KR20230056113A - A composition for antivirus comprising broccoli leaf extract - Google Patents
A composition for antivirus comprising broccoli leaf extract Download PDFInfo
- Publication number
- KR20230056113A KR20230056113A KR1020210139311A KR20210139311A KR20230056113A KR 20230056113 A KR20230056113 A KR 20230056113A KR 1020210139311 A KR1020210139311 A KR 1020210139311A KR 20210139311 A KR20210139311 A KR 20210139311A KR 20230056113 A KR20230056113 A KR 20230056113A
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- KR
- South Korea
- Prior art keywords
- influenza virus
- leaf extract
- broccoli
- broccoli leaf
- present
- Prior art date
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Abstract
Description
본 발명은 브로콜리잎 추출물 또는 이의 분획물을 포함하는 항바이러스 조성물에 관한 것으로, 구체적으로 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 인플루엔자 바이러스에 대한 항바이러스 용도에 관한 것이다.The present invention relates to an antiviral composition comprising a broccoli leaf extract or a fraction thereof, and specifically to an antiviral use against influenza virus comprising the broccoli leaf extract or a fraction thereof as an active ingredient.
인플루엔자 바이러스는 오르쏘믹소바이러스(orthomyxovirus)에 속하며, 인체에 급성 호흡기 질환을 일으키는, 전염성이 매우 강한 바이러스다. 해마다 전세계적으로 집단감염이나 대유행을 야기하여 면역력이 취약한 영유아, 고령자, 기저질 환자의 경우에는 사망에 이르게 하는 바이러스 중 하나이다. 인플루엔자 바이러스는 인플루엔자 바이러스 A, 인플루엔자 바이러스 B, 인플루엔자 바이러스 C의 세 가지 형이 있다. 이 중 사람 간에 전염되어 대유행을 야기하는 인플루엔자 바이러스는 주로 A형이며, B형도 A형만큼은 아니지만 사람간의 전염력이 강하다. Influenza virus belongs to the orthomyxovirus and is a highly contagious virus that causes acute respiratory illness in humans. It is one of the viruses that cause group infections or pandemics worldwide every year, leading to death in infants, the elderly, and patients with underlying diseases with weak immunity. There are three types of influenza viruses: influenza virus A, influenza virus B, and influenza virus C. Among them, the influenza virus that is transmitted between humans and causes a pandemic is mainly type A, and type B is not as contagious as type A, but is highly contagious between humans.
인플루엔자 바이러스의 표면에는 당단백질인 적혈구 응집소(hemagglutinin, HA)와 "뉴라미니데이즈(neuraminidase, NA)"라는 두 종류의 표면 항원이 존재하며, 내부에는 8개의 분절된 RNA가 존재한다. HA는 숙주세포의 표면에 있는 시알산(sialic acid) 잔기와 결합하여 바이러스를 숙주세포에 부착시켜 바이러스가 숙주세포로 침투할 수 있게 만들어주는 역할을 한다. 뉴라미니데이즈는 감염된 숙주 세포내에서 증식한 바이러스가 세포 표면의 올리고사카라이드 부분과 뉴라민산(neuraminic acid) 잔기 사이의 알파-케토시딕 결합(α-ketosidic bond)을 끊어서 바이러스가 숙주세포 밖으로 나갈 수 있게 만들어주는 역할을 한다. 이 두 가지 표면 항원은 여러가지 아형이 발견되어 있으며, 아형에 따라 분류를 하고 있으며, 이 분류법에 따르면 1918년에 유행했던 스페인독감은 H1N1형 인플루엔자 바이러스 A, 1957년에 유행했던 아시아독감은 H2N2형 인플루엔자 바이러스 A, 1968년에 유행 했던 홍콩독감은 H3N2형 인플루엔자 바이러스 A에 해당한다. 요즘은 특히 교통이 발달함에 따라 한 곳에서 독감이 발생하여 전세계적으로 퍼지는데 걸리는 시간이 매우 짧아져서 해마다 계절성 독감이 유행하고 있고, 2009년에는 인플루엔자 바이러스가 대변이를 일으켜, 기존에는 없던 새로운 인플루엔자 바이러스인 신종플루가 발생하는 등 인플루엔자 바이러스는 큰 사회적 문제를 야기하고 있다.Two types of surface antigens, glycoproteins hemagglutinin (HA) and "neuraminidase (NA)" exist on the surface of the influenza virus, and eight segmented RNAs exist inside. HA binds to sialic acid residues on the surface of host cells to attach the virus to the host cell so that the virus can penetrate the host cell. Neuraminidase occurs when the virus propagated in an infected host cell breaks the α-ketosidic bond between the oligosaccharide moiety and the neuraminic acid residue on the cell surface, allowing the virus to escape the host cell. It acts as a way to get out. These two surface antigens have been found in various subtypes, and are classified according to the subtypes. According to this classification, the Spanish flu epidemic in 1918 was H1N1 influenza virus A, and the Asian flu epidemic in 1957 was H2N2 influenza virus. Virus A, the Hong Kong flu epidemic in 1968 corresponds to the H3N2 influenza virus A. These days, especially with the development of transportation, the time it takes for flu to spread worldwide after it occurs in one place is very short, so seasonal flu is prevalent every year. Influenza viruses are causing great social problems, such as the outbreak of the new influenza virus.
이러한 인플루엔자 바이러스의 감염을 예방하거나 치료하기 위한 방법으로는 바이러스 백신의 접종 또는 항인플루엔자 바이러스제 복용이 있다. 바이러스 백신은 바이러스의 감염 자체를 차단한다는 점에서 장점이 있으나, 유행하는 인플루엔자 바이러스의 아형과 백신에 존재하는 인플루엔자 바이러스의 아형이 맞지 않으면 효과가 매우 낮고, 아형이 맞는 경우에도 감염을 완벽히 억제하지 못하기 때문에 항인플루엔자 바이러스제 개발의 필요성이 중요하다. Methods for preventing or treating influenza virus infection include inoculation with a virus vaccine or taking an anti-influenza virus drug. Antiviral vaccines have the advantage of blocking viral infection itself, but if the subtypes of the prevalent influenza virus and the subtypes of the influenza virus present in the vaccine do not match, the effect is very low, and even if the subtypes match, the infection cannot be completely suppressed. Therefore, the need to develop anti-influenza virus agents is important.
인플루엔자 바이러스의 증식을 억제하는 대표적인 항바이러스 제제로는 아만타딘(amantadine)과 리만타딘(rimantadine)이 있으나, 이들 두 가지 항바이 러스 제제들은 혈청형 A형 인플루엔자 바이러스에만 효과적이며, M2 단백질 이 없는 혈청형 B형 인플루엔자 바이러스에는 효과가 없는것으로 확인되었 다(Washington, D.C.: American Society for Microbiology, 1998). 또한, 아만타딘과 리만타딘은 사용시 인플루엔자 바이러스 M2 단백질의 이 온채널기능에 영향을 미치지 못하는 변이 바이러스의 출현이 매우 쉽게 일 어나는 단점이 있는 것으로 확인되고 있다.Representative antiviral agents that inhibit the proliferation of influenza virus include amantadine and rimantadine, but these two antiviral agents are effective only for serotype A influenza virus and are effective only for serotype A viruses without M2 protein. It has been confirmed to be ineffective against influenza B virus (Washington, D.C.: American Society for Microbiology, 1998). In addition, amantadine and rimantadine have been confirmed to have a disadvantage in that the appearance of mutant viruses that do not affect the ion channel function of the influenza virus M2 protein occurs very easily when used.
이러한 단점을 보완하기 위하여 16종의 모든 혈청형 A형 인플루엔자 바이러스와 혈청형 B형 인플루엔자 바이러스에 효과적인 항바이러스 제제로 자나미비르(zanamivir)와 오셀타미비르(oseltamivir)가 개발되었으나, 자나미비르는 흡입 및 정맥 투여해야 하는 단점이 있으며, 오셀타미비르는 경구투여가 가능하나 최근 내성 바이러스의 출현 보고(세계 보건기구 (WHO), 2010)와 경구투여시 구토와 현기증 등의 부작용이 있어 단점으로 지적되고 있다.In order to compensate for these disadvantages, zanamivir and oseltamivir have been developed as antiviral agents effective against all 16 serotype A influenza viruses and serotype B influenza viruses, but zanamivir is inhaled. and intravenous administration, and oseltamivir can be administered orally, but it is pointed out as a disadvantage due to the recent report of the emergence of a resistant virus (World Health Organization (WHO), 2010) and side effects such as vomiting and dizziness when administered orally. there is.
이와 관련하여, 국내에서는 생약 추출물 및 식물 추출물을 대상으로 항바이러스 효능에 대한 연구가 진행 중이기는 하지만, 아직까지는 미비한 실정이다. In this regard, although studies on the antiviral efficacy of crude drug extracts and plant extracts are in progress in Korea, they are still incomplete.
이러한 배경하에, 본 발명자들은 기존의 항바이러스 제제의 단점을 극복하여 독성 및 부작용이 거의 없이 항바이러스 활성을 발휘할 수 있는 생약 추출물을 유효성분으로 하는 조성물을 개발하고자 예의 노력 연구한 결과, 브로콜리잎 추출물의 인플루엔자 바이러스에 대한 항바이러스 효능을 확인함으로써, 본 발명을 완성하였다.Under this background, the present inventors have studied diligently to develop a composition containing herbal extracts as active ingredients, which can exhibit antiviral activity with little toxicity and side effects by overcoming the disadvantages of existing antiviral preparations, and as a result, broccoli leaf extract The present invention was completed by confirming the antiviral efficacy against influenza virus.
본 발명의 하나의 목적은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 항바이러스용 약학 조성물을 제공하는 것이다.One object of the present invention is to provide an antiviral pharmaceutical composition comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
본 발명의 다른 하나의 목적은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 인플루엔자 바이러스 감염질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating influenza virus infectious diseases comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
본 발명의 또 다른 하나의 목적은 상기 약학 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는 인플루엔자 바이러스 감염질환의 예방 또는 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating influenza virus infectious diseases comprising administering the pharmaceutical composition to a non-human subject.
본 발명의 또 다른 하나의 목적은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 항바이러스용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide an antiviral food composition comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
본 발명의 또 다른 하나의 목적은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 항바이러스용 사료 조성물을 제공하는 것이다.Another object of the present invention is to provide an antiviral feed composition comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.
본 발명의 하나의 양태로서, 전술한 목적을 달성하기 위해, 본 발명은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 항바이러스용 약학 조성물을 제공한다.As one aspect of the present invention, in order to achieve the above object, the present invention provides an antiviral pharmaceutical composition comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
본 발명의 용어, "브로콜리(broccoli)"는 배추속의 한 종류인 브라시카 올레라케아(Brassica oleracea var. italica Plenck)에 속하는 채소의 일종이다. 브로콜리는 교목으로서 많이 쓰이며 나무처럼 생겨나서 두껍고 질긴 가운데 부분을 주로 하고 피어나는 모양이다. 꽃 부분을 먹는 배추 중 한 종류가 이탈리아에서 품종 개량되어 지금의 모습이 되었다. 브로콜리는 비타민 C가 풍부하며 항암 물질을 다량 섬유하고 있다. 한번 조리해 먹을 때 30 mg 이상의 비타민 C가 나온다고 한다(Understanding Nutrition, Eleanor N. Whitney and Eva M. N. Hamilton, Table H, supplement, page 373). 입 속에서 먹으면서 발생하는 3.3-디인돌리메탄은 브로콜리가 함유하고 있는 면역물질을 소화체계에서 분비함으로써 항암 치료를 돕는 역할을 한다. 또한, 브로콜리는 글루코사민을 갖고 있어 설포라판이라는 항암 물질을 끌어내는 역할을 하지만 10분 넘게 끓이게 되면 모든 영양소가 파괴된다. 또한, 브로콜리는 미국 타임지에서 선정한 세계 10대 푸드 중 하나이기도 하다.The term of the present invention, "broccoli" is a kind of vegetable belonging to Brassica oleracea var. italica Plenck, a kind of Chinese cabbage. Broccoli is widely used as a tree, and it looks like a tree, so it mainly blooms with a thick and tough middle part. One of the types of cabbage that eats the flower part was bred in Italy and became what it is today. Broccoli is rich in vitamin C and contains a large amount of anti-cancer substances. It is said that more than 30 mg of vitamin C can be obtained when cooked once (Understanding Nutrition, Eleanor N. Whitney and Eva M. N. Hamilton, Table H, supplement, page 373). 3.3-diindolimethane, which is generated while eating in the mouth, plays a role in helping cancer treatment by secreting immune substances contained in broccoli in the digestive system. In addition, broccoli has glucosamine, which plays a role in drawing out an anti-cancer substance called sulforaphane, but all nutrients are destroyed when boiled for more than 10 minutes. In addition, broccoli is also one of the world's top 10 foods selected by the US Times.
또한, "브로콜리잎"은 브로콜리 1개 수확하는데 10장 이상의 잎이 나오는 브로콜리의 부산물로 버려지는 부분이지만, 브로콜리의 영양성분 80% 정도는 함유하고 있다. 특히 비타민 C는 케일보다 15~80% 이상 월등히 높은 것으로 조사되었으며, 설포라판은 위암과 위퀘양을 일으키는 헬리코박터균을 없애주며, 인돌-3-카피놀은 여성호르몬인 에스트로겐을 완화시켜 유방암 발생을 억제할 뿐만 아니라, 식이섬유가 풍부하기 때문에 장 속 유해물질 배출에 도움을 줘 대장암 및 결장암 예방에도 좋다. In addition, "broccoli leaf" is a part that is discarded as a by-product of broccoli, in which 10 or more leaves are produced when one broccoli is harvested, but contains about 80% of the nutritional components of broccoli. In particular, vitamin C was found to be 15-80% higher than that of kale, sulforaphane eliminates Helicobacter pylori, which causes gastric cancer and gastric cancer, and indole-3-capinol relieves estrogen, a female hormone, to suppress breast cancer. In addition, because it is rich in dietary fiber, it helps in the discharge of harmful substances in the intestines and is good for preventing colon and colon cancer.
본 발명에 있어서, 상기 브로콜리잎은 상업적으로 판매되는 것을 구입하여 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수 있으나, 이에 제한되지 않는다.In the present invention, the broccoli leaf may be purchased and used commercially, or collected or cultivated in nature, but is not limited thereto.
본 발명의 용어, "추출물"은 목적하는 물질을 다양한 용매에 침지한 다음, 상온 또는 가온 상태에서 일정시간 동안 추출하여 수득한 액상성분, 상기 액상성분으로부터 용매를 제거하여 수득한 고형분 등의 결과물을 의미한다. 뿐만 아니라, 상기 결과물에 더하여, 상기 결과물의 희석액, 이들의 농축액, 이들의 조정제물, 정제물 등을 모두 포함하는 것으로 포괄적으로 해석될 수 있다. 이에 따라, 본 발명에서 제공하는 브로콜리잎 추출물은 이를 추출 처리하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함하는 것으로 해석될 수 있다.As used herein, the term "extract" refers to a product such as a liquid component obtained by immersing a target material in various solvents and then extracting at room temperature or a warm state for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. it means. In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof. Accordingly, the broccoli leaf extract provided in the present invention is an extract obtained by extracting it, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a purified product or purified product of the extract, or a mixture thereof. It can be interpreted as including extracts of all formulations that can be formed using the extract itself and the extract solution.
본 발명의 브로콜리잎 추출물에 있어서 이의 추출 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 열수 추출법, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다.In the broccoli leaf extract of the present invention, the extraction method thereof is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.
상기 추출에 사용되는 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물, 알코올 또는 이들의 혼합 용매 등을 들 수 있고, 이들은 단독으로 사용되거나 1종 이상 혼합하여 사용될 수 있으며, 구체적으로 알코올이 사용될 수 있으며, 주로 탄소수 1 내지 4의 알코올을 사용할 수 있다.The type of solvent used for the extraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, alcohol, or a mixed solvent thereof, which may be used alone or in combination of one or more, and specifically, alcohol may be used, mainly having 1 to 4 carbon atoms. of alcohol can be used.
본 발명의 일 실시예에서는, 상기 브로콜리잎을 세절하고 10배 부피의 증류수를 가한 후, Pulsed electric field (PEF) 처리하고, 상기 PEF 처리한 현탁액에 10배의 에탄올을 가하고 상온에 3시간 방치 후 여과한 여과액을 동결건조하여 브로콜리잎 추출물로 수득하였다. In one embodiment of the present invention, after cutting the broccoli leaves and adding 10-fold volume of distilled water, pulsed electric field (PEF) treatment, adding 10-fold ethanol to the PEF-treated suspension and leaving it at room temperature for 3 hours The filtered filtrate was freeze-dried to obtain a broccoli leaf extract.
또한, 상기 추출물은 추출 후 건조 분말 형태로 제조되어 사용될 수 있지만, 이제 제한되는 것은 아니다.In addition, the extract may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.
본 발명의 용어, "분획물"은, 여러 다양한 구성 성분들을 포함하는 혼합물로부터 특정 성분 또는 특정 성분 그룹을 분리하기 위하여 분획을 수행하여 얻어진 결과를 의미한다.As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing various components.
본 발명에서 상기 분획물을 얻는 분획 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 수행될 수 있다. 상기 분획 방법의 비제한적인 예로는, 브로콜리잎을 추출하여 얻은 추출물에 소정의 용매를 처리하여 상기 추출물로부터 분획물을 얻는 방법을 들 수 있다.In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. A non-limiting example of the fractionation method may include a method of obtaining fractions from the extract by treating the extract obtained by extracting broccoli leaves with a predetermined solvent.
본 발명에서 상기 분획물을 얻는 데에 사용되는 분획 용매의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 분획 용매의 비제한적인 예로는 물, 알코올 등의 극성 용매; 헥산(Hexan), 에틸아세테이트(Ethyl acetate), 클로로포름(Chloroform), 디클로로메탄(Dichloromethane) 등의 비극성 용매 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상 혼합하여 사용될 수 있다. 상기 분획 용매 중 알코올을 사용하는 경우에는 구체적으로 C1 내지 C4의 알코올을 사용할 수 있다.In the present invention, the type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 alcohols may be specifically used.
본 발명의 용어, "항바이러스"는, 항바이러스 효과 혹은 작용과 같이 사용되며, 식물, 동물, 세균 등의 살아 있는 세포에 기생하고 증식하여 여러 질환을 일으키는 여과성 병원체인 바이러스 감염 및 증식을 억제하는 것을 의미한다.The term of the present invention, "antiviral", is used together with the antiviral effect or action, and inhibits the infection and proliferation of viruses, which are filterable pathogens that parasitize and proliferate in living cells such as plants, animals, bacteria, and cause various diseases. means that
본 발명에 있어서, 상기 바이러스는 인플루엔자 바이러스일 수 있으며, 구체적으로 PR8 인플루엔자 바이러스(Influenza virus; PR8)일 수 있으나, 이에 제한되는 것은 아니다. 상기 PR8 인플루엔자 바이러스는 H1N1 변종인 신종플루를 의미하며, 다양한 타입이 존재하여 해마다 종류가 달라지는 A형 독감 바이러스에 속한다. In the present invention, the virus may be an influenza virus, specifically a PR8 influenza virus (PR8), but is not limited thereto. The PR8 influenza virus refers to H1N1 strain swine flu, and belongs to the type A influenza virus, in which various types exist and the type changes every year.
본 발명의 구체적인 일 실시예에서는, 본 발명의 브로콜리잎 추출물을 처리한 결과, PR8 인플루엔자 바이러스에 대한 우수한 항바이러스 활성을 확인하였다(도 2 내지 도 4). In a specific embodiment of the present invention, as a result of treating the broccoli leaf extract of the present invention, excellent antiviral activity against PR8 influenza virus was confirmed (FIGS. 2 to 4).
이는, 상기 브로콜리잎 추출물이 상기 바이러스에 대한 예방 또는 치료에 유용하게 이용될 수 있음을 시사하는 것이다.This suggests that the broccoli leaf extract can be usefully used for prevention or treatment of the virus.
본 발명의 다른 하나의 양태로서, 전술한 목적을 달성하기 위해, 본 발명은 브로콜리잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 인플루엔자 바이러스 감염질환의 예방 또는 치료용 약학 조성물을 제공한다.As another aspect of the present invention, in order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating influenza virus infectious diseases comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
이때 상기 용어, "브로콜리잎", "추출물" 및 "분획물"은 상기에서 서술한 바와 같다.At this time, the terms "broccoli leaf", "extract" and "fraction" are as described above.
본 발명의 용어, "인플루엔자 바이러스 감염질환"은 인플루엔자 바이러스의 감염으로 유발되는 질환을 의미한다.As used herein, the term "influenza virus infectious disease" refers to a disease caused by infection with an influenza virus.
상기 용어, "감염"은 병원성 미생물이 숙주가 되는 생물체의 체내에 침입하여, 발육 증식한 상태를 의미한다.The term "infection" refers to a state in which a pathogenic microorganism invades the body of a host organism and grows and proliferates.
본 발명의 일 구체예로, 상기 인플루엔자바이러스 감염질환이란, 사람에서는 독감, 감기, 인후염, 기관지염, 폐렴, 급성호흡기질환 또는 중증호흡기질환등 일 수 있으며, 동물에서는 조류독감, 돼지독감, 염소독감 등의 급성호흡기질환, 중증호흡기질환, 설사일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the influenza virus infectious disease may be flu, cold, sore throat, bronchitis, pneumonia, acute respiratory disease or severe respiratory disease in humans, and bird flu, swine flu, chlorine flu in animals, etc. It may be acute respiratory disease, severe respiratory disease, diarrhea, but is not limited thereto.
본 발명의 용어, "예방"은 본 발명에 따른 약학 조성물의 투여에 의해 인플루엔자 바이러스에 의한 감염질환의 발병을 억제시키거나 또는 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of infectious diseases caused by influenza virus by administration of the pharmaceutical composition according to the present invention.
본 발명의 용어, "치료"는 상기 약학 조성물의 투여에 의해 인플루엔자 바이러스 감염질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of suspected or affected subjects of influenza virus infection by the administration of the pharmaceutical composition.
본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 추출물을 0.001 내지 80, 구체적으로 0.001 내지 70, 더욱 구체적으로 0.001 내지 60 중량%로 포함할 수 있으나, 이에 제한되지 않는다.The pharmaceutical composition of the present invention may include 0.001 to 80, specifically 0.001 to 70, and more specifically 0.001 to 60% by weight of the extract based on the total weight of the composition, but is not limited thereto.
또한, 상기 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 약학적으로 허용가능한 담체, 부형제 또는 희석제를 추가로 포함할 수 있고, 상기 담체는 비자연적 담체(non-naturally occuring carrier)를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. there is. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
또한, 상기 약학 조성물은 각각 통상의 방법에 따라 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 또는 좌제의 형태로 제형화하여 사용할 수 있다. 구체적으로, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하지만, 이에 제한되지 않는다. 이러한 고형제제는 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제 등도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In addition, the pharmaceutical composition may be formulated according to conventional methods such as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and transdermal formulations. Absorbents, gels, lotions, ointments, creams, patches, cataplasma agents, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages, or suppositories may be formulated and used. Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. It may be prepared by adding various excipients, for example, wetting agents, sweeteners, aromatics, and preservatives, in addition to liquids and liquid paraffin for oral use. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
본 발명의 다른 하나의 양태로서, 전술한 목적을 달성하기 위해, 상기 약학 조성물을 인간을 제외한 인플루엔자 바이러스 감염질환이 의심되는 개체에 투여하는 단계를 포함하는 인플루엔자 바이러스 감염질환의 예방 또는 치료 방법을 제공한다.As another aspect of the present invention, in order to achieve the above object, to provide a method for preventing or treating influenza virus infectious disease comprising administering the pharmaceutical composition to a subject suspected of having an influenza virus infectious disease other than human do.
이때, 상기 용어, "브로콜리잎", "인플루엔자 바이러스 감염질환", "예방"및"치료"는 상기에서 서술한 바와 같다.At this time, the terms "broccoli leaf", "influenza virus infectious disease", "prevention" and "treatment" are as described above.
본 발명의 용어, "투여"는 적절한 방법으로 개체에게 상기 추출물을 포함하는 조성물을 도입하는 행위를 의미한다.As used herein, the term "administration" refers to the act of introducing a composition containing the extract to a subject in an appropriate manner.
본 발명의 용어, "개체"는 인플루엔자 바이러스 감염질환이 발병하였거나 발병할 수 있는 인간을 포함한 쥐, 생쥐, 가축 등의 모든 동물을 의미한다. 구체적인 예로, 인간을 포함한 포유동물일 수 있다.As used herein, the term "individual" refers to all animals, such as rats, mice, livestock, and the like, including humans who have or may develop influenza virus infectious diseases. As a specific example, it may be mammals including humans.
본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여한다. 상기 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 예를 들면, 상기 브로콜리잎 추출물은 1일 0.01 내지 5000 mg/kg으로, 구체적으로 10 내지 1000 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한 번 또는 수회 나누어 투여할 수도 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. For example, the broccoli leaf extract may be administered at a dose of 0.01 to 5000 mg/kg per day, specifically 10 to 1000 mg/kg, and the administration may be administered once or several times a day.
상기 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
또한, 상기 약학 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.In addition, the pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) according to the desired method, and the dosage is the patient's condition and weight, the degree of disease , Depending on the drug form, administration route and time, it can be appropriately selected by those skilled in the art.
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는, 브로콜리잎 추출물 또는 이의 분획물을 포함하는 항바이러스용 식품 조성물을 제공한다.Another aspect of the present invention for achieving the above object provides an antiviral food composition comprising a broccoli leaf extract or a fraction thereof.
이때, 상기 용어, "브로콜리잎", "추출물", "분획물" 및 "항바이러스"의 정의는 상기에서 서술한 바와 같다.At this time, the definitions of the term "broccoli leaf", "extract", "fraction" and "antiviral" are as described above.
본 발명의 용어, "식품"은 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료, 비타민 복합제, 건강 기능 식품 및 건강 식품 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcoholic beverages. , vitamin complexes, health functional foods and health foods, etc., and include all foods in the conventional sense.
본 발명의 식품 조성물은 일상적으로 섭취가능한 브로콜리잎으로부터 유래되었기 때문에 높은 항바이러스 효과를 기대할 수 있으므로, 건강 증진 목적으로 매우 유용하게 사용될 수 있다.Since the food composition of the present invention is derived from broccoli leaves that can be consumed daily, a high antiviral effect can be expected, so it can be used very usefully for health promotion purposes.
상기 건강 기능(성) 식품(functional food)이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 '기능(성)'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한, 상기 식품의 제형은 식품으로 인정되는 제형이면 제한없이 제조될 수 있다.The health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, and has high medical effect. means food. Here, 'function (sex)' means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food can be prepared without limitation as long as the formulation is recognized as food.
본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 천연물을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나므로, 본 발명의 식품은 항바이러스 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using natural substances as raw materials, and has excellent portability. The food of the invention can be consumed as an adjuvant to enhance the antiviral effect.
상기 건강 식품(health food)은 일반식품에 비해 적극적인 건강유지나 증진 효과를 가지는 식품을 의미하고, 건강보조식품(health supplement food)은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강 기능 식품, 건강식품, 건강보조식품의 용어는 호용된다.The health food (health food) means a food having an active health maintenance or promotion effect compared to general food, health supplement food (health supplement food) means a food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.
구체적으로, 상기 건강 기능 식품은 본 발명의 조성물을 음료, 차류, 향신료, 껌, 과자류 등의 식품 소재에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용이 없는 장점이 있다.Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, chewing gum, confectionery, etc., or encapsulated, powdered, or suspended, and when ingested, certain health-related foods It means to bring about an effect, but unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material.
상기 식품 조성물은 생리학적으로 허용 가능한 담체를 추가로 포함할 수 있는데, 담체의 종류는 특별히 제한되지 않으며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다.The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.
또한, 상기 식품 조성물은 식품 조성물에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu), 크륨(Cr) 등의 미네랄; 및 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다. In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.
또한, 상기 식품 조성물은 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 데히드로초산나트륨 등), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨류엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 글루타민산나트륨 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(food additives)을 포함할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 식품 조성물의 일 예로 건강음료 조성물로 사용될 수 있으며, 이 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 슈크로스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 건강음료 조성물 100 ml 당 일반적으로 약 0.01 내지 0.04 g, 구체적으로 약 0.02 내지 0.03 g이 될 수 있다.An example of the food composition of the present invention may be used as a health beverage composition, and in this case, it may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 ml of the health drink composition of the present invention.
상기 외에 건강음료 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일주스, 과일주스 음료, 또는 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination.
이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 건강음료 조성물 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는, 브로콜리잎 추출물 또는 이의 분획물을 포함하는 항바이러스용 사료 조성물을 제공한다.Another aspect of the present invention for achieving the above object provides an antiviral feed composition comprising a broccoli leaf extract or a fraction thereof.
이때, 상기 용어, "브로콜리잎", "추출물", "분획물" 및 "항바이러스"는 상기에서 서술한 바와 같다.At this time, the terms "broccoli leaf", "extract", "fraction" and "antivirus" are as described above.
본 발명에 따른 브로콜리잎 추출물은 우수한 항바이러스 효과를 나타내므로, 인플루엔자 바이러스 감염질환의 예방 또는 개선을 목적으로 사료 조성물에 포함될 수 있으며, 상기 사료 조성물은 동물이 일상적으로 섭취하는 것이 가능하기 때문에 인플루엔자 바이러스 감염질환의 예방 또는 개선에 대하여 높은 효과를 기대할 수 있다. Since the broccoli leaf extract according to the present invention exhibits an excellent antiviral effect, it can be included in a feed composition for the purpose of preventing or improving influenza virus infectious diseases, and since the feed composition can be routinely consumed by animals, influenza virus A high effect can be expected for the prevention or improvement of infectious diseases.
본 발명의 용어, "사료"는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미한다. As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals.
상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박 류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물 성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feeds such as proteins, inorganic materials, oils, mineral oils, oils, single-celled proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.
본 발명의 브로콜리잎 추출물 또는 이의 분획물을 함유하는 조성물은 독성이나 부작용을 일으키지 않으면서 인플루엔자 바이러스에 대한 감염 억제 효과를 나타내므로, 상기 바이러스 질환의 예방 또는 치료를 위한 항바이러스제 개발에 이용될 수 있다.Since the composition containing the broccoli leaf extract or a fraction thereof of the present invention exhibits an infection inhibitory effect against influenza virus without causing toxicity or side effects, it can be used to develop an antiviral agent for preventing or treating the above viral disease.
도 1은 브로콜리잎 추출물의 마우스 대식 세포 RAW 264.7 세포에 대한 농도별 독성을 확인한 그래프이다.
도 2A는 브로콜리잎 추출물을 인플루엔자 바이러스(PR8-GFP)와 동시 감염시키고, 인플루엔자 바이러스에 대한 억제 효능을 형광현미경으로 관찰한 결과이다 (Medium; 음성 대조군, PR8-GFP; 인플루엔자 바이러스 감염군).
도 2B은 도 2A에서 형광현미경으로 관찰한 각 세포를 유세포 분석기를 사용하여 GFP 발현을 확인한 히스토그램 비교 결과이다.
도 2C는 도 2B에서 유세포 분석기를 통해 발현된 GFP 단백질의 형광값을 수치화하여 비교한 그래프이다.
도 3는 RAW 264.7 세포에 브로콜리잎 추출물 200 μg/ml과 인플루엔자 바이러스를 동시에 감염시키고 세포를 고정한 후, 바이러스 단백질 A) M2, B) NP, C) NS1 각각의 발현을 면역형광염색법으로 확인한 결과이다. (Medium ; 음성 대조군, PR8-GFP;인플루엔자 바이러스 감염군, PR8-GFP/브로콜리잎 추출물; 브로콜리잎 추출물 100 μg/ml 처리군) (M2; Matrix protein 2, NP; Nucleoprotein, NS1 (Non-structural protein 1)
도 4은 RAW 264.7 세포에 브로콜리잎 추출물과 인플루엔자 바이러스를 동시에 감염시키고 세포를 모은 후, 단백질을 분리하여 인플루엔자 바이러스 단백질의 발현 정도를 비교한 결과이다. 마지막 레인은 인플루엔자 바이러스 감염 없이 추출물만 처리한 세포이다.1 is a graph confirming the toxicity of broccoli leaf extract to mouse macrophage RAW 264.7 cells by concentration.
Figure 2A shows the result of co-infecting broccoli leaf extract with influenza virus (PR8-GFP) and observing the inhibitory effect against influenza virus under a fluorescence microscope (Medium; negative control group, PR8-GFP; influenza virus infection group).
Figure 2B is a histogram comparison result of confirming GFP expression using a flow cytometer for each cell observed with a fluorescence microscope in Figure 2A.
FIG. 2C is a graph comparing the fluorescence values of the GFP protein expressed through flow cytometry in FIG. 2B by quantifying them.
Figure 3 is a result of confirming the expression of each of viral proteins A) M2, B) NP, and C) NS1 by immunofluorescence staining after infecting RAW 264.7 cells with 200 μg/ml of broccoli leaf extract and influenza virus at the same time and fixing the cells. . (Medium; negative control group, PR8-GFP; influenza virus infection group, PR8-GFP/broccoli leaf extract;
Figure 4 is a result of comparing the expression levels of influenza virus proteins by simultaneously infecting RAW 264.7 cells with broccoli leaf extract and influenza virus, collecting cells, and isolating proteins. The last lane is cells treated with extract only without influenza virus infection.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.
실시예 1. 브로콜리잎 열수 추출물의 제조Example 1. Preparation of hot water extract of broccoli leaves
브로콜리잎을 세절하여 10배의 증류수를 가한 후 Pulsed electric field (PEF) 처리를 하였다. PEF 처리는 5 kW 에서 7 kJ 의 에너지를 주며 5초간 진행하였다 (out voltage 60%, pulsed width 25 μs, frequency 100 Hz). PEF 처리한 현탁액에 10배의 에탄올을 가하고 상온에 3시간 방치 후 여과를 진행하였다. 여과액을 72시간 동결건조 하고 가루 형태로 분쇄하여 -80℃에 보관 후 실험에 사용하였다. Broccoli leaves were cut, 10 times distilled water was added, and pulsed electric field (PEF) treatment was performed. PEF treatment was performed for 5 seconds with 7 kJ of energy at 5 kW (out voltage 60%, pulsed width 25 μs,
실시예 2. 브로콜리잎 추출물의 세포 독성 분석Example 2. Cytotoxicity analysis of broccoli leaf extract
브로콜리잎 추출물이 세포에 미치는 독성을 평가하기 위해, 상기 실시예 1에서 제조된 추출물을 생쥐 대식세포인 Raw 264.7 세포주에 처리하여 세포 생존율을 측정하였다. 세포 생존율은 CCK-8 assay로 측정하였다. In order to evaluate the toxicity of the broccoli leaf extract to cells, the extract prepared in Example 1 was treated with a mouse macrophage, Raw 264.7 cell line, and cell viability was measured. Cell viability was measured by CCK-8 assay .
구체적으로, 생쥐 대식세포인 RAW 264.7 세포를 96 well 플레이트에 2 x 105 cells/well로 시딩하고, 브로콜리잎 추출물을 1 부터 1000 μg/ml 까지 농도별로 24시간 처리한 후, CCK-8 시약을 10 μl 넣고 2시간 반응시킨 다음 450 nm 파장에서 흡광도를 측정하였다.Specifically, RAW 264.7 cells, which are mouse macrophages, were seeded in a 96 well plate at 2 x 10 5 cells/well, treated with broccoli leaf extract for 24 hours at each concentration from 1 to 1000 μg/ml, and then CCK-8 reagent After adding 10 μl and reacting for 2 hours, absorbance was measured at a wavelength of 450 nm.
그 결과, 도 1에 나타난 바와 같이, 브로콜리잎 추출물의 1, 10, 50, 100, 200, 500 및 1000 μg/ml의 모든 농도에서 100% 이상 생존율을 보임으로써, 세포독성을 나타내지 않는 것을 확인하였다.As a result, as shown in Figure 1, by showing a viability of 100% or more at all concentrations of 1, 10, 50, 100, 200, 500 and 1000 μg / ml of the broccoli leaf extract, it was confirmed that there was no cytotoxicity .
실시예 3. 브로콜리잎 추출물의 인플루엔자 바이러스에 대한 항바이러스 활성 분석 Example 3. Analysis of antiviral activity of broccoli leaf extract against influenza virus
실시예 3-1. PR8 인플루엔자 바이러스에 대한 감염도 확인Example 3-1. Confirmation of infection with PR8 influenza virus
감염 24시간 후의 GFP(green fluorescent protein) 형광 이미지를 이용한 브로콜리잎 추출물의 PR8 인플루엔자 바이러스에 대한 항바이러스 활성을 알아보았다.The antiviral activity of broccoli leaf extract against PR8 influenza virus was investigated using GFP (green fluorescent protein) fluorescence images 24 hours after infection.
구체적으로, 96 well TC 플레이트에 Raw 264.7 세포 (5Х105 cell/well)를 배양한 후, 상기 실시예 1에서 제조한 브로콜리잎 추출물 200 및 400 μg/ml과 PR8-GFP 바이러스를 동시에 처리하고 감염 정도를 확인하였다Specifically, after culturing Raw 264.7 cells (5Х10 5 cell/well) in a 96 well TC plate, 200 and 400 μg/ml of broccoli leaf extract prepared in Example 1 and PR8-GFP virus were simultaneously treated and the degree of infection confirmed
그 결과, 도 2에 나타난 바와 같이, 브로콜리잎 추출물과 PR8-GFP 인플루엔자 바이러스를 동시 처리한 결과, 브로콜리잎 추출물 200 μg/ml 이상의 농도에서 바이러스의 감염률이 현저히 떨어진 것을 확인할 수 있었으며(도 2A), 이는 유세포 분석기를 사용하여 GFP 발현을 확인한 히스토그램 비교(도 2B) 및 GFP 단백질의 형광값을 수치화하여 PR8-GFP 바이러스에 의한 형광값을 1로 기준으로 하여 상대적으로 비교한 그래프를 통해서도 확인할 수 있었다(도 2C).As a result, as shown in FIG. 2, as a result of simultaneous treatment of the broccoli leaf extract and the PR8-GFP influenza virus, it was confirmed that the infection rate of the virus was significantly reduced at a concentration of 200 μg / ml or more of the broccoli leaf extract (FIG. 2A), This was also confirmed through a histogram comparison (Fig. 2B) of GFP expression using a flow cytometer and a graph in which the fluorescence value of the GFP protein was quantified and the fluorescence value by the PR8-GFP virus was compared relative to 1 ( Figure 2C).
실시예 3-2. 면역형광염색법Example 3-2. immunofluorescence staining
인플루엔자 바이러스 단백질 M2, NP 및 NS1의 발현에 대한 브로콜리잎 추출물의 효능을 면역형광염색법으로 확인하였다. The efficacy of broccoli leaf extract on the expression of influenza virus proteins M2, NP and NS1 was confirmed by immunofluorescence staining.
구체적으로, Raw 264.7 세포에 브로콜리잎 추출물과 PR8 인플루엔자 바이러스를 24시간 감염시키고, Raw 264.7 세포를 PBS로 세척한 후 100% 차가운 메탄올 용액에 5분, 3.7% 포르말린 용액에 10분간 고정하였다. 1% BSA와 0.05% tween-20이 함유된 PBS 용액에서 1시간 블로킹하고, 바이러스 단백질 M2, NP, NS1에 대한 1차 항체에 반응시킨 후 PBS로 세척하고, Alexa588이 결합된 2차 항체와 반응시켰다 (Red 형광). 이후, 다시 PBS로 세척한 후 DAPI 용액을 첨가하여 핵을 염색하고(blue 형광), 형광현미경으로 merge하여 두 가지 색의 형광을 겹쳐서 확인하였다.Specifically, Raw 264.7 cells were infected with broccoli leaf extract and PR8 influenza virus for 24 hours, and Raw 264.7 cells were washed with PBS and then fixed in 100% cold methanol solution for 5 minutes and 3.7% formalin solution for 10 minutes. Blocking for 1 hour in a PBS solution containing 1% BSA and 0.05% tween-20, reacting with primary antibodies against viral proteins M2, NP, and NS1, washing with PBS, and reacting with Alexa588-conjugated secondary antibodies (Red fluorescence). Thereafter, after washing with PBS again, DAPI solution was added to stain the nucleus (blue fluorescence), and merged with a fluorescence microscope to confirm that the fluorescence of the two colors was overlapped.
그 결과, 도 3에 나타난 바와 같이, PR8-GFP 인플루엔자 바이러스만을 처리하였을 때보다 브로콜리잎 추출물과 동시에 처리한 세포에서 각 바이러스 단백질 M2, NP 및 NS1의 발현이 현저히 억제됨을 확인하였다. As a result, as shown in FIG. 3, it was confirmed that the expression of each of the viral proteins M2, NP, and NS1 was significantly suppressed in cells treated simultaneously with the broccoli leaf extract, compared to when only the PR8-GFP influenza virus was treated.
실시예 3-3. 웨스턴 블랏 분석Example 3-3. Western blot analysis
인플루엔자 바이러스 단백질 HA, PA, M2, PB2, NA 및 M1의 발현에 대한 브로콜리잎 추출물의 효능을 웨스턴 블랏으로 확인하였다.The effects of broccoli leaf extract on the expression of influenza virus proteins HA, PA, M2, PB2, NA and M1 were confirmed by Western blotting.
구체적으로, RAW 264.7 세포에 브로콜리잎 추출물 200 또는 400 μg/ml과 PR8-GFP 바이러스를 동시에 감염시키고 감염된 세포로부터 단백질을 분리하여 SDS-PAGE를 실시하여, 인플루엔자 바이러스 단백질 HA, PA, M2, PB2, NA, M1의 발현 정도를 비교 확인하였다.Specifically, RAW 264.7 cells were simultaneously infected with 200 or 400 μg/ml of broccoli leaf extract and PR8-GFP virus, and proteins were separated from the infected cells and subjected to SDS-PAGE to obtain influenza virus proteins HA, PA, M2, PB2, The expression levels of NA and M1 were compared and confirmed.
그 결과, 도 4에 나타난 바와 같이, 브로콜리잎 추출물을 바이러스와 동시에 감염시켰을 때, 브로콜리 추출물 200 μg/ml 농도 이상에서 상기 바이러스 단백질 HA, PA, M2, PB2, NA 및 M1의 발현이 모두 현저히 억제됨을 확인하였다.As a result, as shown in Figure 4, when the broccoli leaf extract was infected with the virus at the same time, the expression of the virus proteins HA, PA, M2, PB2, NA and M1 were all significantly suppressed at a concentration of 200 μg / ml or more of the broccoli extract. confirmed.
이를 통해, 브로콜리잎 추출물이 인플루엔자 바이러스에 대한 억제 효능을 있음을 확인하였다.Through this, it was confirmed that the broccoli leaf extract has an inhibitory effect on influenza virus.
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 실시예들은 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, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.
Claims (7)
An antiviral feed composition comprising a broccoli leaf extract or a fraction thereof as an active ingredient.
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