KR20180055402A - Antiviral Composition Comprising Water Soluble Extraction of Saururus chinensis or Fraction of Extraction - Google Patents
Antiviral Composition Comprising Water Soluble Extraction of Saururus chinensis or Fraction of Extraction Download PDFInfo
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- KR20180055402A KR20180055402A KR1020160153228A KR20160153228A KR20180055402A KR 20180055402 A KR20180055402 A KR 20180055402A KR 1020160153228 A KR1020160153228 A KR 1020160153228A KR 20160153228 A KR20160153228 A KR 20160153228A KR 20180055402 A KR20180055402 A KR 20180055402A
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- saururus chinensis
- water
- fraction
- virus
- antiviral
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Abstract
Description
본 발명은 삼백초(Saururus chinensis) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 조성물에 관한 것이다.The present invention relates to an antiviral composition containing an aqueous extract of Saururus chinensis or a fraction thereof as an active ingredient.
바이러스는 매우 간단하고 작은 생명체로, 인체에 감염되어 바이러스성 질환을 일으키기도 한다. 바이러스는 많은 성질이 세균과 달라서 일반적인 항생제로는 증식이 억제되지 않기 때문에, 바이러스 감염증은 기존의 항생제로는 치료가 어려우며, 바이러스의 감염을 막아주거나 바이러스를 죽이는 항바이러스 제제로 치료가 가능하다.Viruses are very simple, small organisms that can infect humans and cause viral diseases. Since viruses differ in many properties from bacteria, they are not inhibited by general antibiotics. Therefore, viral infections are difficult to treat with conventional antibiotics, and can be treated with antiviral agents that prevent virus infection or kill viruses.
그러나 현재까지 바이러스 감염을 치료하는 항바이러스 제제의 종류나 수가 매우 빈약하여, 대부분의 바이러스 감염증은 환자의 면역기능에 의해 스스로 치유되기를 기대하는 형편이다(Rider et al., 2011). 또한, 현재 사용 중인 항바이러스제제들은 특정한 바이러스만 선택적으로 작용하여 그 바이러스의 증식과정 중 특정 단계를 방해하여 바이러스 감염증을 치료하는 약물들로서, 치료할 수 있는 바이러스의 종류가 매우 제한적인 약물이 대부분이다. 특히, 바이러스는 약물의 표적이 될 수 있는 단백질의 종류도 매우 제한적이며, 그 구조도 바이러스마다 매우 상이하기 때문에, 한꺼번에 많은 종의 세균을 죽이는 광범위 항생제와 유사한 광범위 항바이러스제를 개발하기는 매우 어렵다. 따라서 다양한 바이러스를 제어할 수 있는 항바이러스성 약물로 인체의 면역기능을 조절하여 비특이적 바이러스 면역체계 즉 바이러스에 대한 자연면역체계를 자극하는 방법이 가장 현실성이 있는 대안이다. However, until now, the number and types of antiviral agents that treat viral infections are so poor that most viral infections are expected to be healed by the patient's immune function (Rider et al., 2011). In addition, currently used antiviral agents are medicines for treating viral infection by selectively acting only a specific virus and interfering with a specific step in the process of propagating the virus, and most of the drugs have a very limited variety of viruses that can be treated. In particular, it is very difficult to develop a broad-spectrum antiviral agent similar to a broad-spectrum antibiotic that kills many species of bacteria at once because viruses are very limited in the types of proteins that can be targets of drugs and their structures vary greatly from virus to virus. Therefore, as an antiviral drug that can control various viruses, it is the most practical alternative to control the immune function of the human body to stimulate a nonspecific virus immune system, that is, a natural immune system against viruses.
한편, 삼백초(Saururus chinensis)는 삼백초과(Saururaceae)에 속하는 여러해살이풀로 제주도에 자생하고 있으며 남해안지역에서 재배되고 있다. 전통적으로 삼백초의 지상부는 민간에서 풍독, 이뇨, 수종, 간염, 폐렴 등의 치료에 사용되어 왔으며, 식용으로도 사용되는 식물이다. 삼백초의 활성에 대한 연구 중에는 삼백초의 알콜 추출물의 염증억제효과 및 항산화효과, 지질대사개선 및 당뇨억제효과 등이 있다는 연구결과들이 있다 (Kim et al., 2003; Yu et al., 2008). 이처럼, 대부분의 삼백초의 기능성에 대한 연구들은 알콜 등을 이용한 유기 용매 추출물이나 유기분획 등을 이용한 것들로, 아직까지 삼백초의 수용성 추출물이나 분획을 이용한 기능성 연구는 매우 미흡한 실정이다.Meanwhile, Saururus chinensis ) is a perennial plant belonging to Saururaceae. It grows in Jeju Island and is cultivated in south coast region. Traditionally, the top part of Saururus chinensis has been used for treatment of poisoning, diarrhea, species, hepatitis and pneumonia in the private sector and is also used for food. Studies have shown that the extracts of Saururus chinensis have anti-inflammatory and antioxidant effects, lipid metabolism and diabetes suppression (Kim et al., 2003; Yu et al., 2008). Thus, most of the studies on the functionality of Saururus chinensis have been conducted using organic solvent extracts or organic fractions using alcohol and the like. Therefore, functional studies using water soluble extracts or fractions of Saururus chinensis have not been studied very much yet.
본 발명의 목적은 아데노 바이러스 또는 포진 바이러스 억제 효과를 나타낼 수 있는 항바이러스성 약학 조성물을 제공하는 것이다.It is an object of the present invention to provide an antiviral pharmaceutical composition capable of exhibiting an adenovirus or herzin virus inhibitory effect.
본 발명의 다른 목적은 아데노 바이러스 또는 포진 바이러스 억제 효과를 나타낼 수 있는 항바이러스성 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide an antiviral health functional food composition capable of exhibiting an adenovirus or herzin virus inhibitory effect.
본 발명의 또 다른 목적은 포진 바이러스 억제 효과를 나타낼 수 있는 항바이러스성 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide an antiviral cosmetic composition capable of exhibiting herpes virus inhibitory effect.
상기 목적을 달성하기 위하여 본 발명은, 삼백초(Saururus chinensis) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides an antiviral pharmaceutical composition comprising a water-soluble extract of Saururus chinensis or a fraction thereof as an active ingredient.
상기 다른 목적을 달성하기 위하여 본 발명은, 삼백초(Saururus chinensis) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 건강기능식품 조성물을 제공한다.In order to accomplish the above other objects, the present invention provides an antiviral health functional food composition comprising a water-soluble extract of Saururus chinensis or a fraction thereof as an active ingredient.
상기 또 다른 목적을 달성하기 위하여 본 발명은, 삼백초(Saururus chinensis) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 화장료 조성물을 제공한다.According to another aspect of the present invention, there is provided an antiviral cosmetic composition comprising a water-soluble extract of Saururus chinensis or a fraction thereof as an active ingredient.
본 발명에 따른 삼백초 수용성 추출물 또는 그 분획은 항바이러스성 효과를 나타내는 바, 특히, 아데노 바이러스 또는 포진 바이러스 억제 활성을 가지고 있어 아데노 바이러스 또는 포진 바이러스를 효과적으로 억제하여 이의 감염 질환을 예방, 개선 또는 치료할 수 있는 조성물로 유용하게 활용될 수 있다.The water-soluble extract of Saururus cerevisiae according to the present invention or its fraction exhibits an antiviral effect. In particular, it has an adenovirus or herpesvirus-inhibiting activity and can effectively prevent an adenovirus or herpes virus and prevent, The present invention is not limited thereto.
도 1은 본 발명의 하나의 실시예에 따른 삼백초 수용성 추출물 및 그 분획의 제조 과정을 나타낸 것이다.
도 2는 본 발명의 다른 하나의 실시예에 따른 삼백초 수용성 추출물의 아데노 바이러스 억제 효과를 나타낸 것이다.
도 3은 도 2의 실시예에 따른 삼백초 수용성 추출물 중 항바이러스 효과를 나타내는 성분 및 그 성분이 세포독성이 없음을 확인한 결과이다.
도 4는 도 2의 실시예에 따른 삼백초 수용성 추출물의 분획이 나타내는 아데노 바이러스 억제 효과를 정량한 것이다.
도 5는 본 발명의 또 다른 하나의 실시예에 따른 삼백초 수용성 추출물의 포진 바이러스 억제 효과를 나타낸 것이다.
도 6은 도 5의 실시예에 따른 삼백초 수용성 추출물이 세포 독성이 없음을 확인한 결과이다.FIG. 1 shows a preparation process of Saururus chinensis Aqueous Extract and fractions thereof according to one embodiment of the present invention.
FIG. 2 shows the adenosine inhibitory effect of the water-soluble extract of Saururus chinensis according to another embodiment of the present invention.
FIG. 3 is a result of confirming that the components and components of the water-soluble extract of Saururus chinensis according to the embodiment of FIG. 2 have no cytotoxicity.
FIG. 4 is a graph showing the adenosine inhibitory effect of the fraction of Saururus chinensis Aqueous extract according to the embodiment of FIG. 2.
FIG. 5 shows the effect of Saururus chinensis Aqueous extract according to another embodiment of the present invention on herpes zoster virus.
FIG. 6 is a result of confirming that the water-soluble extract of Saururus chinensis according to the embodiment of FIG. 5 is not cytotoxic.
본 발명의 발명자들은 면역 활성을 조절하여 바이러스 감염 질환을 치료할 수 있는 방법을 연구하던 중, 삼백초 수용성 추출물 또는 그 분획이 특정 바이러스의 증식을 억제하는 것을 확인함으로써 본 발명을 완성하였다.The inventors of the present invention completed the present invention by confirming that the water-soluble extract of Saururus chinensis or the fraction thereof inhibits the proliferation of a specific virus while studying a method of treating a virus infectious disease by controlling immunological activity.
따라서 본 발명은 삼백초(Saururus chinensis ) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 약학 조성물을 제공한다.Therefore, the present invention relates to a method for producing Saururus chinensis ) water-soluble extract or a fraction thereof as an active ingredient.
이때, 상기 삼백초 수용성 추출물은 삼백초 지상부를 열수로 추출하여 얻어질 수 있는 바, 삼백초 지상부는 삼백초의 줄기 또는 잎을 포함할 수 있으나 이에 제한되는 것은 아니다.At this time, the water-soluble extract of Saururus chinensis can be obtained by extracting the Saururus chinensis with hot water, and the Saururus chinensis can include the stem or leaf of Saururus chinensis, but is not limited thereto.
상기 삼백초 수용성 추출물의 분획의 평균 중량 분자량은 100 Da 내지 5,000 Da일 수 있고, 바람직하게는 100 Da 내지 3,000 Da일 수 있으며, 보다 바람직하게는 100 Da 내지 1,000 Da일 수 있다.The average molecular weight of the fraction of the water-soluble extract of Saururus chinensis may be 100 Da to 5,000 Da, preferably 100 Da to 3,000 Da, and more preferably 100 Da to 1,000 Da.
한편, 상기 바이러스는 아데노 바이러스 또는 포진 바이러스일 수 있는 바, 상기 삼백초 수용성 추출물 또는 이의 분획은 농도 의존적으로 아데노 바이러스 또는 포진 바이러스의 증식을 효과적으로 억제할 수 있다. On the other hand, the virus may be an adenovirus or a herpesvirus. The water-soluble extract of Saururus chinensis or a fraction thereof may effectively inhibit the proliferation of adenovirus or herpes virus in a concentration-dependent manner.
한편, 상기 삼백초 수용성 추출물 또는 이의 분획은 전체 조성물 100 중량부에 대하여 0.1 내지 50 중량부로 포함될 수 있는 바, 이러한 수치 범위 내에서 항바이러스 효과가 현저히 나타날 수 있으므로 바람직하다.On the other hand, the water-soluble extract of Saururus chinensis or its fraction may be contained in an amount of 0.1 to 50 parts by weight based on 100 parts by weight of the whole composition, and the antiviral effect may be remarkably exhibited within such a range.
본 발명에 따른 약학 조성물은 상기 삼백초 수용성 추출물 또는 이의 분획 외에 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다. 본 발명에서 사용 가능한 담체, 부형제 또는 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 또는 광물유 등을 들 수 있다.The pharmaceutical composition according to the present invention may further comprise an appropriate carrier, excipient or diluent commonly used in the production of the pharmaceutical composition, in addition to the above-mentioned water-soluble extract or the fraction thereof. Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.
본 발명에 따른 약학 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.The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물은 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제할 수 있다. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.
또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol,
또한, 본 발명에 따른 약학 조성물의 투여량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.Further, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.
더불어, 본 발명은 삼백초(Saururus chinensis ) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 건강기능식품 조성물을 제공한다.In addition, the present invention relates to the use of Saururus chinensis ) water-soluble extract or a fraction thereof as an active ingredient.
이때, 상기 삼백초 수용성 추출물은 삼백초 지상부를 열수로 추출하여 얻어질 수 있는 바, 삼백초 지상부는 삼백초의 줄기 또는 잎을 포함할 수 있으나 이에 제한되는 것은 아니다.At this time, the water-soluble extract of Saururus chinensis can be obtained by extracting the Saururus chinensis with hot water, and the Saururus chinensis can include the stem or leaf of Saururus chinensis, but is not limited thereto.
보다 구체적으로, 상기 삼백초 수용성 추출물의 분획의 평균 중량 분자량은 100 Da 내지 5,000 Da일 수 있고, 바람직하게는 100 Da 내지 3,000 Da일 수 있으며, 보다 바람직하게는 100 Da 내지 1,000 Da일 수 있다.More specifically, the average weight molecular weight of the fraction of the water extract of Saururus chinensis may be 100 Da to 5,000 Da, preferably 100 Da to 3,000 Da, and more preferably 100 Da to 1,000 Da.
또한, 상기 바이러스는 아데노 바이러스 또는 포진 바이러스일 수 있는 바, 상기 삼백초 수용성 추출물 또는 이의 분획은 아데노 바이러스 또는 포진 바이러스의 증식 및 활성을 농도 의존적으로 억제할 수 있다.In addition, the virus may be adenovirus or herpesvirus. The water-soluble extract of Saururus chinensis or a fraction thereof may inhibit the proliferation and activity of adenovirus or herpesvirus in a concentration-dependent manner.
상기 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일 주스, 합성 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품 조성물은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알코올 및 비타민 복합제 중 어느 하나의 형태일 수 있다.The health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts , Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, synthetic fruit juices and vegetable drinks. These components may be used independently or in combination. The health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex Lt; / RTI >
또한, 상기 건강기능식품 조성물은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합 여부는 다른 규정이 없는 한 식품의약품 안정청에 승인된 식품첨가물공전의 총칙 및 일반 시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.In addition, the health functional food composition may further include a food additive, and the suitability of the food functional additive as a "food additive" Of the present invention.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산 칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류 첨가 알칼리제, 보존 료제제, 타르색소 제제 등의 혼합 제제류 등을 들 수 있다.Examples of the products that have been used in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, sensory coloring matter, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, and a tar coloring agent.
더욱이, 본 발명은 삼백초(Saururus chinensis ) 수용성 추출물 또는 이의 분획을 유효성분으로 함유하는 항바이러스성 화장료 조성물을 제공한다.Furthermore, the present invention relates to a method for producing Saururus chinensis ) water-soluble extract or a fraction thereof as an active ingredient.
이때, 상기 삼백초 수용성 추출물은 삼백초 지상부를 열수로 추출하여 얻어질 수 있는 바, 삼백초 지상부는 삼백초의 줄기 또는 잎을 포함할 수 있으나 이에 제한되는 것은 아니다.At this time, the water-soluble extract of Saururus chinensis can be obtained by extracting the Saururus chinensis with hot water, and the Saururus chinensis can include the stem or leaf of Saururus chinensis, but is not limited thereto.
보다 구체적으로, 상기 삼백초 수용성 추출물의 분획의 평균 중량 분자량은 100 Da 내지 5,000 Da일 수 있고, 바람직하게는 100 Da 내지 3,000 Da일 수 있으며, 보다 바람직하게는 100 Da 내지 1,000 Da일 수 있다.More specifically, the average weight molecular weight of the fraction of the water extract of Saururus chinensis may be 100 Da to 5,000 Da, preferably 100 Da to 3,000 Da, and more preferably 100 Da to 1,000 Da.
한편, 상기 바이러스는 포진 바이러스일 수 있는 바, 본 발명에 따른 삼백초 수용성 추출물 또는 분획을 포함하는 화장료 조성물은 특히 피부로 그 병증이 나타나는 포진 바이러스 감염 시에 농도 의존적으로 우수한 항바이러스 효과를 나타낼 수 있다.On the other hand, since the virus may be a herpesvirus, the cosmetic composition comprising the Saururus chinensis extract or fraction according to the present invention may exhibit an excellent antiviral effect in a dose-dependent manner upon infection with herpes virus, .
본 발명의 화장료 조성물은 상기 유효성분 이외에 통상적으로 허용되는 성분들을 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition of the present invention may contain ingredients generally accepted in addition to the above-mentioned effective ingredients, and may include conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 제한되는 것은 아니다. 보다 상세하게는, 유연 화장수(스킨), 영양 화장수(밀크로션), 영양크림, 마사지크림, 에센스, 아이크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powdered foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a flexible lotion (skin), a nutritional lotion (milk lotion), a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder .
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성 오일, 식물성 오일, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴라아미드 파우더가 이용될 수 있으며, 특히 스프레이인 경우에는 추가적으로 클로로 플루오로 히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or a polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Propane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예로서 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤지방족에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르를 들 수 있다.When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화이소스테아릴 알코올, 폴리옥시에틸렌소르비톨에스테르 및 폴리옥시에틸렌소르비탄에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시테이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 오일, 라놀린 유도체 또는 에톡실화글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이며, 본 발명의 내용을 예시하는 것일 뿐이므로 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. It is to be understood, however, that these examples are provided for the purpose of providing a more complete understanding of the present invention to those skilled in the art, and that the scope of the present invention is limited only by the following examples It is not.
<< 실시예Example 1> 삼백초 수용성 추출물과 그 분획의 제조 1> Preparation of water-soluble extracts of Saururus chinensis and its fractions
도 1A에 나타난 바와 같이, 본초원(김해시)에서 구입한 건조된 삼백초의 줄기와 잎 40 g을 분쇄하고, 분쇄한 약재에 증류수 300 ml를 넣은 후, 125℃에서 2시간 반응시켜 추출물을 얻었다. 이 추출물을 상온에서 식힌 후 2,000 rpm에서 20분간 원심 분리하고 상등액을 모아 삼백초 수용성 추출물(SC0)을 분리하였다. 이렇게 얻은 SC0 수용성 추출물을 감압농축기로 60℃에서 50 mg/ml이 되도록 농축한 후, 다시 상온으로 식힌 다음, 원심분리하여 침전물을 제거하고 Sephadex G50 colimn chromatography와 물을 이용하여 분자량 크기에 따라 분리하여 분획화하였다. 이후, 도 1B에 나타난 바와 같이, 얻어진 분획들의 260 nm에서의 흡광도를 조사하여 자외선을 흡수하는, 중량 평균 분자량이 1,000 Da 이하인 유기물질이 포함된 분획을 모아 삼백초 수용성 추출물 분획(SC1)을 정제하였다.As shown in FIG. 1A, the dried Saururus chinensis stalks and leaves (40 g) purchased from this grassland (Kimhae city) were pulverized, 300 ml of distilled water was added to the pulverized medicinal material, and the mixture was reacted at 125 ° C for 2 hours to obtain an extract. The extracts were cooled at room temperature, centrifuged at 2,000 rpm for 20 minutes, and the supernatant was collected to separate the water-soluble extract (SC0) of Saururus chinensis. The thus obtained SC0 aqueous extract was concentrated to a concentration of 50 mg / ml at 60 ° C using a vacuum concentrator, and then cooled to room temperature. Then, the precipitate was removed by centrifugation and separated according to molecular weight using Sephadex G50 colimn chromatography and water And fractionated. Then, as shown in Fig. 1B, the obtained fractions were examined for absorbance at 260 nm to collect ultraviolet light-absorbing fractions containing an organic substance having a weight average molecular weight of 1,000 Da or less, and the water-soluble extract fraction (SC1) .
<실시예 2> 삼백초 수용성 추출물(SC0) 및 분획(SC1)의 아데노 바이러스 증식 억제 효과 확인Example 2 Confirmation of Inhibitory Effect of Water-Soluble Extract (SC0) and Fraction (SC1) on Growth of Adenovirus
(1) 감염 단위 측정 및 세포 배양 감염 단위 50(Tissue culture infective does 50, TCID(1) Infection unit measurement and cell culture Infection unit 50 (Tissue culture infective does 50, TCID 5050 ) 방법) Way
AD5 벡터는 E1 유전자가 결실된 아데노 바이러스 지놈(genome, DNA)를 가지고 있는 플라스미드 DNA로, 이 E1이 결실된 자리에 외부 유전자를 삽입하여 외부 유전자를 세포에 전달하는 재조합 바이러스를 만드는 데 사용된다. 이 플라스미드 DNA를 E1 단백질을 발현하는 세포, 즉 HEK 293A 세포에 주입하면, E1 단백질의 도움으로 플라스미드에 있는 Ad5 벡터 지놈이 복제되고, 복제된 지놈이 아데노 바이러스의 빈 캡시드에 들어가 Ad5 벡터 지놈을 가지고 있는 바이러스 입자(Ad5 vector 바이러스 입자)가 만들어진다. 이렇게 얻어진 Ad5 벡터 바이러스 입자는 다시 E1 단백질을 발현하는 HEK 293A 세포에 감염되어 새로운 Ad5 벡터 바이러스 입자를 생산하게 된다. 이러한 Ad5 벡터 바이러스 입자의 증식 과정은 정상적인 아데노 바이러스가 숙주 세포에 감염되어 증식하는 과정과 동일한 것으로 알려져 있다(Catalucci et al., 2005). 이러한 특성 때문에 Ad5 벡터는 아데노 바이러스 증식의 시험관 모델로 이용되고 있으며, 아데노 바이러스의 증식을 조절하는 물질을 탐색하는 데에 활용되고 있다(Matias et al., 2010).The AD5 vector is a plasmid DNA that contains an adenovirus genome (DNA) in which the E1 gene has been deleted. This plasmid DNA is used to create a recombinant virus that inserts an external gene into the deletion site of E1 to deliver an external gene to the cell. When this plasmid DNA is injected into a cell expressing the E1 protein, that is, HEK 293A cells, the Ad5 vector genome in the plasmid is replicated with the help of the E1 protein, and the cloned gene enters the empty capsid of the adenovirus and the Ad5 vector genome Virus particles (Ad5 vector virus particles) are made. The Ad5 vector viral particles thus obtained are again infected with HEK 293A cells expressing the E1 protein to produce new Ad5 vector virus particles. The proliferation of Ad5 vector virus particles is known to be identical to that of normal adenovirus infected by host cells (Catalucci et al., 2005). Because of these properties, the Ad5 vector has been used as an in vitro model for adenovirus proliferation and is being explored for substances that regulate adenovirus proliferation (Matias et al., 2010).
이에, 본 실험에서도 Ad5 벡터를 이용하여, Ad5 벡터 바이러스 입자가 HEK 293A 세포에 감염된 후 자라서 나타나는 배양 세포의 세포변성효과(Cytopathic effect, CPE)를 이용하여 삼백초 수용성 추출물의 바이러스 증식 억제 효과를 조사하였다. 구체적으로, HEK 293A 세포는 배양 시 일반적으로 배양 접시 바닥에 붙어서 길게 늘어지는 모양으로 자라지만, Ad 벡터 바이러스가 감염되면 세포가 배양 접시에서 떨어져 나와 둥근 모양의 세포로 전환되는데, 이러한 현상을 이용하여 Ad5 벡터 바이러스 입자 용액 내의 살아 있는 바이러스 입자 수, 즉 감염 단위(infective unit, IU)를 측정하였다. 또한, 일반적으로 전체 배양 중인 세포의 50% 정도가 바닥에서 떨어져 둥근 모양을 보여줄 때를 기준으로 바이러스 증식을 판단하는 바, 이를 세포 배양 감염 단위 50(Tissue culture infective does 50, TCID50) 방법이라 칭하고, 이를 수행하였다.Thus, in this experiment, the Ad5 vector was used to investigate the inhibitory effect of the water-soluble extract of Saururus chinensis on the viral growth inhibition by using the cytotoxic effect (CPE) of the cultured cells grown after the Ad5 vector virus particles were infected with HEK 293A cells . Specifically, when HEK 293A cells are cultured, they generally grow on the bottom of the culture dish and grow long. However, when the Ad vector virus is infected, the cells are separated from the culture dish and converted into round cells. The number of living virus particles in the Ad5 vector virus particle solution, that is, the infective unit (IU), was measured. In general, virus propagation is judged based on the time when about 50% of the cells in the whole culture are rounded off from the bottom, which is referred to as a cell culture infective does 50 (TCID 50 ) method , Respectively.
먼저, Invitrogen(미국)에서 분양받은 HEK 293A 세포를 배양 접시에 웰당 5×104 cell이 되게 접종하여 하룻밤 동안 배양하여 세포들이 배양 접시에 붙어서 자라도록 하였다. 다음날, 각 웰에 Ad5 벡터 바이러스 입자를 2.5×104개, 1.25×104개, 0.62×104개 등 2배씩 연쇄적으로 희석하여 11개 웰에 접종했다. 이와 같이 Ad5 벡터 바이러스 입자를 접종하면 처음 웰에는 숙주세포와 바이러스의 비율 즉 MOI(multiplicity of infection)가 2:1, 즉 2-1이 되며, 두 번째 웰은 4:1 또는 2-2가 된다. 이와 같이 11번 웰의 MOI가 2-11이 될 때까지 2배씩 희석하여 접종하였다. 이렇게 바이러스를 접종한 직후, 삼백초 수용성 추출물을 처리하고 배양을 시작하였다. 구체적으로, 삼백초 수용성 추출물을 각각 4 mg/ml, 2 mg/ml, 1 mg/ml 또는 0.5 mg/ml가 되도록 처리한 4개의 그룹을 사용하였고, 대조군으로는 삼백초 수용성 추출물 대신 동량의 물(vehicle)을 첨가하여 배양하였다. 배양 후, TCID50 분석법으로 매일 배양 접시 웰을 관찰하여 증식 여부를 조사하였다.First, HEK 293A cells from Invitrogen (USA) were inoculated into a culture dish at 5 × 10 4 cells per well and cultured overnight to allow the cells to grow on the culture dish. The next day, 2.5 × 10 4 dogs Ad5 vector viral particles in each well, 1.25 × 10 4 dogs, 0.62 × 10 to 4, including 2-fold serial dilution ever was inoculated into 11 wells. Thus, when inoculated with vector Ad5 virus particles first well is a host cell with a second ratio that is (multiplicity of infection) of a virus MOI: is a 1, that is 2-1, and the second wells 4: is 1 or 2 -2 . In this manner, the 11th well was inoculated by diluting 2-fold until the MOI reached 2 -11 . Immediately after the virus was inoculated, the aqueous extract of Saururus chinensis was treated and cultivation was started. Specifically, four groups were used in which the aqueous extracts of Saururus chinensis were treated so as to be 4 mg / ml, 2 mg / ml, 1 mg / ml or 0.5 mg / ml, respectively. As a control group, ) Was added and cultured. After incubation, the culture wells were observed daily by TCID 50 assay method to determine whether they were proliferating.
이 방법은 숙주세포 배양과 함께 접종한 바이러스 입자의 비율이 두 배씩 계단식으로 낮아지도록 했기 때문에, 일정 시간이 지난 후 TCID50이 나타나는 배양 웰은 바이러스 증식 속도에 따라 다르게 나타난다. 예를 들어, 1번 웰과 같이 숙주세포 2개당 살아있는 바이러스 입자 1개를 감염시킨 경우에는 2개의 세포 중 하나가 바이러스에 감염되기 때문에 짧은 시간 내에 TCID50을 나타낸다. 즉, 2-1, 2-2 내지 2-11의 순서로 갈수록 TCID50을 나타내는 시간이 길어지며, 또한 같은 시간이더라도 바이러스의 감염 및 증식 속도가 빠르다면 TCID50을 나타내는 시간이 짧아지게 된다.Since this method allows the proportion of virus particles inoculated with the host cell culture to be lowered step by step twice, culture wells in which TCID 50 appears after a certain period of time differ depending on the virus growth rate. For example, when one live virus particle is infected with two host cells, such as the No. 1 well, one of the two cells is infected with a virus, and thus TCID 50 is displayed within a short time. That is, the longer the time of TCID 50 in the order of 2 -1 , 2 -2 to 2 -11 , and the shorter the time of TCID 50 if the infection and proliferation rate of the virus is fast even at the same time.
본 발명의 실험 결과, 도 2에 나타난 바와 같이, 삼백초 수용성 추출물로 처리한 배양에서 TCID50을 나타내는 웰의 MOI 값이 대조군보다 높게 나타났으며, 이러한 차이는 조사한 3일간 계속 확인되었다. 즉, 바이러스의 감염 및 증식 속도가 삼백초 수용성 추출물 처리 시 현저히 감소하였다. 특히, 배양을 시작한 지 3일째 되는 날 관찰한 결과를 보면 배양액 내의 삼백초 수용성 추출물의 농도가 0.5 mg/ml가 되도록 처리한 배양에서는 TCID50을 보여주는 가장 낮은 MOI 값이 2-8로 대조군 배양의 2-10보다 높게 나타났다. 이러한 결과는 0.5 mg/ml의 삼백초 수용성 추출물로 처리한 배양에서는 살아있는 바이러스의 수가 대조군 배양보다 대략 4배 정도 적다는 것을 보여준다. 또한, 모든 실험 내내 이와 같은 삼백초 수용성 추출물의 바이러스 증식 억제 효과는 첨가한 삼백초 수용성 추출물의 농도에 비례하여 강하게 나타났다.As a result of the experiment of the present invention, as shown in Fig. 2, the MOI value of the well showing the TCID 50 in the culture treated with the water-soluble extract of Saururus chinensis was higher than that of the control, and this difference was continuously confirmed for three days. In other words, virus infectivity and proliferation rate were remarkably decreased when the water extract of Saururus was treated. In particular, the results of observation on the third day after the initiation of culture showed that the lowest MOI value of TCID 50 was 2 -8 in cultures treated with 0.5 mg / ml of saururus aqueous extract in the culture, Respectively . These results show that the number of viable viruses in cultures treated with 0.5 mg / ml Saururus chinensis extract is approximately 4 times less than in control cultures. In addition, throughout the entire experiment, the inhibitory effect of the water extract of Saururus chinensis on the virus proliferation was increased in proportion to the concentration of the water extract of Saururus chinensis.
(2) 삼백초 수용성 추출물의 성분 확인(2) Identification of components of water-soluble extract of Saururus chinensis
또한, 삼백초 수용성 추출물의 어떤 성분이 아데노 바이러스의 증식을 억제하는 지를 확인하기 위하여, 삼백초 수용성 추출물을 물로 분획하여 얻은 SC1 분획이 아데노 바이러스의 증식을 억제하는 지를 조사하였다. 상기에서 준비한 HEK 293A 세포에 Ad5 벡터 바이러스를 MOI가 2-1에서 2-11이 될 때 까지 2배씩 희석하여 접종한 후, SC1 분획을 각각 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml 또는 0.125 mg/ml이 되게 첨가하여 배양하였고, 대조군에는 SC1 분획 대신에 동량의 물을 첨가하여 배양하였다.In order to confirm whether or not a component of the water extract of Saururus chinensis inhibits adenovirus proliferation, it was examined whether the SC1 fraction obtained by fractionation of water-soluble extract of Saururus chinensis inhibited adenovirus proliferation. After inoculation was diluted 2 fold until the HEK 293A cells are prepared from the MOI to Ad5 vector virus to be 2-11 in 2 -1, 1 mg / ml for each fraction SC1, 0.5 mg / ml, 0.25 mg / ml Or 0.125 mg / ml, and the same amount of water was added to the control group in place of the SC1 fraction.
배양을 시작한 지 3일 째 되는 날에 TCID50을 보여주는 배양 웰의 가장 낮은 MOI 값을 비교한 결과, 도 3(a)에 나타난 바와 같이, SC1 분획 1 mg/ml으로 처리한 배양에서는 2-5(첫 번째 막대그래프), SC1 분획 0.5 mg/ml으로 처리한 배양에서는 2-7(두 번째 막대그래프), SC1 분획 0.25 mg/ml으로 처리한 배양에서는 2-8(세 번째 막대그래프) 등으로 나타났으며, SC1 분획 0.125 mg/ml으로 처리한 배양과 대조군에서는 2-9(네 번째 및 다섯 번째 막대그래프)으로 나타났다. 이러한 결과는 0.25 mg/ml 이상의 농도에서 SC1 분획은 농도에 비례하여 감염된 바이러스의 증식을 억제하는 기능이 있음을 보여주는 결과로, SC1 분획에는 삼백초 수용성 추출물의 바이러스 증식 억제 물질이 포함되어 있음을 보여준다.Comparison of the lowest MOI value of culture wells showing TCID 50 on days not start a culture that is at 3 days, 3 as shown in (a), in a culture treated with
(3) 숙주세포에 대한 세포독성 확인(3) Identification of cytotoxicity against host cells
한편, 삼백초 수용성 추출물로부터 분획한 SC1의 Ad5 벡터 바이러스 증식 억제 효과는 숙주세포인 HEK 293A 세포의 증식을 억제함으로써, 즉, 숙주세포에 대한 독성을 나타냄으로써 나타날 수도 있는 바, 이러한 가능성을 배제하기 위하여 HEK 293A 세포 배양에 SC1을 첨가한 다음, MTT 어세이를 통해 세포의 생존 정도를 비교하였다.On the other hand, the effect of inhibiting the growth of Ad5 vector virus of SC1 fractionated from the water-soluble extract of Saururus chinensis may be caused by inhibiting the proliferation of host cell HEK 293A cells, that is, showing toxicity to the host cell. After SC1 was added to the HEK 293A cell culture, cell survival was assessed by MTT assay.
구체적으로, HEK 293A 세포 현탁액을 5x105 cell/ml가 되도록 준비한 후, 96 웰 플레이트에 100 ㎕씩 분주한 다음, 하룻밤 배양하여 세포를 배양접시에 부착시켰다. 다음 날, 각 웰에 최종농도가 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml이 되도록 SC1을 각 배양에 5 ㎕씩 첨가한 다음, 3일간 배양하고, 배지에 MTT 시약을 넣어준 후 37℃ 배양기에서 1 내지 4시간동안 반응시켰다. 반응이 완료된 후 595 nm에서 흡광도를 측정하였다. 대조군으로는 SC1 대신 동량의 물을 첨가하였다.Specifically, HEK 293A cell suspension was prepared to have a concentration of 5 × 10 5 cells / ml, and 100 μl of each was dispensed into a 96-well plate. The cells were then cultured overnight to adhere the cells to the culture dish. On the next day, SC1 was added to each well at a final concentration of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml in each well, followed by culturing for 3 days. MTT Reagents were added and incubation was carried out for 1 to 4 hours at 37 ° C in an incubator. After completion of the reaction, the absorbance was measured at 595 nm. As a control, an equal amount of water was added instead of SC1.
그 결과, 도 3(b)에 나타난 바와 같이, SC1을 넣어준 배양액의 흡광도는 대조군 배양액의 흡광도와 큰 차이를 보이지 않는 것으로 나타났다(막대그래프 순서대로 각각 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml 및 대조군이다).As a result, as shown in Fig. 3 (b), the absorbance of the culture solution containing SC1 was not significantly different from the absorbance of the control culture solution (1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml and control group).
상기 결과들은 삼백초 수용성 추출물은 숙주세포에 영향을 미치지 않으면서, 바이러스의 증식을 선택적으로 억제하는 것으로 보여주는 것으로, 삼백초 수용성 추출물에는 아데노 바이러스의 증식을 억제하는 물질이 포함되어 있음을 보여준다.The above results show that the water extract of Saururus chinensis selectively inhibits the proliferation of viruses without affecting the host cells. It shows that the water extract of Saururus chinensis contains a substance inhibiting adenovirus proliferation.
(4) 삼백초 수용성 추출물 분획(SC1)의 아데노 바이러스 억제 효과 정량(4) Quantitative determination of adenovirus inhibitory effect of water-soluble extract fraction (SC1)
Ad5 벡터는 녹색 형광 단백질(GFP) 유전자를 발현하도록 조작되어 있으므로 숙주 세포에서 이 바이러스가 증식하게 되면 세포 내부에 GFP 단백질의 양이 늘어나게 되고, 그 결과 배양 중인 세포의 전체 형광량이 증가하게 된다. 이러한 특성을 이용하여 삼백초 수용성 추출물의 바이러스 증식 억제 효과를 간단하게 정량적으로 비교하였다.Since the Ad5 vector is engineered to express the green fluorescent protein (GFP) gene, when the virus grows in the host cell, the amount of GFP protein inside the cell increases, resulting in an increase in the total fluorescence of the cultured cells. Using these properties, the inhibitory effect of the water - soluble extract of Saururus chinensis was simply and quantitatively compared.
먼저, 상기에서 준비한 HEK 298A 세포를 배양 접시에 5 X 104 cell이 되도록 접종한 후, Ad5 바이러스 입자를 MOI가 0.1이 되도록 5 X 103 개 접종하였다. 이후, 삼백초 수용성 추출물 분획(SC1)을 각각 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml로 첨가하여 배양하였다. 그리고 Ad5 벡터 바이러스 입자 접종 후 매 12시간 마다 배양중인 세포가 보여주는 형광의 양을 Synergy HT multi-mode microplate reader(BioTek Instruments, Inc., USA)을 이용하여 측정하였다.First, the HEK 298A cells prepared in the above was inoculated to a 5
그 결과, 도 4에 나타난 바와 같이, 삼백초 수용성 추출물 분획으로 처리한 세포 배양 시 형광량의 증가 속도가 대조군에 비하여 낮게 나타났으며, 또한 처리한 상기 분획의 농도에 비례하여 상기 분획의 농도가 높을수록 증가 정도가 낮았다.As a result, as shown in FIG. 4, the rate of increase in the amount of fluorescence in cell culture treated with the water-soluble extract fraction of Saururus chinensis was lower than that of the control, and the concentration of the fraction was high in proportion to the concentration of the treated fraction The increase in the number of recorded images was low.
<실시예 3> 삼백초 수용성 추출물(SC0)의 포진 바이러스 증식 억제 효과 확인Example 3 Confirmation of Inhibitory Effect of Saururus chinensis Aqueous Extract (SC0) on Shingles Virus Growth
(1) GFP 형광 측정(1) GFP fluorescence measurement
HSV 앰플리콘(amplicon)은 HSV를 활용한 유전자 전달 벡터(vector) 중 하나로, HSV 앰플리콘을 가지고 있는 바이러스 입자는 HSV처럼 신경세포에 감염되기 때문에, 신경세포에 외부 유전자를 전달하는 수단으로 활용되고 있다(Silva and Bowers. 2009). HSV 앰플리콘 바이러스 입자는 HSV 앰플리콘을 포함하는 플라스미드로부터 만들어지는데, 이 플라스미드에는 HSV의 복제에 필요한 복제기점 DNA(oriS)와 복제된 DNA를 HSV 캡시드 껍질에 넣어주는 데에(packaging) 필요한 DNA(pac)가 존재한다. 이 플라스미드를 HSV의 초기단백질과 캡시드 단백질을 만드는 세포에 넣어주면, 마치 HSV 감염에서처럼 HSV 앰플리콘 DNA가 복제되고 HSV 캡시드 껍질에 들어가 HSV 앰플리콘 바이러스 입자가 만들어진다(Laimbacher et al., 2012). 이러한 특성 때문에 이 바이러스 벡터는 완전한 바이러스 대신에 바이러스의 증식을 시험관에서 연구하는 모델로 이용된다. 이에, 본 실험에서 포진 바이러스에 대한 효과를 확인하기 위해 HSV 앰플리콘을 사용하였다.HSV amplicon is one of the gene transfer vectors that utilize HSV. HSV amlicorin viral particles are used as a means of delivering an external gene to neurons, (Silva and Bowers, 2009). The HSV ampicillin viral particles are made from a plasmid containing an HSV ampicron, which contains the DNA (oriS) and the DNA necessary for packaging the HSV capsid shell pac). Putting this plasmid into the cells that make up the early proteins and capsid proteins of HSV makes HSV ampicillin DNA replicate, as in HSV infection, and into HSV capsid shells to produce HSV ampicillin virus particles (Laimbacher et al., 2012). Because of this nature, this viral vector is used as a model for studying viral growth in vitro instead of a complete virus. In this experiment, HSV amplicon was used to confirm the effect on herpes zoster virus.
구체적으로, HSV-p1005 HSV 앰플리콘 플라스미드는 CMV 프로모터에 GFP 단백질 유전자가 결합되어 있는 플라스미드로서(Russo et al., 2009. Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward. J. Neurosci. 29, 35293537.), HSV의 초기단백질 ICP27을 발현하는 Vero2-2 세포(서울대 세포주은행)에 감염시키면 형광을 확인할 수 있다. 이 HSVp1005 앰플리콘에도 GFP를 발현하는 유전자가 존재하여, 앰플리콘이 증식하면 증식 정도에 비례하여 GFP 단백질이 증가하고, 결과적으로 세포 내의 형광의 양도 늘어나는 특성을 보여준다(Yoon et al., 2009).Specifically, the HSV-p1005 HSV ampicillin plasmid is a plasmid in which the GFP protein gene is bound to the CMV promoter (Russo et al., 2009. Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward. J. Neurosci. 29, 35293537 ) And Vero2-2 cells expressing HSV early protein ICP27 (Seoul National University Cell Line Bank). In this HSVp1005 amplicon, the presence of GFP-expressing genes shows that GFP protein increases in proportion to the degree of proliferation of the ampicillin, resulting in an increase in the amount of fluorescence in the cells (Yoon et al., 2009).
이에, Vero2-2 세포 현탁액을 5x105 cell/ml가 되도록 준비한 후, 96 웰 플레이트에 100 ㎕씩 분주한 다음(5x104 cell/well), 하룻밤 배양하여 세포를 배양 접시에 부착시켰다. 다음날, 각 웰에 삼백초 수용성 추출물을 최종농도가 각각 2 mg/ml, 1 mg/ml, 0.5 mg/ml 되도록 5 ㎕씩 첨가하고, 여기에 HSV 앰플리콘 바이러스 입자를 MOI가 0.4(1.25×104개)가 되는 조건으로 첨가하여 배양하였다. 대조군 배양에는 삼백초 수용성 추출물 대신에 증류수를 처리하였으며, 이렇게 처리한 Vero2-2 세포를 37℃에서 3일간 배양하면서 각 웰에서 보여주는 형광의 양을 측정하였다. 각 실험은 두 번 반복하였다. Then, Vero2-2 cell suspension was prepared so as to have a concentration of 5x10 5 cells / ml. Then, 100 μl of each of the cells was dispensed into a 96-well plate (5x10 4 cells / well) and then the cells were allowed to adhere to the culture dish overnight. The next day, the three hundred seconds aqueous extract to each well respectively, to have a final concentration of 2 mg / ml, 1 mg / ml, 0.5 mg / ml to be added in 5 ㎕, and here the HSV amplicon virus particles the MOI is 0.4 (1.25 × 10 4 ) Were added and cultured. Control cultures were treated with distilled water instead of water-soluble extract of Saururus chinensis. The Vero2-2 cells thus treated were cultured at 37 DEG C for 3 days, and the amount of fluorescence shown in each well was measured. Each experiment was repeated twice.
그 결과, 도 5에 나타난 바와 같이, 삼백초 수용성 추출물을 0.5 mg/ml, 1 mg/ml, 2 mg/ml가 되도록 첨가한 세포 모두에서, 세포 내 형광의 양이 농도 의존적으로 낮게 나타났으며, 그 증가 속도도 농도에 비례하여 낮아지는 것을 확인하였다. As a result, as shown in FIG. 5, the amount of intracellular fluorescence was low in all of the cells added with water-soluble extract of Saururus chinensis to 0.5 mg / ml, 1 mg / ml and 2 mg / ml, It was confirmed that the rate of increase was also decreased in proportion to the concentration.
(2) 숙주세포에 대한 세포독성 확인(2) Cytotoxicity of host cells
이러한 형광량의 증가 억제 효과는 삼백초 수용성 추출물이 바이러스의 증식을 억제하는 대신 숙주세포인 베로 세포(Vero cell)의 증식을 억제함으로써 나타날 수도 있는 바, 이러한 가능성을 배제하기 위하여 베로 세포 배양액에 실험에 사용한 삼백초 수용성 추출물을 첨가한 다음 Vero cell의 생존을 확인하였다. 즉, Cyto X Cell viability assay kit(LPS solution, Daejeon, Korea)를 이용하여 배양중인 세포의 생존율을 조사하였다.In order to suppress the increase of the amount of fluorescence, the water-soluble extract of Saururus chinensis may be suppressed by suppressing the proliferation of the Vero cell, which is a host cell, instead of suppressing the viral proliferation. Survival of Vero cells was confirmed by adding the water-soluble extract of Saururus chinensis. The survival rate of the cultured cells was examined using the Cyto X Cell viability assay kit (LPS solution, Daejeon, Korea).
구체적으로, Vero cell 현탁액을 5 x 105 cell/ml이 되도록 준비한 후, 96 웰 플레이트에 100 ㎕씩 분주한 다음, 하룻밤 배양하여 세포를 배양접시에 부착시켰다. 다음 날, 각 웰에 삼백초 수용성 추출물을 최종농도가 2 mg/ml, 1 mg/ml, 0.5 mg/ml 되도록 각 배양에 5 ㎕씩 첨가한 다음, 24시간 동안 배양하였다. 24시간이 지난 후에, 배양 배지에 Cyto X 10 ㎕를 넣어주고 37℃ 배양기에서 1 내지 4시간 반응시키고 450 nm에서 흡광도를 측정하였다.Specifically, a Vero cell suspension was prepared to have a concentration of 5 × 10 5 cells / ml, and 100 μl of each was dispensed into a 96-well plate. The cells were then cultured overnight to attach the cells to the culture dish. On the next day, 5 μl of the extract was added to each well to give final concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml to each well, and then cultured for 24 hours. After 24 hours, 10 μl of Cyto X was added to the culture medium, reacted for 1 to 4 hours at 37 ° C. in an incubator, and the absorbance at 450 nm was measured.
그 결과 도 6에 나타난 바와 같이, 삼백초 수용성 추출물을 넣어준 배양액의 흡광도는 대조군 배양액의 흡광도와 큰 차이를 보이지 않는 바, 실험에 사용한 농도에서는 삼백초 수용성 추출물이 Vero cell의 증식에 영향을 미치지 않는 것을 알 수 있었다(첫 번째 막대그래프부터 차례대로 각각 물을 투여한 대조군, 삼백초 수용성 추출물 2 mg/ml, 1 mg/ml, 0.5 mg/ml). As a result, as shown in FIG. 6, the absorbance of the culture solution containing the water-soluble extract of Saururus chinensis did not significantly differ from the absorbance of the control culture solution, and the water-soluble extract of Saururus chinensis did not affect Vero cell proliferation (2 mg / ml, 1 mg / ml, 0.5 mg / ml) of the water-soluble extract of Saururus chinensis, respectively.
즉, 이러한 결과는 삼백초 수용성 추출물은 숙주세포의 증식에는 영향을 미치지 않으면서, HSV 바이러스의 증식을 선택적으로 억제하여, 감염된 세포에서 GFP 단백질이 발현되는 것을 방해하고 있음을 보여준다.That is, these results show that the water extract of Saururus chinensis selectively inhibits the proliferation of HSV virus and prevents the expression of GFP protein in infected cells without affecting host cell proliferation.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.
Claims (13)
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