KR101468300B1 - Composition for anti-cancer or anti-oxidation comprising the extract of Salsola collina Pallas as active ingredient - Google Patents
Composition for anti-cancer or anti-oxidation comprising the extract of Salsola collina Pallas as active ingredient Download PDFInfo
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- KR101468300B1 KR101468300B1 KR1020130032935A KR20130032935A KR101468300B1 KR 101468300 B1 KR101468300 B1 KR 101468300B1 KR 1020130032935 A KR1020130032935 A KR 1020130032935A KR 20130032935 A KR20130032935 A KR 20130032935A KR 101468300 B1 KR101468300 B1 KR 101468300B1
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Abstract
본 발명은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 함유하는 항암 또는 항산화용 조성물에 관한 것으로, 보다 구체적으로 본 발명은 솔장다리 추출물을 유효성분으로 포함하는, 암 예방 또는 치료용 약학 조성물, 항산화 또는 항암용 식품 조성물 및 항산화용 화장료 조성물에 관한 것이다.
본 발명의 솔장다리(Salsola collina Pallas) 추출물을 포함하는 조성물은 강한 항암 및 항산화 활성을 나타내므로, 항암 또는 항산화를 목적으로 하는 의약품, 식품 및 화장품 등에 이용될 수 있다.The present invention relates to a process for the preparation of The present invention relates to a pharmaceutical composition for preventing or treating cancer, an antioxidant or anticancer composition, a composition for preventing or treating cancer, and a composition for preventing or treating cancer, which comprises, as an active ingredient, To an antioxidant cosmetic composition.
The Salsola of the present invention collina Pallas) extract has strong anticancer and antioxidant activity and thus can be used for medicines, foods and cosmetics intended for anticancer or antioxidation.
Description
본 발명은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 함유하는 항암 또는 항산화용 조성물에 관한 것으로, 보다 구체적으로 본 발명은 솔장다리 추출물을 유효성분으로 포함하는, 암 예방 또는 치료용 약학 조성물, 항산화 또는 항암용 식품 조성물 및 항산화용 화장료 조성물에 관한 것이다.
The present invention relates to a process for the preparation of The present invention relates to a pharmaceutical composition for preventing or treating cancer, an antioxidant or anticancer composition, a composition for preventing or treating cancer, and a composition for preventing or treating cancer, which comprises, as an active ingredient, To an antioxidant cosmetic composition.
인간의 질병 중 암은 현대의학의 발달에도 불구하고 아직까지 치료에 어려움이 있는 질병으로, 그 발생 요인의 80 ~ 90 %는 환경적 요인에 기인하고, 이 중 30~60%가 식이 및 영양과 연관되어 있으며, 기타 다른 요인으로는 흡연, UV, X-ray와 같은 방사선 조사, 직업적 요인, 음주, 내인성 호르몬 등에 의해 유발된다고 알려져 있다.Cancer among human diseases is a disease that is difficult to treat despite the development of modern medicine. 80 ~ 90% of the causes are caused by environmental factors, and 30 ~ 60% of them are caused by diet and nutrition And other factors are known to be caused by radiation, such as smoking, UV, X-ray, occupational factors, drinking, endogenous hormones, and the like.
이러한 암 가운데 대장암을 치료하는 방법으로서, 수술법이 널리 사용되고 있다. 그러나, 국소적으로 진행된 질병 또는 전이성 질병의 대장암 환자 사이에서, 예후는 높은 발병률과 사망률로 매우 불리하다.As a method of treating colorectal cancer among these cancers, surgical methods are widely used. However, among colorectal cancer patients with locally advanced disease or metastatic disease, prognosis is highly disadvantageous with high incidence and mortality.
한편 대장암을 치료하는 방법으로서, 수술보다는 덜 유리한 결과를 나타내는 약물 요법이 시도되고 있다. 항종양 화합물인 5-FU(5-플루오로우라실)는 대장암의 치료에서 주요 선택 약물이고, 그의 사용은 단지 한계적으로만 효과적인 것으로 증명되었다. 5-FU는 대장암 크기를 일시적으로 감소시킬 수 있지만, 환자의 생존기간이 실질적으로 연장되었거나 "치유"된 것(5년의 경감기를 기준으로)을 나타내는 증거는 거의 없다. 5-FU를 사용한 화학 요법이 질병이 간으로 전이된 환자에서 사용되고 있지만, 그러한 케이스의 단지 25 % 또는 그 미만에서만 일시적인 개선이 관찰되며, 전체적으로 생존율은 유의한 영향을 받지 않는다. 5-FU의 한계적인 효과에도 불구하고, 확실하게 더 효과적인 것으로 나타나는 다른 약물이나 복합 치료는 없었다.On the other hand, as a method of treating colorectal cancer, drug therapy that shows less favorable results than surgery is being tried. 5-FU (5-fluorouracil), an antineoplastic compound, is a major selective agent in the treatment of colorectal cancer and its use has only proven to be marginally effective. Although 5-FU can temporarily reduce colon cancer size, there is little evidence that the patient's survival has been substantially extended or "healed" (based on a 5 year reliever). Although chemotherapy with 5-FU has been used in patients with disease-to-liver metastases, only temporary improvement is observed in only 25% or less of such cases, and overall survival is not significantly affected. Despite the marginal effects of 5-FU, there were no other drugs or combination therapies that appeared to be more effective.
대장암에 대한 종래의 치료, 예를 들면, 외과적 절제술 또는 5-FU를 사용하는 화학 요법을 받고 있는 환자에 있어서의 매우 빈약한 5년 생존율(약 50 % 또는 그 미만)을 고려하면, 수술 이후, 진단을 확정하거나 암의 체적을 제거하는 것을 보조하는 것과 같이, 약물 또는 화합물을 사용하여 사람의 악성 대장암을 효과적으로 치료하는 새로운 방법을 발견하는 것은 매우 유용할 것이다. 수술법이 암 조직 모두를 제거할 수는 없으므로, 약물 치료는 또한 각종 기관으로 전이되거나 퍼진, 진행된 질병의 환자를 위해 매우 유용할 것이다.Considering the very poor 5-year survival rate (about 50% or less) in patients undergoing conventional therapy for colorectal cancer, for example, surgical resection or chemotherapy using 5-FU, Thereafter, it would be very useful to find new ways to effectively treat human malignant colorectal cancer using drugs or compounds, such as assisting in diagnosing or removing the volume of cancer. Since surgery can not remove all of the cancerous tissue, drug therapy will also be very useful for patients with advanced disease, which have spread or spread to various organs.
이에 많은 연구자들이 암을 정복하기 위해 여러 방면으로 다양한 연구를 진행하고 있으며, 이러한 연구의 한 부분으로 식품이나 천연 약제 중에서 암의 증식을 차단하거나 억제하는 성분을 생물체에서 찾으려는 노력이 이루어지고 있다.Many researchers are conducting various studies to conquer cancer. As a part of this research, efforts are being made to find an ingredient in an organism that blocks or inhibits the growth of cancer in foods or natural medicines.
현재 천연물을 중심으로 하는 항암제의 개발은 전통적으로 오랜 기간 민간요법 등으로 사용되어 오던 식물 등의 추출물들을 현대 세포생물학적 연구의 발달로 확립된 암 유전자, 암 억제 유전자, 시그날 전달인자, 약제 내성 감수성 인자 및 세포사멸과 관련된 인자 등 암 분자표적을 중심으로 스크리닝하여 활성물질을 분리, 정제하고 그 활성물질의 구조를 결정하는 항암제의 개발이라 할 수 있다. Currently, the development of anticancer drugs centering on natural products is based on the development of modern cell biology research, which has been used for a long time in folk remedies and the like, as a cancer gene, a cancer suppressor gene, a signal transduction factor, a drug resistance susceptibility factor And screening for cancer target such as factors related to apoptosis, thereby separating and purifying the active substance and determining the structure of the active substance.
한편, 인체는 산화촉진물질과 산화억제물질이 균형을 이루고 있으나, 여러 가지 요인들로 인하여 이러한 균형 상태를 잃고 산화를 촉진하는 방향으로 기울게 되면, 생체 내에 산화적 스트레스(oxidative stress)가 유발되어 세포손상 및 병리적 질환을 유발하게 된다. 이러한 산화적 스트레스의 직접적 원인이 되는 활성 산소종(reactive oxygen species, ROS)은 화학적으로 불안정하고 반응성이 높아 DNA, 단백질, 지질 및 탄수화물과 같은 여러 생체물질과 쉽게 반응할 수 있으며, 생체 내 고분자들을 공격하여 세포와 조직에 비가역적인 손상을 일으키거나 돌연변이, 세포독성 및 암 등을 초래하게 된다.On the other hand, the human body has a balance between the oxidation-promoting substance and the oxidation-inhibiting substance, but if the balance state is lost due to various factors and is inclined to promote oxidation, oxidative stress is induced in the living body, Damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, are chemically unstable and highly reactive, and can easily react with various biomaterials such as DNA, protein, lipid and carbohydrate. Attacks, causing irreversible damage to cells and tissues, or causing mutations, cytotoxicity, and cancer.
이러한 활성산소의 작용을 중화시키는 성질을 가지고 있는 항산화제는 저분자 화합물로서 체내에서 고분자 항산화물(효소)의 작용을 돕거나 자체적으로 활성산소를 제거하기도 하는데, 베타카로틴, 비타민 A, 비타 민C(아스코르빈산), 비타민 E(토코페롤)와 플라보노이드 등이 대표적인 항산화 성분들로서, 강력한 항산화력을 지니고 있으며, 현재 식물영양소(phytonutrients)들이 항산화 성분으로 주목받아 활발하게 연구되고 있다.These antioxidants, which neutralize the action of active oxygen, are low molecular weight compounds that help the body to work with polymeric antioxidants (enzymes) or remove free radicals themselves. Beta-carotene, vitamin A, vitamin C Ascorbic acid), vitamin E (tocopherol) and flavonoids are typical antioxidants and have strong antioxidant activity. Phytonutrients have been actively studied as antioxidants.
한편, 천연소재 중 솔장다리는 수송나물속(Salsola)에 속하며 바닷가 모래땅에서 자라는 한해살이 풀이다. 주로 소금기가 있는 토양이나 해변에 많이 나며 특히 한국, 중국, 동부 시베리아 및 일본에 분포하며 어린 순은 나물로 먹는 염생식물이다. 상기 수송나물 속 추출물은 동물의 혈압저하 효과(중국의 중약대사전) 및 해열, 해독, 이습, 지통의 작용효과(중국의 고원중초약치료수책) 외에도 다양한 생리활성을 가지고 있다고 알려져 있다. On the other hand, among the natural materials, Sol Staple is an annual plant belonging to Salsola in transport or in the sandy areas of the sea. It is mainly found in salty soils and beaches, especially in Korea, China, Eastern Siberia and Japan. It is known that the above-mentioned extracts of the transporter have various physiological activities in addition to blood pressure lowering effect (Chinese herbal medicine metabolism) of animals and action effects of phytotoxicity, detoxification, humidification,
이러한 배경하에, 본 발명자들은 항암 또는 항산화 효과를 나타내면서 부작용이 적은 천연물을 찾고자 예의 연구 노력한 결과, 식용 및 약용으로서 가치가 무궁무진한 솔장다리로부터 추출물을 얻어 항암 및 항산화 활성에 대해 알아보고, 이들 추출물이 세포주기에 미치는 영향과 세포주기에 부가되는 여러 단백질 발현 양상에 미치는 영향을 확인함으로써 본 발명을 완성하였다.
Under these circumstances, the inventors of the present invention have studied the anticancer and antioxidant activity of extracts from Solanaceae, which are valuable as edible and medicinal drugs, And the effect on the expression pattern of various proteins added to the cell cycle, thereby completing the present invention.
본 발명의 하나의 목적은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공하는 것이다.One object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an extract of Salsola collina Pallas as an active ingredient.
본 발명의 다른 목적은 상기 솔장다리 추출물을 유효성분으로 포함하는 항산화 또는 항암용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for antioxidant or anticancer comprising the Solanaceae extract as an active ingredient.
본 발명의 또 다른 목적은 상기 솔장다리 추출물을 유효성분으로 포함하는, 항산화용 화장료 조성물을 제공하는 것이다.
Yet another object of the present invention is to provide a cosmetic composition for antioxidation comprising the Solanaceae extract as an active ingredient.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising an extract of Salsola collina Pallas as an active ingredient.
본 발명의 용어 "솔장다리(Salsola collina Pallasas)"는 명아주과(Chenopodiaceae) 수송나물속(Salsola)에 속하는 1년생 초본식물로 중국, 만주, 몽골, 아무르, 우수리, 티베트, 시베리아, 중앙아시아, 유럽에 분포하고 있다. 높이는 30 ㎝ 정도이며 밑에서 많은 가지가 갈라지고 곧추서거나 비스듬히 자라며 처음에는 연하지만 점차 딱딱해진다. 꽃은 7 ~ 8월에 엷은 녹색으로 피며 잎짬(leaf axil)에 1개씩 달린다. 꽃대는 없고 2개의 포가 있으며 화피편과 수술은 각 5개, 화주는 2개로 갈라진다. 과실은 포과로 짧은 난형이며 화피의 밑부분으로 싸였으며 1개의 종자가 들어 있다. 어린잎은 식용으로 사용한다고 알려져 있으나, 아직까지 구체적인 효능에 대해서는 알려진 바 없었는바, 본 발명에서는 솔장다리의 추출물이 항암 및 항산화 효과가 있음을 확인한 것이다.
The term " Salsola " collina Pallasas "is a perennial herbaceous plant belonging to Chenopodiaceae transport or Salsola, distributed in China, Manchuria, Mongolia, Amur, Tribe, Tibet, Siberia, Central Asia and Europe. The flowers bloom in July and August, and they run one on the leaf axil. There are no flower buds, there are two bolls, The fruit is split into 5 pieces and the shippers divide into 2 pieces. Fruit is short ovate with flecks, wrapped in the bottom part of the furrow and contains one seed. Young leaves are known to be used for food, but the specific efficacy is still known However, in the present invention, it was confirmed that the extract of Brassica juncea had anticancer and antioxidant effect.
본 발명의 용어 "솔장다리 추출물"이란 솔장다리를 추출하여 수득한 추출물을 의미하는데, 본 발명의 목적상, 솔장다리 추출물은 솔장다리를 탄소수 1 내지 4의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택되는 용매, 바람직하게는 메탄올, 에탄올 또는 부탄올로 추출한 것을 포함할 수 있다. 또한, 본 발명에 있어서, 상기 추출물에는, 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물 중 어느 하나도 포함될 수 있다.
For the purpose of the present invention, the Solanum tuberosum extract is obtained by extracting Solanum tuberosum with an alcohol having from 1 to 4 carbon atoms and a mixed solvent thereof. The term " Solanum tuberculosis extract " , Preferably methanol, ethanol or butanol. In addition, in the present invention, the above extract may contain any one of the extract obtained by the extraction treatment, the diluted solution or the concentrate of the extract, the dried product obtained by drying the extract, or the adjusted product or the purified product.
본 발명의 일 실시예에서는 95 % 에탄올이 첨가된 시료를 45 ℃에서 초음파처리를 하여 추출하는 방법으로 솔장다리 추출물을 수득하였다. 상기 추출물은 농축 후 동결건조 하였으며, 동결건조된 시료는 DMSO(Dimethyl sulfoxide)에 녹여 실험에 사용하였다.
In one embodiment of the present invention, a Solanum tuberculosis extract was obtained by subjecting a sample to which 95% ethanol had been added by ultrasonication at 45 ° C. The extract was concentrated and lyophilized. The lyophilized sample was dissolved in DMSO (dimethyl sulfoxide) and used in the experiment.
본 발명에서 예방 및 치료대상 암은 특정 암으로 한정되지 않으며, 바람직하게는 유방암, 폐암, 위암, 간암, 혈액암, 뼈암, 췌장암, 피부암, 머리 또는 목암(head or neck cancer), 피부 또는 안구 흑색종, 자궁육종, 난소암, 직장암, 항문암, 대장암, 난관암, 자궁내막암, 자궁경부암, 소장암, 내분비암, 갑상선암, 부갑상선암, 신장암, 연조직종양, 요도암, 전립선암, 기관지암 또는 골수암을 들 수 있으며, 더욱 바람직하게는 대장암일 수 있다.
The cancer to be prevented and treated in the present invention is not limited to a specific cancer but preferably includes a breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, Endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, uterine cancer, uterine cancer, ovarian cancer, rectal cancer, Cancer or bone marrow cancer, and more preferably it can be a colon cancer.
본 발명에서 "대장암(colon cancer)"이란 대장에 생긴 암세포로 이루어진 악성 종양을 의미하며, 크게 유전적인 요인과 식이로 나눌 수 있다. 음식 그 자체가 직접적인 발암물질을 포함하고 있지 않더라도, 우리가 음식물을 섭취하고 몸에 흡수되는 과정에서 발암물질이 생겨날 수도 있는데, 이는 식이성분을 대사시키는 과정에서 장내세균이 생산하는 효소에 의해 섭취 음식물이 발암물질로 전환될 수 있기 때문이다.
In the present invention, "colon cancer" refers to a malignant tumor consisting of cancer cells in the large intestine, which can be divided into genetic factors and diets. Even though the food itself does not contain any direct carcinogens, it is possible that carcinogens may form during the process of ingesting and absorbing the food, which is caused by the enzymes produced by intestinal bacteria during the metabolism of the dietary components Can be converted to carcinogens.
본 발명의 목적상, 상기 대장암은 본 발명의 솔장다리 추출물에 의해 예방 또는 치료의 목적이 되는 질병으로서, 본 발명의 일 실시예에서는 대장암 세포주인 HT29 세포의 독성 실험 및 항암 활성 기전을 통하여, 상기 솔장다리 추출물이 암세포의 세포주기를 조절하여 높은 항암활성효과를 보유하고 있음을 확인하였다.
For the purpose of the present invention, the colorectal cancer is a disease to be prevented or treated by the Solanaceae extract of the present invention. In one embodiment of the present invention, the toxicity test and the anticancer activity mechanism of the HT29 cell line, , And it was confirmed that the Solanaceae extract had high anticancer activity by controlling the cell cycle of cancer cells.
본 발명에서 솔장다리 추출물은 대장암 세포 HT29에 서로 다른 농도(10, 20, 30, 40, 50, 60, 70 ㎍/mL)로 48시간 동안 처리하여 WST 분석방법으로 세포생존률을 확인한 결과, 30 ㎍/mL 농도부터 투여량에 의존적으로 HT29 세포의 성장을 억제하였으며, 49.1 ㎍/mL 농도에서는 50 % 사멸효과(IC50)가 있음을 확인하였다(도 1의 A).
In the present invention, the cell survival rate was examined by WST analysis for 48 hours in different concentrations (10, 20, 30, 40, 50, 60, 70 ㎍ / mL) From the concentration of / / mL, the growth of HT29 cells was inhibited depending on the dose, and it was confirmed that there was a 50% killing effect (IC 50 ) at the concentration of 49.1 / / mL (Fig.
또한 솔장다리 추출물은 대장암 세포 HT29 에 서로 다른 농도(20, 40, 60, 80, 100 ㎍/mL)별로 48시간 동안 처리한 후 HT29의 형태변화를 관찰한 결과, 투여량에 의존적으로 HT29 세포의 특징인 구형(Sphere)의 형태가 불규칙적으로 변하고, 무리를 이루어 자라는 형태가 단일세포형태로 변하는 것이 관찰되었다(도 1의 B). 따라서 본 발명의 솔장다리 추출물이 대장암 세포의 성장을 억제할 수 있으므로, 대장암 예방 또는 치료에 적합함을 확인하였다.
In addition, after treatment of colon cancer cells HT29 with different concentrations (20, 40, 60, 80, 100 ㎍ / mL) for 48 hours, the morphological changes of HT29 were observed, (Fig. 1B). The shape of the sphere, which is a characteristic of the sphere, changes irregularly. Therefore, it has been confirmed that the extract of Solanaceae according to the present invention can inhibit the growth of colon cancer cells, thus being suitable for prevention or treatment of colon cancer.
본 발명에서 솔장다리 추출물의 항암활성기전을 알아보기 위하여 솔장다리 추출물을 대장암 세포 HT29에 농도(20, 40, 60 ㎍/mL) 별로 24시간 동안 처리한 다음 PI(Propium Iodide) 염색을 실시하여 유세포분석기(Flow cytometry, Quanta SC, Beckman)를 이용한 세포주기변화를 분석한 결과, 농도의존적으로 G2/M arrest가 유도되었고, 상기 G2/M arrest 유도에 의해서 상대적으로 G1기의 세포분포가 점차 감소됨을 확인하였다(도 3). 또한 웨스턴 블롯 혼성화(Western blot hybridization)의 결과, 솔장다리 추출물을 처리한 경우, G2기에서 M기로의 전이에 필수적인 단백질인 Cyclin-dependent kinase(CDK) 활성인자인 Cdc25C, cyclin A의 발현이 감소되었으며, CDK 억제인자인 p21 및 Wee1, Cdc2의 inactive form인 phospho-Cdc2, Cdc25C의 inactive form인 phospho-Cdc25C의 발현이 증가됨을 확인하였다(도 4). 이러한 결과를 통하여 솔장다리 추출물의 처리에 의한 p21과 Wee 1의 발현 증가 및 Cdc25C의 발현 억제에 의하여 Cdc2/Cyclin complex의 활성화가 저해되어 HT29 세포의 G2/M 정지가 유도되어 높은 항암활성을 보유하고 있음을 확인하였다.
In order to investigate the anticancer activity of the extracts of Solanum tuberculosis in the present invention, the Solanum tuberculosis extract was treated with colorectal cancer cell HT29 (20, 40, 60 ㎍ / mL) for 24 hours and then PI Cell cycle analysis using flow cytometry (Quanta SC, Beckman) revealed that concentration-dependent G2 / M arrest was induced, and G2 / M arrest induction led to a gradual decrease in G1 cell distribution (Fig. 3). Western blot hybridization revealed that Cdc25C, cyclin A, which is an essential protein for the G2-to-M transition, was reduced by treatment with Solanum tuberculosis extract. , The expression of phospho-Cdc2, an inactive form of CDK inhibitor p21 and Wee1 and Cdc2, and phospho-Cdc25C, an inactive form of Cdc25C (FIG. 4). These results suggest that treatment of Cdc2 / Cyclin complex by inhibition of expression of p21 and
본 발명에서 용어 "예방"은 상기 조성물의 투여로 상기 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, "치료"는 상기 조성물의 투여로 상기 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.
The term "prevent" in the present invention means all the actions of inhibiting or delaying the onset of the disease upon administration of the composition, and "treatment" means any action that alleviates or alleviates symptoms of the disease do.
본 발명의 상기 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 보다 효과적인 예방 또는 치료를 위하여, 본 발명의 솔장다리 추출물은 1일 0.0001 내지 100 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여함이 바람직하고, 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다.
The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for more effective preventive or therapeutic treatment, it is preferable to administer the Solanaceae extract of the present invention at a dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day, It may be administered or divided into several doses.
본 발명의 약학적 조성물은 쥐, 마우스, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구,직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 (intracerebroventricular) 주사에 의해 투여될 수 있다.
The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
본 발명의 상기 솔장다리 추출물을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 바람직하게 상기 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 경피흡수제, 겔제, 로션제, 연고제, 크림제, 첩부제, 카타플라스마제, 페이스트제, 스프레이, 피부 유화액, 피부 현탁액, 경피 전달성 패치, 약물 함유 붕대 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있으나, 이에 제한되지 않는다.
The pharmaceutical composition for preventing or treating cancer, which comprises the Solanaceae extract of the present invention as an active ingredient, may further comprise an appropriate carrier, excipient or diluent commonly used in the production of a pharmaceutical composition. Preferably, the composition is in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze- The composition may have any one of formulations selected from the group consisting of ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal patches, drug-containing dressings and suppositories, It may, but is not limited to, various forms of parenteral administration.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propyleneglycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61(tween 61), 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.
In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
다른 하나의 양태로서, 본 발명은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 포함하는, 항암 또는 항산화용 식품 조성물을 제공한다. 상기 솔장다리 추출물에 관해서는 전술한 바와 같으며, 본 발명의 추출물은 항암 또는 항산화 효과를 갖기 때문에 식품조성물에 첨가할 수 있다.
In another aspect, the present invention provides a method for the treatment of Salsola collina Pallas) extract as an active ingredient. The extract of the present invention is as described above, and the extract of the present invention can be added to a food composition since it has anticancer or antioxidative effect.
본 발명의 용어 "항암"은 암세포의 증식을 억제하거나 암세포를 죽이는 등의 역할을 하는 것을 의미하며, 본 발명에서 암의 예방, 치료 또는 개선 등의 용어와 혼용할 수 있다. 본 발명의 솔장다리 추출물의 항암활성에 대해서는 상기에서 설명한 바와 같다.
The term "anti-cancer" of the present invention means inhibiting the proliferation of cancer cells or killing cancer cells, and may be used in combination with terms such as prevention, treatment, or improvement of cancer in the present invention. The anticancer activity of the Solanaceae extract of the present invention is as described above.
본 발명의 용어 "항산화"는 산화를 억제하는 작용을 의미하는 것으로, 인체는 산화촉진물질(prooxidant)과 산화억제물질(antioxidant)이 균형을 이루고 있으나 여러 가지 요인들에 의하여 이런 균형상태가 불균형을 이루게 되고 산화 촉진 쪽으로 기울게 되면, 산화적 스트레스(oxidative stress)가 유발되어 잠재적인 세포손상 및 병리적 질환을 일으키게 된다. 이러한 산화적 스트레스의 직접적 원인이 되는 활성 산소종(reactive oxygen species, ROS)은 불안정하고 반응성이 높아 여러 생체물질과 쉽게 반응하고, 체내 고분자들을 공격하여 세포와 조직에 비가역적인 손상을 일으키거나 돌연변이, 세포독성 및 발암 등을 초래하게 된다. NO, HNO2, ONOO-와 같은 활성 질소종(reactive nitrogen species, RNS)은 염증 반응 시 대식세포 호중구 및 다른 면역 세포들의 면역반응으로 인해 다량 생성되며, 이때 ROS도 같이 생성된다. 상기와 같은 활성산소는 체내에서 세포를 산화시켜 파괴시키며, 그에 따라 각종 질환에 노출되게 된다. 따라서, 본 발명의 솔장다리 추출물을 식품에 포함시키면 항산화 효과를 달성함으로써, 건강증진에 기여할 수 있다.
The term "antioxidant" of the present invention means an antioxidant action. The human body has a balance between prooxidant and antioxidant, but due to various factors, And leaning towards oxidative stimulation leads to oxidative stress leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. The reactive nitrogen species (RNS) such as NO, HNO2, and ONOO- are produced in large quantities by the immune response of macrophage neutrophils and other immune cells during the inflammatory reaction. Such active oxygen oxidizes and destroys cells in the body, thereby exposing them to various diseases. Therefore, the antioxidative effect can be achieved by incorporating the extract of the present invention into a food, thereby contributing to health promotion.
본 발명의 식품조성물은 항산화효과를 통해 상기 활성산소로 인해 발생하는 질환의 예방, 치료 또는 개선효과를 가질 수 있다. 상기 활성산소로 인해 발생하는 질환들은 동맥경화증, 루게릭병, 파킨슨병, 알츠하이머, 근위축색경화증 및 헌팅톤병을 포함하는 퇴행성 신경질환, 심근경색, 협심증, 관상동맥질환, 허혈성 심장질환을 포함하는 심혈관 질환, 뇌졸중을 포함하는 허혈성 뇌질환, 당뇨병, 위염 및 위암을 포함하는 소화기계 질환, 암, 백혈병, 노화, 류마티스 관절염, 간염, 아토피성 피부염 등 다양한 질환을 포함할 수 있으며, 바람직하게는 활성산소에 의해 발생되는 노화일 수 있다.
The food composition of the present invention may have an effect of preventing, treating or improving a disease caused by the active oxygen through an antioxidative effect. The diseases caused by the active oxygen include, but are not limited to, degenerative neurological diseases including arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, proximal axillary sclerosis and Huntington's disease, cardiovascular diseases including myocardial infarction, angina pectoris, coronary artery disease, , Arthritis, rheumatoid arthritis, hepatitis, atopic dermatitis, and the like, preferably arthritis, arthritis, osteoarthritis, osteoporosis, osteoporosis, ≪ / RTI >
본 발명의 일실시예에서는 솔장다리 추출물의 DPPH 라디칼 소거 활성을 분석한 결과, 농도의존적으로 DPPH 라디칼 억제능이 증가함을 확인하였다(도 5의 A). 또한 DPPH 억제율 비율이 50 %(IC50)일 ,때 상기 추출물의 농도가 4.829 μg/mL로 나타나 본 발명의 솔장다리 추출물이 강한 항산화능을 보유하고 있음을 확인하였다(도 5의 B).
In one embodiment of the present invention, the DPPH radical scavenging activity of the Solanum tuberosum extract was analyzed and it was confirmed that the DPPH radical scavenging ability was increased in a concentration-dependent manner (FIG. 5A). In addition, when the DPPH inhibitory ratio was 50% (IC 50 ), the extract had a concentration of 4.829 μg / mL, indicating that the extract of the present invention had a strong antioxidant ability (FIG. 5B).
본 발명의 조성물을 식품 첨가물로 사용할 경우, 상기 솔장다리 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있으며, 식품학적으로 허용가능한 식품 보조 첨가제를 추가로 포함할 수 있다. 본 발명의 조성물은 천연물로부터 유래한 추출물을 유효성분으로 하므로 안정성 면에서 문제가 없기 때문에 혼합량에 큰 제한은 없다.
When the composition of the present invention is used as a food additive, the extract can be used as it is or can be used in combination with other food or food ingredients, and can be suitably used according to ordinary methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment), and may further include a food-acceptable food-aid additive. Since the composition of the present invention contains an extract derived from a natural substance as an active ingredient, there is no problem in terms of stability, so there is no great limitation on the amount of the mixture.
본 발명의 식품 조성물은 통상적인 의미의 식품을 모두 포함할 수 있으며, 기능성 식품, 건강기능식품 등 당업계에 알려진 용어와 혼용 가능하다.
The food composition of the present invention may include all foods having a conventional meaning, and may be mixed with terms known in the art such as functional foods, health functional foods, and the like.
본 발명의 용어 "기능성 식품"은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The term "functional food" in the present invention means a food prepared and processed by using a raw material or ingredient having functionality useful to the human body according to Law No. 6727 on health functional foods, and " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.
또한, 본 발명의 용어 "건강기능식품"은 건강보조의 목적으로 특정성분을 원료로 하거나 식품 원료에 들어있는 특정성분을 추출, 농축, 정제, 혼합 등의 방법으로 제조, 가공한 식품을 말하며, 상기 성분에 의해 생체방어, 생체리듬의 조절, 질병의 방지와 회복 등 생체조절기능을 생체에 대하여 충분히 발휘할 수 있도록 설계되고 가공된 식품을 말하는 것으로서, 상기 건강식품용 조성물은 질병의 예방 및 질병의 회복 등과 관련된 기능을 수행할 수 있다.
The term "health functional food" of the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in a food raw material, Refers to a food which is designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease and the like can be sufficiently exhibited to the living body by the above components. Recovery, and so on.
본 발명의 조성물이 사용될 수 있는 식품의 종류에는 제한이 없다. 아울러 본 발명의 솔장다리 추출물을 활성성분으로 포함하는 조성물은 당업자의 선택에 따라 식품에 함유될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림 류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다.
There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising the Solanum tuberculosis extract of the present invention as an active ingredient can be prepared by admixing other appropriate auxiliary ingredients that can be contained in the food and known additives according to the selection of the person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.
또한, 본 발명에 적용될 수 있는 식품에는 예컨대, 특수영양식품(예: 조제유류, 영,유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예: 라면류, 국수류 등), 건강보조식품, 조미식품(예: 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예:스낵류), 유가공품(예: 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예: 과실, 채소류 음료, 두유류, 발효음료류 등), 천연조미료(예, 라면스프 등) 등 모든 식품을 포함할 수 있다.
Examples of foods that can be used in the present invention include special nutritional foods such as crude oil, milk, baby food, meat products, fish meat products, tofu, mushrooms, noodles such as ramen noodles, (Such as soy sauce, doenjang, kochujang, mixed potatoes), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickles ), Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).
본 발명의 건강기능식품 조성물이 음료의 형태로 사용될 경우에는 통상의 음료와 같이 여러 가지 감미제, 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 외에 본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다
When the health functional food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It can also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks
또 하나의 양태로서, 본 발명은 솔장다리(Salsola collina Pallas) 추출물을 유효성분으로 포함하는, 항산화용 화장료 조성물을 제공한다. 상기 솔장다리 추출물에 관해서는 전술한 바와 같으며, 본 발명의 추출물은 항산화 효과를 갖기 때문에 항산화 효과를 목적으로 화장료 조성물에 첨가할 수 있다.
In another aspect, the present invention provides a method of producing a compound of formula collina Pallas) extract as an active ingredient. Since the extract of the present invention has an antioxidative effect, it can be added to the cosmetic composition for the purpose of antioxidative effect.
본 발명의 화장료 조성물은 일반적인 유화 제형 및 가용화 제형의 형태로 제조할 수 있다. 상기 유화 제형으로는 영양화장수, 크림, 에센스 등이 있으며, 상기 가용화 제형으로는 유연화장수 등이 있다. 적합한 제형은 이에 제한되지는 않으나, 예를 들어 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀), 바이온형의 소낭 분산제의 형태, 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태일 수 있다. 또한, 포말(foam)의 형태 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태일 수 있다.
The cosmetic composition of the present invention can be produced in the form of a general emulsified formulation and a solubilized formulation. Examples of the emulsified formulations include nutritive lotions, creams, essences, and the like, and the solubilization formulations include softening longevity. Suitable formulations include, but are not limited to, solutions, gels, solid or paste anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) A cream, a skin, a lotion, a powder, an ointment, a spray, or a conical stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
상기 화장료 조성물은 추가적으로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제, 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장료 조성물에 통상적으로 사용되는 임의의 다른 성분과 같은 통상적으로 사용되는 보조제를 함유할 수 있다.
The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, surfactants, water, ionic or nonionic emulsifiers, Auxiliaries such as ionic sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients conventionally used in cosmetic compositions ≪ / RTI >
본 발명의 솔장다리(Salsola collina Pallas) 추출물을 포함하는 조성물은 강한 항암 및 항산화 활성을 나타내므로, 항암 또는 항산화를 목적으로 하는 의약품, 식품 및 화장품 등에 이용될 수 있다.
The Salsola of the present invention collina Pallas) extract has strong anticancer and antioxidant activity and thus can be used for medicines, foods and cosmetics intended for anticancer or antioxidation.
도 1은 솔장다리 에탄올 추출물이 HT29 및 3T3-L1 세포 생존율에 미치는 영향을 나타낸다.
도 2는 솔장다리 에탄올 추출물을 처리한 후 HT29 세포의 형태변화를 나타낸다.
도 3은 솔장다리 에탄올 추출물이 HT29 세포의 세포주기에 미치는 영향을 나타낸다. (A) 솔장다리 에탄올 추출물을 처리한 후 세포주기 분포에 대한 히스토그램을 나타낸다. M1, DNA 합성 부주기(sub G1); M2, (DNA 합성 준비기(G1); M3, DNA 합성기(S); M4, 분열준비기/분열기(G2/M). (B) 솔장다리 추출물을 처리한 후 세포주기 분포율을 나타낸다.
도 4는 HT29 세포에서 솔장다리 추출물에 의한 세포주기 관련 단백질 발현의 조절을 나타낸다.
도 5는 DPPH 라디칼 소거활성분석에 의한 솔장다리 에탄올 추출물의 항산화 활성을 나타낸다. (A) 여러 가지 농도의 솔장다리 추출물에 의한 DPPH 억제율(%). (B) 솔장다리 추출물의 50 % 저해농도(IC50) 값.Figure 1 shows the effect of the ethanol extract of Brassica juncea on HT29 and 3T3-L1 cell survival.
FIG. 2 shows morphological changes of HT29 cells after treatment with ethanol extract of Brassica juncea.
FIG. 3 shows the effect of ethanol extract of Brassica juncea on the cell cycle of HT29 cells. (A) Histogram of cell cycle distribution after treatment with Solanum radix extract. M1, DNA synthesis cycle (sub G1); M2, (DNA synthesis preparator (G1), M3, DNA synthesizer (S), M4, cleavage preparation / cleavage (G2 / M), and (B)
Figure 4 shows the regulation of cell cycle-related protein expression by Solanum tuberculosis extracts in HT29 cells.
FIG. 5 shows the antioxidative activity of ethanol extract of Solanum tuber according to DPPH radical scavenging activity assay. (A)% inhibition of DPPH by various concentrations of Solanum tuberculosis extract. (B) 50% inhibitory concentration (IC 50 ) value of Solanum tuberculosis extract.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예Example 1: One: 솔장다리Sol Staple 추출물의 제조 Preparation of extract
중국 유래 자생식물 중 하나인 솔장다리(Salsola collina Pallas.) 추출물은 한국생명공학연구원 해외생물소재허브센터의 한국식물추출물은행을 통해 분양받았다. 상기 솔장다리를 95 % 에탄올 용매를 사용하여 추출하였다. 구체적으로는 95 %에탄올이 첨가된 시료를 45 ℃에서 초음파처리를 하여 추출하였고 농축 후 동결건조하였다. 동결건조된 시료는 Dimethyl sulfoxide (DMSO)에 녹여 실험에 사용하였다.One of the Chinese native plants, Salsola Collina Pallas.) extracts were distributed through the Korea Plant Extract Bank at the Korea Biotech Research Center's Overseas Biomaterials Hub Center. The brassieres were extracted using a 95% ethanol solvent. Specifically, a sample containing 95% ethanol was extracted by ultrasonication at 45 ° C, concentrated and lyophilized. The lyophilized samples were dissolved in dimethyl sulfoxide (DMSO) and used in the experiment.
실시예Example 2: 세포배양 2: Cell culture
솔장다리 추출물의 세포독성 실험을 위해 대장암 세포주 HT29 및 정상 세포주 3T3-L1을 American Type Culture Collection (ATCC)로부터 구입하여 사용하였다. HT29 세포주는 10% 우태혈청(fetal bovine serum, FBS) 및 페니실린/스트렙토마이신(penicillin/streptomycin)이 포함된 DMEM(Dulbecco's modified Eagle's medium) 배지에서, 3T3-L1 세포주의 배양은 10 % 우아혈청(bovine calf serum, BCS) 및 페니실린/스트렙토마이신이 포함된 DMEM 배지를 사용하여 배양하였다.
Colon cancer cell line HT29 and normal cell line 3T3-L1 were purchased from the American Type Culture Collection (ATCC) for the cytotoxicity test of Brassica sativa L. extract. HT29 cell line was cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum (FBS) and penicillin / streptomycin, and 3T3-L1 cell line was cultured in 10% calf serum, BCS) and penicillin / streptomycin.
실시예Example 3: 대장암세포 사멸 효과 분석 3: Analysis of colon cancer cell killing effect
솔장다리 추출물이 대장암 세포의 생존률에 미치는 영향을 알아보기 위하여 WST(Water soluble tetrazolium) 분석을 통해 분석하였으며, 솔장다리 추출물을 농도별로 처리 한 다음 대장암 세포의 형태변화는 위상차현미경 아래에서 확인하였다.Water soluble tetrazolium (WST) analysis was carried out to investigate the survival rate of colon cancer cells. The morphological changes of colorectal cancer cells were analyzed under a phase contrast microscope .
구체적으로 대장암 세포주와 정상 세포주를 24 웰 조직배양접시(24-well tissue culture plate)에 웰 당 5 X 104 세포를 부착시킨 후 솔장다리 추출물을 농도(10, 20, 30, 40, 50, 60, 70 μg/mL) 별로 처리하여 배양하였다. 48시간 배양 후 세포 사멸 유발 유무를 WST 분석을 통해 분석하였으며 대장암 세포의 형태변화는 위상차현미경 아래에서 확인하였다(도 1 및 도 2).Specifically, colon cancer cell lines and normal cell lines were plated on a 24-well tissue culture plate at 5 × 10 4 cells / well, and then the extracts were incubated at 10, 20, 30, 40, 50, 60, 70 μg / mL). After 48 hours incubation, cell death or not induction was analyzed by WST analysis and morphological changes of colon cancer cells were confirmed under a phase contrast microscope (Fig. 1 and Fig. 2).
본 발명의 솔장다리 추출물이 대장암세포의 생존율에 미치는 영향을 살펴본 결과 도 1에 나타난 바와 같이 HT29의 세포증식 억제능은 30 μg/mL에서부터 나타났으며 49.1 μg/mL에서는 50 % 사멸효과를 보였다. 반면 정상 세포주인 3T3-L1에서는 세포독성을 보이지 않았다. 이러한 결과는 솔장다리 추출물이 암세포 사멸효과가 있음을 보여주는 것이다.As shown in FIG. 1, the inhibitory activity of HT29 on cell proliferation was found to be 30 μg / mL and the 50% kill effect was observed at 49.1 μg / mL. On the other hand, 3T3-L1, a normal cell line, was not cytotoxic. These results show that the extract of Brassica juncea has cancer-killing effect.
또한 HT29 세포에 솔장다리 추출물을 농도(20, 40, 60, 80, 100 μg/mL) 별로 처리한 후 세포의 형태변화를 위상차 현미경으로 사진을 관찰하여 도 2에 나타내었다. 도 2에서 볼 수 있듯이 솔장다리 추출물의 처리 농도에 따라 점차적으로 HT29 세포의 특징적인 구형(sphere)의 형태가 불규칙적으로 변하고, 또한 무리를 이루어 자라는 형태가 단일세포형태로 변하는 것을 관찰할 수 있었다. 이 역시 솔장다리 추출물의 암세포에 대한 효과를 뒷받침해주는 결과이다.
In addition, HT29 cells were treated with Solanum tuberculosis extract at various concentrations (20, 40, 60, 80, and 100 μg / mL), and the morphological changes of the cells were photographed with a phase contrast microscope. As can be seen from FIG. 2, the characteristic sphere morphology of HT29 cells was gradually changed according to the treatment concentration of Solanum tuberculosis extract, and the morphology changed into a single cell morphology. This also supports the effect of Solanum tuberculosis extract on cancer cells.
실시예Example 4: 세포주기 변화 분석 4: Cell cycle analysis
실시예 3에서 솔장다리 추출물의 암세포 사멸효과를 확인하였는 바, 이러한 효과의 기작을 확인하기 위하여 세포주기 분석을 실시하였다.In Example 3, the cancer cell killing effect of the Solanum tuberculosis extract was confirmed. To confirm the mechanism of this effect, cell cycle analysis was performed.
T29 세포에 솔장다리 추출물을 처리한 후 세포주기의 변화를 확인하기 위하여 PI(Propium Iodide) 염색을 실시하여 유세포분석기(Flow cytometry, Quanta SC, Beckman)를 이용하여 분석하였다. T29 cells were stained with PI (Propidium iodide) and analyzed by flow cytometry (Quanta SC, Beckman).
구체적으로 HT29 세포를 6 웰 조직배양 접시(6-well tissue culture plate)에 웰 당 5 X 105 세포를 부착시킨 후 솔장다리 추출물을 농도(20, 40, 60 μg/mL)별로 처리하였다. 상기 세포를 24시간 배양한 후 세포를 회수하고 PI 염색을 실시하여 세포주기를 분석하였다. Specifically, HT29 cells were plated on a 6-well tissue culture plate at 5 × 10 5 cells / well and treated with Solanum tuberosum extract (20, 40, 60 μg / mL). After culturing the cells for 24 hours, the cells were recovered and PI staining was performed to analyze the cell cycle.
상기 HT29 세포에 솔장다리 추출물을 처리한 후 세포주기의 변화를 유세포분석기로 분석한 결과, 도 3에 나타난 바와 같이 농도의존적으로 G2/M 정지(arrest)가 유도됨을 확인하였다. 또한 G2/M 정지가 유도되면서 상대적으로 G1기의 세포 분포는 점차 감소함을 확인하였다. 이러한 결과를 통하여 솔장다리 추출물은 암세포의 G2/M기의 정지를 유발하여 암세포의 증식을 억제함을 확인할 수 있었다.
As shown in FIG. 3, it was confirmed that G2 / M arrest was induced in a concentration-dependent manner by the flow cytometric analysis of the HT29 cells after treatment with Solanum tuberculosis extract. In addition, G2 / M arrest was induced, and the cell distribution of G1 phase was gradually decreased. From these results, it was confirmed that the Solanum radix extract inhibited the proliferation of cancer cells by inducing the G2 / M phase arrest of cancer cells.
실시예Example 5: 항암활성 관련 단백질 발현 5: Expression of anticancer activity-related proteins 조절능Control ability 분석 analysis
솔장다리 추출물의 항암 활성 기전을 밝히기 위해 세포주기 중 G2/M기 조절에 관여하는 단백질 발현을 웨스턴 블롯 혼성화(Western blot hybridization)로 분석하였다. To elucidate the antitumor activity of the extracts, the expression of proteins involved in G2 / M regulation in the cell cycle was analyzed by Western blot hybridization.
추출물 시료 처리가 끝난 HT29 배양세포에서 단백질을 추출하여 브래드포드 분석(Bradford assay)으로 단백질 농도를 결정한 후 30 μg의 단백질을 10% SDS-PAGE로 전기영동하고 니트로셀룰로오스 멤브레인(Nitrocellulose membrane)에 블롯팅(Botting) 한 후 대상 단백질의 항체와 혼성화 하였다. 멤브레인 세척 후 HRP가 태깅(tagging)된 이차항체로 한 시간 동안 반응시킨 후 화학발광 검출 시스템을 이용하여 단백질 발현을 분석하였다(도 4).Proteins were extracted from the HT29 cultured cells after the treatment of the extract samples, and the protein concentration was determined by Bradford assay. Then, 30 μg of the protein was electrophoresed on 10% SDS-PAGE and blotted on a nitrocellulose membrane And then hybridized with the antibody of the target protein. After washing the membrane, HRP was reacted with a tagged secondary antibody for one hour, and protein expression was analyzed using a chemiluminescence detection system (FIG. 4).
본 발명의 솔장다리 추출물이 HT29 세포의 G2/M 중지를 유도하는 것을 확인하였는바, 관련 단백질 발현에 미치는 영향을 웨스턴 블롯 혼성화로 분석하였다. 그 결과 도 4에 제시된 바와 같이 Cyclin-dependent kinase(CDK) 억제인자로 알려진 p21 및 Wee1의 발현이 증가되었고, Cdc2(Cell division cycle 2)의 불활성 형태인 phosphor-Cdc2, Cdc25C(cell division cycle 25 homologue C)의 불활성 형태인인 phosphor-Cdc25C의 발현이 증가되었다. 이 때 p53의 발현에는 변화가 없는 것으로 보아 p53에 비의존적인 p21의 발현 증가임을 확인할 수 있었다. 반면 Cdc25C 및 cyclin A의 발현은 감소되었다. 이러한 결과들을 종합해 볼 때, HT29 세포에서 솔장다리 추출물 처리에 의한 G2/M 중지는 p21 및 Wee 1의 발현 증가와 Cdc25C의 발현 감소에 따른 G2/M기 체크 포인트(check point)인 Cdc2/cyclin complex의 활성 저해에 의한 것임을 알 수 있었다.
It was confirmed that the extract of Solanaceae according to the present invention induced G2 / M arrest in HT29 cells, and the effect on the expression of the related proteins was analyzed by Western blot hybridization. As shown in FIG. 4, the expression of p21 and Wee1, which are known as cyclin-dependent kinase (CDK) inhibitors, was increased and phosphor-Cdc2, Cdc25C (cell division cycle 25 homologue C), which is an inactive form of phosphor-Cdc25C. At this time, there was no change in the expression of p53, indicating that the expression of p21 in p53 was increased. On the other hand, the expression of Cdc25C and cyclin A was decreased. These results suggest that G2 / M arrest by treatment with Solanum tuberculosis extract in HT29 cells is associated with increased expression of p21 and
실시예Example 6: 6: DPPHDPPH 라디칼Radical 소거 활성 분석 Scavenging activity analysis
DPPH(2,2-diphenyl-1-picrylhydrazyl) 라디칼 소거 활성(DPPH radical scavenging activity) 측정을 이용하여 솔장다리 추출물의 항산화능 분석을 다음과 같이 실시하였다.The DPPH radical scavenging activity was measured by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. Respectively.
전자공여능은 항산화작용의 지표로 사용되고 있으며 식물 추출물의 항산화능 측정에 많이 사용되고 있다. 전자공여작용은 인체 내에서 생성되는 프리라디칼의 전자를 공여하여 프리라디칼에 의한 노화와 질병을 억제하는 작용으로 이용되고 있다. 전자공여능 측정은 DPPH radical 소거법을 이용하여 측정하였다. DPPH는 비교적 안정한 자유 라디칼(free radical)로써, 아스코르브산(ascorbic acid), 토코페롤(tocopherol), 폴리히드록시(polyhydroxy) 방향족 화합물, 방향족 아민류에 의해 환원되어 짙은 자색이 탈색되는 원리를 이용하여 항산화 활성을 간단히 측정할 수 있는 동시에 식물체의 항산화 활성과도 연관성이 매우 높기 때문에 많이 이용되고 있는 방법이다.The electron donating ability is used as an index of antioxidant activity and is widely used for measuring the antioxidant activity of plant extracts. The electron donating action is used to donate electrons of free radicals generated in the human body, thereby suppressing aging and disease by free radicals. The electron donating ability was measured using the DPPH radical scavenging method. DPPH is a relatively stable free radical which is reduced by ascorbic acid, tocopherol, polyhydroxy aromatic compounds and aromatic amines and decolorizes deep purple, Can be easily measured, and at the same time, it has a high correlation with the antioxidant activity of plants.
상기 실시예 1의 방법으로 제조한 솔장다리 추출물을 농도별 (2.56 ~ 320 μg/mL)로 메탄올에 녹여 준비한 후, 96 well plate에 메탄올에 용해된 1.5 
The sole extract prepared by the method of Example 1 was dissolved in methanol at a concentration of 2.56 to 320 μg / mL and prepared. Then, a solution of 1.5
DPPH 라디칼 소거 활성 (%)={1-(A-B)/C}×100
DPPH radical scavenging activity (%) = {1- (AB) / C} 100
상기 A는 520 nm에서 시료의 흡광도, B는 520 nm에서 색 대조군 흡광도, C는 520 nm에서 대조군 흡광도이다.
A is the absorbance of the sample at 520 nm, B is the color control absorbance at 520 nm, and C is the control absorbance at 520 nm.
본 발명의 솔장다리 추출물의 항산화능을 DPPH 라디칼 소거 활성 측정을 통해 분석한 결과, 도 5의 A에 나타난 바와 같이 솔장다리 추출물의 농도가 증가함에 따라 DPPH 라디칼 억제능이 증가함을 확인하였다. 억제능 비율이 50 %일 때, 솔장다리 추출물의 농도(IC50)는 4.829 μg/ml로 양성 대조군인 아스코르브산(1.318 μg/ml)보다는 다소 높았으나 매우 강한 항산화 활성을 지니는 것을 알 수 있었다 (도 5의 B). As a result of analyzing the antioxidative activity of the extract of the present invention by measuring the DPPH radical scavenging activity, it was confirmed that the DPPH radical inhibition was increased as the concentration of the extract was increased as shown in FIG. 5A. When the inhibitory ratio was 50%, the concentration (IC 50 ) of Brassica sativa extract was 4.829 μg / ml, which was slightly higher than that of ascorbic acid (1.318 μg / ml) 5 of B).
따라서 본 연구 결과를 통해 솔장다리의 추출물이 높은 항산화 활성을 지니고 있으며 암세포의 세포주기를 조절하여 높은 항암 활성을 보유함을 확인할 수 있었다.
Therefore, the results of this study suggest that the extract of Solanaceae has a high antioxidant activity and has a high anticancer activity by controlling the cell cycle of cancer cells.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
Claims (7)
A pharmaceutical composition for preventing or treating colorectal cancer, comprising an extract of Salsola collina Pallas as an active ingredient.
상기 추출물은 물, 탄소수 1 내지 4의 알코올 또는 이들의 혼합용매로 추출한 것인 조성물.
The method according to claim 1,
Wherein the extract is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
상기 알코올은 에탄올인 것인 조성물.
3. The method of claim 2,
Wherein the alcohol is ethanol.
상기 대장암의 예방 또는 치료효과는 암세포의 세포주기 조절에 의해 달성되는 것인 조성물.
The method according to claim 1,
Wherein the preventive or therapeutic effect of the colon cancer is achieved by controlling the cell cycle of cancer cells.
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| Magnetic Resonance in Chemistry. Vol. 46. No. 1, pp.94-98 * |
| Magnetic Resonance in Chemistry. Vol. 46. No. 1, pp.94-98* |
| Pharmaceutical Chemistry Journal. Vol. 36, No. 10, pp.544-545 * |
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| Zeitschrift fur Naturforschung C-Journal of Biosciences. Vol. 63, No. 5-6, pp.347-354 * |
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