KR20240059259A - Composition for preventing or treating radiation-resistant head and neck cancer comprising Spatholobus suberectus Dunn extract as an active ingredient - Google Patents
Composition for preventing or treating radiation-resistant head and neck cancer comprising Spatholobus suberectus Dunn extract as an active ingredient Download PDFInfo
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- KR20240059259A KR20240059259A KR1020220140310A KR20220140310A KR20240059259A KR 20240059259 A KR20240059259 A KR 20240059259A KR 1020220140310 A KR1020220140310 A KR 1020220140310A KR 20220140310 A KR20220140310 A KR 20220140310A KR 20240059259 A KR20240059259 A KR 20240059259A
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- cancer
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- head
- neck cancer
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
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Abstract
본 발명은 계혈등 추출물을 유효성분으로 포함하는 방사선 내성 두경부암의 예방 또는 치료용 조성물에 관한 것으로서, 본 발명의 계혈등 추출물은 방사선 내성 두경부암 세포주를 효과적으로 사멸시키는 것을 확인하였다. 또한, 세포사멸과 관련된 PARP의 발현은 감소시키며, ROS 생성을 유도하여, 방사선 내성 두경부암 세포주의 세포사멸을 유도하는 것을 확인하였다. 또한, 방사선 처리와 병용하면, 계혈등 추출물을 단독으로 처리하는 것과 비교하여, 효과적으로 방사선 내성 두경부암 세포주를 사멸시킬 수 있는 것을 확인하였다.The present invention relates to a composition for the prevention or treatment of radiation-resistant head and neck cancer containing the extract of Gyehyeol, etc. as an active ingredient. It was confirmed that the extract of Gyehyeol, etc. of the present invention effectively kills radiation-resistant head and neck cancer cell lines. In addition, it was confirmed that it reduced the expression of PARP, which is related to apoptosis, and induced ROS production, thereby inducing apoptosis of radiation-resistant head and neck cancer cell lines. In addition, it was confirmed that when used in combination with radiation treatment, radiation-resistant head and neck cancer cell lines can be killed more effectively than when the extract such as Gyehyeol is treated alone.
Description
본 발명은, 계혈등 추출물을 유효성분으로 포함하는 방사선 내성 두경부암의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating radiation-resistant head and neck cancer, comprising an extract such as Gyehyeol as an active ingredient.
암은 세계적으로 높은 사망률을 보이고 있으며, 서구 사회에서는 심혈관 질환 다음으로 가장 일반적인 사망 원인이다. 특히, 인구의 고령화와 더불어 흡연 인구의 증가 및 대기 오염으로 인해 폐암이 증가하고 있으며, 식생활이 서구화되어 고지방식의 섭취가 일반화되고, 환경오염 물질의 급격한 증가, 음주량의 증가 등으로 대장암, 유방암, 전립선암 등이 지속적으로 증가하는 추세에 있다. 이러한 실정에서 암의 조기 예방 및 치료를 가능하게 하여 인간 건강의 증진, 건강한 삶의 질 향상 및 인류 보건 증진에 기여할 수 있는 항암 물질의 창출이 절실히 요구되고 있다. 암을 정복하기 위해 DNA 손상/복구의 기본적인 메커니즘, 아폽토시스, 종양형성, 및 분자 측면에서의 치료의 더 깊은 이해에 초점을 맞춘 혁신적인 암 연구가 강력하게 요구되며, 이는 결국 암 요법에서의 돌파구가 될 수 있을 것이다.Cancer has a high mortality rate worldwide and is the most common cause of death in Western societies after cardiovascular disease. In particular, with the aging of the population, lung cancer is on the rise due to an increase in the number of smokers and air pollution, and the consumption of high-fat diets is becoming more common due to the westernization of eating habits, and the rapid increase in environmental pollutants and increased drinking volume are leading to colon cancer and breast cancer. , prostate cancer, etc. are continuously increasing. In this situation, there is an urgent need for the creation of anticancer substances that can contribute to the improvement of human health, healthy quality of life, and human health by enabling early prevention and treatment of cancer. To conquer cancer, there is a strong need for innovative cancer research that focuses on a deeper understanding of the fundamental mechanisms of DNA damage/repair, apoptosis, tumorigenesis, and treatment from a molecular perspective, which will eventually lead to a breakthrough in cancer therapy. You will be able to.
두경부암(Head and Neck Cancer)은 전세계적으로 매년 600,000명 정도 발생하는, 전체암 중에 6번째로 흔한 암으로 알려져 있다. 두경부란 뇌 아래에서 가슴 윗 부분 사이를 말하고, 두경부에는 비강, 혀, 입, 후두, 침샘등 음식을 먹거나 목소리를 내는 등의 기능을 하는 기관이 많이 있으며 이런 기관에 생긴 암을 두경부암이라고 통틀어 말한다. 유전적인 요인과 흡연, 음주가 두경부암을 일으킨다고 알려져 있다. 두경부암은 타 장기의 암보다 안면과 목이라는 특수성 때문에 치료상의 제약이 많고 진행 시 치명적이고 치료 후 기능상 외견상 장애가 많이 나타나는 곳이기도 하다. 발생되는 병의 병리적 기원도 다양하고 동일병리의 병이라고 하더라도 발생위치에 따라 전혀 다른 예후를 보이는 특징을 가지고 있다. 인구의 노령화와 음주와 흡연이 증가하면서 두경부암의 발생률도 증가 추세여서, 전체 암의 4~5%를 차지하며, 통계적으로 볼 때 장기별 암발생 기준으로 7위, 남자환자 기준으로 5위에 해당하는 비교적 흔한 암에 속한다. 두경부암의 종류를 구체적으로 살펴보면, 크게 비강 및 부비동암, 구강암, 비인두암, 구인두암, 하인두암, 후두암, 경부 식도암, 침샘암, 타액선암, 악성 림프종, 악성 흑색종, 각종 연부조직 암 등이 있다. 두경부암의 또 다른 중요한 특징은 다른 장기에 이차암이 발병하는 경우가 빈번하다는 것이다. 보고에 따라 다르지만 평생에 걸쳐 약 10~40%에서 두경부 외 다른 부위 폐, 위, 식도 등에 이차암이 발병하는 것으로 알려졌다. 이처럼 이차암의 발병률이 높은 이유는 두경부암과 폐암, 위암, 식도암이 발암 원인 인자를 공유하기 때문이다. 두경부암의 치료 방법으로는 수술, 방사선치료, 항암화학요법을 주로 사용한다.Head and neck cancer is known to be the 6th most common cancer among all cancers, occurring in approximately 600,000 cases annually worldwide. The head and neck region refers to the area between the lower part of the brain and the upper part of the chest. There are many organs in the head and neck region that function such as the nasal cavity, tongue, mouth, larynx, and salivary glands, and other functions such as eating and vocalization. Cancers that occur in these organs are collectively called head and neck cancer. . It is known that genetic factors, smoking, and drinking cause head and neck cancer. Compared to cancers of other organs, head and neck cancer has many limitations in treatment due to the special characteristics of the face and neck, is fatal when it progresses, and often causes apparent functional impairment after treatment. The pathological origins of the diseases that occur are diverse, and even the same pathology has the characteristic of showing completely different prognoses depending on the location of occurrence. As the population ages and drinking and smoking increase, the incidence of head and neck cancer is also increasing, accounting for 4-5% of all cancers. Statistically, it ranks 7th in cancer occurrence by organ and 5th in male patients. It is a relatively common cancer. Looking at the types of head and neck cancer in detail, they largely include nasal cavity and paranasal sinus cancer, oral cancer, nasopharyngeal cancer, oropharynx cancer, hypopharyngeal cancer, laryngeal cancer, cervical esophagus cancer, salivary gland cancer, salivary gland cancer, malignant lymphoma, malignant melanoma, and various soft tissue cancers. . Another important characteristic of head and neck cancer is that secondary cancer frequently develops in other organs. Depending on the report, it is known that approximately 10 to 40% of people develop secondary cancer in areas other than the head and neck, such as the lungs, stomach, and esophagus, throughout their lifetime. The reason why the incidence of secondary cancer is so high is because head and neck cancer, lung cancer, stomach cancer, and esophageal cancer share the causative factor of carcinogenesis. The main treatment methods for head and neck cancer are surgery, radiation therapy, and chemotherapy.
한편, 생체 내의 세포들은 세포사멸(apoptosis)의 과정을 거쳐 제거된다. 그러나 암세포는 계속된 분열로 인해 무한으로 증식한다. 이러한 암세포를 제거하기 위해서는 사멸을 유도해야 한다. PARP는 DNA가 손상되었을 때 복구를 하는 역할을 한다. 이는 세포사멸이 일어나면 기능을 하지못한다. 때문에 사멸이 일어나는 세포에서 PARP의 양은 줄어드는 것으로 나타난다. ROS(Reactive oxygen species)는 암세포에서 세포사멸을 일으키는 요소 중 하나이다. DCF-DA를 이용한 염색은 ROS의 발생 정도를 확인할 수 있게 한다. ROS의 증가는 암세포의 사멸이 증가함을 확인할 수 있게 한다.Meanwhile, cells in the living body are eliminated through the process of apoptosis. However, cancer cells proliferate infinitely due to continuous division. To remove these cancer cells, death must be induced. PARP plays a role in repairing DNA when it is damaged. It ceases to function when cell death occurs. Therefore, the amount of PARP appears to decrease in cells undergoing death. ROS (Reactive oxygen species) is one of the factors that cause apoptosis in cancer cells. Staining using DCF-DA allows the level of ROS generation to be confirmed. An increase in ROS confirms an increase in the death of cancer cells.
한편, 계혈등은 콩과의 밀화두(Spatholobus suberectus Dunn : 密花豆)의 덩굴줄기를 말한다. 계혈등은 신선한 줄기를 자르면 적갈색의 닭의 피와 같은 액즙이 흘러나와 계혈등이라 불리게 되었으며, 혈액순환촉진 작용이 있어, 생리통, 월경불순 등에 효과가 있고, 사지마비동통에도 효과가 있다. 약리작용으로는 소염, 진통작용, 신장과 자궁대사 활성화, 자궁흥분작용, 관상동맥 확장, 백혈구 증가 작용등이 보고되었다.Meanwhile, Gyehyeoldeung refers to the vine stem of wheat flower bean ( Spatholobus suberectus Dunn: 密花豆) of the legume family. When the fresh stem of Gyehyeoldeung is cut, a reddish-brown juice like chicken blood flows out, giving it the name Gyehyeoldeung. It has the effect of promoting blood circulation and is effective in menstrual cramps, menstrual irregularities, and quadriplegic pain. Pharmacological effects include anti-inflammatory, analgesic, activation of kidney and uterine metabolism, uterine stimulation, coronary artery expansion, and white blood cell increase.
이에, 본 발명자들은 계혈등 추출물이, 방사선 내성 두경부암의 세포사멸을 유도하는 것을 확인하여, 본 발명을 완성하였다.Accordingly, the present inventors confirmed that the extract of Gyehyeol, etc., induces cell death in radiation-resistant head and neck cancer, and completed the present invention.
본 발명의 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는 두경부암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 항암보조제를 제공하는 것이다.Another object of the present invention is to provide an anti-cancer adjuvant for head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 또 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 또 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating radiation resistant head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 또 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 항암보조제를 제공하는 것이다.Another object of the present invention is to provide a radiation resistant anti-cancer adjuvant for head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 또 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving radiation resistant head and neck cancer, which contains Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 또 다른 목적은 시험관 내(in vitro)에서, 두경부암 세포주에 계혈등 추출물을 처리하는 단계;를 포함하는, 두경부암 세포주의 증식 억제 방법을 제공하는 것이다.Another object of the present invention is to provide a method of inhibiting the proliferation of head and neck cancer cell lines, comprising the step of treating the head and neck cancer cell lines with an extract such as Gyehyeol, in vitro.
상기 목적을 달성하기 위하여, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는 두경부암의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 항암보조제를 제공한다.In addition, the present invention provides an anti-cancer supplement for head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 예방 또는 개선용 식품조성물 제공한다.In addition, the present invention provides a food composition for preventing or improving head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating radiation resistant head and neck cancer, comprising an extract of Spatholobus suberectus Dunn as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 항암보조제를 제공한다.In addition, the present invention provides an anti-cancer adjuvant for radiation resistant head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 개선용 식품조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving radiation resistant head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 시험관 내(in vitro)에서, 두경부암 세포주에 계혈등 추출물을 처리하는 단계;를 포함하는, 두경부암 세포주의 증식 억제 방법을 제공한다.In addition, the present invention provides a method of inhibiting the proliferation of head and neck cancer cell lines, comprising the step of treating the head and neck cancer cell lines with an extract such as Gyehyeol, in vitro.
본 발명은 계혈등 추출물을 유효성분으로 포함하는 두경부암의 예방 또는 치료용 조성물에 관한 것으로서, 본 발명의 계혈등 추출물은 방사선 내성 두경부암 세포주를 효과적으로 사멸시키는 것을 확인하였다. 또한, 세포사멸과 관련된 PARP의 발현은 감소시키며, ROS 생성을 유도하여, 두경부암 세포주의 세포사멸을 유도하는 것을 확인하였다. 또한, 방사선 처리와 병용하면, 계혈등 추출물을 단독으로 처리하는 것과 비교하여, 효과적으로 방사선 내성 두경부암 세포주를 사멸시킬 수 있는 것을 확인하여, 방사선 내성 두경부암의 치료에 유용하게 이용할 수 있다.The present invention relates to a composition for the prevention or treatment of head and neck cancer containing an extract of Gyehyeol, etc. as an active ingredient. It was confirmed that the extract of Gyehyeol, etc. of the present invention effectively kills radiation-resistant head and neck cancer cell lines. In addition, it was confirmed that it reduced the expression of PARP, which is related to apoptosis, and induced ROS production, thereby inducing apoptosis of head and neck cancer cell lines. In addition, it was confirmed that when used in combination with radiation treatment, it can effectively kill radiation-resistant head and neck cancer cell lines compared to treatment with extracts such as Gyehyeol alone, so it can be usefully used in the treatment of radiation-resistant head and neck cancer.
도 1은 본 발명의 계혈등 추출물 처리에 따른, 방사선 내성 두경부암에서의 세포독성을 확인한 도이다.
도 2는 본 발명의 계혈등 추출물의 처리에 따른, 방사선 내성 두경부암 세포주에서의 PARP 발현 억제를 확인한 도이다.
도 3은 본 발명의 계혈등 추출물의 처리에 따른, 방사선 내성 두경부암 세포주에서의 ROS 생성 유도를 확인한 도이다.
도 4는 본 발명의 계혈등 추출물과 방사선 조사의 병용 처리에 따른, 방사선 내성 두경부암 세포주의 세포사멸을 확인한 도이다.Figure 1 is a diagram confirming cytotoxicity in radiation-resistant head and neck cancer according to treatment with the extract of the present invention.
Figure 2 is a diagram confirming the inhibition of PARP expression in radiation-resistant head and neck cancer cell lines according to treatment with the extract of the present invention.
Figure 3 is a diagram confirming the induction of ROS production in radiation-resistant head and neck cancer cell lines according to treatment with the extract of Gyehyeol, etc. of the present invention.
Figure 4 is a diagram confirming the apoptosis of radiation-resistant head and neck cancer cell lines following the combined treatment of the extract of Gyehyeol, etc. of the present invention and radiation irradiation.
이하 첨부된 도면을 참조하여 본 발명의 실시예들을 상세히 설명한다. 이하의 설명에 있어, 당업자에게 주지 저명한 기술에 대해서는 그 상세한 설명을 생략할 수 있다. 또한, 본 발명을 설명함에 있어서, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있다. 또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다.Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.
따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는 두경부암의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating head and neck cancer, comprising an extract of Spatholobus suberectus Dunn as an active ingredient.
본 발명에서 사용되는 용어 “예방”은 본 발명의 조성물의 투여로 특정 질환의 증상을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.The term “prevention” used in the present invention refers to all actions that suppress the symptoms or delay the progression of a specific disease by administering the composition of the present invention.
본 발명에서 사용되는 용어 “치료”는 본 발명의 조성물의 투여로 특정 질환의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.
본 발명의 약학 조성물에는 유효성분 이외에 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 효과를 증가시킬 수 있다.The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.
본 발명에 따른 약학 조성물의 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 약학 조성물 중 포함되는 유효성분의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 약학 조성물에0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 인체에 독성을 나타낼 수 있다.The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.
본 발명의 일실시예에 따르면, 상기 계혈등 추출물은, 물, C1 내지 C4의 저급 알코올, 저급 알코올 수용액, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군에서 선택된 용매로 추출되는 것일 수 있고, 바람직하게는 물이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the extract such as gyehyeol may be extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohol, lower alcohol aqueous solution, ethyl acetate, hexane and chloroform, preferably Water, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 두경부암은, 방사선 내성(radiationresistant) 두경부암을 더 포함하는 것일 수 있다.According to one embodiment of the present invention, the head and neck cancer may further include radiation resistant head and neck cancer.
본 발명의 “방사선 내성(radiationresistant)”은, 방사선 조사에서, 높은 단위로 종양 치유가 가능한 정도를 종양의 치유선량이라고 하며, 방사선 치유선량이 높아진 종양의 상태를 뜻한다.“Radiation resistant” of the present invention refers to the degree to which a tumor can be cured in high units by radiation irradiation, which is referred to as the tumor healing dose, and refers to the state of a tumor in which the radiation healing dose is increased.
본 발명의 일실시예에 따르면, 상기 두경부암은 구강암, 구인두암, 하인두암, 후두암, 침샘암, 비강암, 부비동암, 비인두암, 경부 식도암, 타액선암, 악성 림프종, 악성 흑색종 및 연부조직 암으로 이루어진 군에서 선택된 것일 수 있고, 바람직하게는 구강암이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the head and neck cancer includes oral cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, salivary gland cancer, nasal cavity cancer, paranasal cancer, nasopharyngeal cancer, cervical esophagus cancer, salivary gland cancer, malignant lymphoma, malignant melanoma, and soft tissue cancer. It may be selected from the group consisting of, and is preferably oral cancer, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 계혈등 추출물은, 두경부암의 세포사멸을 유도하는 것일 수 있고, 상기 세포사멸을 유도하는 것은, PARP(poly (ADP-ribose) polymerase)의 발현을 억제시키는 것일 수 있으며, ROS(Reactive oxygen species)의 생성을 유도하는 것일 수 있다.According to one embodiment of the present invention, the extract such as Gyehyeol may induce apoptosis of head and neck cancer, and inducing the apoptosis may inhibit the expression of PARP (poly (ADP-ribose) polymerase). It may be that it induces the generation of ROS (Reactive oxygen species).
본 발명의 일실시예에 따르면, 상기 계혈등 추출물은 방사선 조사와 병용 처리되는 것일 수 있다.According to one embodiment of the present invention, the extract such as Gyehyeol may be treated in combination with radiation irradiation.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 항암보조제를 제공한다.In addition, the present invention provides an anti-cancer supplement for head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
본 발명의 "항암 보조제"는 항암제의 항암효과를 개선, 향상 또는 증대시킬 수 있는 제제로서, 그 자체로는 항암활성을 나타내지 않으나 항암제와 함께 사용될 경우, 상기 항암제의 항암효과를 개선, 향상 또는 증대시킬 수 있는 제제일 수 있다. 또한, 농도의존적인 항암활성을 나타내는 제제를 그 자체로는 항암활성을 나타내지 않은 수준으로 항암제와 함께 사용할 경우, 상기 항암제의 항암효과를 개선, 향상 또는 증대시킬 수 있는 제제일 수 있다. The "anti-cancer adjuvant" of the present invention is an agent that can improve, enhance or increase the anti-cancer effect of an anti-cancer agent. It does not show anti-cancer activity by itself, but when used together with an anti-cancer agent, it improves, enhances or increases the anti-cancer effect of the anti-cancer agent. It may be an agent that can do it. In addition, when an agent that exhibits concentration-dependent anticancer activity is used together with an anticancer agent at a level that does not show anticancer activity on its own, it may be an agent that can improve, enhance, or increase the anticancer effect of the anticancer agent.
항암 보조제의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 본 발명의 항암 보조제는 목적하는 바에 따라 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 경구 투여, 비 내 투여, 폐 내 투여, 직장 내 투여될 수 있으나, 이에 제한되지는 않는다. 또한, 상기 항암 보조제는 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.Anticancer adjuvants can be administered through any general route as long as they can reach the target tissue. The anti-cancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, pulmonaryly, or rectally, depending on the purpose, but is not limited thereto. No. Additionally, the anti-cancer adjuvant can be administered by any device that allows the active substance to move to target cells.
본 발명의 또 다른 목적은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 두경부암의 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
본 발명에서 사용되는 용어 “개선”은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term “improvement” refers to any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.
본 발명의 식품 조성물은 본 발명의 유효성분을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 본 발명의 식품 조성물은 상기 약학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.
또한 상기 식품 조성물은 유효성분인 추출물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition, the food composition contains, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.
본 발명의 기능성 식품 조성물은 두경부암의 예방 또는 치료 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공될 수 있다. 본 발명에서 '건강기능성 식품 조성물'이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. 본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 '식품 첨가물 공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다. 예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다. 캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다. 환 형태의 건강기능식품은 본 발명의 유효성분과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다. 과립 형태의 건강기능식품은 본 발명의 유효성분의 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating head and neck cancer. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions of the food additive code and general test methods approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agents. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating radiation resistant head and neck cancer, comprising an extract of Spatholobus suberectus Dunn as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 항암보조제를 제공한다.In addition, the present invention provides an anti-cancer adjuvant for radiation resistant head and neck cancer containing Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 계혈등(Spatholobus suberectus Dunn) 추출물을 유효성분으로 포함하는, 방사선 내성(radiationresistant) 두경부암의 예방 또는 개선용 식품조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving radiation resistant head and neck cancer, comprising Spatholobus suberectus Dunn extract as an active ingredient.
또한, 본 발명은 시험관 내(in vitro)에서, 두경부암 세포주에 계혈등 추출물을 처리하는 단계;를 포함하는, 두경부암 세포주의 증식 억제 방법을 제공한다.In addition, the present invention provides a method of inhibiting the proliferation of head and neck cancer cell lines, comprising the step of treating the head and neck cancer cell lines with an extract such as Gyehyeol, in vitro.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
<제조예 1> 계혈등 추출물의 제조<Preparation Example 1> Preparation of extract such as chicken blood
본 말명의 계혈등 추출물의 두경부암에 대한 항암 효과를 확인하기 위하여, 계혈등 추출물을 제조하였다. 구체적으로, 강원도 홍천에서 재배한 계혈등을 100% distilled water로 48시간 동안 탕전 추출하였다. 그 후 추출액을 여과한 후 감압 농축하여, 열수 추출물을 수득하였다(수득율 7.6%).In order to confirm the anticancer effect of the extract of the present name, Gyehyeol, etc., on head and neck cancer, an extract of Gyehyeol, etc. was prepared. Specifically, Gyehyeol, etc. grown in Hongcheon, Gangwon-do were extracted with 100% distilled water for 48 hours. Afterwards, the extract was filtered and concentrated under reduced pressure to obtain a hot water extract (yield 7.6%).
<실시예 1> 계혈등 추출물의 방사선 내성 두경부암 세포주의 세포 독성 확인<Example 1> Confirmation of cytotoxicity of extracts such as Gyehyeol on radiation-resistant head and neck cancer cell lines
본 발명의 계혈등 추출물의 방사선 내성 두경부암에 대한 세포 독성을 확인하였다. 구체적으로, 방사선 내성 두경부암 세포주인 Moc1-RR(경희의료원, 대한민국, 서울)를 사용하였으며, 배양 배지는 31.3% nutrient mixture, 5% inactivated fetal bovine serum, 1% penicillin-streptomycin를 포함한 IMDM배지를 필터링 한 후 5mg/mL Insulin, Hydrocortisone, EGF를 추가된 배지를 이용하였다. Moc1-RR 세포주 100 μl를 96-well plate에 1.0 × 104 세포농도가 되도록 분주한 뒤, 37℃의 5% CO2 인큐베이터(MCO-15AC, Sanyo, Osaka, Japan)에서 배양하였다. 배양된 세포에, 배지를 제거하고, 상기 제조예 1의 계혈등 추출물을 0, 6.25, 12.5, 25 및 50 μg/ml의 농도로 각각 100 μl씩 처리한 뒤, 24시간동안 추가로 배양하였다. 그 후 배양된 세포에 EZ-Cytox 시약 (DoGen)을 10 μl 처리하고 1시간 동안 반응시켰다. 반응 종료 후 마이크로플레이트 리더기(BKMPR-1101)를 이용하여 450nm 파장대에서 흡광도를 측정하여, 세포 생존율을 확인하였다.The cytotoxicity of the extract of the present invention against radiation-resistant head and neck cancer was confirmed. Specifically, the radiation-resistant head and neck cancer cell line Moc1-RR (Kyung Hee Medical Center, Seoul, Korea) was used, and the culture medium was filtered IMDM medium containing 31.3% nutrient mixture, 5% inactivated fetal bovine serum, and 1% penicillin-streptomycin. After that, a medium containing 5mg/mL Insulin, Hydrocortisone, and EGF was used. 100 μl of the Moc1-RR cell line was dispensed into a 96-well plate at a cell concentration of 1.0 × 10 4 and cultured in a 5% CO 2 incubator (MCO-15AC, Sanyo, Osaka, Japan) at 37°C. The medium was removed from the cultured cells, and the extract of Preparation Example 1 was treated with 100 μl each at concentrations of 0, 6.25, 12.5, 25, and 50 μg/ml, and then cultured for an additional 24 hours. Afterwards, the cultured cells were treated with 10 μl of EZ-Cytox reagent (DoGen) and reacted for 1 hour. After completion of the reaction, absorbance was measured in the 450 nm wavelength range using a microplate reader (BKMPR-1101) to confirm cell viability.
그 결과, 도 1에 나타낸 바와 같이, 본 발명의 계혈등 추출물은, 농도의존적으로, 방사선 내성 두경부암 세포주인 Moc1-RR을 사멸시키는 것을 확인하여, 암세포주에 대한 세포 독성을 확인하였다.As a result, as shown in Figure 1, the extract of the present invention was confirmed to kill Moc1-RR, a radiation-resistant head and neck cancer cell line, in a concentration-dependent manner, confirming its cytotoxicity against cancer cell lines.
<실시예 2> 계혈등 추출물의 세포사멸 유도 효과 확인<Example 2> Confirmation of apoptosis-inducing effect of extract such as Gyehyeol
본 발명의 계혈등 추출물이, 세포사멸과 관련된 PARP의 발현을 조절하는지 확인하였다. 구체적으로, Moc1-RR 세포주에, 계혈등 추출물을 0 및 25 μg/ml의 농도로 각각 처리하고, 24시간 동안 37℃의 5% CO2의 조건으로 배양하였다. 그 후 세포를 수득하여, PBS로 세척한 뒤, CETI Lysis Buffer with Inhibitor(TransLab, Korea)를 첨가하여 세포를 파쇄 후 30분 동안 ice에서 반응시켰다. 그 후 세포파쇄물을 13,000 rpm에서 10분간 원심 분리하여 세포막 성분 등을 제거한 후, RC DC™ Protein Assay Kit II(protein assay kit) (Bio-rad, USA)를 사용하여 단백질을 정량하여 시료별 (계혈등 추출물 0, 25 ㎍/ml 처리군들)로 동일한 단백질의 양이 포함되도록 정량하였다. 그 후 단백질 시료에 5×로딩 다이를 넣고 5분 동안 95℃로 가열하여 단백질을 변성시키고, 10% SDS-PAGE 젤에 시료를 로딩하여 95V로 110분 동안 전기영동을 수행하였으며, 젤 위에서 분리된 단백질들을 290mA로 2시간 동안 멤브레인으로 트랜스퍼하였다. 그 후 멤브레인을 5% 탈지유가 포함된 TBST (tris buffered saline, pH 7.5) 용액으로 상온에서 1시간 동안 블로킹한 뒤 1차 항체 항-PARP(cell signaling)을 1:1,000으로 희석하여 블로킹한 멤브레인과 4℃에서 오버나잇으로 반응시켰다. 반응 종료 후 TBST로 3회 세척한 뒤, horseradish peroxidase가 결합된 항-rabbit IgG를 1:10,000 으로 희석하여 상온에서 멤브레인과 1시간 동안 반응시킨 다음 TBST로 3회 세척하였다. 세척된 멤브레인을 chemiluminescent reagent와 1분동안 반응시킨 후, chemiluminescent western blot 이미징 장비(Corebiosystem, Korea)를 통해 단백질 밴드를 가시화하였다.It was confirmed whether the extract of the present invention regulates the expression of PARP, which is related to apoptosis. Specifically, the Moc1-RR cell line was treated with Gyehyeol extract at concentrations of 0 and 25 μg/ml, respectively, and cultured at 37°C in 5% CO 2 conditions for 24 hours. Afterwards, the cells were obtained, washed with PBS, and CETI Lysis Buffer with Inhibitor (TransLab, Korea) was added, and the cells were disrupted and reacted on ice for 30 minutes. Afterwards, the cell lysate was centrifuged at 13,000 rpm for 10 minutes to remove cell membrane components, and then the protein was quantified using RC DC™ Protein Assay Kit II (protein assay kit) (Bio-rad, USA) and analyzed for each sample (measured by blood test). The extract was quantified to contain the same amount of protein (groups treated with 0 and 25 μg/ml). After that, a 5×loading die was placed in the protein sample and heated to 95°C for 5 minutes to denature the protein. The sample was loaded on a 10% SDS-PAGE gel and electrophoresis was performed at 95V for 110 minutes. Proteins were transferred to the membrane at 290 mA for 2 hours. Afterwards, the membrane was blocked with TBST (tris buffered saline, pH 7.5) solution containing 5% skim milk at room temperature for 1 hour, and then the primary antibody anti-PARP (cell signaling) was diluted 1:1,000 to block the membrane and The reaction was performed overnight at 4°C. After completion of the reaction, the reaction was washed three times with TBST, then horseradish peroxidase-conjugated anti-rabbit IgG was diluted 1:10,000, reacted with the membrane at room temperature for 1 hour, and then washed three times with TBST. After reacting the washed membrane with chemiluminescent reagent for 1 minute, protein bands were visualized using chemiluminescent western blot imaging equipment (Corebiosystem, Korea).
그 결과, 도 2에 나타낸 바와 같이, 계혈등 추출물은 농도 의존적으로 PARP의 발현을 감소시키는 것을 확인하여, 계혈등 추출물이 방사선 내성 두경부암 세포주의 세포사멸을 유도하는 것을 확인하였다.As a result, as shown in Figure 2, it was confirmed that the extract of Gyehyeol, etc. reduced the expression of PARP in a concentration-dependent manner, confirming that the extract of Gyehyeol, etc., induced apoptosis of radiation-resistant head and neck cancer cell lines.
<실시예 3> 계혈등 추출물의 방사선 내성 두경부암 세포주에서의 ROS 생성 확인<Example 3> Confirmation of ROS production in radiation-resistant head and neck cancer cell lines from extracts of Gyehyeol, etc.
본 발명의 계혈등 추출물이, 방사선 내성 두경부암 세포주에서, 세포 사멸을 유도하는 ROS 생성을 촉진하는지 확인하였다. 구체적으로, ROS의 생성을 확인할 수 있는 DCF-DA assay를 수행하였으며, Moc1-RR 세포주 100 μl를 96-well plate에 1.0 × 104 세포농도가 되도록 분주한 뒤, 37℃의 5% CO2 인큐베이터에서 배양하였다. 배양된 세포에, 배지를 제거하고, 20 μM의 DCF-DA를 처리하여 45분동안 반응시켰다. 반응 종료 후 배지를 제거한 후 페놀 레드가 포함되지 않은 IMDM 배지로 세척하였다. 그 후 계혈등 추출물을 0 및 25 μl의 농도로 100 μl씩 처리한 뒤 4시간동안 추가로 배양하였다. 배양 종료 후 마이크로플레이트 리더기를 이용하여 485nm와 535nm에서 형광을 확인하여, ROS 생성을 확인하였다.It was confirmed whether the extract of the present invention promotes the production of ROS, which induces cell death, in radiation-resistant head and neck cancer cell lines. Specifically, DCF-DA assay was performed to confirm the generation of ROS, and 100 μl of Moc1-RR cell line was dispensed into a 96-well plate to reach a cell concentration of 1.0 × 10 4 and incubated in a 5% CO 2 incubator at 37°C. It was cultured in . To the cultured cells, the medium was removed, and 20 μM of DCF-DA was treated and reacted for 45 minutes. After completion of the reaction, the medium was removed and washed with IMDM medium that does not contain phenol red. Afterwards, 100 μl each of the extracts of Gyehyeol, etc. were treated at concentrations of 0 and 25 μl and cultured for an additional 4 hours. After completion of incubation, ROS generation was confirmed by checking fluorescence at 485 nm and 535 nm using a microplate reader.
그 결과, 도 3에 나타낸 바와 같이, 계혈등 추출물은 방사선 내성 두경부암 세포주에서 ROS의 생성을 유도하여, 방사선 두경부암 세포주의 사멸을 유도하는 것을 확인하였다.As a result, as shown in Figure 3, it was confirmed that the extract of Gyehyeol et al. induced the production of ROS in the radiation-resistant head and neck cancer cell line, thereby inducing the death of the radiation-resistant head and neck cancer cell line.
<실시예 4> 계혈등 추출물과 방사선 병용 효과 확인<Example 4> Confirmation of the effect of combined use of gyehyeol extract and radiation
본 발명의 계혈등 추출물이 방사선과 병용 처리로, 방사선 내성 두경부암을 사멸시키는지 확인하였다. 구체적으로, Moc1-RR 세포주 100 μl를 96-well plate에 1.0 × 104 세포농도가 되도록 각각 두 개의 plate에 분주한 뒤, 37℃의 5% CO2 인큐베이터에서 배양하였다. 배양 종료 후 배지를 제거하고, 계혈등 추출물을 각각 0 및 25 μl의 농도로 100 μl 처리한 뒤, 24시간 동안 배양하였다. 배양 종료 후 새로운 IMDM 배지로 교체한 뒤, 한 plate에는 방사선을 4 Gy로 처리하였다. 그 후 두 plate 모두 추가로 48시간 배양하였다. 배양 종료 후 상기 실시예 1과 동일한 방법으로, 세포 생존율을 확인하였다.It was confirmed whether the extract of the present invention kills radiation-resistant head and neck cancer by treatment in combination with radiation. Specifically, 100 μl of the Moc1-RR cell line was dispensed into two plates in a 96-well plate at a cell concentration of 1.0 × 10 4 and then cultured in a 5% CO 2 incubator at 37°C. After completion of the culture, the medium was removed, and 100 μl of Gyehyeol, etc. extract was treated at a concentration of 0 and 25 μl, respectively, and cultured for 24 hours. After the culture was completed, the medium was replaced with new IMDM medium, and one plate was treated with 4 Gy of radiation. Afterwards, both plates were cultured for an additional 48 hours. After completion of culture, cell viability was confirmed in the same manner as in Example 1 above.
그 결과, 도 4에 나타낸 바와 같이, 본 발명의 계혈등 추출물은, Moc1-RR 세포주에서, 방사선과 병용처리되면, 계혈등 추출물 단독 처리와 비교하여, 세포사멸 유도 효과가 유의적으로 증가하는 것을 확인하여, 방사선과 병용처리 효과를 확인하였다.As a result, as shown in Figure 4, when the extract of the present invention was treated in combination with radiation in the Moc1-RR cell line, it was confirmed that the apoptosis inducing effect was significantly increased compared to the treatment of the extract alone. Thus, the effect of combined treatment with radiation was confirmed.
따라서, 본 발명의 계혈등 추출물은 방사선 내성 두경부암 세포주를 효과적으로 사멸시키는 것을 확인하였다. 또한, 세포사멸과 관련된 PARP의 발현은 감소시키며, ROS 생성을 유도하여, 방사선 내성 두경부암 세포주의 세포사멸을 유도하는 것을 확인하였다. 또한, 방사선 처리와 병용하면, 계혈등 추출물을 단독으로 처리하는 것과 비교하여, 효과적으로 방사선 내성 두경부암 세포주를 사멸시킬 수 있는 것을 확인하였다.Therefore, it was confirmed that the extract of the present invention effectively kills radiation-resistant head and neck cancer cell lines. In addition, it was confirmed that it reduced the expression of PARP, which is related to apoptosis, and induced ROS production, thereby inducing apoptosis of radiation-resistant head and neck cancer cell lines. In addition, it was confirmed that when used in combination with radiation treatment, radiation-resistant head and neck cancer cell lines can be killed more effectively than when the extract such as Gyehyeol is treated alone.
Claims (14)
상기 계혈등 추출물은, 물, C1 내지 C4의 저급 알코올, 저급 알코올 수용액, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군에서 선택된 용매로 추출되는 것인, 조성물.According to clause 1,
The composition, wherein the extract such as gyehyeol is extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohol, lower alcohol aqueous solution, ethyl acetate, hexane, and chloroform.
상기 두경부암은, 방사선(Radioactive ray) 내성 두경부암을 더 포함하는 것인, 조성물.According to clause 1,
The composition, wherein the head and neck cancer further includes radioactive ray-resistant head and neck cancer.
상기 두경부암은 구강암, 구인두암, 하인두암, 후두암, 침샘암, 비강암, 부비동암, 비인두암, 경부 식도암, 타액선암, 악성 림프종, 악성 흑색종 및 연부조직 암으로 이루어진 군에서 선택된 것인, 조성물.According to clause 1,
The head and neck cancer is selected from the group consisting of oral cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, salivary gland cancer, nasal cavity cancer, paranasal cancer, nasopharyngeal cancer, cervical esophageal cancer, salivary gland cancer, malignant lymphoma, malignant melanoma, and soft tissue cancer. .
상기 계혈등 추출물은, 두경부암의 세포사멸을 유도하는 것인, 조성물.According to clause 1,
A composition wherein the extract such as Gyehyeol induces apoptosis of head and neck cancer.
상기 세포사멸을 유도하는 것은, PARP(poly (ADP-ribose) polymerase)의 발현을 억제시키는 것인, 조성물.According to clause 5,
A composition that induces apoptosis by inhibiting the expression of PARP (poly (ADP-ribose) polymerase).
상기 세포사멸을 유도하는 것은, ROS(Reactive oxygen species)의 생성을 유도하는 것인, 조성물.According to clause 5,
A composition that induces cell death by inducing the production of ROS (Reactive oxygen species).
상기 계혈등 추출물은 방사선 조사와 병용 처리되는 것인, 조성물.According to clause 1,
A composition in which the extract such as gyehyeol is treated in combination with irradiation.
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| PCT/KR2023/016564 WO2024090954A1 (en) | 2022-10-27 | 2023-10-24 | Composition for preventing or treating radioresistant head and neck cancer, comprising extract of spatholobi caulis as active ingredient |
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