KR102070040B1 - Composition comprising extract of Sargassum fusiforme for preventing or treating influenza - Google Patents
Composition comprising extract of Sargassum fusiforme for preventing or treating influenza Download PDFInfo
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- KR102070040B1 KR102070040B1 KR1020180054108A KR20180054108A KR102070040B1 KR 102070040 B1 KR102070040 B1 KR 102070040B1 KR 1020180054108 A KR1020180054108 A KR 1020180054108A KR 20180054108 A KR20180054108 A KR 20180054108A KR 102070040 B1 KR102070040 B1 KR 102070040B1
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- KR
- South Korea
- Prior art keywords
- ethyl acetate
- hexane
- extract
- acetone
- influenza
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/202—Algae extracts
Abstract
본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 조성물에 관한 것으로, 톳 추출물이 인플루엔자 바이러스에 대한 항바이러스 활성이 있으며, 동속이종인 다른 해조류 추출물에 비해 항바이러스 활성이 우수하다는 것을 확인함으로써, 본 발명의 톳 추출물을 인플루엔자의 예방 또는 치료용 의약품, 건강기능식품, 동물사료 첨가제 또는 천연소독제 등의 개발시에 유용하게 이용할 수 있을 것으로 기대된다. The present invention relates to a composition for preventing or treating influenza, comprising the extract of Sargassum fusiforme as an active ingredient, the extract having antiviral activity against influenza virus, and excellent antiviral activity compared to other algae extracts of the same species. By confirming that, it is expected that the extract of the present invention can be usefully used in the development of medicines for preventing or treating influenza, health functional foods, animal feed additives or natural antiseptics.
Description
본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating influenza, comprising the extract of Sargassum fusiforme as an active ingredient.
일반적으로 독감으로 알려져 있는 인플루엔자(influenza)는 인플루엔자 바이러스에 의한 급성 호흡기 질환이다. 인플루엔자는 우리나라를 비롯하여 전 세계에서 발생하며, 계절 구분이 있는 지역에서는 매년 겨울에 소규모로 유행되고 있다. 인플루엔자는 전염성이 강하고, 노인이나 소아, 다른 질환을 앓고 있는 사람이 걸리면 사망률이 증가하고 합병증의 발생이 증가하고, 일부 지역에 한정된 발병이 아닌, 새로운 종류의 인플루엔자 바이러스에 의해 짧은 시간에 넓은 지역에서 유행하게 되면 전 세계적인 문제가 될 수 있어 인플루엔자의 예방 및 치료는 중요한 사회적 이슈 중 하나이다. Influenza, commonly known as the flu, is an acute respiratory disease caused by the influenza virus. Influenza occurs all over the world, including Korea, and is spreading in small scale every winter in regions with seasonal divisions. Influenza is highly contagious, affecting elderly people, children, and other people with other diseases, resulting in increased mortality, increased incidence of complications, and a wider area in a shorter period of time due to a new type of influenza virus, rather than a limited outbreak. The epidemic can be a global problem, preventing and treating influenza is one of the major social issues.
인플루엔자 바이러스(influenza virus)는 오르소믹소비리대(orthomyxoviridae)과에 속하는 RNA 바이러스로서 A, B 및 C형으로 분류된다. 특히, A형 바이러스의 경우 사람에게 주로 감염이 확인되고, B 또는 C형에 비하여 돼지, 기타 포유류 및 다양한 야생조류에서 감염이 확인되는 바이러스이며, 최근 전 세계적으로 문제가 되고 있는 조류 인플루엔자 바이러스(avian influenza virus), 돼지 인플루엔자 바이러스(swine influenza virus) 및 신종 인플루엔자 바이러스(influenza A virus subtype H1N1) 등이 이에 속한다. Influenza virus is an RNA virus belonging to the orthomyxoviridae family and is classified into types A, B and C. In particular, the type A virus is a virus that is confirmed mainly in humans, the infection is confirmed in pigs, other mammals and a variety of wild birds compared to the type B or C, avian influenza virus (avian) has become a problem worldwide recently influenza virus, swine influenza virus and swine influenza virus (influenza A virus subtype H1N1).
인플루엔자 바이러스의 표면에는 헤마글루티닌(hemagglutinin, HA, H)과 뉴라미니다아제(neuraminidase, NA, N)라는 두 가지의 단백질이 존재하는데, 헤마글루티닌 및 뉴라미니다아제의 조합에 의해 인플루엔자 바이러스의 아형(subtype)이 결정된다(Schnitzler, S.U., et al., 2009; Wright, P.F., et al., 2007). 즉, 헤마글루티닌은 16종이 있고, 뉴라미니다아제에는 9종이 있어, 총 144종류의 인플루엔자 바이러스가 발생할 수 있는 것이다. 조류의 경우, 인플루엔자 바이러스 감염은 주로 H5형, H7형 및 H9형이 관련되어 있고, 인간의 경우, H1형, H2형 및 H3형과 2가지 형태의 뉴라미니다아제(N1 및 N2)만이 관련되어 있으므로, 이론적으로는 인간은 조류 인플루엔자 바이러스에 감염되지 않아야 한다. 그러나 최근에 조류 인플루엔자 바이러스가 유전자 재조합 과정을 거치지 않은 상태에서 직접적으로 사람에게 전파된 예가 발생하고 있다. On the surface of the influenza virus there are two proteins, hemagglutinin (HA, H) and neuraminidase (NA, N), which are combined with hemagglutinin and neuraminidase. Subtypes of influenza viruses are determined (Schnitzler, SU, et al., 2009; Wright, PF, et al., 2007). That is, there are 16 types of hemagglutinin, and 9 types of neuraminidase, and a total of 144 types of influenza viruses can occur. In birds, influenza virus infection is mainly associated with H5, H7 and H9 types, and in humans, only H1, H2 and H3 types and only two types of neuraminidase (N1 and N2) In theory, humans should not be infected with avian influenza virus. Recently, however, avian influenza viruses have been directly transmitted to humans without undergoing genetic recombination.
돼지 인플루엔자(swine influenza)는 1918년에 처음으로 보고되었으며, 인간이 가지고 있는 인플루엔자 바이러스 수용체(N-acetylneuraminic acid α2,6 galactose)와 조류가 가지고 있는 인플루엔자 바이러스 수용체(N-acetylneuraminic acid α2,3 galactose)를 동시에 가지고 있어, 조류 인플루엔자 바이러스가 돼지의 몸 안에서 재배열(reassortment) 또는 적응(adaptation)을 통해 사람에게 감염될 수 있는 바이러스 혼합용기(mixing vessels)의 역할을 한다고 알려져 있다. 2009년 멕시코에서 처음 발병하여 전 세계로 전염되었던 신종 인플루엔자(novel influenza)는 돼지 인플루엔자 바이러스 H1N1형이 돼지에게 감염되어 인간에게 전염이 가능한 바이러스로 변이된 것이다(Dawood, F.S., et al., 2009; Zimmer, S.M., et al., 2009). The swine influenza was first reported in 1918, and the human influenza virus receptor (N-acetylneuraminic acid α2,6 galactose) and the bird influenza virus receptor (N-acetylneuraminic acid α2,3 galactose) At the same time, avian influenza viruses are known to act as viral mixing vessels that can infect humans through reassortment or adaptation in pigs. Novel influenza, which first developed in Mexico in 2009 and has been spread around the world, is due to the swine influenza virus H1N1 strain that has been infected with swine and becomes a virus that can be transmitted to humans (Dawood, FS, et al., 2009; Zimmer, SM, et al., 2009).
신종 인플루엔자(신종플루)는 기본적으로 감기처럼 고열, 기침 등의 증상이 나타나는데 현재 상태보다 인간에게 치명적인 바이러스로 변할 가능성이 높다. 세계보건기구(WHO)가 집계하는 신종플루의 감염자 수는 2009년 10월 초까지 그 수가 34만 3200명에 달하였고, 사망자는 4,000명을 넘어섰으며, 2010년 세계보건기구는 신종플루의 현황을 6단계인 대유행 상태로 격상한 사례가 있다. 신종플루는 일반 독감과 비슷하게 발열, 인후통, 기침, 콧물 또는 코막힘의 증상을 보여 주었으며, 2009년 발생한 H1N1의 신종플루는 일반 독감보다 심한 치명적인 폐 손상을 유발하는 병리적 현상을 보여주었다. Swine influenza (H1N1) is basically a symptom of a fever and cough, like a cold, and is more likely to become a fatal virus than humans. The number of people infected with the H1N1 flu recorded by the World Health Organization (WHO) reached 34,200 by the beginning of October 2009, and the death toll exceeded 4,000. There is an example of a six-step pandemic. H1N1 flu showed symptoms of fever, sore throat, cough, runny nose or stuffy nose, similar to that of the common flu. H1N1 H1N1 influenza showed pathological phenomena that resulted in more severe lethal lung damage than the common flu.
인플루엔자 바이러스에 의한 인플루엔자의 발병을 예방하기 위해서는 백신의 확보가 필수적이나 예방적 차원의 국가적 백신 대책으로는 매번 항원변이가 일어나는 RNA 바이러스를 모두 예방할 수 있는 백신을 만드는 것이 거의 불가능하다. 따라서 조류 인플루엔자, 돼지 인플루엔자 및 신종 인플루엔자 또는 이들의 변이 및 혼합에 의한 새로운 병원체에 의한 질병에 대비하기 위해서는 이를 예방하고 치료할 수 있는 치료제의 개발이 필수적이다. In order to prevent influenza caused by influenza virus, it is necessary to secure a vaccine, but as a preventive national vaccine, it is almost impossible to make a vaccine that can prevent all the RNA viruses that occur every time. Therefore, in order to prepare for a disease caused by avian influenza, swine influenza and new influenza or a new pathogen caused by mutation and mixing thereof, it is necessary to develop a therapeutic agent that can prevent and treat it.
현재 인플루엔자의 치료제로는 바이러스 기원의 뉴라미니다아제에 대한 선택적인 저해물질로 개발된 경구용 치료제인 타미플루(tamiflu, 성분명: oseltamivir phosphate)와 흡입형인 리렌자(relenza, 성분명: zanamivir) 등이 있다. 타미플루의 경우에는 오심, 구토, 신경계나 정신계의 이상과 같은 부작용이 보고되었으며, 일본의 경우 타미플루 승인 후 확인된 소아 환자의 사망이 15건 정도였다. 또한, 경구용 치료제인 타미플루는 내성 바이러스의 출현에 대한 신속한 대응이 어렵다는 문제점을 가지고 있으며, 최근에는 타미플루에 대한 내성 바이러스의 출현이 보고되고 있으며(Lee, J.Y., 2009; Hauge, S.H., et al., 2009) 타미플루 내성 바이러스의 인간 대 인간 전염도 발견되고 있는 실정이다. 따라서 신규의 인플루엔자 치료제 개발은 인간의 보건 안전에 필수적인 사항이다. Current treatments for influenza include oral therapeutics such as tamiflu (component name: oseltamivir phosphate) and inhaled relenza (component name: zanamivir) developed as selective inhibitors of neuraminidase of viral origin. Tamiflu has reported side effects such as nausea, vomiting, and nervous or mental problems. In Japan, 15 cases of pediatric patients confirmed after Tamiflu approval. In addition, Tamiflu, an oral therapeutic agent, has a problem that it is difficult to respond quickly to the emergence of resistant virus, and recently, the emergence of resistant virus to Tamiflu has been reported (Lee, JY, 2009; Hauge, SH, et al. , 2009) Human-to-human transmission of Tamiflu-resistant viruses has also been found. Therefore, the development of new influenza treatments is essential for human health and safety.
일반적으로 새로운 성분의 약제를 개발하기 위해 기존 약제를 실험적으로 변형시키는 것보다는 전통 의학에서 사용되고 있는 천연물 약제들로부터 새로운 활성 성분을 찾는 것이 여러 가지 면에서 장점이 많다. 특히 이러한 활성 성분들은 오랫동안 사용되어 왔기 때문에 약물에 의한 독성 염려가 적다. 이러한 사실은 인플루엔자 바이러스 치료제로 사용되는 타미플루의 출발물질이 중국의 토착식물인 '스타아니스' 열매가 주원료가 되어 개발된 것으로부터 알 수 있다. 또한, 천연물 자체를 바이러스 치료제로 사용할 수 있다면 수집 및 분리 과정을 거치지 않으므로 대량 시료를 확보하기가 용이하며 가축 사료에 혼합하여 공급할 수 있는 장점을 갖고 있다. In general, finding new active ingredients from natural medicines used in traditional medicine has many advantages, rather than experimentally modifying existing drugs to develop new ingredients. In particular, since these active ingredients have been used for a long time, there is little concern about toxicity due to drugs. This can be seen from the fact that Tamiflu's starting material, which is used as a treatment for influenza virus, was developed mainly from the Chinese native 'Staranis' fruit. In addition, if the natural product itself can be used as a virus treatment, it does not go through the collection and separation process, so it is easy to secure a large sample and has the advantage that it can be mixed and supplied to livestock feed.
톳(Sargassum fusiforme; Hizikia fusiforme)은 다년생 갈조류에 속하는 해조류로, 중국, 일본 및 우리나라 조간대 하부에 분포하고 있으며, 주로 제주도와 남해안에서 자연산을 채취하여 이식 양식을 통하여 생산되고 있다. 톳에는 칼슘, 요오드, 철 등의 무기 염류가 많이 포함되어 혈관경화를 막아주고, 상용으로 섭취할 시, 치아가 건강해지고 머리털이 윤택해지며, 임산부에게는 태아의 뼈를 튼튼하게 해주는 등, 예로부터 기호식품의 하나로 알려져 있다. 현재까지 보고된 톳의 생리 활성으로는 항암, 항산화, 면역억제, HIV-1 억제 작용 등이 보고되고 있다(Fan, S., et al., 2017; Zhou, J., et al., 2008; Chen, X., et al., 2012; Paskaleva, E.E., et al., 2006; Paskaleva, E.E., et al., 2008). Sargassum fusiforme (Hizikia fusiforme ) is a perennial brown algae, distributed in the lower tidal flats of China, Japan, and Korea. Contains a lot of inorganic salts such as calcium, iodine, iron, etc. to prevent vascular hardening, and when consumed commercially, teeth are healthy and hair is moisturized, and fetal bones are strengthened to pregnant women. It is known as one of the favorite foods. Physiological activities of so far reported to be anti-cancer, antioxidant, immunosuppressive, HIV-1 inhibitory activity (Fan, S., et al., 2017; Zhou, J., et al., 2008; Chen, X., et al., 2012; Paskaleva, EE, et al., 2006; Paskaleva, EE, et al., 2008).
이에, 본 발명자들은 톳 추출물을 이용하여 항바이러스 활성을 연구하는 과정에서 톳 추출물이 인플루엔자 바이러스에 대한 항바이러스 활성이 우수함을 확인함으로써 본 발명을 완성할 수 있었다. Thus, the present inventors were able to complete the present invention by confirming that the extract 우수 is excellent in the antiviral activity against influenza virus in the course of studying the antiviral activity using the extract 톳.
종래 선행기술로서 중국공개특허 제105853670호 및 제106309679호에는 해조(Sargassum)가 포함된 중국 약재 혼합물의 조류 인플루엔자 치료 효과가 기재되어 있으나, 본 발명의 톳 추출물의 인플루엔자 치료 효과는 기재되어 있지 않다. 또한, 중국공개특허 제106309470호에는 톳의 다당류의 호흡기세포융합바이러스(RSV) 및 인간 엔터로바이러스(EV71)에 대한 항바이러스 효과가 기재되어 있으나, 본 발명의 인플루엔자 바이러스에 대한 항바이러스 효과는 기재되어 있지 않다. In the prior art, Chinese Patent Publication Nos. 105853670 and 106309679 describe algal influenza treatment effects of a Chinese medicinal mixture containing seaweed (Sargassum), but do not describe the influenza therapeutic effect of the extract of the present invention. In addition, Chinese Patent Publication No. 106309470 describes the antiviral effect on polysaccharide respiratory syncytial virus (RSV) and human enterovirus (EV71), but the antiviral effect on the influenza virus of the present invention is described. It is not.
비특허문헌인 Paskaleva, E.E., et al., 2006 및 Paskaleva, E.E., et al., 2008에는 톳 추출물의 HIV-1에 대한 항바이러스 효과가 기재되어 있고, Park, S.Y., et al., 2016에는 톳 추출물의 노로바이러스에 대한 항바이러스 효과가 기재되어 있으나, 본 발명의 인플루엔자 바이러스에 대한 항바이러스 효과는 기재되어 있지 않다. Non-patent literature Paskaleva, EE, et al., 2006 and Paskaleva, EE, et al., 2008 describe the antiviral effects of 톳 extract on HIV-1, and Park, SY, et al., 2016 Although the antiviral effect of the extract of against norovirus is described, the antiviral effect of the influenza virus of the present invention is not described.
본 발명의 목적은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 조성물을 제공하는 데 있다. It is an object of the present invention to provide a composition for preventing or treating influenza, comprising the extract of Sargassum fusiforme as an active ingredient.
본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 약학 조성물 또는 동물 약품에 관한 것이다.The present invention relates to a pharmaceutical composition or animal medicine for influenza prophylaxis or treatment containing Sargassum fusiforme extract as an active ingredient.
또한, 본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 개선용 건강기능식품, 동물사료 첨가제 또는 예방용 천연소독제에 관한 것이다. In addition, the present invention relates to a health functional food for improving influenza, an animal feed additive, or a natural antiseptic for preventing influenza, which contains the extract of Sargassum fusiforme as an active ingredient.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물일 수 있다. The 톳 extract may be an extract of 톳 with one or more solvents selected from the group consisting of water, lower alcohols of C1 to C4, acetone, acetone, ethyl acetate, and hexane.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물일 수 있다. The 톳 extract is suspended in distilled water after extracting 톳 with water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and at least one solvent selected from the group consisting of hexane (hexan) It may be a butanol fraction obtained by fractionation in the order of hexane, ethyl acetate, butanol or ethyl acetate fraction.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating influenza, comprising the extract of Sargassum fusiforme as an active ingredient.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤, 에틸아세테이트 및 헥산으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물일 수 있으며, 상기 C1 내지 C4의 저급 알코올은 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등일 수 있다. 바람직하게는 에탄올, 메탄올 및 부탄올이다. 더 바람직하게는 50%(v/v) 메탄올 및 50%(v/v) 에탄올이다. The 톳 extract may be an extract of 로 with one or more solvents selected from the group consisting of water, lower alcohols of C1 to C4, acetone, ethyl acetate and hexane, wherein the lower alcohols of C1 to C4 are methanol, ethanol, Propanol, isopropanol, butanol and the like. Preferably ethanol, methanol and butanol. More preferably 50% (v / v) methanol and 50% (v / v) ethanol.
상기 용매는 톳 중량의 2배 내지 500배의 중량을 가할 수 있다.The solvent may add a weight of 2 to 500 times the weight of Kw.
또한, 상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤, 에틸아세테이트 및 헥산으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 유기용매로 분획하여 얻은 분획물일 수 있다. In addition, the 톳 extract is a fraction obtained by distilling the extract extracted with 이상의 water, at least one solvent selected from the group consisting of water, C1 to C4 lower alcohol, acetone, ethyl acetate and hexane with distilled water, and then fractionated with an organic solvent Can be.
상기 유기용매는 C1 내지 C4의 저급 알코올, 에틸아세테이트, 헥산 및 아세톤 등일 수 있다. 상기 C1 내지 C4의 저급 알코올은 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등 일 수 있다. 바람직하게는 헥산, 에틸아세테이트, 부탄올일 수 있고, 더 바람직하게는 에틸아세테이트 또는 부탄올이며, 가장 바람직하게는 부탄올이다. The organic solvent may be C1 to C4 lower alcohols, ethyl acetate, hexane and acetone. The lower alcohol of C1 to C4 may be methanol, ethanol, propanol, isopropanol, butanol and the like. Preferably hexane, ethyl acetate, butanol, more preferably ethyl acetate or butanol, most preferably butanol.
상기 톳 추출물을 포함하는 조성물은 약학적 조성물 일 수 있다. The composition comprising the 톳 extract may be a pharmaceutical composition.
상기 약학적 조성물은 상기 톳 추출물 및 약학적으로 허용 가능한 부형제 또는 담체를 포함할 수 있다. The pharmaceutical composition may comprise the 톳 extract and a pharmaceutically acceptable excipient or carrier.
상기 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 톳 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토즈, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다. The pharmaceutical composition may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. . Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose in the extract of the present invention. Mixed with gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
상기 약학적 조성물의 투여량은 치료 받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 0.1㎎/㎏/일 내지 500㎎/㎏/일이다. 투여는 하루에 한 번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. The dosage of the pharmaceutical composition will vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
상기 약학적 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 점막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. The pharmaceutical composition may be administered to mammals such as rats, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine mucosal or cerebrovascular injections.
또한, 상기 톳 추출물을 포함하는 조성물은 건강기능식품일 수 있다. In addition, the composition containing the 톳 extract may be a health functional food.
상기 건강기능식품은 상기 톳 추출물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함할 수 있다. The health functional food may include the 톳 extract and a food supplement acceptable food supplement.
상기 건강기능식품은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 본 발명의 톳 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성식품류 등이 있다. The health functional food includes the form of tablets, capsules, pills or liquids, and the food to which the extract of the present invention may be added, for example, various foods, beverages, gums, teas, vitamin complexes, Health functional foods.
상기 톳 추출물을 포함하는 조성물은 또한, 동물사료 첨가제일 수 있다. The composition comprising the 톳 extract may also be an animal feed additive.
상기 동물사료 첨가제는 본 발명의 톳 추출물이 동물용 사료에 0.001중량% 내지 30중량%, 바람직하게는 0.001중량% 내지 10중량%, 가장 바람직하게는 0.001중량% 내지 5중량%로 첨가될 수 있다. The animal feed additive may be added to the animal feed of 0.001% to 30% by weight, preferably 0.001% to 10% by weight, most preferably 0.001% to 5% by weight of the extract of the present invention. .
또한, 상기 톳 추출물을 포함하는 조성물은 천연소독제일 수 있다. In addition, the composition comprising the extract 톳 may be a natural antiseptic.
상기 천연소독제는 인플루엔자 바이러스의 소독용 조성물로, 액제, 정제, 과립제, 산제 등의 다양한 형태로의 제제로 제조가 가능하다. The natural antiseptic is a composition for disinfecting influenza virus, it can be prepared in a variety of forms, such as liquids, tablets, granules, powders.
본 발명은 톳(Sargassum fusiforme) 추출물을 유효성분으로 포함하는 인플루엔자 예방 또는 치료용 조성물에 관한 것으로, 톳 추출물이 인플루엔자 바이러스에 대한 항바이러스 활성이 있으며, 동속이종인 다른 해조류 추출물에 비해 항바이러스 활성이 우수하다는 것을 확인하였다. The present invention relates to a composition for preventing or treating influenza, comprising the extract of Sargassum fusiforme as an active ingredient, the extract having antiviral activity against influenza virus, and excellent antiviral activity compared to other algae extracts of the same species. It was confirmed that.
이를 통해, 본 발명의 톳 추출물을 인플루엔자의 예방 또는 치료용 의약품, 건강기능식품, 동물사료 첨가제 또는 천연소독제 등의 개발시에 유용하게 이용할 수 있을 것으로 기대된다. Through this, it is expected that the extract of the present invention can be usefully used in the development of medicines for preventing or treating influenza, health functional foods, animal feed additives or natural antiseptics.
도 1은 톳 추출물의 돼지 인플루엔자 바이러스에 대한 항바이러스 활성을 확인한 결과를 보여주고 있다.
도 2는 톳 추출물의 세포독성을 확인한 결과를 보여주고 있다.
도 3은 톳 분획물의 돼지 인플루엔자 바이러스에 대한 항바이러스 활성을 확인한 결과를 보여주고 있다.
도 4는 톳 분획물의 세포독성을 확인한 결과를 보여주고 있다.
도 5는 톳 추출물 또는 분획물의 돼지 인플루엔자 바이러스 표면 단백질인 뉴라미다아제 단백질 발현 억제 효과를 확인한 결과를 보여주고 있다.
도 6은 톳 추출물 또는 분획물의 돼지 인플루엔자 바이러스 표면 단백질인 뉴라미다아제 및 헤마글루티닌 단백질 발현 억제 효과를 웨스턴 블롯으로 확인한 결과로, (A)는 각 단백질 발현을 밴드로 확인한 결과를, (B)는 단백질 발현 정도를 수치화한 결과를 보여주고 있다.
도 7은 톳과 동속이종인 해조류 추출물과 톳 추출물의 세포독성(A) 및 돼지 인플루엔자 바이러스에 대한 항바이러스 활성(B)을 비교한 결과를 보여주고 있다.
도 8은 톳 추출물의 항바이러스 활성이 코로나 바이러스 대비, 인플루엔자바이러스에 현저히 높음을 보여주고 있다. Figure 1 shows the results of confirming the antiviral activity of swine influenza virus of the extract.
Figure 2 shows the results confirming the cytotoxicity of the extract 톳.
Figure 3 shows the results of confirming the antiviral activity against swine influenza virus of the 톳 fraction.
Figure 4 shows the results confirming the cytotoxicity of the 톳 fraction.
Figure 5 shows the results of confirming the effect of inhibiting the expression of neuramidase protein, a surface protein of swine influenza virus of the extract or fraction 톳.
6 is a Western blot confirming the effect of inhibiting the expression of porcine influenza virus surface proteins neuramidase and hemagglutinin protein of 톳 extract or fraction, (A) is the result of confirming each protein expression in a band, ( B) shows the result of quantifying the degree of protein expression.
Figure 7 shows the results of comparing the antiviral activity (B) and the cytotoxicity (A) and swine influenza virus of the algae extract of the same species as murine and murine extract.
Figure 8 shows that the antiviral activity of the extract extract is significantly higher in influenza virus, compared to the corona virus.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.
<실시예 1. 톳 추출물의 제조 및 항바이러스 활성 측정><Example 1. Preparation of 톳 extract and measurement of antiviral activity>
실시예Example 1-1. 톳 추출물의 제조 1-1. Preparation of 톳 Extract
본 발명에서 사용된 톳(Sargassum fusiforme)은 광주광역시에 소재하는 조선대학교 해양갈조식물자원 은행에서 분양받았다. Sargassum fusiforme ( 에서 ) used in the present invention was distributed by the marine reed plant resource bank of Chosun University located in Gwangju.
톳 3g에 하기 표 1에 해당되는 각각의 용매 15㎖씩을 가한 후 초음파 추출기로 2시간씩 총 3회에 걸쳐 추출하였다. 이후, 고형분을 제거하여 추출액을 얻었고, 얻은 추출액을 감압 농축하여 추출물을 제조한 후, 추출량을 확인하였고, 그 결과를 표 1에 나타내었다. 톳 3 g of each solvent corresponding to Table 1 was added to 3 g, and extracted three times for 2 hours using an ultrasonic extractor. Thereafter, the solid was removed to obtain an extract. The extract was concentrated under reduced pressure to prepare an extract, and then the amount of the extract was checked. The results are shown in Table 1 below.
실시예Example 1-2. 톳 추출물의 항바이러스 활성 확인 1-2. 항 Antiviral Activity of Extracts
상기 실시예 1-1에서 추출한 톳 추출물의 항바이러스 활성을 확인하기 위해 중앙백신연구소로부터 제공받은 돼지 인플루엔자 바이러스 H1N1(A/Sw/Kor/CAN1/04, KCTC11165BP)을 이용하였고, 바이러스 감염에 의한 감염세포의 생존율을 분석함으로써 항바이러스 활성을 확인하였다. In order to confirm the antiviral activity of the extract 에서 in Example 1-1, swine influenza virus H1N1 (A / Sw / Kor / CAN1 / 04, KCTC11165BP) provided from the central vaccine research institute was used, and infection by virus infection Antiviral activity was confirmed by analyzing the viability of the cells.
96웰 플레이트(96 well plate)에 웰 당 1×104개의 MDCK(Madin-Darby, canine kidney) 세포를 분주한 후 5% FBS(fetal bovine serum), 1% 페니실린/스트렙토마이신(penicillin/streptomycin) 및 0.4% L-글루타민(L-glutamine)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium) 배지를 넣고 하루 동안 배양한 후, 배양액을 버리고 PBS(phosphate buffer saline) 용액으로 세척하였다. 이후 세포에 감염배지(infection media, DMEM+0.5% BSA(bovine serum albumin)+1㎍/㎖ trypsin)를 넣고, 돼지 인플루엔자 바이러스를 50 TCID50/100㎕/웰의 농도가 되도록 접종하고, 상기 실시예 1-1에서 제조한 톳 추출물을 농도별(1, 2, 5, 10, 20㎍/㎖)로 처리한 후 5일 동안 배양하였다. 이때, 세포에 아무것도 처리하지 않은 것을 정상 대조군으로 이용하였고, 바이러스만을 처리한 것을 음성 대조군으로, 항바이러스제인 리바비린(ribavirin) 10uM을 처리한 것을 양성 대조군으로 이용하였다. Dispense 1 × 10 4 MDCK (Madin-Darby, canine kidney) cells per well into a 96-well plate, followed by 5% fetal bovine serum (FBS), 1% penicillin / streptomycin (penicillin / streptomycin). And Dulbeco's Modified Eagle's Medium (DMEM) medium containing 0.4% L-glutamine (L-glutamine) was added and cultured for one day, the culture was discarded and washed with PBS (phosphate buffer saline) solution. Infection in cell culture medium after the insert (infection media, DMEM + 0.5% BSA (bovine serum albumin) + 1㎍ / ㎖ trypsin), was inoculated so that the swine influenza virus concentration of 50
5일 동안 배양한 세포를 이용하여 MTT(3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) 어세이를 수행하였다. 배양한 세포에 MTT 용액을 넣고 37℃에서 반응시킨 후 배양액을 제거하고 DMSO(dimethyl sulfoxide) 200㎕를 넣었다. 피펫팅으로 잘 섞어 준 다음 10분 동안 방치한 후 550㎚에서 흡광도를 측정하여 세포의 생존율을 확인하였다. 이때, 정상 대조군의 세포 생존율 100%를 기준으로 각 용매에 따른 추출물에 의한 세포 생존율을 계산하였고, 그 결과를 도 1 및 표 2에 나타내었다. 표 2의 경우, 각 용매에 따른 추출물의 항바이러스 활성으로 인해 인플루엔자 바이러스에 의한 세포사멸이 억제되었으므로, 추출물에 의한 세포 생존율을 항바이러스 활성과 동일한 개념으로 사용하였다. MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed using cells cultured for 5 days. MTT solution was added to the cultured cells, and the reaction was performed at 37 ° C., the culture solution was removed, and 200 μl of DMSO (dimethyl sulfoxide) was added thereto. After mixing well by pipetting and left for 10 minutes, the absorbance was measured at 550 nm to confirm cell viability. At this time, the cell viability by the extract according to each solvent was calculated based on the cell survival rate of 100% of the normal control, the results are shown in Figure 1 and Table 2. In Table 2, apoptosis by influenza virus was inhibited due to the antiviral activity of the extract according to each solvent, and thus cell viability by the extract was used in the same concept as the antiviral activity.
도 1 및 상기 표 2에서 보여주듯이, 돼지 인플루엔자 바이러스만을 처리한 음성 대조군의 경우, 바이러스에 의해 세포사멸이 이루어지는 반면에, 항바이러스제인 리바비린을 처리한 양성 대조군의 경우에는 바이러스에 의한 세포사멸이 억제되는 것을 알 수 있었다. 또한, 본 발명의 톳 추출물을 처리한 경우에는 50%(v/v) 에탄올, 100%(v/v) 에탄올, 50%(v/v) 메탄올, 100%(v/v) 메탄올, 부탄올 및 에틸아세테이트 톳 추출물이 인플루엔자 바이러스에 의한 세포사멸 억제 활성, 즉 항바이러스 활성이 가장 좋으며, 아세톤 및 헥산 톳 추출물의 경우에도 인플루엔자 바이러스에 대한 항바이러스 활성이 있는 것을 확인하였다. 반면에 증류수를 이용하여 추출한 톳 추출물의 경우에는 추출량이 많았지만(상기 표 1 참조) 항바이러스 활성은 약하게 나타났다. 이는, 항바이러스 활성을 갖는 톳 추출물을 확보하기 위해서는 유기용매를 이용하는 것이 필요하다는 것을 보여주고 있다. As shown in FIG. 1 and Table 2, in the negative control group treated with the swine influenza virus only, apoptosis was performed by the virus, whereas the positive control group treated with the antiviral agent ribavirin inhibited cell death by the virus. I could see. In addition, when the extract of the present invention was treated, 50% (v / v) ethanol, 100% (v / v) ethanol, 50% (v / v) methanol, 100% (v / v) methanol, butanol and It was confirmed that ethyl acetate 이 extract had the best anti-apoptotic activity by influenza virus, that is, antiviral activity, and that acetone and hexane 항 extracts also had antiviral activity against influenza virus. On the other hand, in case of extract extracted using distilled water, the extraction amount was large (see Table 1 above), but the antiviral activity was weak. This shows that it is necessary to use an organic solvent in order to secure the extract of antiviral activity.
실시예 1-3. 톳 추출물의 세포독성 확인Example 1-3. 세포 Confirm cytotoxicity of extract
상기 실시예 1-1의 톳 추출물에 의한 세포독성 여부를 확인하기 위해 MTT 어세이를 진행하였다. MTT assay was performed to confirm the cytotoxicity of the extract of Example 1-1.
96웰 플레이트(96 well plate)에 웰 당 1×104개의 MDCK 세포를 분주한 후 5% FBS, 1% 페니실린/스트렙토마이신 및 0.4% L-글루타민이 포함된 DMEM 배지를 넣고 하루 동안 배양한 후, 배양액을 버리고 PBS 용액으로 세척하였다. 이후 세포에 새 배지를 넣고, 상기 실시예 1-1에서 제조한 톳 추출물을 각각 5, 10, 20㎍/㎖이 되도록 처리한 후 5일 동안 배양하였다. 이때, 세포에 아무것도 처리하지 않은 것을 정상 대조군으로 이용하였다. Dispense 1 × 10 4 MDCK cells per well into a 96 well plate, add DMEM medium containing 5% FBS, 1% penicillin / streptomycin, and 0.4% L-glutamine, and incubate for one day. The cultures were discarded and washed with PBS solution. Thereafter, fresh cells were added to the cells, and the extracts prepared in Example 1-1 were treated to 5, 10, and 20 µg / ml, respectively, and then cultured for 5 days. At this time, the cells were not treated anything was used as a normal control.
5일 동안 배양한 세포를 이용하여 상기 실시예 1-2와 동일한 방법으로 MTT 어세이를 수행하였다. 이때, 정상 대조군의 세포 생존율 대비 각각의 추출물에 의한 세포 생존율을 %로 환산하였고, 그 결과를 도 2에 나타내었다. MTT assay was performed using the cells cultured for 5 days in the same manner as in Example 1-2. At this time, the cell viability of each extract compared to the cell viability of the normal control group was converted into%, and the results are shown in FIG. 2.
도 2에서 보여주듯이, 상기 실시예 1-1의 톳 추출물 대부분이 10㎍/㎖ 이하의 농도에서 세포독성이 거의 없었고, 헥산 추출물, 에틸아세테이트 추출물 및 아세톤 추출물의 경우에는 20㎍/㎖의 농도에서 세포 독성이 있는 것으로 나타났다. As shown in FIG. 2, most of the extracts of Example 1-1 had little cytotoxicity at concentrations of 10 μg / ml or less, and in the case of hexane extract, ethyl acetate extract and acetone extract, at a concentration of 20 μg / ml. It has been shown to be cytotoxic.
상기 표 1의 용매에 따른 추출량, 도 1 및 표 2의 항바이러스 활성 및 도 2의 세포독성 결과를 통해, 50%(v/v) 메탄올이 최적의 용매이지만, 식품에 사용 가능한 용매 조건을 고려하여 50%(v/v) 에탄올을 용매로 하여 추출하는 것을 추출 조건으로 결정하였다. Through the extraction amount according to the solvent of Table 1, the antiviral activity of Figure 1 and Table 2 and the cytotoxicity results of Figure 2, 50% (v / v) methanol is the optimum solvent, but considering the solvent conditions that can be used in food Extraction with 50% (v / v) ethanol as a solvent was determined as extraction conditions.
<실시예 2. 톳 분획물의 제조> Example 2. Preparation of Cellular Fractions
상기 실시예 1-1에서 추출한 톳 추출물을 용매 분획방법을 적용하여 물, 부탄올, 에틸아세테이트 및 헥산 용매를 이용하여 극성 순으로 성분을 용출시켜 분획물을 제조하였다. The extract was extracted in Example 1-1 by applying a solvent fractionation method to prepare a fraction by eluting the components in the order of polarity using water, butanol, ethyl acetate and hexane solvent.
상기 실시예 1-1에서 제조한 톳 추출물 중 50%(v/v) 에탄올을 용매로 하여 추출한 톳 추출물 730㎎을 증류수에 현탁하고, 이후 헥산, 에틸아세테이트, 부탄올 순으로 각 용매를 1㎖씩 가하여 각 용매에 해당되는 분획물을 확보하였고, 확보한 분획물의 분획량 및 추출함량을 하기 표 3에 나타내었다. 730 mg of the extract extracted using 50% (v / v) ethanol as a solvent among the extracts prepared in Example 1-1 was suspended in distilled water, and then 1 ml of each solvent was added in the order of hexane, ethyl acetate and butanol. The fractions corresponding to each solvent were obtained by addition, and the fractions and extraction contents of the obtained fractions are shown in Table 3 below.
실시예Example 2-2. 톳 2-2. 톳 분획물의Fraction 항바이러스 활성 확인 Confirm antiviral activity
상기 2-1에서 제조한 톳 분획물을 이용하여 상기 실시예 1-2와 동일한 방법으로 톳 분획물의 항바이러스 활성을 확인하였다. 이때, 각 분획물의 농도가 2, 5, 10, 20㎍/㎖이 되도록 처리하였고, 그 결과를 도 3 및 하기 표 4에 나타내었다. Using the 톳 fraction prepared in 2-1, the antiviral activity of the 톳 fraction was confirmed in the same manner as in Example 1-2. At this time, the concentration of each fraction was treated to 2, 5, 10, 20 ㎍ / ㎖, the results are shown in Figure 3 and Table 4 below.
도 3 및 상기 표 4에서 보여주듯이, 부탄올 분획물을 20㎍/㎖으로 처리한 경우, 50%(v/v) 에탄올 톳 추출물 및 양성 대조군으로 이용한 리바비린과 유사한 정도의 항바이러스 활성을 나타내는 것을 확인하였고, 에틸아세테이트 분획물의 경우에도 항바이러스 활성이 높게 나타났다. As shown in FIG. 3 and Table 4, when the butanol fraction was treated with 20 μg / ml, it was confirmed that the antiviral activity was similar to that of ribavirin used as a 50% (v / v) ethanol 톳 extract and a positive control. The antiviral activity was also high in the ethyl acetate fraction.
실시예 2-3. 톳 분획물의 세포독성 확인Example 2-3. 확인 Confirmation of cytotoxicity of fractions
상기 실시예 2-1의 톳 분획물에 의한 세포독성 여부를 확인하기 위해 상기 실시예 1-3과 동일한 방법으로 MTT 어세이를 진행하였고, 상기 실시예 2-1의 톳 분획물을 5, 10, 20㎍/㎖이 되도록 처리하였고, 그 결과를 도 4에 나타내었다. MTT assay was performed in the same manner as in Example 1-3 to determine whether cytotoxicity was caused by the VIII fraction of Example 2-1, and the VIII fraction of Example 2-1 was 5, 10, 20 Treatment was performed to μg / ml and the results are shown in FIG. 4.
도 4에서 보여주듯이, 상기 실시예 2-1의 톳 분획물 대부분이 10㎍/㎖ 이하의 농도에서 세포독성이 거의 없었고, 에틸아세테이트 분획물 및 헥산 분획물의 경우에는 20㎍/㎖의 농도에서 세포 독성이 있는 것으로 나타났다. As shown in FIG. 4, most of the 톳 fractions of Example 2-1 showed little cytotoxicity at concentrations of 10 μg / ml or less, and cytotoxicity at concentrations of 20 μg / ml for ethyl acetate and hexane fractions. Appeared to be.
상기 표 3의 용매에 따른 추출량, 도 3 및 표 4의 항바이러스 활성 및 도 4의 세포독성 결과를 통해, 부탄올을 용매로 하여 분획한 톳 분획물이 가장 우수한 항바이러스 활성을 가지고 있음을 알 수 있었다. According to the extraction amount according to the solvent of Table 3, the antiviral activity of Figure 3 and Table 4 and the cytotoxicity results of Figure 4, it was found that the 톳 fraction fractionated with butanol as the solvent has the best antiviral activity. .
상기 실시예 1 및 실시예 2의 결과를 통해, 본 발명의 톳 추출물 및 분획물이 인플루엔자 바이러스에 대한 항바이러스 활성이 우수함을 알 수 있었다. Through the results of Examples 1 and 2, it was found that the extract and fractions of the present invention has excellent antiviral activity against influenza virus.
<실시예 3. 톳 추출물의 인플루엔자 바이러스 표면 단백질 억제 효과 확인><Example 3. Confirmation of influenza virus surface protein inhibitory effect of the extract
인플루엔자 바이러스의 표면에는 헤마글루티닌(hemagglutinin, HA)과 뉴라미니다아제(neuraminidase, NA)라는 두 가지 단백질이 존재하며, 이러한 바이러스 표면 단백질에 의해 감염이 일어난다. 따라서, 본 발명의 톳 추출물의 항바이러스 활성을 확인하기 위해 인플루엔자 바이러스 표면 단백질 발현에 있어서 본 발명의 톳 추출물의 억제 효과를 형광 현미경과 웨스턴 블롯(Western blot) 방법을 이용하여 확인하였다. There are two proteins on the surface of the influenza virus, hemagglutinin (HA) and neuraminidase (NA), which are caused by the viral surface proteins. Therefore, in order to confirm the antiviral activity of the extract of the present invention, the inhibitory effect of the extract of the present invention on the expression of influenza virus surface protein was confirmed using a fluorescence microscope and Western blot method.
실시예 3-1. 형광 현미경을 이용한 바이러스 표면 단백질 억제 효과 확인 Example 3-1. Confirmation of Virus Surface Protein Inhibition by Fluorescence Microscopy
8웰 챔버 슬라이드(LAB-TEK, NUNC, Thermo Fisher Scientific, USA)에 MDCK 세포를 분주하여 배양하고, 0.01 MOI(multiplicity of infection)로 돼지 인플루엔자 바이러스(H1N1 바이러스)를 2시간 동안 처리하여 세포에 감염시켰다. 이후, 세포를 PBS로 세척한 후, 세포에 상기 실시예 1의 톳 추출물 또는 실시예 2의 톳 분획물이 하기 표 5에 기재된 농도로 포함되어 있는 DMEM 배지를 넣고 24시간 동안 배양하였다. 이때, 아무것도 처리하지 않은 세포를 정상 대조군으로, 돼지 인플루엔자 바이러스만을 처리한 세포를 음성 대조군으로, 리바비린을 처리한 세포를 양성 대조군으로 이용하였다. MDCK cells were aliquoted and cultured in 8-well chamber slides (LAB-TEK, NUNC, Thermo Fisher Scientific, USA), and infected with cells by treatment of swine influenza virus (H1N1 virus) for 2 hours with 0.01 multiplicity of infection (MOI). I was. Thereafter, the cells were washed with PBS, and the cells were placed in DMEM medium containing the extract of Example 1 or the fraction of Example 2 at the concentrations described in Table 5 below, and cultured for 24 hours. At this time, cells treated with nothing were used as normal controls, cells treated with swine influenza virus only as negative controls, and cells treated with ribavirin as positive controls.
배양한 세포를 4℃에 냉장 보관된 PBS를 이용하여 3회 세척한 뒤, 4% 파라포름알데히드(paraformaldehyde) 용액을 30분 동안 처리하여 세포를 고정시켰다. 고정시킨 세포에 1% BSA(bovine serum albumin) 용액을 처리하여 1시간 동안 블로킹(blocking)시킨 후, 돼지인플루엔자 바이러스의 뉴라미니다아제 단백질을 인식하는 단일클론 항체(Gene Tex Co., San Antonio, TX, USA)를 처리하였다. 이후, 형광물질이 결합되어 있는 2차 항체(FITC-conjugated goat anti-Rb lgG 항체, Abcam, Cambridge, UK)를 처리하고, 500nM DAPI 용액을 이용하여 추가적으로 세포 내 DNA를 염색하였다. 염색한 세포를 마운팅 용액(mounting solution)(Vectashield, Vector Laboratories Inc., USA)으로 고착 시킨 후, 형광현미경(Olympus ix70 Fluorescence Microscope, Olympus Corporation, Tokyo, Japan)을 이용하여 돼지 인플루엔자 바이러스의 뉴라미니다아제 단백질의 발현 정도를 관찰하였고, 그 결과를 도 5에 나타내었다. The cultured cells were washed three times using PBS stored at 4 ° C., and then fixed with cells treated with 4% paraformaldehyde solution for 30 minutes. After blocking for 1 hour by treatment with 1% BSA (bovine serum albumin) solution on the immobilized cells, a monoclonal antibody (Gene Tex Co., San Antonio, Co., Ltd.) that recognizes the neuraminidase protein of swine influenza virus TX, USA). Subsequently, the secondary antibody (FITC-conjugated goat anti-Rb lgG antibody, Abcam, Cambridge, UK) to which the fluorescent material is bound was treated, and further intracellular DNA was stained using a 500 nM DAPI solution. The stained cells are fixed with a mounting solution (Vectashield, Vector Laboratories Inc., USA) and then neuraminas of swine influenza virus using a fluorescence microscope (Olympus ix70 Fluorescence Microscope, Olympus Corporation, Tokyo, Japan). The degree of expression of the azetin protein was observed, and the results are shown in FIG. 5.
도 5에서 보여주듯이, 아무것도 처리하지 않은 정상 대조군에 비해 돼지 인플루엔자 바이러스를 처리한 음성 대조군에서는 뉴라미다아제 단백질의 발현이 강하게 나타나는 것을 확인하였다. 반면에, 상기 실시예 1의 톳 추출물 또는 실시예 2의 부탄올 톳 분획물 및 에틸아세테이트 톳 분획물을 처리한 경우에는 뉴라미다아제 단백질의 발현이 억제되는 것을 확인하였다. 그러나 상기 실시예 2의 증류수 톳 분획물 및 헥산 톳 분획물의 경우에는 돼지 인플루엔자 바이러스의 뉴라미다아제 단백질 발현 억제 효과가 거의 없는 것으로 나타났다. 이러한 결과는 상기 실시예 2-2의 톳 분획물의 항바이러스 효과를 확인한 결과와 일치한다는 것을 알 수 있었다. As shown in FIG. 5, it was confirmed that the expression of the neuramidase protein was strongly expressed in the negative control group treated with swine influenza virus compared to the normal control group which was not treated with anything. On the other hand, when the 톳 extract of Example 1 or the butanol 톳 fraction and the ethyl acetate 톳 fraction of Example 2 was confirmed that the expression of the neuramidase protein is inhibited. However, the distilled water 톳 fraction and hexane 톳 fraction of Example 2 were found to have little effect of inhibiting the expression of the neuraminidase protein of the swine influenza virus. These results were found to be in agreement with the results of confirming the antiviral effect of the 톳 fraction of Example 2-2.
실시예 3-2. 웨스턴 블롯을 이용한 바이러스 표면 단백질 억제 효과 확인 Example 3-2. Confirmation of Virus Surface Protein Inhibition by Western Blot
6웰 플레이트에 MDCK 세포를 분주하고 플레이트 상에 90% 정도로 세포가 차도록 배양하였다. 배양한 세포에 돼지 인플루엔자 바이러스(H1N1 바이러스)를 0.01 MOI로 2시간 동안 처리하여 세포에 감염시켰다. 이후, 세포를 PBS로 세척한 후, 세포에 상기 실시예 1의 톳 추출물 또는 실시예 2의 톳 분획물이 상기 표 5에 기재된 농도가 되도록 포함된 DMEM를 넣고 처리하고 24시간 동안 배양하였다. 이때, 아무것도 처리하지 않은 세포를 정상 대조군으로, 돼지 인플루엔자 바이러스를 처리한 세포를 음성 대조군으로, 리바비린을 처리한 세포를 양성 대조군으로 이용하였다. MDCK cells were aliquoted into 6-well plates and cultured so that the cells were about 90% full on the plates. The cultured cells were infected with porcine influenza virus (H1N1 virus) at 0.01 MOI for 2 hours. Then, after washing the cells with PBS, the cells were added to the DMEM contained in the extract of Example 1 or 톳 fraction of Example 2 to the concentration described in Table 5, and incubated for 24 hours. At this time, cells treated with nothing were used as normal controls, cells treated with swine influenza virus as negative controls, and cells treated with ribavirin as positive controls.
배양한 세포를 PBS로 세척하고, 100㎕의 세포 용해 버퍼(cell lysis buffer)(50mM NaF, 0.5%[v/v] NP-40, 1mM EDTA, 120mM NaCl, 50mM Tris-HCl, pH 7.6)를 이용하여 세포를 파쇄하고 원심분리하여 세포 용출액을 확보하였다. 확보한 세포 용출액을 이용하여 SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10-12%[w/v] acrylamide gel)를 실시한 뒤, 세미-드라이 트랜스퍼(semi-dry transfer) 기기를 이용하여 PVDF(polyvinylidene fluoride) 멤브레인에 단백질을 이동(transfer)시켰다. 멤브레인은 5%[w/v] 탈지유로 1시간 동안 상온에서 처리하여 블로킹(blocking)시키고, 돼지 인플루엔자 바이러스의 뉴라미니다아제와 헤마글루티닌을 인식하는 1차 항체(AbFrontier Co., Ltd., Seoul, Korea)를 처리하여 반응 시킨 후, 각각의 1차 항체를 인식하는 2차 항체를 처리하고 ECL 용액을 이용하여 단백질 밴드를 확인하였다. 또한, 확인된 단백질 밴드의 밀도를 측정하여 단백질 발현 정도를 수치화 하였고, 그 결과를 도 6에 나타내었다. The cultured cells were washed with PBS, and 100 μl of cell lysis buffer (50 mM NaF, 0.5% [v / v] NP-40, 1 mM EDTA, 120 mM NaCl, 50 mM Tris-HCl, pH 7.6) was added. Cells were disrupted and centrifuged to secure cell eluate. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10-12% [w / v] acrylamide gel) was performed using the obtained cell eluate, followed by PVDF using a semi-dry transfer device. (polyvinylidene fluoride) proteins were transferred to the membrane. The membrane was blocked by treatment at room temperature for 1 hour with 5% [w / v] skim milk, and the primary antibody (AbFrontier Co., Ltd.) that recognizes neuraminidase and hemagglutinin of swine influenza virus. , Seoul, Korea) and reacted, and then treated with a secondary antibody that recognizes each primary antibody and confirmed the protein band using the ECL solution. In addition, the degree of protein expression was quantified by measuring the density of the identified protein band, and the results are shown in FIG. 6.
도 6(A)의 단백질 발현 밴드 결과 및 6(B)의 발현량을 수치화한 결과에서 보여주듯이, 아무것도 처리하지 않은 정상 대조군에 비해 돼지 인플루엔자 바이러스를 처리한 음성 대조군에서 바이러스 표면 단백질인 뉴라미다아제 및 헤마글루티닌 단백질 모두 발현이 증가하였다. 반면에, 상기 실시예 1의 톳 추출물 또는 실시예 2의 톳 분획물 중 부탄올 분획물 및 에틸아세테이트 분획물을 처리한 경우에는 뉴라미다아제 및 헤마글루티닌 단백질의 발현이 현저히 억제된 것을 확인하였다. As shown in the results of the protein expression band of FIG. 6 (A) and the results of quantifying the expression level of 6 (B), the neuramida which is the virus surface protein in the negative control group treated with swine influenza virus compared to the normal control group treated with nothing Both aze and hemagglutinin protein increased expression. On the other hand, when the butanol fraction and ethyl acetate fraction of the extract of Example 1 or the extract of Example 2 were treated with ethyl acetate, it was confirmed that the expression of neuramidase and hemagglutinin protein was significantly suppressed.
상기 실시예 3-1 및 실시예 3-2의 결과를 통해, 본 발명의 톳 추출물과 분획물이 인플루엔자 바이러스에 대한 항바이러스 활성이 우수함을 알 수 있었고, 분획물의 경우에는 부탄올 분획물 및 에틸아세테이트 분획물이 항바이러스 활성이 가장 우수하다는 것을 알 수 있었다. Through the results of Examples 3-1 and 3-2, it can be seen that the extract and fraction of the present invention has excellent antiviral activity against influenza virus, but in the case of the fraction, butanol fraction and ethyl acetate fraction are The antiviral activity was found to be the best.
<실시예 4. 톳 추출물 및 다른 해조류 추출물의 항바이러스 활성 비교><Example 4. Comparison of antiviral activity of 톳 extract and other seaweed extracts>
본 발명의 톳 추출물의 인플루엔자 바이러스에 대한 항바이러스 활성이 우수함을 확인하기 위해 톳과 동속이종인 다른 해조류 추출물과 항바이러스 활성을 비교하였다. In order to confirm that the antiviral activity against the influenza virus of the extract of the present invention is excellent, the antiviral activity was compared with that of other algae extracts of the same species.
비교에 사용된 해조류는 6종으로 짝잎모자반(Sargassum hemiphyllum), 괭생이모자반(Sargassum horneri), 경단구슬모자반(Sargassum muticum), 알쏭이자반(Sargassum confusum), 큰열매모자반(Sargassum macrocarpum), 왜모자반(Sargassum yezoense)으로, 광주광역시에 소재하는 조선대학교 해양갈조식물자원은행에서 분양받았다. 분양 받은 각 해조류는 상기 실시예 1-1의 톳 추출물 제조 방법과 동일한 방법으로 추출하였고, 추출 용매는 50%(v/v) 에탄올을 이용하였다. Six species of algae used in comparison were Sargassum hemiphyllum , Sargassum horneri , Sargassum muticum , Sargassum confusum , Sargassum macrocarpum , Dwarf hat Sargassum yezoense was distributed by the Marine Brown Algae Plant Resource Bank of Chosun University in Gwangju. Each seaweed that was distributed was extracted in the same manner as in the method of preparing the extract of Example 1-1, and 50% (v / v) ethanol was used as the extraction solvent.
각 해조류 추출물의 항바이러스 활성을 확인하기에 앞서 상시 실시예 1-3과 동일한 방법으로 각 추출물에 의한 세포독성을 확인하였고, 그 결과를 도 7(A)에 나타내었다. Prior to confirming the antiviral activity of each seaweed extract, cytotoxicity was confirmed by each extract in the same manner as in Example 1-3, and the results are shown in FIG. 7 (A).
도 7(A)에서 보여주듯이, 짝잎모자반, 괭생이모자반, 경단구슬모자반, 알쏭이자반의 경우에는 세포독성이 거의 나타나지 않는 반면에, 큰열매모자반과 왜모자반은 10㎍/㎖ 이상의 농도에서 세포독성이 있음을 알 수 있었다. As shown in FIG. 7 (A), cytotoxicity was not observed in the case of a pair of leaf leaf hats, a hawthorn hair band, a dandelion ball hair band, and a round head band, whereas a large hawk barn and a dwarf barn had cells at a concentration of 10 µg / ml or more. It was found to be toxic.
이를 바탕으로, 항바이러스 활성 확인 실험 진행시 세포독성이 관찰되지 않은 짝잎모자반, 괭생이모자반, 경단구슬모자반, 알쏭이자반은 최종 처리농도가 5, 10, 20㎍/㎖이 되도록 처리하였고, 세포독성이 있는 큰열매모자반과 왜모자반은 최종 처리농도가 1, 2, 5㎍/㎖이 되도록 처리하였다. On the basis of this, the antiviral activity was confirmed to be treated with the final treatment concentration of 5, 10, 20 ㎍ / ㎖ of the leaf leaf patch, Hoengsae amoeba, dandelion moss ball, and albinizaban, in which no cytotoxicity was observed. Toxic large barberry and dwarf hatchlings were treated to final treatment concentrations of 1, 2 and 5 ㎍ / mL.
상기 해조류 추출물의 항바이러스 활성 확인은 상기 실시예 1-2과 동일한 방법을 수행하여 확인하였고, 그 결과를 도 7(B) 및 하기 표 6에 나타내었다. Confirmation of the antiviral activity of the seaweed extract was confirmed by performing the same method as in Example 1-2, the results are shown in Figure 7 (B) and Table 6 below.
도 7(B) 및 상기 표 6에서 보여주듯이, 본 발명의 톳 추출물의 돼지 인플루엔자 바이러스에 대한 항바이러스 활성이 톳과 동속이종인 해조류 추출물들에 비해 높게 나타났고, 양성 대조군으로 이용한 리바비린과 비슷한 항바이러스 활성을 나타내는 것을 확인하였다. As shown in Figure 7 (B) and Table 6, the antiviral activity of swine influenza virus of the 톳 extract of the present invention was higher than that of seaweed extracts of the same species as 톳, and antiviral similar to ribavirin used as a positive control It was confirmed to exhibit activity.
이를 통해, 본 발명의 톳 추출물이 세포 독성이 거의 없으며, 인플루엔자 바이러스에 대한 항바이러스 활성이 높다는 것을 알 수 있었다. 또한, 톳은 현재 다른 모자반류에 비하여 대량 양식을 하고 있으므로, 산업적으로 적용 가능한 우수한 해조류임을 보여주는 것이다. Through this, it was found that the extract of the present invention has little cytotoxicity and high antiviral activity against influenza virus. In addition, it is showing that it is an excellent seaweed that can be applied industrially because it is currently a large-scale aquaculture compared to other mothers.
<실시예 5. 톳 추출물의 바이러스 종류에 따른 항바이러스 활성 비교>Example 5 Comparison of Antiviral Activity According to Virus Types of Extracts
톳 추출물이 노로바이러스-1(norovirus-1), HIV-1(human immunodeficiency virus-1) 등에 항바이러스 활성이 있음이 보고되었다. 따라서, 본 발명의 톳 추출물이 인플루엔자 바이러스 특이적 항바이러스 활성을 나타내는지 확인하기 위해 코로나 바이러스의 한 종류인 돼지 유행성 설사증 바이러스(porcine epidemic diarrhea virus, PEDV)를 이용하여 항바이러스 활성을 비교하였다. 톳 extract has been reported to have antiviral activity in norovirus-1 and human immunodeficiency virus-1. Therefore, antiviral activity was compared using porcine epidemic diarrhea virus (PEDV), which is a type of corona virus, to confirm whether the extract of the present invention exhibits influenza virus specific antiviral activity.
톳 추출물의 항바이러스 활성을 확인하기 위해서, 상기 실시예 1-2의 방법과 동일하게 진행하였으며, PEDV를 감염시키기 위한 세포로는 MDCK 세포 대신 베로(VERO) 세포를 이용하였다. 톳 추출물의 경우에는 50% 에탄올 톳 추출물을 10㎍/㎖ 및 20㎍/㎖이 되도록 처리하였고, 코로나 바이러스에 대한 양성 대조군으로는 아자우리딘 5㎍/㎖을, 인플루엔자 바이러스에 대한 양성 대조군으로는 리바비린 10uM을 처리하였고, 그 결과를 도 8에 나타내었다. In order to confirm the antiviral activity of the extract 진행, it proceeded in the same manner as in Example 1-2, Vero cells were used instead of MDCK cells as cells for infecting PEDV. In the case of 톳 extract, 50% ethanol 톳 extract was treated to 10 µg / ml and 20 µg / ml, and 5 µg / ml of azauridine as a positive control for coronavirus and 5 µg / ml as a positive control for influenza virus.
도 8에서 보여주듯이, 50%(v/v) 에탄올 톳 추출물의 항바이러스 활성이 돼지 유행성 설사증 바이러스(PEDV) 대비 돼지 인플루엔자바이러스(H1N1)에서 더 높게 나타나는 것을 확인하였다. As shown in Figure 8, it was confirmed that the antiviral activity of 50% (v / v) ethanol 톳 extract is higher in swine influenza virus (H1N1) compared to swine epidemic diarrhea virus (PEDV).
이를 통해, 본 발명의 톳 추출물은 인플루엔자 바이러스에 의한 세포 사멸을 저해함으로써, 인플루엔자 바이러스에 대한 선택적이고 우수한 항바이러스 활성을 나타냄을 알 수 있었다. Through this, the extract of the present invention inhibited cell death by influenza virus, it can be seen that the selective and excellent antiviral activity against influenza virus.
<제제예 1. 약학적 제제>Preparation Example 1. Pharmaceutical Formulations
제제예 1-1. 정제의 제조Formulation Example 1-1. Manufacture of tablets
본 발명의 톳 추출물 20g을 각각 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다. 20 g of the extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, respectively. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.
제제예 1-2. 주사액제의 제조Formulation Example 1-2. Preparation of Injection Solution
본 발명의 톳 추출물 100㎎, 염화나트륨 0.6g 및 아스코르브산 0.1g을 증류수에 용해시켜서 100㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.100 mg of the extract of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C for 30 minutes.
<제제예 2. 건강기능식품의 제조>Preparation Example 2 Preparation of Health Functional Food
제제예 2-1. 건강기능식품의 제조Formulation Example 2-1. Preparation of Health Functional Food
본 발명의 톳 추출물 20g, 비타민 혼합물 적량, 비타민 A 아세테이트 70㎍, 비타민 E 1.0㎎, 비타민 B1 0.13㎎, 비타민 B2 0.15㎎, 비타민 B6 0.5㎎, 비타민 B12 0.2㎍, 비타민 C 10㎎, 비오틴 10㎍, 니코틴산아미드 1.7㎎, 엽산 50㎍, 판토텐산 칼슘 0.5㎎, 무기질 혼합물 적량, 황산제1철 1.75㎎, 산화아연 0.82㎎, 탄산 마그네슘 25.3㎎, 제1인산칼륨 15㎎, 제2인산칼슘 55㎎, 구연산칼륨 90㎎, 탄산칼슘 100㎎, 염화마그네슘 24.8㎎을 섞어 과립으로 제조하였으나, 용도에 따라 다양한 제형으로 변형시켜 제조할 수 있다. 또한, 상기의 비타민 및 미네랄 혼합물의 조성비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합하여 제조할 수 있다.20 g of extract of the present invention, vitamin mixture proper amount, vitamin A acetate 70 ㎍, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 ㎍,
제제예 2-2. 건강기능성 음료의 제조Formulation Example 2-2. Preparation of Health Functional Drink
본 발명의 톳 추출물 1g, 구연산 0.1g, 프락토올리고당 100g, 정제수 900g을 섞어 통상의 음료 제조방법에 따라 교반, 가열, 여과, 살균, 냉장하여 음료를 제조하였다.1g of the extract of the present invention, citric acid 0.1g, fructooligosaccharide 100g, purified water 900g was mixed, stirring, heating, filtration, sterilization, refrigerated according to the conventional beverage production method to prepare a beverage.
<제제예 3. 동물사료첨가제 제조>Preparation Example 3 Preparation of Animal Feed Additives
본 발명의 톳 추출물 10g을 사료용 부형제 50g과 섞어 사료첨가제를 제조하였다. 그러나, 상기 배합비는 임의로 변형 실시하여도 무방하며, 통상의 사료조성물 제조방법에 따라 상기의 성분을 혼합하여 제조에 사용할 수 있다.10 g of the extract of the present invention was mixed with 50 g of a feed excipient to prepare a feed additive. However, the blending ratio may be modified arbitrarily, and the above ingredients may be mixed and used in preparation according to a conventional feed composition manufacturing method.
<제제예 4. 소독제의 제조>Preparation Example 4 Preparation of Disinfectant
본 발명의 톳 추출물 3g을 정제수 1,000㎖에 가하여 무방부제용 천연소독제를 제조하였다.3 g of the extract of the present invention was added to 1,000 ml of purified water to prepare a natural antiseptic for antiseptic.
Claims (15)
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는 인플루엔자 예방 또는 치료용 약학 조성물.The method of claim 1,
The 톳 extract is suspended in distilled water after extracting 톳 with water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and at least one solvent selected from the group consisting of hexane (hexan) , Hexane, ethyl acetate, butanol fraction butanol fraction obtained by fractionation or ethyl acetate fraction, characterized in that the pharmaceutical composition for preventing or treating influenza.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는 인플루엔자 개선용 건강기능식품. The method of claim 4, wherein
The 톳 extract is suspended in distilled water after extracting 톳 with water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and at least one solvent selected from the group consisting of hexane (hexan) , Hexane, ethyl acetate, butanol fraction butanol fraction obtained by fractionation in order or the functional food for improving influenza, characterized in that the ethyl acetate fraction.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는 인플루엔자 예방 또는 치료용 동물 약품.The method of claim 7, wherein
The 톳 extract is suspended in distilled water after extracting 톳 with water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and at least one solvent selected from the group consisting of hexane (hexan) , Hexane, ethyl acetate, butanol fraction butanol fraction obtained by fractionation or ethyl acetate fraction, characterized in that the animal medicine for preventing or treating influenza.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexan)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는 인플루엔자 개선용 동물 사료 첨가제. The method of claim 10,
The 톳 extract is suspended in distilled water after extracting 톳 with water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and at least one solvent selected from the group consisting of hexane (hexan) , Hexane, ethyl acetate, butanol fraction butanol fraction obtained by dividing in the order or the animal feed additive for improving influenza, characterized in that the ethyl acetate fraction.
상기 톳 추출물은 톳을 물, C1 내지 C4의 저급 알코올, 아세톤(acetone), 에틸아세테이트(ethyl acetate) 및 헥산(hexane)으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출한 추출물을 증류수로 현탁한 후, 헥산, 에틸아세테이트, 부탄올 순으로 분획하여 얻은 부탄올 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는 인플루엔자 예방용 천연소독제. The method of claim 13,
The 톳 extract is suspended in distilled water after extracting the 톳 extract with at least one solvent selected from the group consisting of water, C1 to C4 lower alcohol, acetone (acetone), ethyl acetate (ethyl acetate) and hexane (hexane) , Hexane, ethyl acetate, butanol, butanol fraction obtained by fractionation or ethyl acetate fraction, characterized in that the natural antiseptic agent.
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| CN103977059A (en) | 2014-05-15 | 2014-08-13 | 长沙绿叶生物科技有限公司 | Special vein nutrient liquid for treating hundestaupe virus and parvovirus infected dog and application method thereof |
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| CN103977059A (en) | 2014-05-15 | 2014-08-13 | 长沙绿叶生物科技有限公司 | Special vein nutrient liquid for treating hundestaupe virus and parvovirus infected dog and application method thereof |
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