KR100471580B1 - Using for a mixture of Duchesnea extract and Ganoderma extract as an apoptosis enhancer - Google Patents
Using for a mixture of Duchesnea extract and Ganoderma extract as an apoptosis enhancer Download PDFInfo
- Publication number
- KR100471580B1 KR100471580B1 KR10-2002-0005021A KR20020005021A KR100471580B1 KR 100471580 B1 KR100471580 B1 KR 100471580B1 KR 20020005021 A KR20020005021 A KR 20020005021A KR 100471580 B1 KR100471580 B1 KR 100471580B1
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- KR
- South Korea
- Prior art keywords
- extract
- snakeberry
- ganoderma lucidum
- apoptosis
- alcohol
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
Abstract
본 발명은 뱀딸기 (Duchesnea chrysantha) 추출물 및 영지버섯 (Ganoderma lucidum) 추출물 및 이들의 혼합물의 용도에 관한 것으로, 구체적으로 용매 추출법을 이용하여 뱀딸기로부터 다당류를, 영지버섯으로부터 고미성분 (triterpenoid 계)을 추출하고, 각 추출물 및 추출 혼합물이 암 세포의 세포사멸을 촉진하는 효과; 암 세포 증식을 억제하는 효과; 및 방사선과 함께 처리할 경우 더 낮은 선량에서도 암 세포의 세포사멸을 촉진하는 효과가 우수함에 따라, 세포증식 억제제, 세포사멸제 뿐 아니라 백혈병 치료제로 이용될 수 있으며, 방사선 치료와 더불어 상기 추출물을 같이 처리하여 백혈병 치료 효과를 더욱 높일 수 있다.The present invention relates to the use of Duchesnea chrysantha extract and Ganoderma lucidum extract and mixtures thereof. Specifically, polysaccharides are extracted from snake berries and triterpenoids from Ganoderma lucidum using solvent extraction. And the effect of each extract and extract mixture promoting apoptosis of cancer cells; Effect of inhibiting cancer cell proliferation; And when treated with radiation, the effect of promoting the apoptosis of cancer cells at a lower dose is excellent, can be used as a therapeutic agent for leukemia as well as cell proliferation inhibitors, apoptosis, and treatment with the extract together with radiation therapy It is possible to further increase the treatment effect of leukemia.
Description
본 발명은 뱀딸기 (Duchesnea chrysantha) 추출물, 영지버섯 (Ganoderma lucidum) 추출물 및 상기 추출물의 혼합물의 용도 관한 것이다.The present invention relates to the use of Duchesnea chrysantha extract, Ganoderma lucidum extract, and mixtures of the extracts.
모든 생명체의 세포들은 기본적으로 유한한 생명을 갖고 생성된 후 성장, 분화를 거쳐 사멸한다. 또한 세포에 심한 손상이 생기거나, 더 이상 생존할 필요가 없을 경우에 세포 자체 내의 내재적인 프로그램이 활성화되어 스스로 파괴하는 작용을 한다. 이와 같은 프로그램된 세포의 죽음의 과정을 세포사멸 (apoptosis)이라고 칭한다. Cells of all living things are basically created with finite life, grow, differentiate and die. In addition, when a cell is severely damaged or no longer needs to survive, the intrinsic program in the cell itself is activated to destroy itself. This process of programmed cell death is called apoptosis.
세포의 예정된 사멸 과정 중 특히 면역체계에 관련된 세포들은 세포사멸 수용체 (death receptor)를 통하여, 또한 DNA 손상이나 스트레스와 관련된 세포들은 미토콘드리아를 통하여 죽음의 신호를 전달하고 증폭시키는 것으로 알려져 있다. 이 두 가지 경로 모두 세포사멸 자극에 의해서 공통적으로 카스파제-3이 활성화될 수 있으며, 활성화된 카스파제-3은 여러 종류의 세포내 단백질을 분해하고 세포사멸의 전형적인 특징인 DNA 절편화 (fragmentation)와 형태적인 변화를 유도함으로써 더 이상 쓸모가 없거나 비정상적인 세포들을 제거하는 세포사멸의 매개체로 작용하는 것으로 알려져 있다.During the predetermined killing process, cells related to the immune system, in particular, are known to transmit and amplify death signals through death receptors, and cells related to DNA damage or stress, through the mitochondria. Both of these pathways can be commonly caspase-3 activated by apoptosis stimulation, and activated caspase-3 breaks down various intracellular proteins and DNA fragmentation is a hallmark of apoptosis. It is known that it acts as a mediator of apoptosis by removing the unwanted or abnormal cells by inducing morphological changes.
세포사멸은 항상성 유지와 발생, 세포 분화 등 생명체의 여러 다양한 생리작용을 정상적으로 유지하는데 중요한 역할을 할 뿐만 아니라, 바이러스가 감염된 세포나 암 등과 같은 악성 세포를 제거하는 체내의 주요 방어 기작으로도 작용한다. 따라서 이들 세포사멸 과정의 조절의 실패가 병리적 원인이 되는 퇴행성 신경질환, 면역계 질환 및 암 등 여러 질병들의 질환에 관련된 예방 및 치료방법의 새로운 방안으로써 약용 식물을 이용한 신기능성 식품의약 등이 개발되고 있다. Apoptosis not only plays an important role in maintaining various physiological functions of life such as homeostasis and development and cell differentiation, but also acts as a major defense mechanism in the body to remove malignant cells such as virus infected cells or cancer. . Therefore, as a new way of preventing and treating diseases related to various diseases such as neurodegenerative diseases, immune system diseases and cancers, in which the failure of the regulation of apoptosis process is a pathological cause, new functional food medicines using medicinal plants are developed. have.
한편, 뱀딸기 (Duchesnea chrysantha)는 우리나라 전지역에서 흔히 서식 분포하고 있는 다년생 초본으로서 일년 내내 재배 가능할 뿐 아니라 민간 약물로 이용되어 왔으며, 본 발명자는 상기 뱀딸기의 줄기와 잎으로부터 용매추출법으로 다당류가 함유된 분획을 얻었으며, 얻어진 분획이 항산화 효과가 우수하여 항산화제로서의 용도를 특허출원한 바 있다 (국내특허출원 제 99-48507호). 또한 뱀딸기의 과실이나 씨로부터 얻은 추출물이 모발강장제와 피부 및 외상 (trauma) 약의 재료로서 특허출원된 바 있으며 (JP-09009952, JP-0318015, JP-03188008), 뱀딸기 초본으로부터 분리한 페놀 화합물들이 항종양 기능을 나타내는 것이 이미 밝혀진 바 있다 (Lee, I.R. and Y.H., Arch. Pharm. Res., 9 (1):1-4, 1986; Lee, I.R. and Yang M.Y, Arch. Pharm. Res., 17 (6):476-479, 1994).On the other hand, Duchesnea chrysantha is a perennial herb commonly inhabited throughout the country and can be grown throughout the year, and has been used as a folk drug. The obtained fraction has an excellent antioxidant effect and has been patented for use as an antioxidant (Domestic Patent Application No. 99-48507). In addition, extracts from the fruit or seeds of the snakeberry have been patented as ingredients for hair tonics and skin and trauma drugs (JP-09009952, JP-0318015, JP-03188008), and phenolic compounds isolated from the snakeberry herb. It has already been shown to exhibit anti-tumor function (Lee, IR and YH, Arch. Pharm. Res ., 9 (1): 1-4, 1986; Lee, IR and Yang MY, Arch. Pharm. Res ., 17 (6): 476-479, 1994).
또한 예로부터 귀중한 민간 치료약으로 사용되어 왔고, 최근에는 인공 재배에 성공한 영지버섯 (Ganoderma Iucidum)은 독성이나 부작용이 거의 없는 항암 또는 면역증강제 (Wang S-Y et al, Int.J.Cancer, 70:699-705, 1997)로서의 작용 및 항방사선 및 항산화기능 (Kim, K.C and I.G.,Int.J.Mol.Med.,4 (3):273-277, 1999) 이 있는 것으로 알려져 있다.In addition, Ganoderma Iucidum , which has been used as a valuable folk remedy since ancient times, and recently succeeded in artificial cultivation, is an anticancer or immunostimulant with little toxicity or side effects (Wang SY et al, Int . J. Cancer , 70: 699- 705, 1997) and anti-radiation and antioxidant functions (Kim, KC and IG, Int . J. Mol . Med ., 4 (3): 273-277, 1999).
그러나 상기 뱀딸기 추출물 및 영지버섯 추출물은 아직까지 세포사멸에 대한 연구는 보고된 바 없다.However, the snakeberry extract and Ganoderma lucidum extract have not been reported for apoptosis.
이에 본 발명자는 용매 추출법을 통해 뱀딸기 및 영지버섯 각각으로부터 추출물을 분리하고, 상기 추출물이 암 세포 증식을 억제하고, 세포사멸을 촉진하며 방사선과 함께 처리하면 방사선의 선량을 낮추면서 세포사멸을 더욱 증진시킬 수 있고, 또한 상기 추출물을 혼합하여 사용하는 경우 그 효과가 극대화가 됨을 알아내어 본 발명을 완성하였다.Therefore, the present inventors separate the extract from each of the raspberry and ganoderma lucidum through a solvent extraction method, and the extract inhibits cancer cell proliferation, promotes cell death, and further enhances cell death while lowering the dose of radiation when treated with radiation. It can also be found that the effect is maximized when used in combination with the extract to complete the present invention.
본 발명의 목적은 뱀딸기의 추출물, 영지버섯 추출물 및 이들의 혼합물의 용도를 제공하는 것이다. It is an object of the present invention to provide the use of the extract of snakeberry, ganoderma lucidum extract and mixtures thereof.
상기한 목적을 달성하기 위하여, 본 발명은 용매 추출법을 얻어진 뱀딸기 또는 영지버섯으로부터 분리한 추출물 및 이들의 혼합물의 용도를 제공한다.In order to achieve the above object, the present invention provides a use of an extract and a mixture thereof separated from the snakeberry or ganoderma lucidum obtained by the solvent extraction method.
이하 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 뱀딸기 추출물은 도 1에 도시한 바와 같은 순서로 수행하여 얻어진다.Mock strawberry extract of the present invention is obtained by performing the same order as shown in Fig.
구체적으로,1) 뱀딸기를 물에 넣고 고온에서 추출한 후 농축시키고; Specifically, 1) the snake berries in water and extracted at high temperature and then concentrated;
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2) 얻어진 잔사에 유기용매를 가하여 수층/유기용매층으로 분리하고;2) adding an organic solvent to the obtained residue and separating the aqueous layer / organic solvent layer;
3) 얻어진 수층에 알콜을 가하여 알콜 분획물을 얻고; 및3) alcohol was added to the obtained aqueous layer to obtain an alcohol fraction; And
4) 상기 알콜 분획물에 추가로 알콜을 가하여 침전시켜 뱀딸기 추출물을 얻는다.4) Further alcohol is added to the alcohol fraction to precipitate to obtain a snakeberry extract.
상기 추출과정에서 사용되는 유기용매는 디클로로메탄, 클로로포름, 테트라하이드로클로라이드 등 할로겐화 탄화수소가 사용가능하며, 바람직하기로는 클로로포름을 사용한다.As the organic solvent used in the extraction process, halogenated hydrocarbons such as dichloromethane, chloroform and tetrahydrochloride can be used. Preferably, chloroform is used.
알콜은 C1∼C4의 저급 알콜, 바람직하기로는 메탄올, 에탄올, 프로판올이 사용되고, 가장 바람직하기로는 에탄올이 사용된다.The alcohol is C 1 -C 4 lower alcohols, preferably methanol, ethanol, propanol, and most preferably ethanol.
추출공정에서 사용되는 알콜의 함량은 추출효과를 최대로 하기 위하여 단계 3에서는 잔사에 대하여 부피비로 0.5∼1.5 배, 바람직하기로는 동일한 부피로 사용하며, 단계 4에서는 알콜 분획물에 대하여 부피비로 2∼4배, 바람직하기로는 3배로 첨가한다.In order to maximize the extraction effect, the alcohol content used in the extraction process is 0.5 to 1.5 times by volume, preferably the same volume as the residue in step 3, and 2 to 4 by volume relative to the alcohol fraction in step 4. Pear, preferably 3 times, is added.
상기 침전된 뱀딸기 추출물은 이 분야에서 널리 알려져 있는 방법에 의해 후처리하여 정제된 상태로 얻을 수 있으며, 일예로 증류수를 가한 후 겔 투과 크로마토그래피를 통과시켜 정제할 수 있다.The precipitated snakeberry extract can be obtained in a purified state by post-treatment by a method well known in the art, for example, can be purified by passing through gel permeation chromatography after adding distilled water.
또한 본 발명의 영지버섯 추출물은 도 2에 도시한 바와 같은 순서로 수행하여 영지버섯 자실체로부터 얻어지며 고미성분 (triterpenoid)을 포함한다.In addition, the Ganoderma lucidum mushroom extract of the present invention is obtained from Ganoderma lucidum fruiting body by carrying out in the order as shown in FIG . 2 and contains a triterpenoid.
구체적으로,1) 영지버섯을 물에 넣고 고온에서 추출하고; 2) 얻어진 상기 추출물에 알콜을 가하여 상등액을 얻고;3) 상기 상등액을 농축한 후 유기용매를 가하여 수층/유기용매층으로 분리하고; 및4) 상기 유기용매층을 감압농축하여 용매를 제거하여 영지버섯 추출물을 얻는다.Specifically, 1) putting the Ganoderma lucidum in water and extracted at high temperature; 2) adding an alcohol to the obtained extract to obtain a supernatant; 3) concentrating the supernatant and then adding an organic solvent to separate the aqueous layer / organic solvent layer; And 4) concentrating the organic solvent layer under reduced pressure to remove the solvent to obtain Ganoderma lucidum extract.
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상기 추출공정에서 사용되는 유기용매 및 알콜은 전술한 바의 뱀딸기 추출공정에서 언급된 용매가 사용가능하고, 바람직하기로는 유기용매는 클로로포름을, 알콜은 에탄올을 사용한다.As the organic solvent and the alcohol used in the extraction process, the solvent mentioned in the above-mentioned snakeberry extraction process can be used. Preferably, the organic solvent uses chloroform and the alcohol uses ethanol.
알콜의 함량은 추출효과를 최대로 하기 위하여 단계 2에서는 잔사에 대하여 부피비로 2∼4 배, 바람직하기로는 4배로 사용한다. In order to maximize the extraction effect, the alcohol content is used in 2 to 4 times, preferably 4 times, by volume ratio with respect to the residue.
단계 3에서 유기용매의 함량은 알콜 분획물에 대하여 부피비로 1∼2배, 바람직하기로는 1.5배로 첨가한다.In step 3, the content of the organic solvent is added 1 to 2 times, preferably 1.5 times, by volume relative to the alcohol fraction.
상기 농축된 영지버섯 추출물은 이 분야에서 널리 알려져 있는 방법에 의해 후처리하여 정제된 상태로 얻을 수 있으며, 일예로 유기용매를 가한 후 겔 투과 크로마토그래피를 통과시켜 정제할 수 있다.The concentrated ganoderma lucidum extract may be obtained by post-treatment by a method well known in the art, and may be purified by, for example, adding an organic solvent and passing through gel permeation chromatography.
전술한 바에서 얻어진 뱀딸기 추출물 및 영지버섯 추출물은 통상적인 방법으로 서로 혼합하여 사용 가능하며, 0.5:1∼1:0.5의 비율로 한다.Snakeberry extract and Ganoderma lucidum extract obtained in the above bar can be used by mixing with each other in a conventional manner, the ratio of 0.5: 1 to 1: 0.5.
또한 본 발명에서는 상기 얻어진 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물을 유효성분으로 함유하는 세포증식 억제용 약학적 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for inhibiting cell proliferation containing the obtained snakeberry extract, Ganoderma lucidum extract and mixtures thereof as an active ingredient.
바람직한 실시예에 따르면, 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물의 암세포 증식억제 효과를 알아보기 위하여, 백혈병 세포 HL-60 및 U937을 배양하고 상기 추출물 각각을 첨가한 후 상기 세포의 생존률 (viability)을 측정한 결과 (실험예 1 및 2), 도 3a 및 도 3b에 도시한 바와 같이 추출물을 첨가하지 않은 대조군 (A)과 비교하여 본 발명의 추출물 (B, C, D)로 처리한 경우 HL-60 및 U937 세포의 생존률이 감소함을 알 수 있었고, 특히 상기 추출물을 혼합한 경우 (D) 생존율이 크게 감소함을 알 수 있었다.According to a preferred embodiment, in order to determine the cancer cell proliferation inhibitory effect of the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention, the survival rate of the cells after culturing leukemia cells HL-60 and U937 and adding each of the extracts As a result of measuring the viability (Experimental Examples 1 and 2), as shown in FIGS . 3A and 3B , the extract (B, C, D) of the present invention was treated as compared to the control (A) without the extract. In one case, it was found that the survival rate of HL-60 and U937 cells was reduced, and in particular, when the extract was mixed, (D) survival rate was greatly reduced.
또한 본 발명에서는 상기 얻어진 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물을 유효성분으로 함유하는 세포사멸 증진용 약학적 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for enhancing apoptosis containing the obtained snakeberry extract, Ganoderma lucidum extract and mixtures thereof as an active ingredient.
바람직한 실시예에 따르면, 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물의 암세포 세포사멸 (apoptosis) 효과를 알아보기 위하여, 백혈병 세포 HL-60 및 U937을 24h, 48h 및 72h 동안 배양하고 상기 추출물 각각을 첨가한 후 유세포 측정기 (flow cytometer)로 세포사멸을 측정한 결과 (실험예 3 및 4), 도 4 및 도 5에 도시한 바와 같이 추출물을 첨가하지 않은 대조군 (A)과 비교하여 본 발명의 추출물 (B, C, D)로 처리한 경우 HL-60 및 U937 세포의 세포사멸이 더욱 강하게 나타나고, 특히 상기 추출물을 혼합한 경우 (D) 세포사멸이 유도되는 동시에 가속화됨을 알 수 있었다.According to a preferred embodiment, in order to determine the cancer cell apoptosis (apoptosis) effect of the snakeberry extract, Ganoderma lucidum extract of the present invention and mixtures thereof, leukemia cells HL-60 and U937 for 24h, 48h and 72h and the extract After adding each of them, apoptosis was measured using a flow cytometer (Experimental Examples 3 and 4), as shown in FIGS . 4 and 5 , compared to the control group (A) without the extract as shown in FIGS . 4 and 5 . When treated with extracts (B, C, D) of HL-60 and U937 cells apoptosis appeared to be stronger, especially when the extract was mixed (D) was found to be induced and accelerated at the same time.
본 발명에 따른 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물의 세포사멸효과는 방사선을 동시에 조사한 경우 그 효과가 현저히 증가함을 알 수 있다.The apoptosis effect of the snakeberry extract, Ganoderma lucidum extract and mixtures thereof according to the present invention can be seen that the effect is significantly increased when irradiated at the same time.
바람직한 실시예에 따르면, 뱀딸기 및 영지버섯 추출물의 혼합물의 방사선 조사에 따른 세포사멸 효과를 알아보기 위하여, 백혈병 세포 HL-60 및 U937을 2 Gy로 감마선을 조사후 24h, 48h 및 72h 동안 배양하고 상기 추출물 각각을 첨가한 후 유세포 측정기로 세포사멸을 측정한 결과 (실험예 5 및 6), 도 6 및 도 7에 도시한 바와 같이 방사선만 조사한 대조군과 비교하여 본 발명의 뱀딸기 및 영지버섯 추출물의 혼합물 및 방사선을 동시에 처리한 경우, 세포사멸효과가 월등히 우수함을 알 수 있었다.According to a preferred embodiment, in order to determine the apoptosis effect of the mixture of the snakeberry and Ganoderma lucidum extract upon irradiation, leukemia cells HL-60 and U937 were irradiated with gamma rays at 2 Gy for 24h, 48h and 72h and then As a result of measuring the apoptosis by flow cytometry after each addition of the extract (Experimental Examples 5 and 6), as shown in Figure 6 and Figure 7 compared to the control group irradiated with only the mixture of the snakeberry and Ganoderma lucidum extract of the present invention And when treated simultaneously with the radiation, the cell death effect was found to be excellent.
또한 방사선을 조사하지 않은 도 4의 D 및 도 5의 D와 비교하면, 사멸된 세포 비율이 증가함을 알 수 있는 바, 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물의 세포사멸 효과는 방사선을 조사하는 경우 더욱 효과적이며, 방사선의 선량을 낮추면서 세포사멸을 증진시킴을 알 수 있다. 상기 방사선은 감마선 또는 전자선으로 치료에 사용되는 통상적인 것이 사용된다.In addition, compared with D of FIG . 4 and D of FIG. 5 which did not irradiate, it can be seen that the percentage of dead cells increased. The apoptosis effect of the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof of the present invention It can be seen that it is more effective when irradiated with radiation and promotes cell death while lowering the dose of radiation. The radiation is a conventional one used for treatment with gamma rays or electron beams.
이러한 효과에 의거하여 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물은 또한 백혈병 치료용 약학적 조성물로 유용하게 사용할 수 있다. 특히, 종래 백혈병 치료시 방사선 치료가 병행됨을 고려하여 볼 때 본 발명의 추출물은 항암 치료제로서 적합하게 사용할 수 있다. Based on these effects, the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof of the present invention can also be usefully used as pharmaceutical compositions for treating leukemia. In particular, in view of the parallel treatment of radiation in the treatment of conventional leukemia, the extract of the present invention can be suitably used as an anticancer agent.
따라서, 본 발명은 세포사멸 및 세포증식억제 효과가 우수한 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물로 이루어진 군에서 선택된 추출물을 방사선 치료와 병행하는 항암 치료방법을 제공할 수 있다.Accordingly, the present invention may provide an anticancer treatment method in which the extract selected from the group consisting of snakeberry extract, ganoderma lucidum extract, and mixtures thereof having excellent cell death and cell proliferation inhibitory effects in combination with radiation therapy.
또한 바람직한 실시예에 따르면, 세포사멸에 관계하여 신호 전달 및 증폭의 역할을 하는 미토콘드리아에 대한 막 전위를 측정한 결과 (실험예 7 및 8), 도 8 및 도 9에 도시한 바와 같이 추출물을 처리하지 않은 대조군과 비교하여 본 발명의 뱀딸기 (B), 영지버섯 추출물 (C)을 처리한 경우 미토콘드리아 막 전위가 감소 (세포사멸 과정의 초기 변화의 하나로서 미토콘드리아 막 전위가 감소함)하였으며, 특히 상기 추출물의 혼합물 (D)이 그 감소 효과가 월등히 우수함을 알 수 있었다.According to a preferred embodiment, as a result of the film measured the potential of the mitochondria relative to the cell death that acts as a signal transduction and amplification (Example 7) and (8) processing the extract as shown in Fig. 8 and 9 The mitochondrial membrane potential was reduced (the mitochondrial membrane potential was reduced as one of the initial changes in the apoptosis process) when the snakeberry (B) and ganoderma lucidum extract (C) of the present invention were treated as compared to the control group. It was found that the mixture (D) of the extract was much better in its reducing effect.
또한 바람직한 또다른 실시예에 따르면, 세포사멸의 매개체로 알려져 있는 카스파제-3의 활성을 백혈병 세포인 HL-60 및 U937에 대하여 측정한 결과 (실험예 9), 도 10에 도시한 바와 같이 추출물을 처리하지 않은 대조군과 비교하여 본 발명의 뱀딸기 및 영지버섯 추출물의 혼합물을 동시에 처리한 경우 상기 세포 모두 카스파제-3의 활성이 증가하여 그 효과가 월등히 우수함을 알 수 있었다.According to another preferred embodiment, the activity of caspase-3, a mediator of apoptosis, was measured against leukemia cells HL-60 and U937 (Experimental Example 9), as shown in FIG . 10 . Compared to the control group did not process the treatment of the mixture of the snakeberry and Ganoderma lucidum extract of the present invention at the same time it was found that the effect of the caspase-3 increased in all of the cells is excellent.
상술한 바와 같이, 본 발명의 뱀딸기의 잎과 줄기부위로부터 얻은 추출물과 영지버섯의 자실체로부터 얻은 추출물 및 이들의 혼합물을 처리했을 경우, 암 세포의 증식을 억제시키고 세포사멸 기능을 증진시키며 방사선과 함께 처리할 경우 더 낮은 선량의 방사선으로 백혈병 세포의 증식 억제 및 세포사멸을 촉진시키는 작용을 한다. 따라서, 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물을 포함하는 조성물은 세포사멸 증진제 및 세포증식 억제제로 유용하며, 방사선 처리와 더불어 수행하는 치료방법 및 백혈병 치료제로서 유용하게 사용될 수 있다.As described above, when the extract obtained from the leaves and stems of the snakeberry of the present invention and the extract obtained from the fruiting body of Ganoderma lucidum and mixtures thereof, inhibits the proliferation of cancer cells, enhances apoptosis and is treated with radiation In this case, the lower dose of radiation acts to inhibit the proliferation and apoptosis of leukemia cells. Therefore, the composition comprising the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention is useful as apoptosis enhancers and cell proliferation inhibitors, and can be usefully used as a therapeutic method and radiation therapy for leukemia.
본 발명의 추출물은 임상투여시에 경구 또는 비경구로 투여가 가능하며 일반적인 의약품제제의 형태로 사용될 수 있다.The extract of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.
즉, 상기 추출물을 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. That is, the extract can 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., which are commonly used, are used. It is prepared by.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose) 또는 락토오스 (Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.
경구투여를 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. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have.
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 사용 될 수 있다.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 a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
일반적으로 의약품에 있어서, 본 발명에 의한 추출물의 유효 용량은 뱀딸기 추출물의 경우 성인 기준 1 g/day이고, 하루 1∼3회, 영지버섯 추출물의 경우 성인 기준 0.85 g/day이고, 하루 1∼3회, 및 상기 추출물의 혼합물의 경우 성인 기준 0.8 g/day이고, 하루 1∼3회 투여될 수 있다. 그러나 상기 투여약량은 변화시킬 필요가 있으며, 특히 치료할 객체의 체질 및 특이성 및 체중, 질병의 종류 및 심도, 제형의 성질, 의약품 투여의 성질 및 투여기간 또는 간격을 고려하여 변화시킬 수 있다.In general, in the pharmaceutical product, the effective dose of the extract according to the present invention is 1 g / day for adults, 1 to 3 times a day for the snakeberry extract, 0.85 g / day for adults for the Ganoderma lucidum extract, and 1 to 3 per day. Times, and the mixture of the extract is 0.8 g / day for adults, may be administered 1 to 3 times a day. However, the dosage amount needs to be changed, and in particular, may be changed in consideration of the constitution and specificity and weight of the object to be treated, the type and depth of the disease, the nature of the formulation, the nature of the drug administration and the duration or interval of administration.
또한 본 발명에서는 상기 얻어진 뱀딸기 추출물 및 영지버섯 추출물의 혼합물을 유효성분으로 함유하는 기능성 식품, 건강보조 식품 또는 특수영양식품을 제공한다. In another aspect, the present invention provides a functional food, health supplement food or special nutrition products containing a mixture of the obtained snakeberry extract and Ganoderma lucidum extract as an active ingredient.
본 명세서에서 '기능성 식품'이란, 일반 식품에 상기 뱀딸기 추출물 및 영지버섯 추출물의 혼합물을 첨가함으로써 일반 식품의 기능성을 향상시킨 식품을 의미한다. As used herein, the term "functional food" means a food product having improved functionality of a general food product by adding a mixture of the snakeberry extract and Ganoderma lucidum extract to the general food product.
기능성 식품과 구별하여, 본 명세서에서 '건강보조식품' 또는 '특수영양식품'이란, 상기 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물을 일반식품에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 건강식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하는 것으로 해석된다. 즉, 건강보조식품은 특정의 약리기능을 가지는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기복용시 발생할 수 있는 부작용등이 없는 장점이 있다.Distinguishing from functional foods, the term 'health supplement' or 'special dietary supplement' herein means adding the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof to general foods, or by encapsulating, powdering, suspension, etc. As a health food, ingesting it means that it has a specific health effect. That is, the health supplement means that it has a specific pharmacological function, but unlike general medicine, it has the advantage that there are no side effects that may occur when the long-term use of the drug as a food raw material.
본 발명에 따른 추출 혼합물을 함유하는 기능성 식품, 건강보조식품 및 특수건강식품의 함량은 사용되는 식품군에 따라 다양하게 변화시킬 수 있으며, 그 함량은 상기 약학적 조성물로의 용도시 측정된 독성 범위내에서 수행한다.The content of functional foods, dietary supplements and specialty health foods containing the extract mixture according to the present invention may vary depending on the food group used, the content of which is within the range of toxicity measured in the use of the pharmaceutical composition. Perform on
이하 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
그러나 하기 실시예는 하나의 예시일 뿐 본 발명의 범위가 이에 한정되는 것은 아니다.However, the following examples are only examples and the scope of the present invention is not limited thereto.
<실시예 1> 뱀딸기 추출물의 분리Example 1 Isolation of Snakeberry Extract
뱀딸기의 잎과 줄기부위 50 g 건조중량을 증류수 300 ml에서 1시간 끓였다. 이 증류수 추출물에 같은 부피의 클로로포름을 첨가하여 1시간 동안 흔들어 혼합한 다음 4 oC에서 8시간 방치하였다. 이어서 증류수 층을 취하고 동일 부피의 에탄올을 첨가하여 침전물을 제거하였다. 상등액에 다시 3배 부피의 에탄올을 첨가하고 10,000 g로 원심 분리하여 획득한 침전물을 시료를 사용하였다 (도 1 참조).50 g dry weight of leaf and stem of snakeberry was boiled in 300 ml of distilled water for 1 hour. An equal volume of chloroform was added to the distilled water extract, shaken for 1 hour, mixed, and left at 4 ° C. for 8 hours. The layer of distilled water was then taken and an equal volume of ethanol was added to remove the precipitate. Three times the volume of ethanol was added to the supernatant again, and the precipitate obtained by centrifugation at 10,000 g was used as a sample (see FIG. 1 ).
얻어진 시료를 동결 건조시켜 정확한 무게를 측정한 후 추출물의 암세포의 증식 억제 및 세포 사멸 기능을 조사하였다.The obtained sample was lyophilized to determine the correct weight, and then the function of the extract was investigated to inhibit the proliferation and apoptosis of cancer cells.
<실시예 2> 영지버섯 추출물의 분리Example 2 Isolation of Ganoderma lucidum Extract
영지버섯 자실체 50 g 건조중량을 증류수 2 L에서 2시간 끓였다. 얻어진 증류수 추출물에 4배 부피의 에탄올을 첨가하여 4 oC에서 하룻밤 동안 방치한 다음 10,000 g로 원심 분리하여 상등액을 취하여 증발기로 감압 농축시켰다. 이 농축액에 1.5배 부피의 클로로포름을 첨가하여 1시간 동안 흔들어 혼합한 다음 방치하였다. 수용액 층은 제거하고 클로로포름 층을 취하여 증발기로 감압 농축시킨 시료를 사용하였다 (도 2 참조).The dried weight of Ganoderma lucidum fruit body 50 g was boiled in 2 L of distilled water for 2 hours. Four times the volume of ethanol was added to the obtained distilled water extract, and the mixture was allowed to stand at 4 ° C. overnight, then centrifuged at 10,000 g to obtain a supernatant, and concentrated under reduced pressure with an evaporator. 1.5-fold volume of chloroform was added to the concentrate, shaken for 1 hour, and allowed to stand. The aqueous layer was removed, the chloroform layer was taken and a sample was concentrated under reduced pressure with an evaporator (see FIG. 2 ).
얻어진 시료를 동결 건조시켜 정확한 무게를 측정한 후 추출물의 암세포의 증식 억제 및 세포 사멸 기능을 조사하였다.The obtained sample was lyophilized to determine the correct weight, and then the function of the extract was investigated to inhibit the proliferation and apoptosis of cancer cells.
<제제예 1> 주사액제의 제조방법Preparation Example 1 Preparation of Injection Solution
유효성분을 함유하는 주사액제는 다음과 같은 방법으로 제조하였다. Injection solution containing the active ingredient was prepared by the following method.
뱀딸기 추출물 1 g, 염화나트륨 0.6 g 및 아스코르브산 0.1 g을 증류수에 용해시켜서 100 ㎖을 만들었다. 이 용액을 병에 넣고 20℃에서 30 분간 가열하여 멸균시켰다.100 g of 1 g of snakeberry extract, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.
상기 주사액제의 구성성분은 다음과 같다. The components of the injection solution are as follows.
뱀딸기 추출물··················1 gSnakeberry Extract ················ 1 g
염화나트륨···················0.6 gSodium Chloride ・ ・ ・ ・ 0.6 g
아스코르브산··················0.1 g0.1 g of ascorbic acid
증류수·····················정량Distilled water ··················
<제제예 2> 정제의 제조방법Preparation Example 2 Preparation of Tablet
유효성분 함유된 정제는 다음과 같은 방법으로 제조한다.Tablets containing the active ingredient are prepared by the following method.
영지버섯 추출물 250 g을 락토오스 175.9 g, 감자전분 180 g 및 콜로이드성 규산 32 g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160 g, 활석 50 g 및 스테아린산 마그네슘 5 g을 첨가해서 얻은 혼합물을 정제로 만들었다. 250 g of ganoderma lucidum extract was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.
상기 정제의 구성성분은 다음과 같다. The components of the tablet are as follows.
영지버섯 추출물················ 250 gGanoderma lucidum mushroom extract ... 250 g
락토오스 ···················175.9 gLactose ········ 175.9 g
감자전분 ····················180 gPotato starch ········· 180 g
콜로이드성 규산 ················ 32 gColloidal silicic acid 32 g
10% 젤라틴 용액10% gelatin solution
감자전분 ····················160 gPotato starch · 160 g
활석 ······················ 50 gTalc · 50 g
스테아르산 마그네슘 ··············· 5 Magnesium stearate ·········· 5
<제제예 3> 캡슐의 제조방법Preparation Example 3 Manufacturing Method of Capsule
뱀딸기 추출물 및 영지버섯 추출물을 1:1 부피비로 혼합하여 연질 캡슐에 충진시켜 캡슐화하였다.Snakeberry extract and Ganoderma lucidum extract were mixed in a 1: 1 volume ratio to encapsulate the soft capsule.
<실험예 1> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 증식 억제 작용 : 백혈병 세포 HL-60의 생존률 (viability) 감소 효과Experimental Example 1 Effect of Snakeberry Extract and Ganoderma Lucidum Extract on Cancer Cell Proliferation Inhibition: Viability Reduction Effect of Leukemia Cell HL-60
상기 실시예에서 얻은 뱀딸기와 영지버섯의 용매추출물이 암세포의 증식 억제 효과를 가지는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 그 영향을 알아보았다.In order to determine whether the solvent extracts of the snakeberry and Ganoderma lucidum obtained in the above examples had a cancer cell proliferation inhibitory effect, the effects of the extracts were included in the culture medium and then examined.
구체적으로, 10％ Fetal Bovine Serum (송아지 태아의 혈액에서 추출한 혈청, Sigma)이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 HL-60을 2 x 105 세포/ml의 농도로 접종시키고 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 0.4% trypan blue로 염색하여 도립현미경 (inverted microscope) 상에서 혈구계산기 (hemocytometer)를 사용하여 세포수를 측정하였다.Specifically, leukemia cells HL-60 were inoculated at a concentration of 2 x 10 5 cells / ml in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum (serum extracted from calf fetal blood, Sigma). After 24 hours, 48 hours and 72 hours of incubation at (CO 2 5%, 37 ° C.), the cells were measured using a hemocytometer on a inverted microscope by staining with 0.4% trypan blue.
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 생존률을 비교하였다. 그 결과, 두 가지 추출물이 함께 처리된 경우 HL-60 세포의 생존률이 크게 감소하므로, 이들 추출물의 혼용은 백혈병 세포 HL-60의 증식을 억제하는 효과를 가짐이 확인되었다 (도 3a 참조).To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 100 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum extract) Survival was compared with the control group without the extract. As a result, since the survival rate of HL-60 cells is greatly reduced when the two extracts are treated together, it was confirmed that the combination of these extracts has the effect of inhibiting the proliferation of leukemia cells HL-60 (see Fig. 3a ).
<실험예 2> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 증식 억제 작용 : 백혈병 세포 U937의 생존률 (viability) 감소 효과Experimental Example 2 Effect of Snakeberry Extract and Ganoderma Lucidum Extract on Cancer Cell Proliferation Inhibition: Viability Reduction Effect of Leukemia Cell U937
상기 실시예에서 얻은 뱀딸기와 영지버섯의 용매추출물이 암세포의 증식 억제 효과를 가지는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 그 영향을 알아보았다.In order to determine whether the solvent extracts of the snakeberry and Ganoderma lucidum obtained in the above examples had a cancer cell proliferation inhibitory effect, the effects of the extracts were included in the culture medium and then examined.
구체적으로, 10％ Fetal Bovine Serum (송아지 태아의 혈액에서 추출한 혈청, Sigma)이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 U937을 2 x 105 세포/ml의 농도로 접종시키고 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 0.4% trypan blue로 염색하여 도립현미경 (inverted microscope) 상에서 혈구계산기 (hemocytometer)를 사용하여 세포수를 측정하였다.Specifically, the leukemia cells U937 were inoculated at a concentration of 2 × 10 5 cells / ml in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum (serum extracted from calf fetal blood, Sigma), followed by incubation (CO 2 5%, 37 ℃) 24 hours, 48 hours, 72 hours after incubation with 0.4% trypan blue staining was measured by using a hemocytometer (hemocytometer) on an inverted microscope (inverted microscope).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 생존률을 비교하였다. 그 결과, 두 가지 추출물이 함께 처리된 경우U937 세포의 생존률이 크게 감소하므로, 이들 추출물의 혼용은 백혈병 세포 U937의 증식을 억제하는 효과를 가짐이 확인되었다 (도 3b 참조).To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 200 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum mushroom extract) Survival was compared with the control group without the extract. As a result, since the survival rate of U937 cells is greatly reduced when the two extracts are treated together, it was confirmed that the combination of these extracts has the effect of inhibiting the proliferation of leukemia cells U937 ( see Fig. 3b ).
<실험예 3> 뱀딸기 추출물과 영지버섯 추출물 혼용시 암세포의 세포사멸 (apoptosis) 효과Experimental Example 3 Apoptosis Effect of Cancer Cells When Mixed with Snakeberry Extract and Ganoderma Lucidum Extract
뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물이 암세포의 세포사멸 (apoptosis) 효과를 가지는지 확인하기 위해, 상기 추출물을 배지에 포함시켜 배양한 후 그 영향을 분석하였다. To determine whether the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof have an apoptosis effect of cancer cells, the extract was included in the culture medium and then analyzed.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 HL-60을 2 x 105 세포/ml의 농도로 접종시키고 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 400 g로 10분간 원심 분리하였다. 침전된 세포에 저장성 형광색소 용액 (hypotonic fluorochrome solution; 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 혼합 용액에 propidium iodide가 50 ㎍/ml이 되도록 첨가) 1ml를 첨가하고 4℃에서 3-4시간 방치한 후 유세포 측정기 (Flow cytometer, Coulter)로 형광을 측정하였다.Specifically, leukemia cells HL-60 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 × 10 5 cells / ml and incubator (CO 2 5%, 37 ° C.), respectively. After incubation for 24 hours, 48 hours and 72 hours, centrifugation was performed at 400 g for 10 minutes. 1 ml of hypotonic fluorochrome solution (addition of propidium iodide to 50 μg / ml to 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 mixed solution) was added to the precipitated cells. After standing for 3-4 hours at ℃ fluorescence was measured by flow cytometer (Coulter).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 세포사멸 정도를 비교하기 위해 유세포 측정기 (Flow cytometer)를 사용하여 측정하였으며, 이러한 결과를 하기 표 1 및 도 4에 나타내었다.To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 100 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum extract) In order to compare the degree of apoptosis with the control group without addition of the extract to the medium was measured using a flow cytometer (Flow cytometer), these results are shown in Table 1 and FIG .
상기 표 1 및 도 4에 따르면, 대조군은 세포사멸이 거의 일어나지 않았고, 이에 비하여 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물은 세포사멸효과가 나타남을 알 수 있었으며, 특히 두 가지 추출물이 함께 처리된 경우 HL-60 세포의 세포사멸이 유도되는 동시에 세포사멸이 가속화된다는 사실이 확인되었다.According to Table 1 and Figure 4 , the control group was almost no cell death, compared to the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention showed a cell death effect, in particular two extracts together When treated, it was confirmed that apoptosis of HL-60 cells was induced and accelerated.
<실험예 4> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 증식 억제 작용 : 백혈병 세포 U937의 세포사멸 (apoptosis) 증진 효과Experimental Example 4 Effect of Snakeberry Extract and Ganoderma Lucidum Extract on Cancer Cell Proliferation Inhibition: Apoptosis Enhancing Effect of Leukemia Cell U937
뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물이 암세포의 세포사멸 (apoptosis) 효과를 가지는지 확인하기 위해, 상기 추출물을 배지에 포함시켜 배양한 후 그 영향을 분석하였다.To determine whether the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof have an apoptosis effect of cancer cells, the extract was included in the culture medium and then analyzed.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 U937을 2 x 105 세포/ml의 농도로 접종시키고 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 400 g로 10분간 원심 분리하였다. 침전된 세포에 저장성 형광색소 용액 (hypotonic fluorochrome solution; 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 혼합 용액에 propidium iodide가 50 ㎍/ml이 되도록 첨가) 1 ml를 첨가하고 4℃에서 3-4시간 방치한 후 유세포 측정기 (Flow cytometer, Coulter)로 형광을 측정하였다.Specifically, leukemia cells U937 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 × 10 5 cells / ml and incubator (CO 2 5%, 37 ° C.) for 24 hours each. After 48 hours, 72 hours of incubation was centrifuged for 10 minutes at 400 g. 1 ml of hypotonic fluorochrome solution (addition of propidium iodide to 50 μg / ml to 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 mixed solution) was added to the precipitated cells. After standing at 4 ° C. for 3-4 hours, fluorescence was measured by a flow cytometer (Coulter).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 세포사멸 정도를 비교하기 위해 유세포 측정기 (Flow cytometer)를 사용하여 측정하였으며, 이러한 결과를 하기 표 2 및 도 5에 나타내었다.To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 200 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum mushroom extract) In order to compare the degree of apoptosis with the control group without addition of the extract to the medium was measured using a flow cytometer (Flow cytometer), these results are shown in Table 2 and FIG .
상기 표 2 및 도 5에 따르면, 실험예 3의 결과와 마찬가지로 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물이 세포사멸효과가 나타남을 알 수 있었으며, 특히 두 가지 추출물이 함께 처리된 경우 U937 세포의 세포사멸이 유도되는 동시에 세포사멸이 가속화된다는 사실이 확인되었다.According to Table 2 and Figure 5 , as in the results of Experimental Example 3, it was found that the snakeberry extract, Ganoderma lucidum mushroom extract, and mixtures thereof showed apoptosis effect, especially when the two extracts were treated together U937 It has been confirmed that cell death is induced at the same time as cell death is induced.
<실험예 5> 뱀딸기 추출물과 영지버섯 추출물을 방사선과 함께 처리했을 때 백혈병 세포 HL-60의 세포사멸 (apoptosis) 효과Experimental Example 5 Apoptosis Effect of Leukemia Cell HL-60 When Snakeberry Extract and Ganoderma Lucidum Extract were Treated with Radiation
본 발명의 뱀딸기와 영지버섯의 용매추출물을 방사선과 함께 처리했을 때 세포사멸이 가속화되는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 그 영향을 알아보았다. In order to confirm that apoptosis is accelerated when the solvent extract of the snakeberry and ganoderma lucidum of the present invention is treated with radiation, the effects of the extracts were included in the culture medium and then examined.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 HL-60을 2 x 105 세포/ml의 농도로 접종시키고 감마선을 상온에서 10분 동안 2 Gy 조사시킨 다음 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 400 g로 10분간 원심 분리하였다. 침전된 세포에 저장성 형광색소 용액 (hypotonic fluorochrome solution; 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 혼합 용액에 propidium iodide가 50 ㎍/ml이 되도록 첨가) 1ml를 첨가하고 4℃에서 3∼4시간 방치한 후 유세포 측정기 (Flow cytometer, Coulter)로 형광을 측정하였다.Specifically, leukemia cells HL-60 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 x 10 5 cells / ml, and gamma rays were irradiated with 2 Gy at room temperature for 10 minutes. After 24 hours, 48 hours, 72 hours of incubation in a incubator (CO 2 5%, 37 ° C.), each was centrifuged at 400 g for 10 minutes. 1 ml of hypotonic fluorochrome solution (addition of propidium iodide to 50 μg / ml to 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 mixed solution) was added to the precipitated cells. After standing for 3 to 4 hours at ℃ fluorescence was measured by flow cytometer (Coulter).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 2 Gy의 감마선을 조사시킨 경우와 배지에 추출물을 첨가하지 않고 2 Gy의 감마선만을 조사시킨 대조군과 세포사멸 효과를 유세포 측정기로 분석하여 비교하였으며, 이러한 결과를 하기 표 3 및 도 6에 나타내었다.To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 100 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum extract) In the case of irradiating 2 Gy gamma rays and the control group irradiated with only 2 Gy gamma rays without adding an extract to the medium, the effect of apoptosis was analyzed by flow cytometry. The results are shown in Table 3 and FIG. 6 .
상기 표 3 및 도 6에 따르면, 두 가지 추출물을 방사선과 함께 처리했을 때 방사선만 처리한 경우보다 HL-60 세포의 세포사멸이 크게 증가하므로, 이들 추출물을 방사선과 함께 처리하면 방사선의 선량을 낮추면서 백혈병 세포 HL-60의 세포사멸을 증진시킬 수 있음이 확인되었다.According to Table 3 and FIG. 6 , when the two extracts were treated with radiation, apoptosis of HL-60 cells was significantly increased than when only the radiation was treated. When these extracts were treated with radiation, leukemia was reduced while lowering the dose of radiation. It was confirmed that the cell death of HL-60 could be enhanced.
<실험예 6> 뱀딸기 추출물과 영지버섯 추출물을 방사선과 함께 처리했을 때 백혈병 세포 U937의 세포사멸 (apoptosis) 증진 효과Experimental Example 6 Apoptosis Enhancement Effect of Leukemia Cell U937 When Treated with Snakeberry Extract and Ganoderma Lucidum Extract with Radiation
본 발명의 뱀딸기와 영지버섯의 용매추출물을 방사선과 함께 처리했을 때 세포사멸이 가속화되는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 그 영향을 알아보았다.In order to confirm that apoptosis is accelerated when the solvent extract of the snakeberry and ganoderma lucidum of the present invention is treated with radiation, the effects of the extracts were included in the culture medium and then examined.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 U937을 2 x 105 세포/ml의 농도로 접종시키고 감마선을 상온에서 10분 동안 2 Gy 조사시킨 다음 배양기 (CO2 5％, 37 ℃)에서 각각 24시간, 48시간, 72시간 배양 후 400 g로 10분간 원심 분리하였다. 침전된 세포에 저장성 형광색소 용액 (hypotonic fluorochrome solution; 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 혼합 용액에 propidium iodide가 50 ㎍/ml이 되도록 첨가) 1ml를 첨가하고 4℃에서 3-4시간 방치한 후 유세포 측정기 (Flow cytometer, Coulter)로 형광을 측정하였다.Specifically, inoculating leukemia cells U937 at a concentration of 2 × 10 5 cells / ml in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum, and irradiating gamma rays for 2 minutes at room temperature for 10 minutes, followed by incubation ( After 24 hours, 48 hours, 72 hours of incubation at 5% CO 2 , 37 ° C.), the cells were centrifuged at 400 g for 10 minutes. 1 ml of hypotonic fluorochrome solution (addition of propidium iodide to 50 μg / ml to 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, 0.1% TritonX-100 mixed solution) was added to the precipitated cells. After standing for 3-4 hours at ℃ fluorescence was measured by flow cytometer (Coulter).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 2 Gy의 감마선을 조사시킨 경우와 배지에 추출물을 첨가하지 않고 2 Gy의 감마선만을 조사시킨 대조군과 세포사멸 효과를 유세포 측정기로 분석하여 비교하였으며, 이러한 결과를 하기 표 4 및 도 7에 나타내었다.To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 200 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum mushroom extract) In the case of irradiating 2 Gy gamma rays and the control group irradiated with only 2 Gy gamma rays without adding an extract to the medium, the effect of cell death was analyzed by flow cytometry. The results are shown in Table 4 and FIG. 7 .
상기 표 4 및 도 7에 따르면, 두 가지 추출물을 방사선과 함께 처리했을 때 방사선만 처리한 경우보다 U937 세포의 세포사멸이 최대 4배 이상 증가하였으며, 상기 추출물을 방사선과 함께 처리하면 방사선의 선량을 낮추면서 백혈병 세포 U937의 세포사멸을 증진시킬 수 있음이 확인되었다.According to Table 4 and FIG. 7 , when the two extracts were treated with radiation, the apoptosis of U937 cells was increased up to four times or more than when only the radiation was treated. When the extract was treated with the radiation, the dose of radiation was lowered. It has been confirmed that the apoptosis of leukemia cells U937 can be enhanced.
<실험예 7> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 사멸 작용 : 백혈병 세포 HL-60의 미토콘드리아 막 전위 (mitochondrial membrane potential) 감소 효과Experimental Example 7 Cancer Cell Killing Effect of Snake Strawberry Extract and Ganoderma Lucidum Extract: Effect of Reducing Leukemia Cell HL-60 on Mitochondrial Membrane Potential
본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물이 암세포의 세포사멸 (apoptosis) 효과를 가지는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 미토콘드리아 막 전위를 분석하였다. In order to confirm whether the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention have apoptosis effect of cancer cells, these extracts were included in the culture medium and analyzed for mitochondrial membrane potential.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 HL-60을 2 x 105 세포/ml의 농도로 접종시킨 후 배양기 (CO2 5％, 37 ℃)에서 48시간 배양시킨 세포배양액 250 ㎕를 취하여 JC-1 (최종농도 10 ㎍/ml, Molecular Probes)을 첨가하고 37℃에서 암소에서 30분간 반응시켰다. 반응액을 400 g에서 10분간 원심 분리하여 침전된 세포를 PBS 500㎕에 현탁시킨 다음 유세포 측정기 (Flow cytometer)를 사용하여 JC-1의 응집형태 (aggregate form)인 적색 형광 강도 (red fluorescence intensity)를 측정하였다.Specifically, leukemia cells HL-60 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 × 10 5 cells / ml, followed by incubator (CO 2 5%, 37 ° C.). 250 µl of the cell culture cultured for 48 hours was taken, and JC-1 (final concentration 10 µg / ml, Molecular Probes) was added and reacted in the dark at 37 ° C for 30 minutes. The reaction solution was centrifuged at 400 g for 10 minutes, and the precipitated cells were suspended in 500 µl of PBS, and then red fluorescence intensity, which is an aggregate form of JC-1, using a flow cytometer. Was measured.
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 (최종첨가액, 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 미토콘드리아 막 전위를 비교하기 위해 유세포 측정기 (Flow cytometer)를 사용하여 형광을 측정하였으며, 이러한 결과를 하기 표 5 및 도 8에 나타내었다.To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 100 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum extract) Fluorescence was measured using a flow cytometer to compare the mitochondrial membrane potential with the control group without the extract. The results are shown in Table 5 and FIG. 8 .
상기 표 5 및 도 8에 따르면, 두 가지 추출물이 함께 처리된 경우 HL-60 세포의 미토콘드리아 막 전위가 대조군에 비해 2배 이상 감소하므로, 세포사멸이 유도되는 동시에 세포사멸이 가속화될 수 있다는 사실이 확인되었다.According to Table 5 and FIG. 8 , when the two extracts were treated together, the mitochondrial membrane potential of HL-60 cells was reduced by more than two times compared to the control group. Thus, the fact that apoptosis was induced could be accelerated at the same time. Confirmed.
<실험예 8> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 사멸 작용 : 백혈병 세포 U937의 미토콘드리아 막 전위 (mitochondrial membrane potential) 감소 효과Experimental Example 8 Cancer Cell Killing Effect of Snakeberry Extract and Ganoderma Lucidum Extract: Effect of Reducing the Mitochondrial Membrane Potential of Leukemia Cell U937
본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물이 암세포의 세포사멸 (apoptosis) 효과를 가지는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 미토콘드리아 막 전위를 분석하였다.In order to confirm whether the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention have apoptosis effect of cancer cells, these extracts were included in the culture medium and analyzed for mitochondrial membrane potential.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 U937을 2 x 105 세포/ml의 농도로 접종시킨 후 배양기 (CO2 5％, 37 ℃)에서 48시간 배양시킨 세포배양액 250 ㎕를 취하여 JC-1 (최종농도 10 ㎍/ml, Molecular Probes)을 첨가하고 37 ℃에서 암소에서 30분간 반응시킨다. 반응액을 400 g에서 10분간 원심 분리하여 침전된 세포를 PBS 500 ㎕에 현탁시킨 다음 유세포 측정기 (Flow cytometer)를 사용하여 JC-1의 응집형태 (aggregate form)인 적색 형광 강도 (red fluorescence intensity)를 측정하였다.Specifically, leukemia cells U937 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 × 10 5 cells / ml, followed by 48 hours in an incubator (CO 2 5%, 37 ° C.). Take 250 µl of the cultured cell culture solution, add JC-1 (final concentration 10 µg / ml, Molecular Probes) and react in the dark at 37 ° C for 30 minutes. The reaction solution was centrifuged at 400 g for 10 minutes, and the precipitated cells were suspended in 500 µl of PBS, and then red fluorescence intensity, which is an aggregate form of JC-1, using a flow cytometer. Was measured.
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가한 경우와 (최종 첨가액, 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물) 배지에 추출물을 첨가하지 않은 대조군과 미토콘드리아 막 전위를 비교하기 위해 유세포 측정기 (Flow cytometer)를 사용하여 형광을 측정하였으며, 이러한 결과를 하기 표 6 및 도 9에 나타내었다.In order to investigate the function of the solvent extract when the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together (final addition solution, 200 ㎍ / ml snakeberry extract + 150 ㎍ / ml Ganoderma lucidum mushroom Extract) The fluorescence was measured using a flow cytometer to compare the mitochondrial membrane potential with the control group without addition of the extract, and the results are shown in Table 6 and FIG. 9 .
상기 표 6 및 도 9에 따르면, 두 가지 추출물이 함께 처리된 경우 U937 세포의 미토콘드리아 막 전위가 2배 이상 감소하므로, 세포사멸이 유도되는 동시에 세포사멸이 가속화될 수 있다는 사실이 확인되었다.According to Table 6 and FIG. 9 , when the two extracts were treated together, the mitochondrial membrane potential of U937 cells was reduced by more than two times, and it was confirmed that apoptosis was induced and cell death could be accelerated.
<실험예 9> 뱀딸기 추출물과 영지버섯 추출물 혼용의 암세포 사멸 작용 : 백혈병 세포 HL-60과 U937의 카스파제-3 활성도 (caspase-3 activity) 증진 효과Experimental Example 9 Mixture of Snakeberry Extract and Ganoderma Lucidum Extract for Cancer Cell Death: Enhancement of Caspase-3 Activity of Leukemia Cells HL-60 and U937
본 발명의 뱀딸기와 영지버섯 추출물이 암 세포의 세포사멸 (apoptosis) 효과를 가지는지 확인하기 위해, 이들 추출물을 배지에 포함시켜 배양한 후 카스파제-3의 활성도를 분석하였다.In order to confirm whether the snakeberry and Ganoderma lucidum extract of the present invention have an apoptosis effect of cancer cells, these extracts were included in the culture medium and analyzed for the activity of caspase-3.
구체적으로, 10％ Fetal Bovine Serum이 포함된 RPMI-1640 배지 (Gibco BRL)에 백혈병 세포 HL-60과 U937을 각각 2 x 105 세포/ml의 농도로 접종시킨 후 배양기 (CO2 5％, 37 ℃)에서 48시간 배양시킨다. 이 세포배양액을 400 g에서 10분간 원심 분리하여 세포를 침전시킨 후 세포 분해용 용액 (Cell Lysis Buffer, Alexis Biochemicals) 50㎕를 첨가하고 어름에서 10분간 반응시킨 다음 10,000 g에서 5분간 원심 분리하여 침전물은 제거시킨다. 96-well plate (Corning)에 일정량의 단백질 (50-200 ㎍)이 포함된 상등액을 취하고 세포 분해용 용액으로 최종 부피가 50㎕이 되도록 채운다. 이 용액에 10 mM의 dethiothreitol이 포함된 반응용액 (2XReaction Buffer, Alexis Biochemicals) 50㎕를 첨가하고 4 mM DEVD-pNA 기질 (Alexis Biochemicals)의 최종농도가 200 μM이 되도록 첨가한 (최종반응액 5 ㎕) 후 37 ℃에서 1 시간 동안 반응시킨 다음 microplate reader (Bio-Rad)에서 흡광도 (405 nm)를 측정하였다.Specifically, leukemia cells HL-60 and U937 were inoculated in RPMI-1640 medium (Gibco BRL) containing 10% Fetal Bovine Serum at a concentration of 2 × 10 5 cells / ml, respectively, followed by incubator (CO 2 5%, 37 Incubate for 48 hours). The cell culture solution was centrifuged at 400 g for 10 minutes to precipitate the cells, and then 50 µl of a cell lysis solution (Cell Lysis Buffer, Alexis Biochemicals) was added and reacted for 10 minutes in ice, followed by centrifugation at 10,000 g for 5 minutes. Is removed. Take a supernatant containing a certain amount of protein (50-200 μg) in a 96-well plate (Corning) and fill it with a final volume of 50 μl with a solution for cell digestion. To this solution, 50 μl of reaction solution containing 10 mM dethiothreitol (2XReaction Buffer, Alexis Biochemicals) was added and the final concentration of 4 mM DEVD-pNA substrate (Alexis Biochemicals) was added to 200 μM (5 μl of final reaction solution). After the reaction at 37 ° C for 1 hour, the absorbance (405 nm) was measured in a microplate reader (Bio-Rad).
용매추출물의 기능을 조사하기 위하여 상기 실시예 1에서 얻은 뱀딸기 추출물과 상기 실시예 2에서 얻은 영지버섯 추출물을 함께 첨가하고 [최종 첨가액, HL-60의 경우 100 ㎍/ml 뱀딸기 추출물＋150 ㎍/ml 영지버섯추출물 (도 10의 A참조); U937의 경우 200 ㎍/ml 뱀딸기 추출물＋150 ㎍/ml 영지버섯 추출물 (도 10의 B 참조)] 배지에 추출물을 첨가하지 않은 대조군과 카스파제-3의 활성도를 비교하기 위해 카스파제-3 비색분석용 키트 (caspase-3 colorimetric assay kit, Alexis Biochemicals)를 사용하여 흡광도를 측정하였다. To investigate the function of the solvent extract, the snakeberry extract obtained in Example 1 and the Ganoderma lucidum extract obtained in Example 2 were added together [final addition solution, 100 ㎍ / ml snakeberry extract +150 ㎍ / ml for HL-60 Ganoderma lucidum extract (see FIG. 10A); In case of U937, 200 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum Extract (see FIG. 10B)] for the caspase-3 colorimetric analysis to compare the activity of caspase-3 and the control group without adding the extract to the medium. Absorbance was measured using a kit (caspase-3 colorimetric assay kit, Alexis Biochemicals).
그 결과, 두 가지 추출물이 함께 처리된 경우 HL-60 세포와 U937 세포 모두 카스파제-3의 활성이 크게 증가하므로, 세포사멸이 유도되는 동시에 세포사멸이 가속화될 수 있다는 사실이 확인되었다.As a result, when both extracts were treated together, it was confirmed that the caspase-3 activity was greatly increased in both HL-60 cells and U937 cells, thereby inducing apoptosis and accelerating apoptosis.
한편 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물의 급성 독성을 알아보기 위하여 하기와 같은 실험을 수행하였다.On the other hand, the following experiment was carried out to determine the acute toxicity of the snakeberry extract, Ganoderma lucidum extract and mixtures thereof of the present invention.
<실험예 10> 랫트에 대한 경구투여 급성 독성실험Experimental Example 10 Oral Acute Toxicity in Rats
6주령의 특정병원부재 (SPF) SD계 랫트를 사용하여 급성독성실험을 실시하였다. 군당 2 마리씩의 동물에 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물을 각각 0.5% 메틸셀룰로즈 용액에 현탁하여 1g/kg/15ml의 용량으로 단회 경구투여하였다. 시험물질 투여후 동물의 폐사여부, 임상증상, 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적검사를 실시하였으며, 부검하여 육안으로 복강장기와 흉강장기의 이상여부를 관찰하였다. 시험결과, 시험물질을 투여한 모든 동물에서 특기할 만한 임상증상이나 폐사된 동물은 없었으며, 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다. 이상의 결과 실험된 화합물은 모두 랫트에서 2 g/kg까지 독성변화를 나타내지 않으며 경구 투여 최소치사량 (LD50)은 6 g/kg이상인 안전한 물질로 판단되었다.Acute toxicity test was performed using 6-week-old SPF SD rats. Snakeberry extract, Ganoderma lucidum extract, and mixtures thereof were suspended in 0.5% methylcellulose solution and administered orally at a dose of 1 g / kg / 15 ml to two animals per group. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 2 g / kg in rats, and the minimum lethal dose (LD 50 ) of 6 g / kg or more was determined to be a safe substance.
상술한 바와 같이, 본 발명이 제공하는 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물은 암 세포의 증식을 억제시키고, 암 세포의 세포사멸 과정을 촉진시키는 기능을 가지며, 방사선과 함께 처리할 경우 더 효율적으로 암 세포의 사멸을 촉진하는 기능도 가지고 있다. 특히 상기 추출물을 혼합하여 처리하는 경우 더욱 향상된 암 세포의 세포사멸 기능을 가지고 있을 뿐 아니라, 용이하게 재배 및 입수할 수 있으므로 이로부터 추출물을 대량 분리할 수 있는 장점이 있다. 따라서 본 발명의 뱀딸기 추출물, 영지버섯 추출물 및 이들의 혼합물은 백혈병 예방과 치료에 사용될 수 있는 약학적 조성물, 세포사멸 및 세포증식 억제제 등에 유용하게 사용될 수 있으며, 기능성 식품으로도 이용이 가능하다.As described above, the snakeberry extract, Ganoderma lucidum extract and mixtures thereof provided by the present invention have the function of inhibiting the proliferation of cancer cells, promoting the process of apoptosis of cancer cells, and more efficiently when treated with radiation It also has the function of promoting the death of cancer cells. In particular, when the extract is treated by mixing, as well as having a more improved apoptosis function of cancer cells, it can be easily grown and obtained, there is an advantage that can be separated from the extract in large quantities. Therefore, the snakeberry extract, Ganoderma lucidum extract, and mixtures thereof of the present invention can be usefully used in pharmaceutical compositions, apoptosis and cell proliferation inhibitors, etc., which can be used for the prevention and treatment of leukemia, and can be used as functional foods.
도 1은 용매 추출법에 의하여 뱀딸기 추출물을 분리하는 과정을 보여주는 블럭도이다. 1 is a block diagram showing a process of separating a snakeberry extract by a solvent extraction method.
도 2는 용매 추출법에 의하여 영지버섯 추출물을 분리하는 과정을 보여주는 블럭도이다. Figure 2 is a block diagram showing a process for separating the Ganoderma lucidum extract by the solvent extraction method.
도 3은 뱀딸기 추출물과 영지버섯 추출물을 단독으로 처리하거나 또는 함께 처리했을 때 백혈병 세포 HL-60 (도 3a)과 U937 (도 3b)의 생존률 (viability)이 감소되고 있음을 보여주는 그래프이다: FIG. 3 is a graph showing that the viability of leukemia cells HL-60 ( FIG. 3A ) and U937 ( FIG. 3B ) is reduced when the snakeberry extract and Ganoderma lucidum extract are treated alone or together:
도 3a A: 대조군; B: 100 ㎍/ml 뱀딸기 추출물; 3A A: control; B: 100 μg / ml Snakeberry Extract;
C: 150 ㎍/ml 영지버섯 추출물; C: 150 μg / ml Ganoderma lucidum extract;
D: 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물,D: 100 µg / ml Snakeberry Extract + 150 µg / ml Ganoderma lucidum extract,
도 3b A: 대조군; B: 200 ㎍/ml 뱀딸기 추출물; 3B A: control; B: 200 μg / ml Snakeberry Extract;
C: 150 ㎍/ml 영지버섯 추출물; C: 150 μg / ml Ganoderma lucidum extract;
D: 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.D: 200 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum extract.
도 4는 뱀딸기 추출물과 영지버섯 추출물을 단독으로 처리하거나 또는 함께 처리했을 때 백혈병 세포 HL-60의 세포사멸 (apoptosis)이 촉진되고 있음을 보여주는 그래프이다: FIG. 4 is a graph showing that apoptosis of leukemia cells HL-60 is promoted when the snakeberry extract and Ganoderma lucidum extract are treated alone or together:
A: 대조군; B: 100 ㎍/ml 뱀딸기 추출물; A: control; B: 100 μg / ml Snakeberry Extract;
C: 150 ㎍/ml 영지버섯 추출물; C: 150 μg / ml Ganoderma lucidum extract;
D: 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.D: 100 µg / ml Snakeberry Extract + 150 µg / ml Ganoderma lucidum extract.
도 5는 뱀딸기 추출물과 영지버섯 추출물을 단독으로 처리하거나 또는 함께 처리했을 때 백혈병 세포 U937의 세포사멸 (apoptosis)이 촉진되고 있음을 보여주는 그래프이다: FIG. 5 is a graph showing that apoptosis of leukemia cells U937 is promoted when the snakeberry extract and Ganoderma lucidum extract are treated alone or together:
A: 대조군; B: 200 ㎍/ml 뱀딸기 추출물; A: control; B: 200 μg / ml Snakeberry Extract;
C: 150 ㎍/ml 영지버섯 추출물; C: 150 μg / ml Ganoderma lucidum extract;
D: 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.D: 200 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum extract.
도 6은 뱀딸기 추출물과 영지버섯 추출물을 방사선 (2 Gy)과 함께 처리했을 때 백혈병 세포 HL-60의 세포사멸 (apoptosis)이 촉진되고 있음을 보여주는 그래프이다: FIG. 6 is a graph showing that apoptosis of leukemia cells HL-60 is promoted when snakeberry extract and Ganoderma lucidum extract are treated with radiation (2 Gy):
A: 2 Gy 감마선; A: 2 Gy gamma rays;
B: 2 Gy 감마선 ＋ 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.B: 2 Gy gamma ray + 100 µg / ml snakeberry extract + 150 µg / ml ganoderma lucidum extract.
도 7은 뱀딸기 추출물과 영지버섯 추출물을 방사선 (2 Gy)과 함께 처리했을 때 백혈병 세포 U937의 세포사멸 (apoptosis)이 촉진되고 있음을 보여주는 그래프이다: 7 is a graph showing that apoptosis of leukemia cells U937 is promoted when the snakeberry extract and Ganoderma lucidum extract are treated with radiation (2 Gy):
A: 2 Gy 감마선; A: 2 Gy gamma rays;
B: 2 Gy 감마선 ＋ 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.B: 2 Gy gamma rays + 200 μg / ml snakeberry extract + 150 μg / ml ganoderma lucidum extract.
도 8은 뱀딸기 추출물과 영지버섯 추출물을 단독으로 처리하거나 또는 함께 처리했을 때 백혈병 세포 HL-60의 미토콘드리아 막 전위 (mitochondrial membrane potential)가 감소되고 있음을 보여주는 그래프이다: FIG. 8 is a graph showing that the mitochondrial membrane potential of leukemia cells HL-60 is reduced when the snakeberry extract and Ganoderma lucidum extract are treated alone or together:
A: 대조군; B: 100 ㎍/ml 뱀딸기 추출물; C: 150 ㎍/ml 영지버섯 추출물; A: control; B: 100 μg / ml Snakeberry Extract; C: 150 μg / ml Ganoderma lucidum extract;
D: 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.D: 100 µg / ml Snakeberry Extract + 150 µg / ml Ganoderma lucidum extract.
도 9는 뱀딸기 추출물과 영지버섯 추출물을 단독으로 처리하거나 또는 함께 처리했을 때 백혈병 세포 U937의 미토콘드리아 막 전위 (mitochondrial membrane potential)가 감소되고 있음을 보여주는 그래프이다: FIG. 9 is a graph showing that the mitochondrial membrane potential of leukemia cells U937 is reduced when the snakeberry extract and Ganoderma lucidum extract are treated alone or together:
A: 대조군; B: 200 ㎍/ml 뱀딸기 추출물; C: 150 ㎍/ml 영지버섯 추출물; A: control; B: 200 μg / ml Snakeberry Extract; C: 150 μg / ml Ganoderma lucidum extract;
D: 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.D: 200 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum extract.
도 10은 뱀딸기 추출물과 영지버섯 추출물을 함께 처리했을 때 백혈병 세포 HL-60과 U937의 카스파제-3의 활성 (caspase-3 activity)이 증가되고 있음을 보여주는 그래프이다: FIG. 10 is a graph showing that caspase-3 activity of leukemia cells HL-60 and U937 is increased when the snakeberry extract and Ganoderma lucidum extract are treated together:
A: 100 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물;A: 100 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum extract;
B: 200 ㎍/ml 뱀딸기 추출물 ＋ 150 ㎍/ml 영지버섯 추출물.B: 200 μg / ml Snakeberry Extract + 150 μg / ml Ganoderma lucidum extract.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20240019908A (en) | 2022-08-05 | 2024-02-14 | 제너럴바이오(주) | A composition including Ganoderma lucidum fruit body extract for anti-cancer |
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