포의 면역조절효과 등 항암효과와 면역효과를 발현므로 항암제 및 건강보조제로서 사용할 수 있다.In addition, the pharmaceutical composition containing the situation mushroom extract can be used as an anticancer agent and a health supplement because it expresses anticancer effects and immune effects, such as inhibiting the metastasis of cancer cells to the liver and immune regulation effects of immune cells.
본 발명의 상황버섯 추출물을 함유하는 약학적 조성물은 암세포에 직접적으로 작용하여 암세포를 파괴하는 세포사멸 또는 세포독성의 효과에 의해서가 아니라 체내 면역조절을 통한 항상성 증가에 의해 암세포의 간으로의 전이를 억제 및 예방할 수 있는 것이다. The pharmaceutical composition containing the extract of the situation mushroom of the present invention acts directly on the cancer cells to prevent metastasis of the cancer cells to the liver by homeostasis through immune regulation, not by the effects of apoptosis or cytotoxicity that destroys the cancer cells. It can be suppressed and prevented.
이하에서는 본 발명의 바람직한 실시예를 통해 본 발명을 더욱 상세히 설명한다. 다만, 이하의 실시예는 본 발명에 대한 이해를 돕기 위한 것일 뿐, 본 발명의 범위가 이하의 실시예로만 제한되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the following examples are merely to help the understanding of the present invention, the scope of the present invention is not limited only to the following examples.
<실시예><Example>
상황버섯 추출물을 함유한 약학적 조성물의 제조법Preparation of pharmaceutical composition containing situation mushroom extract
동충하초(10 g), 길경(6 g), 진피(5 g), 산약(6 g), 당귀(6 g), 황기(6 g), 대추(5 g), 하수오(6 g), 익지인(4 g), 백출(10 g), 백복령(6 g), 산수유(6 g)의 12가지 약재들을 1L의 물로 3시간을 달인 후 이 한약물에 상황버섯의 에탄올 추출물(0.14 g)과 홍삼엑기스(0.13 g)를 배합하여 상황버섯 추출물을 함유한 약학적 조 성물 18.4 g을 제조하였다. Cordyceps sinensis (10 g), Gilyeong (6 g), dermis (5 g), powder (6 g), Angelica (6 g), Astragalus (6 g), jujube (5 g), sewage (6 g), ripe (4 g), Baekchul (10 g), Baekbokryeong (6 g), Cornus (6 g), and 12 hours of decoction of 1 liter of water, and the ethanol extract (0.14 g) of red mushrooms and red ginseng Extract (0.13 g) was combined to prepare 18.4 g of a pharmaceutical composition containing a mushroom extract.
하기의 다이어그램은 상황버섯을 함유한 약학적 조성물의 제조방법을 나타낸 것이고 표 1은 상황버섯을 함유한 약학적 조성물의 제조방법을 나타낸 것이다.The following diagram shows a method for preparing a pharmaceutical composition containing a mushroom, and Table 1 shows a method for preparing a pharmaceutical composition containing a mushroom.
* 상황버섯 23.7g에 에탄올 0.14g을 가함* 0.14 g of ethanol is added to 23.7 g of mushrooms
** 홍삼사포닌 160mg/g(0.13g)** Red Ginseng Saponin 160mg / g (0.13g)
가루제제의 제조Preparation of Powder
상기 배합물 76.44 g을 감압농축기로 농축, 동결건조기를 이용하여 건조시켜 가루제제(18.4 g)로 만들었다. 76.44 g of the compound was concentrated in a vacuum condenser and dried using a lyophilizer to obtain a powder (18.4 g).
본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물을 액제로 사용할 경우, 동물에 경구투여 시에는 본 발명의 가루제제를 물에 10 mg/mouse, 50 mg/mouse의 농도로 용해시켜 사용하였으며, 세포 처리 시에는 본 발명의 가루제제를 물에 10 mg/ml의 농도로 용해시켜 사용하였다. 또한 필요한 경우 하기 실시예에서 사용되는 농도가 되도록 배지로 희석하였다. When using the pharmaceutical composition containing the situation mushroom extract according to the present invention as a liquid, oral administration to the animal was used to dissolve the powder formulation of the present invention in water at a concentration of 10 mg / mouse, 50 mg / mouse, In the cell treatment, the powder formulation of the present invention was dissolved in water at a concentration of 10 mg / ml and used. It was also diluted with medium to the concentration used in the following examples, if necessary.
세포주의 배양Culture of Cell Lines
쥐의 대장암 세포주인 Colon26-L5를 다음과 같이 배양하였다. 10% 소 태아 혈청을 포함하는 RPMI 1640(GIBCO-BRL, Richmond, U.S.A.) 배지에 37℃, 5% CO2 및 95% 공기 상태로 상기 세포주를 배양기 안에서 배양하였다.Colon colorectal cancer cell line Colon26-L5 was cultured as follows. The cell lines were cultured in an incubator at 37 ° C., 5% CO 2 and 95% air in RPMI 1640 (GIBCO-BRL, Richmond, USA) medium containing 10% fetal bovine serum.
암세포 독성효과 실험Cancer Cell Toxicity Effect Experiment
본 발명의 상황버섯 추출물을 함유하는 약학적 조성물의 암세포주에서의 세포독성을 확인하기 위하여, 쥐 대장암 세포주인 Colon26-L5 세포를 사용하였다. 세포독성 확인을 위해 MTT 어세이를 실시하였다. 배양된 암세포를 96웰 플레이트(96well plate)에 각 웰당 10,000여개씩 분주하고 24시간 경과 후 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물의 액제를 농도별로 계대 희석하여 100㎕씩 첨가하였다. 상기 액제를 첨가한 후 24시간이 경과하였을 때, MTT 용액을 넣고 반응시켰다. 그 후 4시간 동안 배양을 한 후, 마이크로플레이트 리더(파장:570nm)로 흡광도를 측정하여 생존율을 측량하였다. 도 1은 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물의 농도에 따른 쥐 대장암 세포주 Colon26-L5의 생존율 측정결과를 나타낸 것이다. 도 1에서 알 수 있는 것과 같이, 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 암세포 자체에 대한 세포독성의 효과는 없었다.In order to confirm the cytotoxicity in the cancer cell line of the pharmaceutical composition containing the situation mushroom extract of the present invention, the colon colorectal cell line Colon26-L5 cells were used. MTT assay was performed to confirm cytotoxicity. Cultured cancer cells were dispensed about 10,000 cells per well into a 96 well plate (96 well plate), and after 24 hours, 100 μl of the pharmaceutical composition containing the situation mushroom extract of the present invention was passage-diluted for each concentration. After 24 hours after the addition of the liquid, MTT solution was added and reacted. After 4 hours of incubation, the absorbance was measured by a microplate reader (wavelength: 570 nm) to measure the survival rate. Figure 1 shows the results of measuring the survival rate of the colon colorectal cancer cell line Colon26-L5 according to the concentration of the pharmaceutical composition containing the situation mushroom extract according to the present invention. As can be seen in Figure 1, the pharmaceutical composition containing the situation mushroom extract according to the present invention did not have a cytotoxic effect on the cancer cells themselves.
Colon26-L5 암세포주에 의한 간전이 억제 작용Hepatic Metastasis Inhibition by Colon26-L5 Cancer Cell Line
먼저 실험군에는 검액으로 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물을 10mg/mouse, 50㎎/mouse농도로 1주일간 경구투여하고 대조군에는 PBS를 투여한다. 일주일 후 in vitro에서 배양한 colon26-L5 대장암 세포주를 5×104 cells/㎖의 농도가 되도록 세포현탁액을 만들어 18∼20ｇ의 BALB/c mice에 1×104cells/0.2㎖을 간문맥을 통해 주사하였다. 암세포 이식 18일 후에 치사시킨 다음 개복하여 간에 전이된 전이암을 정상군과 그 무게를 비교하여 평가했다. 간전이에 미치는 효과를 관찰한 결과, 도 2에서와 같이 colon26-L5 암세포주를 사용한 실험적 간전이 모델에서 농도의존적으로 간전이 억제 효과를 나타내었다. 또한, 실험기간 중 생쥐의 체중 변화를 관찰한 결과, colon26-L5 암세포주를 이식하지 않은 정상군에서는 꾸준히 체중증가를 나타내는 반면에 대조군과 실험군에서는 체중이 증가하다가 colon26-L5 암세포주를 이식한 이후에는 체중이 감소되었으며, 시일이 지 남에 따라 체중이 다시 회복하였다. 또한 암세포 이식 전에 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물을 투여한 실험군에서는 대조군에 비해 체중이 빨리 회복되었으나, 시일이 지남에 따라 그 차이는 감소되어 대조군과 실험군 사이에 체중의 차이는 없었다. First, the experimental group was orally administered to the pharmaceutical composition containing the situation mushroom extract according to the present invention as a test solution at a concentration of 10 mg / mouse and 50 mg / mouse for 1 week, and the control group was administered with PBS. A week later, a cell suspension was prepared in a colon26-L5 colorectal cancer cell line cultured in vitro to a concentration of 5 × 10 4 cells / ml, and 1 × 10 4 cells / 0.2ml in 18-20 g BALB / c mice through the portal vein. Injection. Eighteen days after cancer cell transplantation, the metastasized cancer metastasized after liver failure was evaluated by comparing its weight with that of the normal group. As a result of observing the effect on liver metastasis, the experimental liver metastasis model using colon26-L5 cancer cell line as shown in FIG. In addition, as a result of observing the weight change of the mice during the experiment period, the normal group without the colon26-L5 cancer cell line showed a steady weight gain, while the weight gain was increased in the control group and the experimental group after the colon26-L5 cancer cell line was transplanted. The body weight was reduced and body weight recovered over time. In addition, in the experimental group administered the pharmaceutical composition containing the situation mushroom extract according to the present invention before cancer cell transplantation, the body weight was recovered faster than the control group, but the difference was reduced over time, so that the difference in weight between the control group and the experimental group was There was no.
따라서 본 발명의 상황버섯 추출물 함유 약학적 조성물의 외형상의 부작용은 관찰되지 않았으며, 암세포주의 이식수술이 체중에 영향을 미쳤다고 볼 수 있다. Therefore, no apparent side effects of the pharmaceutical composition containing the situation mushroom extract of the present invention was observed, and it can be seen that the transplantation of the cancer cell line affected the body weight.
Colon26-L5 암주에 의한 간전이 모델에 미치는 면역계의 역할과 생쥐에 대한 간전이 억제효과Role of the Immune System in the Liver Metastasis Model of Colon26-L5 Strains and Its Inhibitory Effect on Mice
실험군은 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물의 액제를 1주일간 경구투여하고 대조군은 PBS를 투여한다. 일주일 후 in vitro에서 배양한 colon26-L5 대장암세포주를 5×104 cells/㎖이 되도록 세포현탁액을 만들어 18∼20ｇ인 BALB/c mice에 1×104cells/0.2㎖을 간문맥을 통해 주사한다. Natural killer (NK) 세포의 depletion을 위해 rabbit anti-asialo GM1 serum (200㎍/mouse; Wako pure chemicals co. Ltd., Japan)을 암세포 주사 1일 전에 복강투여 하며, 마크로파지 depletion을 위해서는 2-클로로아데노신 (50㎍/mouse; Sigma, MO, USA)을 암세포 주사 1일 전에 정맥주사한다. 암세포 이식 18일 후에 경추탈구로 치사시킨 다음 개복하여 간에 전이된 전이암을 정상군과 그 무게를 비교하여 평가한다. 도 3a,3b, 3c와 같이 NK 세포가 없는 상황에서 초기 면역반응에 관계하는 마크로파지가 간전이에 미치는 효과를 생체 내에서 관찰하였다. NK 세포를 선택적으로 제거 한 상황에서는 면역반응에 관여하는 세포가 줄어들게 되므로 간전이가 증가하게 대조군에 비해 유의적으로 증가하게 된다. 그러나 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물은 NK 세포가 없는 상황에서도 암세포의 간 전이를 유의적으로 억제하는 효과를 나타내었다. 이는 본 발명에 의한 상황버섯 추출물을 함유하는 조성물이 마크로파지에 의해 유도되는 면역반응에 관여함을 나타낸다. 도 3b와 같이 마크로파지가 없는 상황에서 초기 면역반응에 관계하는 NK 세포가 간전이에 미치는 효과를 생체 내에서 관찰하였다. 마크로파지를 선택적으로 제거한 상황에서는 면역반응에 관여하는 세포가 줄어들게 되므로 암세포의 간전이가 대조군에 비해 유의적으로 증가하게 된다. 그러나 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 마크로파지가 없는 상황에서도 간전이를 유의적으로 억제하는 효과를 나타내었다. 이는 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물이 NK 세포에 의해 유도되는 면역반응에 관여함을 나타낸다. 다만 이러한 간전이 억제효과는 NK 세포가 제거된 모델에서 더욱 강하게 나타났으므로 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물의 간전이 억제효과는 NK 세포보다는 마크로파지에 의한 효과가 더욱 강함을 알 수 있다.The experimental group was orally administered a solution of the pharmaceutical composition containing the situation mushroom extract of the present invention for 1 week, and the control group was administered with PBS. A week later make the cell suspension to be 5 × 10 4 cells / ㎖ a colon26-L5 colon cancer cell lines cultured in vitro, and injected with 1 × 10 4 cells / 0.2㎖ the 18~20g of BALB / c mice via the portal vein . Rabbit anti-asialo GM1 serum (200 µg / mouse; Wako pure chemicals co. Ltd., Japan) was intraperitoneally administered 1 day prior to cancer cell injection for depletion of natural killer (NK) cells, and 2-chloroadenosine for macrophage depletion. (50 μg / mouse; Sigma, MO, USA) is injected intravenously 1 day prior to cancer cell injection. Eighteen days after cancer cell transplantation, the carcinoma of the cervical dislocation and then laparotomy metastasis to the liver are evaluated by comparing their weight with the normal group. 3A, 3B and 3C, the effect of macrophages involved in the initial immune response in the absence of NK cells in liver metastasis was observed in vivo. In the situation where the NK cells are selectively removed, the cells involved in the immune response are reduced so that the liver metastasis is increased significantly compared to the control group. However, the pharmaceutical composition containing the mushroom extract of the present invention showed an effect of significantly inhibiting liver metastasis of cancer cells even in the absence of NK cells. This indicates that the composition containing the situation mushroom extract according to the present invention is involved in the immune response induced by macrophages. In the absence of macrophages as shown in Figure 3b the effect of NK cells involved in the initial immune response on liver metastasis was observed in vivo. In the case where the macrophages are selectively removed, the cells involved in the immune response are reduced, so the liver metastasis of the cancer cells is significantly increased compared to the control group. However, the pharmaceutical composition containing the situation mushroom extract according to the present invention showed an effect of significantly inhibiting liver metastasis even in the absence of macrophages. This indicates that the pharmaceutical composition containing the situation mushroom extract according to the present invention is involved in the immune response induced by NK cells. However, since the effect of inhibiting liver metastasis was stronger in a model in which NK cells were removed, the effect of inhibiting liver metastasis of the pharmaceutical composition containing the situation mushroom extract according to the present invention is stronger than that of NK cells. Can be.
상황버섯 추출물을 함유하는 약학적 조성물의 비장세포에 대한 면역조절효과 Immunomodulatory Effect on Spleen Cells of Pharmaceutical Compositions Containing Extracts of Sacral Mushrooms
기존에 알려진 대부분의 항암제는 면역기능을 저하시키는 단점이 있는 것으로 알려져 있으므로 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 면역기능 조절에 어떠한 역할을 하는지 관찰하기 위해 면역 관련실험을 행하였다. 그 첫 단계로서 BALB/c mice에 실험군은 본 발명에 의한 상황버섯 추출물을 함유하 는 약학적 조성물을 1주일간 경구투여하고 대조군에는 PBS를 투여한다. 일주일 후 mice를 치사시킨 후 비장을 적출하여 electronic chemical balance(MC1, sartolius)로 비장무게를 측정하고 난 후 PBS(Phosphate buffered saline)로 세척하였다. 그 후 후 RPMI 배지가 들어있는 Petri dish (35×100㎜)에서 작은 해부 가위로 절단한 다음 cell strainer (40㎛, Becton Dickinson, USA)로 여과하여 조직편 및 분리되지 않은 세포덩어리를 제거한 후 800rpm에서 3분간 3회 원심분리하여 세척하고 complete RPMI 배지에 부유시킨다. BALB/c mice에 실험군은 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물을 1주일간 경구투여하고 대조군에는 PBS를 투여한다. 마지막 약물투여로부터 1일 후 2㎖의 3% thioglycolate broth를 복강투여하고 4일 후 복강세포를 복강세척을 통하여 회수한다. Saiki 등의 방법에 의해 복강세포 중의 마크로파지의 함량을 확인한 후 실험에 사용한다. 비장세포 현탁액을 1×106/㎖ 씩 카운팅하여 5㎖ tube에 100㎕씩 분주한다. 이후 CD3, CD4, Mac-1 FITC-conjugated antibody를 50㎕씩 가한 후 실온에서 15분 동안 반응시킨 후 double staining으로 CD19, CD8, NK1.1 PE-conjugated antibody를 50㎕ 씩 가한 후 실온에서 15분 반응시켰다. 이상의 면역 형광염색 과정은 빙욕 상에서 시행하였다. 염색이 완료된 세포들을 0.5㎖의 완충액에 부유시켜 FACS vantage (Becton Dickinson Co., USA)를 이용하여 분석한다. 시료당 5,000개의 세포에 대하여 리스트모드로 자료를 취합하였으며 Cell Quest 프로그램을 이용, 분석하였다. 데이터의 분석은 forward scatter (FSC)와 side scatter (SSC)의 dual parameter를 이용한 dot plot 상에서 전체 비장세포와 small lymphocyte 영역 및 lymphoblast 영역을 구분하여 그 중의 B cell, Total T cell, CD4+ T cell, CD8+ T cell, Mac-1+
cell, NK1.1 cell의 비율을 산출한다.Since most of the known anticancer drugs are known to have a disadvantage of lowering immune function, the pharmaceutical composition containing the situation mushroom extract according to the present invention was subjected to immune-related experiments to observe what role it plays in regulating immune function. As a first step, the experimental group was orally administered to BALB / c mice for 1 week orally with the pharmaceutical composition containing the situation mushroom extract according to the present invention, and the control group was administered with PBS. One week later, mice were killed and spleens were extracted, and the spleen weight was measured using an electronic chemical balance (MC1, sartolius), followed by washing with PBS (Phosphate buffered saline). Thereafter, the petri dish (35 × 100㎜) containing RPMI medium was cut with small dissecting scissors, filtered through a cell strainer (40 μm, Becton Dickinson, USA) to remove tissue fragments and undifferentiated cell masses, and then at 800 rpm. Wash by
세포와 소림프구 영역 및 임파아구 영역을 구분하여 그 중의 B 세포, T 세포, CD4+, CD8+, Mac-1+, NK1.1 세포의 비율(gated %)을 산출하였다. 마크로파지와 NK 세포는 이물질이 체내에 침입하였을 경우 가장 먼저 일어나는 면역반응이다. 따라서 도 4a에서와 같이 마크로파지의 표면항원인 Mac-1과, NK cell의 표면항원인 NK1.1의 비율을 측정하였다. 그 결과, 본발명의 상황버섯 추출물을 함유하는 약학적 조성물을 투여한 군은 대조군과 비교하여 마크로파지는 10㎎, 50㎎에서 각각 30%, 18%의 높은 증가율을 보이며, NK 세포는 같은 농도에서 5%, 1%의 증가율을 나타내었다. 따라서 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 자연면역을 증진시키는 효과가 있음을 알 수 있으며 그 효과는 마크로파지 의존적인 자연면역을 증진시키는데 더욱 효과적임을 알 수 있었다. 마크로파지와 NK세포에 의해 자연면역이 일어난 후에는 T 세포가 관여하는 세포성 면역과 B 세포가 관여하는 체액성 면역의 특이면역이 관여하는 면역반응이 일어나게 된다. 따라서 본 발명에 의한 상황버섯 추출물을 함유한 약학적 조성물의 특이적 면역반응을 관찰하고자 세포성 면역을 매개하는 total T 세포의 표면항원인 CD3와 체액성 면역을 매개하는 total B 세포의 표면항원인 CD19의 비율을 측정하였다. 그 결과, 도 4b와 같이 대조군과 비교하여 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물 을 투여한 군에서는 10㎎, 50㎎에서 total T 세포가 14% 정도 증가하였으며 total B 세포가 15%, 18% 정도 증가하였다. 따라서 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물이 자연면역을 증진시킬 뿐만 아니라 특이적 면역반응인 세포성 면역과 체액성 면역에도 역시 효과적임을 알 수 있었다. T 세포에 의한 특이적 면역반응은 크게 세포독성 T 세포에 의해 감염된 세포를 직접적으로 죽이는 반응과 보조 T세포에 의해 마크로파지나 다른 림프구를 활성화시키는 반응의 두 가지로 나눌 수 있다. 두 가지 면역세포에 의해 나타나는 면역반응의 양상이 달라지게 될 뿐만 아니라 활성화되는 cytokine도 달라지게 된다. 따라서 상황배합 복합처방(SBP)가 세포독성T 세포와 보조 T세포의 어느 쪽을 활성화시키는데 관여하는지 관찰하기 위하여 세포독성 T세포의 표면항원인 CD8와 보조T세포의 표면항원인 CD4의 비율을 관찰하였다. 그 결과, 도 4c와 같이 대조군과 비교하여 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물을 투여한 군에서는 10㎎, 50㎎에서 보조 T세포가 5%, 18% 정도 증가하였으며 세포독성 T세포가 15%, 18% 정도 증가하였다. 따라서 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물은 세포성 면역의 세포독성 T세포, 보조 T세포 모두를 활성화시킴을 확인하였다.By dividing the cells from the lymphocyte region and lymphocyte region, the ratio (gated%) of B cells, T cells, CD4 + , CD8 + , Mac-1 + , and NK1.1 cells was calculated. Macrophages and NK cells are the first immune response to foreign body invasion. Therefore, as shown in FIG. 4a, the ratio of Mac-1, the surface antigen of macrophages, and NK1.1, the surface antigen of NK cells, were measured. As a result, the group administered with the pharmaceutical composition containing the extract of the situation mushroom of the present invention showed a high increase rate of 30% and 18% at 10 mg and 50 mg, respectively, compared to the control group, and NK cells at the same concentration The rate of increase was 5% and 1%. Therefore, it can be seen that the pharmaceutical composition containing the situation mushroom extract according to the present invention has an effect of enhancing natural immunity, and the effect is more effective in enhancing macrophage-dependent natural immunity. After spontaneous immunization by macrophages and NK cells, immune responses involving the specific immunity of cellular immunity involving T cells and humoral immunity involving B cells occur. Therefore, in order to observe the specific immune response of the pharmaceutical composition containing the situation mushroom extract according to the present invention, the surface antigen of CD3 and total B cells that mediate humoral immunity The proportion of CD19 was measured. As a result, in the group administered with the pharmaceutical composition containing the situation mushroom extract according to the present invention as compared to the control as shown in Figure 4b, total T cells increased by 14% at 10 mg, 50 mg and total B cells 15% , 18% increase. Therefore, it was found that the pharmaceutical composition containing the situation mushroom extract according to the present invention not only enhances natural immunity but also is effective for cellular immunity and humoral immunity, which are specific immune responses. Specific immune responses by T cells can be broadly divided into those that directly kill cells infected by cytotoxic T cells and those that activate macrophages or other lymphocytes by helper T cells. Not only will the immune response caused by the two immune cells change, but also the cytokine that is activated. Therefore, we observed the ratio of CD8, the surface antigen of cytotoxic T cells, and CD4, the surface antigen of helper T cells, to observe whether SBP is involved in activating cytotoxic T cells and helper T cells. It was. As a result, in the group administered with the pharmaceutical composition containing the situation mushroom extract of the present invention as compared to the control as shown in Figure 4c the secondary T cells increased by 5%, 18% at 10 mg, 50 mg and cytotoxic T cells Increased by 15% and 18%. Therefore, it was confirmed that the pharmaceutical composition containing the situation mushroom extract of the present invention activates both cytotoxic T cells and helper T cells of cellular immunity.
비장세포의 증식 어세이Splenocyte Proliferation Assays
BALB/c mice에 실험군은 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물을 1주일간 경구투여하고 대조군은 PBS를 투여한다. 일주일 후 mice를 치사시킨 후 비장을 적출하여 electronic chemical balance(MC1, sartolius)로 비장무게를 측정했다. 그 후 PBS(Phosphate buffered saline)로 세척한 후 RPMI 배지가 들어있는 Petri dish (35×100㎜)에서 작은 해부가위로 절단한 다음 cell strainer (40㎛, Becton Dickinson, USA)로 여과하여 조직편 및 분리되지 않은 세포덩어리를 제거하였다. 800rpm에서 3분간 3회 원심분리하여 세척하고 complete RPMI 배지에 부유시킨다. 마우스로부터 분리한 비장세포를 2×106/㎖로 complete RPMI 배지에 부유시킨 후 100㎕씩 96-well U-bottom culture plate에 가하고 1㎍/㎖ 의 Con A(concanavalin A, Sigma), LPS(lipopolysaccharide, Sigma)와 함께 72시간 배양한 후 XTT법에 의해 증식을 측정하였다. ELISA-reader를 이용하여 570nm에서 흡광도를 측정하고, 대조군의 흡광도와 비교하여 세포성장률을 %로 환산하였다. The experimental group is administered orally to the BALB / c mice for a week containing the pharmaceutical composition containing the situation mushroom extract according to the present invention, the control group is administered PBS. One week later, the mice were killed and the spleen was extracted and the spleen weight was measured by electronic chemical balance (MC1, sartolius). After washing with PBS (Phosphate buffered saline), cut in small dissecting scissors in a Petri dish (35 × 100 mm) containing RPMI medium, and then filtered with a cell strainer (40 μm, Becton Dickinson, USA) to isolate tissues and isolate them. Uncollected cell mass was removed. Wash by
in vitro에서 실시된 비장세포에 대한 분열유도 반응실험에 사용된 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 비장세포에 아무런 독성도 나타내지 않는 농도로 사용되었다. 비장세포에 대한 분열유도 반응은 외부의 자극에 대한 면역세포의 활성화 및 증식을 나타내는 것으로 인식될 수 있는데 본 실험에 의해 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물이 면역세포의 증식에 어떤 효과가 있는지를 관찰한 결과, 도 5와 같이 처리하지 않은 대조군에 비해 본 발명에 의한 약학적 조성물을 투여한 군에서 10㎎, 50㎎에서 각 20%를 36%, 44%로 농도가 증가함에 따라 유의적으로 면역세포의 증식이 증가하였으며 LPS와 Con A를 처리하였을 경우의 분열유도반응은 100%에서 140%, 146%로 대조군과 실험군 간에 더욱 큰 차이를 보이며 증가하는 것으로 나타났다. 따라서 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 면역세포의 활성화를 유도할 뿐만 아니라 또한 외부자극에 대한 면역반응으로서 면역세포의 활성화를 더욱 강하게 유도함을 확인 수 있었다. The pharmaceutical composition containing the situation mushroom extract according to the present invention used in the test for inducing division of splenocytes conducted in vitro was used at a concentration that shows no toxicity to splenocytes. The division-induced response to splenocytes can be recognized to indicate the activation and proliferation of immune cells against external stimuli. According to the present experiment, the pharmaceutical composition containing the situation mushroom extract according to the present invention was used for the proliferation of immune cells. As a result of observing the effect, in the group administered with the pharmaceutical composition according to the present invention as compared to the control group not treated as shown in FIG. The proliferation of immune cells was significantly increased, and the induction of fission induction with LPS and Con A was increased from 100% to 140% and 146% with a greater difference between control and experimental groups. Therefore, the pharmaceutical composition containing the situation mushroom extract according to the present invention could not only induce the activation of immune cells but also induce the activation of immune cells more strongly as an immune response to external stimulation.
이상에서 살펴본 바와 같이, 본 발명의 상황버섯 추출물을 함유하는 약학적 조성물은 암세포의 간 전이 억제 및 면역세포의 면역조절 활성화에 의한 면역조절효과를 가진다. 그러므로 본 발명에 의한 상황버섯 추출물을 함유하는 약학적 조성물은 암치료제로서 사용될 수 있으며, 암예방을 위한 건강보조식품으로 유용하게 사용될 수 있다. As described above, the pharmaceutical composition containing the situation mushroom extract of the present invention has an immunomodulatory effect by inhibiting liver metastasis of cancer cells and activating immune regulation of immune cells. Therefore, the pharmaceutical composition containing the situation mushroom extract according to the present invention can be used as a cancer treatment, and can be usefully used as a dietary supplement for cancer prevention.
본 발명에 따른 조성물은 탕재, 환약, 산제로서 경구투여되며, 성인의 경우 조성물의 중량으로 100g을 1일, 2회 복용한다.The composition according to the present invention is orally administered as a hot powder, a pill, a powder, and in the case of an adult, 100 g is taken twice a day by the weight of the composition.
앞에서 설명된 본 발명의 일실시예는 본 발명의 기술적 사상을 한정하는 것으로 해석되어서는 안 된다. 본 발명의 보호범위는 청구범위에 기재된 사항에 의하여만 제한되고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명의 기술적 사상을 다양한 형태로 개량 변경하는 것이 가능하다. 따라서 이러한 개량 및 변경은 통상의 지식을 가진 자에게 자명한 것인 한 본 발명의 보호범위에 속하게 될 것이다. One embodiment of the present invention described above should not be construed as limiting the technical spirit of the present invention. The scope of protection of the present invention is limited only by the matters described in the claims, and those skilled in the art can change and change the technical idea of the present invention in various forms. Therefore, such improvements and modifications will fall within the protection scope of the present invention, as will be apparent to those skilled in the art.
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| KR101160943B1 (en) * | 2009-11-25 | 2012-06-28 | 주식회사한국신약 | Health functional foods compositions for the prevention and amelioration of cancer containing the mixed extract of Phellinus linteus mycelium and cultured Panax Ginseng Cameyer as an active ingredient |
| CN102824517A (en) * | 2012-09-24 | 2012-12-19 | 南京正亮医药科技有限公司 | Application of suoquan capsules in preparing medicines for suppressing OS-732 cell proliferation |
| KR101257909B1 (en) * | 2011-10-17 | 2013-04-24 | 주식회사한국신약 | Pharmaceurtical compositions for the prevention or treatment of cancer containing the mixed extract of Phellinus linteus mycelium and cultured Panax Ginseng Cameyer as an active ingredient |
| CN103285103A (en) * | 2013-06-20 | 2013-09-11 | 周义 | Antitumor medicinal powder |
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| KR20030002213A (en) * | 2001-06-30 | 2003-01-08 | 알앤엘생명과학주식회사 | Compositions having Anti-cancer Activities |
| KR20020093147A (en) * | 2002-05-30 | 2002-12-13 | 지니스생명공학 주식회사 | Preventive and Dietary Supplement for adult chronic disease |
| KR20040039940A (en) * | 2002-11-05 | 2004-05-12 | 학교법인 경희대학교 | Pharmaceutical compositions having anti-cancer and antimetastatic activities |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010076920A1 (en) * | 2008-12-31 | 2010-07-08 | 주식회사한국신약 | Anticancer health food containing phellinus linteus and vegetable worms |
| KR101033671B1 (en) * | 2008-12-31 | 2011-05-12 | 주식회사한국신약 | Anticancer health foods including Phellinus linteus and vegetable worms |
| KR101160943B1 (en) * | 2009-11-25 | 2012-06-28 | 주식회사한국신약 | Health functional foods compositions for the prevention and amelioration of cancer containing the mixed extract of Phellinus linteus mycelium and cultured Panax Ginseng Cameyer as an active ingredient |
| KR101257909B1 (en) * | 2011-10-17 | 2013-04-24 | 주식회사한국신약 | Pharmaceurtical compositions for the prevention or treatment of cancer containing the mixed extract of Phellinus linteus mycelium and cultured Panax Ginseng Cameyer as an active ingredient |
| CN102824517A (en) * | 2012-09-24 | 2012-12-19 | 南京正亮医药科技有限公司 | Application of suoquan capsules in preparing medicines for suppressing OS-732 cell proliferation |
| CN102824517B (en) * | 2012-09-24 | 2015-07-08 | 扬州国综生物科技有限公司 | Application of suoquan capsules in preparing medicines for suppressing OS-732 cell proliferation |
| CN103285103A (en) * | 2013-06-20 | 2013-09-11 | 周义 | Antitumor medicinal powder |
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| KR20060072887A (en) | 2006-06-28 |
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