KR20210141341A - Composition for the prevention or treatment of SARS-CoV-2 infection, comprising the extract of Agrimonia pilosa as an active ingredient - Google Patents
Composition for the prevention or treatment of SARS-CoV-2 infection, comprising the extract of Agrimonia pilosa as an active ingredient Download PDFInfo
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- KR20210141341A KR20210141341A KR1020210049299A KR20210049299A KR20210141341A KR 20210141341 A KR20210141341 A KR 20210141341A KR 1020210049299 A KR1020210049299 A KR 1020210049299A KR 20210049299 A KR20210049299 A KR 20210049299A KR 20210141341 A KR20210141341 A KR 20210141341A
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- extract
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- yongacho
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Abstract
Description
본 발명은 용아초(Agrimonia pilosa) 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating a corona virus (SARS-CoV-2) infection comprising an extract of Agrimonia pilosa as an active ingredient.
현재 전세계적으로 문제되고 있는 코로나 바이러스는 SARS-CoV-2 감염에 의한 호흡기 증후군이라 정의되고 있으며, 질병 분류는 법정감염병 제1급감염병 신종감염병증후군 및 질병 코드는 U07.1이다. 병원체는 SARS-CoV-2 : Coronaviridae에 속하는 RNA 바이러스이고, 전파 경로는 현재까지 비말(침방울), 접촉을 통한 전파로 알려져있다. 따라서, 기침이나 재채기를 할 때 생긴 비말(침방울)을 통한 전파 등 또는 코로나19 바이러스에 오염된 물건을 만진 뒤 눈, 코, 입을 만짐으로써 전파되고 있다고 알려져 있다. 전세계 치명률은 약 3.4%(WHO, 3.5 기준)이고, 단 국가별 · 연령별 치명률 수준은 매우 상이하다. 고령, 면역기능이 저하된 환자, 기저질환을 가진 환자가 주로 중증, 사망을 초래한다.The coronavirus, which is currently a worldwide problem, is defined as a respiratory syndrome caused by SARS-CoV-2 infection. The pathogen is SARS-CoV-2: RNA virus belonging to Coronaviridae, and the transmission route is known to be spread through droplets (saliva) and contact. Therefore, it is known that spread through droplets (saliva drops) generated when coughing or sneezing or by touching an object contaminated with the Corona 19 virus and then touching your eyes, nose, and mouth. The global fatality rate is about 3.4% (based on WHO, 3.5), however, the level of fatality rate by country and by age is very different. The elderly, immunocompromised patients, and patients with underlying diseases mainly cause severe and death.
코로나 바이러스의 잠복기는 1~14일(평균 4~7일)이고, 진단 기준으로는 진단을 위한 검사기준에 따라 감염병병원체 감염이 확인된 사람의 검체에서 바이러스 분리 또는 검체에서 특이 유전자 검출하여 진단하고 있다. 코로나 바이러스의 증상으로는 발열, 권태감, 기침, 호흡곤란 및 폐렴 등 경증에서 중증까지 다양한 호흡기감염증이 나타나며, 그 외 가래, 인후통, 두통, 객혈과 오심, 설사 등도 나타난다. 현재 수액 보충, 해열제 등 보존적 치료를 하는 것 외에 특이적인 항바이러스제가 없는 것이 현실이다.The incubation period of corona virus is 1 to 14 days (average 4 to 7 days), and as a diagnostic standard, it is diagnosed by isolating the virus from a sample of a person who has been infected with an infectious disease pathogen or detecting a specific gene from the sample according to the diagnostic test standards. have. Corona virus symptoms include fever, malaise, cough, shortness of breath and pneumonia, and various respiratory infections ranging from mild to severe. Currently, there is no specific antiviral drug other than conservative treatment such as fluid supplementation and antipyretic drugs.
한편, 용아초(Agrimonia pilosa)는 한국, 일본, 중국, 인도 등지에 분포하는 여러해살이풀이다. 이는 짚신나물 또는 선학초라고도 불린다. 굵은 뿌리줄기에 줄기가 나오고 높이 30~100cm이며 전체에 털이 있다. 잎은 어긋나고 우상복엽이며 작은잎은 5~7개로 타원형이고 가장자리에 톱니가 있다. 끝에 달린 3개의 작은잎은 크기가 비슷하고 작은잎 사이에 작은잎 같은 것이 달린다. 꽃은 6~8월에 피고 황색이며 총상꽃차례에 달린다. 꽃받침 통은 길이 3mm 정도이고 세로줄이 있으며 위 끝이 5개로 갈라진다. 꽃잎은 5개, 수술은 12개이며 열매는 꽃받침통 안에 들어 있다. 어린순을 나물로 하고 한방에서 선초를 이질, 위궤양, 자궁출혈에 사용한다. 용아초를 자세히 살펴보면, 갈고리 같은 털들이 있다. 사람들의 옷이나 신발에 잘 달라붙는 성향이 있어 짚신에 붙어 이곳저곳을 붙어 다녔다는 데서 '짚신나물'이라는 이름이 유래되었다.On the other hand, dragonfly ( Agrimonia pilosa ) is a perennial herb distributed in Korea, Japan, China, and India. It is also called straw sandalwood or seonhakcho. Stems appear on thick rhizomes, 30-100 cm high, and hairy throughout. The leaves are alternate phyllotaxis, and the small leaves are 5-7, oval, with sawtooth on the edge. The three small leaves at the end are similar in size, and there is something like a small leaf between the small leaves. Flowers bloom in June-August, yellow, and hang in raceme. The calyx tube is about 3mm long, has a vertical line, and the upper end is divided into 5 pieces. There are 5 petals and 12 stamens, and the fruit is in the calyx tube. Young shoots are used as herbs, and in oriental medicine, seoncho is used for dysentery, gastric ulcer, and uterine bleeding. If you look closely at the dragonfly, there are hook-like hairs. The name 'Salmaceous Vegetables' was derived from the tendency to stick to people's clothes and shoes, so it was attached to sandals and hung around here and there.
따라서, 본 발명은 용아초 추출물을 유효성분으로 하여, 코로나 바이러스를 예방, 개선 또는 치료할 수 있는 조성물에 관한 것으로서, 용아초 추출물을 사용하여 SARS-CoV-2 바이러스에 대한 플라크 감소 실험을 진행한 결과 농도의존적으로 유의적인 결과를 확인하였다. 또한, SARS-CoV-2의 spike protein(스파이크 단백질)의 RNA 복제억제 실험을 진행한 결과 매우 우수하게 바이러스의 복제를 억제함을 확인하였다. 따라서, 용아초 추출물은 SARS-CoV-2의 RNA복제를 억제하여 코로나 바이러스를 억제하는 것으로 나타났다. 이를 통하여 고농도에서 독성이 없으면서 치료효과가 우수한 코로나 바이러스 감염의 예방 또는 개선용 조성물의 개발이 가능할 것이며, 이를 통하여 코로나 바이러스의 예방 또는 개선용 조성물로서 추가 연구를 통해 건강기능식품 또는 의약품으로 개발 가능할 것이다.Therefore, the present invention relates to a composition capable of preventing, improving, or treating a corona virus by using the Yongacho extract as an active ingredient. Significant results were confirmed in a concentration-dependent manner. In addition, as a result of the RNA replication inhibition experiment of the spike protein of SARS-CoV-2, it was confirmed that it inhibited virus replication very well. Therefore, it was shown that the Yongacho extract inhibits the RNA replication of SARS-CoV-2 to suppress the corona virus. Through this, it will be possible to develop a composition for the prevention or improvement of corona virus infection, which is non-toxic at high concentration and has excellent therapeutic effect, and through this, it will be possible to develop as a health functional food or drug through additional research as a composition for preventing or improving corona virus. .
본 발명의 목적은, 용아초(Agrimonia pilosa) 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.It is an object of the present invention, to provide a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection comprising an extract of dragon fruit ( Agrimonia pilosa ) as an active ingredient.
또한, 본 발명의 다른 목적은 용아초(Agrimonia pilosa) 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating corona virus (SARS-CoV-2) infection comprising an extract of yongacho ( Agrimonia pilosa ) as an active ingredient.
또한, 용아초에서 분리된 우르솔산(ursolic acid) 또는 케르세틴(quercetin)를 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for preventing or treating coronavirus (SARS-CoV-2) infection comprising ursolic acid or quercetin as an active ingredient isolated from Yongacho.
즉, 본 발명은 용아초 추출물 또는 이로부터 분리된 우르솔산(ursolic acid) 또는 케르세틴(quercetin)를 이용하여 코로나 바이러스(SARS-CoV-2) 감염에 대한 예방, 개선 또는 치료 효과를 확인하고, 이를 통해 용아초추출물 또는 우르솔산(ursolic acid) 또는 케르세틴(quercetin)를 이용한 코로나 바이러스(SARS-CoV-2)를 예방, 개선 또는 치료할 수 있는 건강기능식품 또는 의약품으로 사용 가능한 조성물을 제조하는 것을 목적으로 한다.That is, the present invention confirms the prevention, improvement, or therapeutic effect on corona virus (SARS-CoV-2) infection by using Yongacho extract or ursolic acid or quercetin isolated therefrom, and For the purpose of preparing a composition that can be used as a health functional food or medicine that can prevent, improve, or treat coronavirus (SARS-CoV-2) using dragon fruit extract or ursolic acid or quercetin through do.
상기 과제를 해결하기 위하여, 본 발명은 용아초 추출물을 유효성분으로 포함하는, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In order to solve the above problems, the present invention provides a health functional food composition for the prevention or improvement of corona virus (SARS-CoV-2) infection, comprising Yongacho extract as an active ingredient.
본 발명의 일 실시예에 있어서, 본 발명의 ‘용아초 추출물’은 물, C1 내지 C4의 알코올, 에틸아세테이트, 클로로포름 및 헥산 중에서 선택된 적어도 어느 하나의 용매로 추출된 것일 수 있고, 바람직하게는 30 내지 70% 에탄올을 사용하여 추출된 것일 수 있으며, 더욱 바람직하게는 50% 에탄올을 사용하여 추출된 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'Yongacho extract' of the present invention is water, C 1 to It may be extracted with at least one solvent selected from C 4 alcohol, ethyl acetate, chloroform, and hexane, preferably extracted using 30 to 70% ethanol, more preferably 50% ethanol It may be extracted using, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘코로나 바이러스’는 베타-코로나 바이러스일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'corona virus' of the present invention may be a beta-coronavirus, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘용아초 추출물’은 코로나 바이러스(SARS-CoV-2)에 대한 플라크 감소 효과를 가지는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'Yongacho extract' of the present invention may have a plaque reducing effect on coronavirus (SARS-CoV-2), but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘용아초 추출물’은 코로나 바이러스(SARS-CoV-2) 복제를 억제시키는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'Yongacho extract' of the present invention may inhibit the corona virus (SARS-CoV-2) replication, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘용아초 추출물’은 우르솔산(ursolic acid) 또는 케르세틴(quercetin) 성분을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'Yongacho extract' of the present invention may include a component of ursolic acid or quercetin, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘용아초 추출물’은 코로나 바이러스로 인한 발열을 억제하는 효과를 가지는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'Yongacho extract' of the present invention may have an effect of suppressing fever due to corona virus, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 ‘조성물’은 코로나 바이러스 스파이크 단백질의 세포막 결합을 차단하여 감염을 억제하는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the 'composition' of the present invention may inhibit infection by blocking the cell membrane binding of the coronavirus spike protein, but is not limited thereto.
또한, 본 발명은 용아초 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of corona virus (SARS-CoV-2) infection comprising a dragon fruit extract as an active ingredient.
또한, 본 발명은 용아초에서 분리된 우르솔산(ursolic acid) 또는 케르세틴(quercetin)를 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus (SARS-CoV-2) infection comprising ursolic acid or quercetin as an active ingredient isolated from Yongacho.
2002년 유행한 사스(SARS), 2012년 유행한 메르스(MERS), 그리고 2020년 시작하여 지금까지 유행하고 있는 SARS-CoV2(Covid-19)까지 다양한 코로나 바이러스에 의하여 전세계적으로 많은 사람이 사망하였다. 또한, 코로나 바이러스는 RNA 바이러스의 특성상 돌연변이가 쉽게 일어나기 쉽기 때문에 변형된 코로나 바이러스가 발생될 확률이 높다. 따라서 본 발명에 따른, 천연물인 용아초 추출물을 사용하여 SARS-CoV-2 바이러스에 대한 플라크 감소 실험을 진행한 결과 농도의존적으로 유의적인 결과를 확인하였다. 또한, SARS-CoV-2의 spike protein(스파이크 단백질)의 RNA 복제억제 실험을 진행한 결과 매우 우수하게 바이러스의 복제를 억제하였다. 따라서, 용아초 추출물은 SARS-CoV-2의 RNA복제를 억제하여 코로나 바이러스를 억제하는 효과를 확인하여 본 발명을 완성하였다.Many people worldwide have died due to various coronaviruses, from the SARS epidemic in 2002, the MERS epidemic in 2012, and the SARS-CoV2 (Covid-19) epidemic that started in 2020 until now. did. In addition, since the coronavirus is prone to mutation due to the nature of the RNA virus, there is a high probability that a modified corona virus will occur. Therefore, according to the present invention, a concentration-dependently significant result was confirmed as a result of a plaque reduction experiment on SARS-CoV-2 virus using a natural product, Yongacho extract. In addition, as a result of the RNA replication inhibition experiment of the spike protein of SARS-CoV-2, the virus replication was very well inhibited. Therefore, the Yongacho extract inhibited RNA replication of SARS-CoV-2 to confirm the effect of suppressing the corona virus, thereby completing the present invention.
도 1은 본 발명의 용아초 추출물을 이용하여 플라크 감소 분석을 실험하여 결과를 나타낸 도이다.
도 2는 본 발명의 용아초 추출물을 이용하여 플라크 감소 분석을 실험하여 결과를 나타낸 도이다.
도 3은 본 발명의 용아초 추출물을 이용하여 플라크 감소 분석을 실험하여 결과를 나타낸 도이다.
도 4는 본 발명의 용아초 추출물을 이용하여 플라크 감소 분석을 실험하여 결과를 나타낸 도이다.
도 5는 본 발명의 용아초 추출물을 이용하여 용아초 추출물의 바이러스 복제억제 실험하여 결과를 나타낸 도이다.
도 6은 본 발명의 용아초 성분인 우르솔산(ursolic acid) 및 케르세틴(quercetin)을 이용하여 플라크감소 분석을 실험하여 결과를 나타낸 도이다.
도 7은 본 발명의 용아초 성분인 우르솔산(ursolic acid) 및 케르세틴(quercetin)과 SARS-CoV-2 spike RBD의 molecular docking을 분석하여 나타낸 도이다.
도 8은 본 발명의 용아초 추출물을 이용하여 체온 상승 유발물질(LPS)에 의한 발열에 대한 해열효과를 실험하여 나타낸 도이다.1 is a diagram showing the results of an experiment for plaque reduction analysis using the Yongacho extract of the present invention.
2 is a diagram showing the results of an experiment for plaque reduction analysis using the Yongacho extract of the present invention.
3 is a diagram showing the results of an experiment for plaque reduction analysis using the Yongacho extract of the present invention.
4 is a diagram showing the results of an experiment for plaque reduction analysis using the Yongacho extract of the present invention.
Figure 5 is a diagram showing the results of a virus replication inhibition experiment of the extract of the dragon fruit of the present invention using the extract.
6 is a diagram showing the results of a plaque reduction assay using ursolic acid and quercetin, which are components of dragon fruit of the present invention.
7 is a diagram showing the analysis of molecular docking of ursolic acid and quercetin and SARS-CoV-2 spike RBD, which are Yongacho components of the present invention.
FIG. 8 is a diagram showing the antipyretic effect on fever caused by a body temperature increase inducer (LPS) by using a dragon fruit extract of the present invention.
이하, 첨부된 도면을 참조하여 본 발명의 실시예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 실시예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허 청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, with reference to the accompanying drawings, the present invention will be described in detail by way of embodiments of the present invention. However, the following examples are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of a user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 '%'는 별도의 언급이 없는 경우, 고체/고체는(w/w) %, 고체/액체는(w/v) %, 그리고 액체/액체는(v/v) %이다.Throughout this specification, '%' used to indicate the concentration of a specific substance is, unless otherwise stated, solid / solid (w / w) %, solid / liquid (w / v) %, and Liquid/liquid is (v/v) %.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.
일 측면에서, 본 발명은 용아초 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것을 목적으로 한다.In one aspect, it is an object of the present invention to provide a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection comprising a dragon fruit extract as an active ingredient.
본 발명에 따른 추출물은 당업계에 공지된 추출 및 분리방법을 사용하여 천연 용아초로부터 추출 및 분리하여 수득한 것을 사용할 수 있으며, 본 발명에서 정의된 “추출물”은 적절한 용매를 이용하여 용아초로부터 추출한 것이며, 예를 들어, 조추출물, 극성용매 가용 추출물 또는 비극성용매 가용 추출물을 모두 포함한다. 상기 용아초로부터 추출물을 추출하기 위한 적절한 용매로는 약학적으로 허용되는 유기용매라면 어느 것을 사용해도 무방하며, 물 또는 유기용매를 사용할 수 있으며, 이에 제한되지는 않으나, 예를 들어, 정제수, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol) 등을 포함하는 탄소수 1 내지 4의 알코올, 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있고, 바람직하게는 에탄올 30 내지 70%를 용매로 사용하였으며, 더욱 바람직하게는 에탄올 50%를 용매로 사용하였다. 추출 방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법 중 어느 하나를 선택하여 사용할 수 있다. 또한, 목적하는 추출물은 추가로 통상의 분획 공정을 수행할 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다.The extract according to the present invention may be obtained by extraction and separation from natural dragon fruit using extraction and separation methods known in the art, and the "extract" as defined in the present invention is obtained from Yonga extract using an appropriate solvent. It is extracted, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. As a suitable solvent for extracting the extract from Yongacho, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto, for example, purified water, methanol (methanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), alcohol having 1 to 4 carbon atoms including butanol (butanol), acetone (acetone), ether (ether), benzene (benzene), chloroform (chloroform), ethyl acetate (ethyl acetate), methylene chloride (methylene chloride), hexane (hexane) and various solvents such as cyclohexane (cyclohexane) can be used alone or in mixture, preferably 30 to 70% ethanol was used as a solvent, more preferably 50% ethanol was used as a solvent. As the extraction method, any one of methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method.
본 발명의 추출물의 제조방법에는 제한이 없으며, 공지되어있는 어떠한 방법도 이용될 수 있다. 예를 들면, 본 발명의 조성물에 포함되는 추출물은 상기한 열수 추출 또는 용매 추출법으로 추출된 1차 추출물을, 감압 증류 및 동결건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말상태로 제조할 수 있다. 또한 상기 1차 추출물을 실리카 겔 컬럼 크로마토그래피(silica gel column chromatography), 박층 크로마토그래피(thin layer chromatography), 고성능 액체 크로마토그래피(high performance liquid chromatography) 등과 같은 다양한 크로마토그래피를 이용하여 추가로 정제된 분획을 얻을 수도 있다. 따라서 본 발명에 있어서 추출물은 추출, 분획 또는 정제의 각 단계에서 얻어지는 모든 추출액, 분리된 화합물, 분획 및 정제물, 그들의 희석액, 농축액 또는 건조물을 모두 포함하는 개념이다.There is no limitation on the method for preparing the extract of the present invention, and any known method may be used. For example, the extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the primary extract extracted by the hot water extraction or solvent extraction method described above. In addition, the primary extract was further purified using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. can also get Therefore, in the present invention, the extract is a concept including all extracts, isolated compounds, fractions and purified substances obtained in each step of extraction, fractionation or purification, and dilutions, concentrates or dried products thereof.
본 발명의 용아초 추출물을 건강기능식품 조성물로 사용하는 경우, 상기 용아초 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 상기 조성물은 유효성분 이외에 식품학적으로 허용가능한 식품보조첨가제를 포함할 수 있으며, 유효성분의 혼합량은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the Yongacho extract of the present invention is used as a health functional food composition, the Yongacho extract can be added as it is or used together with other foods or food ingredients, and can be appropriately used according to a conventional method. In addition to the active ingredient, the composition may contain a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).
본 발명에서 사용되는 용어 "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.The term "food supplement additive" used in the present invention refers to a component that can be added to food as an auxiliary, and is added to the manufacture of health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에서 사용되는 용어 "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 '기능성'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 통상의 기술분야에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 통상의 기술분야에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한없이 제조될 수 있다. 본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 앙염증 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the ordinary technical field, and at the time of the preparation, it can be prepared by adding raw materials and components commonly added in the conventional technical field. In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time using food as a raw material, and has excellent portability, and the present invention health functional food can be taken as a supplement to enhance the anti-inflammatory effect.
또한, 본 발명의 조성물이 사용될 수 있는 건강식품의 종류에는 제한이 없다. 아울러 본 발명의 용아초 추출물을 유효성분으로 포함하는 조성물은 당업자의 선택에 따라 건강기능식품에 함유될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림 류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다.In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition comprising the extract of the dragon fruit of the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.
이하 본 발명의 용아초 추출물을 유효성분으로 포함하는, 코로나 바이러스 감염의 예방 또는 개선용 건강기능식품의 제조예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, a manufacturing example of a health functional food for the prevention or improvement of corona virus infection, comprising the extract of the present invention as an active ingredient, will be described, but the present invention is not intended to limit it, but to describe it in detail.
<제조예 1><Production Example 1>
용아초 추출물 200 ㎎Yongacho extract 200 mg
비타민 A 아세테이트 70 ㎍70 μg vitamin A acetate
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B 1 0.13 ㎎
비타민 B 2 0.15 ㎎
비타민 B 6 0.5㎎
비타민 B 12 0.2 ㎍0.2 μg of vitamin B 12
비타민 C 10 ㎎Vitamin C 10 mg
비오틴10 ㎍Biotin 10 μg
니코틴산아미드 1.7 ㎎Nicotinamide 1.7 mg
엽산 50 ㎍50 μg folic acid
판토텐산 칼슘 0.5 ㎎Calcium pantothenate 0.5 mg
황산제1철 1.75 ㎎Ferrous sulfate 1.75 mg
산화아연 0.82 ㎎Zinc oxide 0.82 mg
탄산마그네슘 25.3 ㎎Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎Potassium monophosphate 15 mg
제2인산칼슘 55 ㎎Dibasic calcium phosphate 55 mg
구연산칼륨 90 ㎎Potassium citrate 90 mg
탄산칼슘 100 ㎎
염화마그네슘 24.8 ㎎Magnesium chloride 24.8 mg
일 측면에서, 본 발명은 용아초 추출물을 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물 또는 용아초에서 분리된 우르솔산(ursolic acid) 또는 케르세틴(quercetin)를 유효성분으로 포함하는 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.In one aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of corona virus (SARS-CoV-2) infection comprising a Yongacho extract as an active ingredient, or ursolic acid or quercetin isolated from Yongacho. ) An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of corona virus (SARS-CoV-2) infection comprising as an active ingredient.
본 발명에서 사용된 용어 "치료"란 본 발명의 조성물의 투여로 코로나 바이러스 감염, 바람직하게는 코로나 바이러스(SARS-CoV-2) 감염의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본 발명의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.As used herein, the term "treatment" refers to any action that improves or advantageously changes the symptoms of a coronavirus infection, preferably a coronavirus (SARS-CoV-2) infection, by administration of the composition of the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact criteria of the disease for which the composition of the present invention is effective, and can determine the degree of improvement, improvement and treatment There will be.
일 구현예에서, 상기 약학 조성물은 경구형 제형, 외용제, 좌제, 멸균 주사용액 및 분무제를 포함하는 군으로부터 선택되는 하나 이상의 제형일 수 있으며, 주사 제형이 더욱 바람직하다.In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injection solutions and sprays, and injection formulations are more preferred.
본 발명에서, 용어 "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 코로나 바이러스 감염, 바람직하게는 코로나 바이러스(SARS-CoV-2) 감염의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread and recurrence of a coronavirus infection, preferably a coronavirus (SARS-CoV-2) infection by administration of the pharmaceutical composition according to the present invention. means
본 발명에서 유효성분과 결합하여 사용된 "치료학적으로 유효한 양"이란 용어는 대상 질환을 예방 또는 치료하는데 유효한 조성물의 약학적으로 허용가능한 염의 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.에 기술되어있다.The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention is It may vary depending on several factors, for example, the method of administration, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well-known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 약학 조성물은 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약제학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약제학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, see, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 주사 제형으로 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.0001 ~ 10 ㎎/㎖이며, 바람직하게는 0.0001 ~ 5 ㎎/㎖이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or locally as an injection formulation) or orally, depending on the desired method, and the dosage may vary depending on the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.
본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid preparations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
또한, 본 발명의 약학적 조성물은 기존의 코로나 바이러스 감염 치료용 조성물과 함께 투여함으로써, 코로나 바이러스 감염 치료 효과를 증가시킬 수 있다. 이때 병용 투여는 기존의 코로나 바이러스 감염 치료용 조성물과 동시에 또는 순차적으로 이루어질 수 있다.In addition, by administering the pharmaceutical composition of the present invention together with the existing composition for treating coronavirus infection, it is possible to increase the therapeutic effect of coronavirus infection. In this case, the combined administration may be performed simultaneously or sequentially with the existing composition for treating coronavirus infection.
본 발명의 용아초 추출물의 용아초(Agrimonia pilosa)는 한방에서 예로부터 널리 사용된 약재로 어린순을 나물로 하고 한방에서 선초를 이질, 위궤양, 자궁출혈에 사용한다. 즉, 약학적 조성물 또는 건강기능식품 조성물로 사용할 경우에 일반적인 합성 화합물에 비하여 부작용이 덜할 수 있으므로, 안전하게 약학적 조성물 및 건강기능식품 조성물에 포함되어 유용하게 사용될 수 있다.Yongacho extract of the present invention ( Agrimonia pilosa ) is a medicinal herb widely used in oriental medicine since ancient times. That is, when used as a pharmaceutical composition or a health functional food composition, side effects may be less than that of a general synthetic compound, so it can be safely included in a pharmaceutical composition and a health functional food composition to be usefully used.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.
<준비예 1> 실험의 재료<Preparation Example 1> Experimental material
DMEM(-/-), Overlay media(0.6% agarose in DMEM), 12 well plate, Vero E6, 1x PBS(Cell culture grade)을 사용하여 하기의 실험방법에 의해 실험을 진행하였다.DMEM (-/-), overlay media (0.6% agarose in DMEM), 12 well plate, Vero E6, 1x PBS (cell culture grade) was used and the experiment was performed according to the following experimental method.
<준비예 2> 용아초 추출물의 제조방법<Preparation Example 2> Preparation method of extract of yongacho extract
용아초(Agrimonia pilosa)의 잎은 서울의 BioKorea Co. LTd(Seoul)에서 구입하고, 바우처 표본(BMRI-RG-1601)은 한국 용인 경희대학교 바이오메디컬연구센터에 기탁하였다. 건조된 샘플(20 kg)을 80±2℃에서 6시간 동안 50% 에탄올로 추출한 다음 여과하였다. 그 후, 회전 증발기를 사용하여 농축시키고 동결건조하였다. 최종 생성물 수율은 용아초 1.57 kg이었다. 용아초 추출물은 사용전까지 4 ℃에서 보관하였다. 여기에서 상기 에탄올 50% 용아초 추출물은 물, 메탄올 10%, 메탄올 30%, 메탄올 50%, 메탄올 70%, 메탄올 90%, 에탄올 10%, 에탄올 30%, 에탄올 50%, 에탄올 70%, 에탄올 90% 추출물을 이용한 예비실험에서의 최적화된 농도로 하기 실험에서는 에탄올 50% 용아초 추출물을 이용하여 실험을 진행하였다.The leaves of the dragonfly plant ( Agrimonia pilosa ) were obtained from BioKorea Co., Ltd. in Seoul. It was purchased from LTd (Seoul), and the voucher sample (BMRI-RG-1601) was deposited at the Biomedical Research Center of Kyunghee University, Yongin, Korea. The dried sample (20 kg) was extracted with 50% ethanol at 80±2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and lyophilized. The final product yield was 1.57 kg of dragonfly. Yongacho extract was stored at 4 ℃ until use. Here, the
<실시예 1> 실험방법<Example 1> Experimental method
① Vero E6 cells을 12웰-플레이트에 3.0 x 105 cells/well로 seeding하고, ② 37℃, CO2 인큐베이터에서 배양하였다. ③ 상등액을 제거하고, 1x PBS로 2번 wash 단계를 진행하였다. ④ Pretreatment(추출, 37℃, CO2 인큐베이터, 2시간), ⑤ 상등액을 제거하고, 바이러스 흡착(virus adsorption) (50pfu/well, shake/10min), ⑥ 상등액을 제거하고, add 1.5㎖ overlay media containing extracts), ⑦ 3일(72시간) 뒤에 결과를 확인하였다.① Vero E6 cells were seeded in a 12-well-plate at 3.0 x 10 5 cells/well, and ② cultured at 37°C, CO 2 in an incubator. ③ The supernatant was removed, and the wash step was performed twice with 1x PBS. ④ Pretreatment (extraction, 37℃, CO 2 incubator, 2 hours), ⑤ Remove supernatant, virus adsorption (50pfu/well, shake/10min), ⑥ Remove supernatant, add 1.5ml overlay media containing extracts), ⑦ 3 days (72 hours) later, the results were confirmed.
<실시예 2> 용아초 추출물의 플라크 감소 분석 실험 - 1<Example 2> Plaque reduction assay of Yongacho extract-1
현재 SARS-CoV-2의 치료 효과가 있다고 알려진 렘데시비르(Remdesivir, Rem)를 비교군으로 설정하여 동일 농도에 대하여 SARS-CoV-2의 플라크 감소 효과를 측정한 결과 도 1에서 나타낸 것과 같이 비교군보다는 약간 낮지만 농도의존적으로 플라크 감소 효과가 증가하는 것으로 나타났다.Remdesivir (Rem), which is currently known to have a therapeutic effect on SARS-CoV-2, was set as a comparison group, and the plaque reduction effect of SARS-CoV-2 was measured at the same concentration as shown in FIG. 1 . Although it was slightly lower than the group, it was found that the plaque reduction effect increased in a concentration-dependent manner.
<실시예 3> 용아초 추출물의 플라크 감소 분석 실험 - 2<Example 3> Plaque reduction analysis experiment of Yongacho extract-2
현재 SARS-CoV-2의 치료 효과가 있다고 알려진 비교군으로 렘데시비르(Remdesivir, Rem) 5 μM와 클로로퀴논 포스페이트(chloroquine phosphate, C.P) 10 μM을 사용하여 실험을 진행하였다. 도 2 내지 도 4에서 나타낸 것과 같이 용아초 추출물은 농도의존적으로 플라크 감소 효과가 우수하며, 고농도에서도 세포독성이 없이 항바이러스 효과를 나타냄을 알 수 있었고, 용아초 추출물을 고농도로 사용하는 경우에는 렘데시비르를 저농도 사용한 것보다 더 우수한 결과를 나타내었다. 도 4의 경우 바이러스 감염시간인 1시간동안 용아초 추출물을 동시 처리한 실험결과로, 용아초 추출물에 의해 바이러스 엔트리 자체가 억제되는 것을 확인하였다. 하기 표 1은 이에 대한 값을 수치로 나타낸 것이다.As a comparison group that is currently known to have a therapeutic effect on SARS-CoV-2, an experiment was conducted using 5 μM of Remdesivir (Rem) and 10 μM of chloroquine phosphate (C.P). As shown in FIGS. 2 to 4 , it was found that the Yongacho extract exhibited an antiviral effect without cytotoxicity even at a high concentration, and exhibited an antiviral effect without cytotoxicity even at a high concentration. It showed better results than the use of low concentration of desivir. In the case of Figure 4, it was confirmed that the virus entry itself was suppressed by the extract of Yongacho as the result of the simultaneous treatment of the extract of Yongacho for 1 hour, which is the virus infection time. Table 1 below shows the values for this numerically.
<실시예 4> 용아초 추출물의 바이러스 복제억제 실험<Example 4> Virus Replication Inhibition Experiment of Yongacho Extract
현재 SARS-CoV-2의 치료 효과가 있다고 알려진 비교군으로 렘데시비르(Remdesivir, Rem) 5 μM와 클로로퀴논 포스페이트(chloroquine phosphate, C.P) 10 μM을 사용하여 실험을 진행하였다. 도 5에서 나타낸 것과 같이 용아초 추출물은 농도의존적으로 바이러스의 스파이크 단백질 발현 RNA복제 억제효과가 우수하며, 고농도에서도 세포독성이 없이 항바이러스 효과를 나타냄을 알 수 있었고, 용아초 추출물을 고농도로 사용하는 경우에는 렘 데 시비르를 저농도 사용한 것보다 더 우수한 결과를 나타내었다.As a comparison group that is currently known to have a therapeutic effect on SARS-CoV-2, an experiment was conducted using 5 μM of Remdesivir (Rem) and 10 μM of chloroquine phosphate (C.P). As shown in FIG. 5, it was found that the Yongacho extract exhibited an antiviral effect without cytotoxicity even at a high concentration, and had an excellent effect of inhibiting the RNA replication of spike protein expression of the virus in a concentration-dependent manner. In this case, it showed better results than the low concentration use of rem des sivir.
<실시예 5> 용아초 성분 우르솔산(ursolic acid) 및 케르세틴(quercetin)의 SARS-CoV-2 복제억제 실험<Example 5> SARS-CoV-2 replication inhibition experiment of Yongacho component ursolic acid and quercetin
용아초 성분의 항바이러스 효과 검증을 위해, Vero E6 세포에 SARS-CoV-2를 감염시킨 후 용아초 성분에서 분리한 우르솔산(ursolic acid) 및 케르세틴(quercetin)을 처리하여 바이러스 복제억제 효능을 측정하였다. 그 결과 도 6와 같이 우르솔산(ursolic acid)는 각각 15.2%, 31.6%, 50.1%, 케르세틴(quercetin)은 각각 28.5%, 40.3%, 49.3%의 바이러스 복제억제 효과를 나타냈다.To verify the antiviral effect of Yongacho components, Vero E6 cells were infected with SARS-CoV-2 and then treated with ursolic acid and quercetin isolated from Yongacho components to measure the virus replication inhibitory efficacy. did. As a result, as shown in FIG. 6 , ursolic acid showed 15.2%, 31.6%, and 50.1%, respectively, and quercetin had 28.5%, 40.3%, and 49.3% virus replication inhibitory effects, respectively.
<실시예 6> 용아초 성분 우르솔산(ursolic acid) 및 케르세틴(quercetin)과 SARS-CoV-2 spike RBD의 molecular docking<Example 6> Molecular docking of yongacho component ursolic acid and quercetin and SARS-CoV-2 spike RBD
용아초 추출물이 SARS-CoV-2의 absorption을 방해하는 것으로 나타났기 때문에, viral spike RBD가 숙주세포에 결합하는 것을 용아초 성분에 의해 방해를 받을수 있다. 이에 용아초 성분인 우르솔산(ursolic acid) 및 케르세틴(quercetin) 구조에 SARS-CoV-2 spike receptor-binding domain(RBD)의 molecular docking을 분석하였다. 또한 SARS-CoV-2 spike RBD가 지속적으로 돌연변이를 일으켜 효율적인 백신 및 항바이러스 치료제 개발을 방해하기 때문에 SARS-CoV-2 spike RBD의 변형인 B.1.1.7 spike RBD에 대한 우르솔산(ursolic acid) 및 케르세틴(quercetin)의 결합 친화도를 분석하여, 우르솔산(ursolic acid) 및 케르세틴(quercetin)의 잠재적 항바이러스 효과를 평가하였다. Molecular docking 분석에서 계산된 결합 에너지는 하기 표 2과 같다. 우르솔산(ursolic acid)는 SARS-CoV-2 spike RBD와 B.1.1.7 spike RBD 모두 비슷한 수치를 나타냈으며, 케르세틴(quercetin)은 B.1.1.7 spike RBD에 더 안정한 것으로 나타났다. 이러한 결과는 용아초 추출물이 SARS-CoV-2 spike RBD 및 B.1.1.7 spike RBD 모두에 항바이러스 치료제로서의 잠재력을 가지고 있음을 나타낸다(도 7 참조).Since Yongacho extract has been shown to interfere with the absorption of SARS-CoV-2, binding of viral spike RBD to host cells may be hindered by Yongacho components. Accordingly, molecular docking of the SARS-CoV-2 spike receptor-binding domain (RBD) was analyzed in the structures of ursolic acid and quercetin, which are components of Yongacho. In addition, ursolic acid for B.1.1.7 spike RBD, a variant of SARS-CoV-2 spike RBD, because SARS-CoV-2 spike RBD continuously mutates and interferes with the development of efficient vaccines and antiviral therapeutics. And by analyzing the binding affinity of quercetin (quercetin), ursolic acid (ursolic acid) and the potential antiviral effect of quercetin (quercetin) was evaluated. Binding energies calculated in molecular docking analysis are shown in Table 2 below. Ursolic acid showed similar values in both SARS-CoV-2 spike RBD and B.1.1.7 spike RBD, and quercetin was found to be more stable in B.1.1.7 spike RBD. These results indicate that Yongacho extract has potential as an antiviral therapeutic agent for both SARS-CoV-2 spike RBD and B.1.1.7 spike RBD (see FIG. 7 ).
<실시예 7> 용아초 추출물의 체온 상승 억제 효과 확인<Example 7> Confirmation of the body temperature increase inhibitory effect of the extract of dragon fruit
고열 동물 모델에서 상기 준비예 2의 제조방법으로 제조된 용아초 추출물의 해열 효과를 확인하였다. 구체적으로, 실험에 사용된 Sprague-Dawley(SD) rats는 일주일간 사육실 환경에 적응시킨 후 사용하였다 사육실 온도는 25±1℃, 습도는 50~60%로 유지하였다. Light/dark cycle이 12시간 주기로 조절되게 한 후, 고형사료와 물을 제한 없이 공급하였다. 경희대학교 동물실험윤리위원회에서 제공한 지짐서의 원리에 의해서 동물실험을 수행하였다. In the high fever animal model, the antipyretic effect of the Yongacho extract prepared by the preparation method of Preparation Example 2 was confirmed. Specifically, the Sprague-Dawley (SD) rats used in the experiment were used after acclimatization to the breeding room environment for a week. The breeding room temperature was maintained at 25±1℃ and humidity at 50-60%. After allowing the light/dark cycle to be adjusted to a 12-hour cycle, solid feed and water were supplied without restriction. Animal experiments were performed according to the guidelines provided by Kyunghee University's Animal Experiment Ethics Committee.
체온 상승 유발물질(lipopolysaccharides, LPS; sigma, USA) 2.5 mg/kg로 생리식염수에 녹여 상기 실험동물의 복강내로 투여하여 발열을 유발하였다. 실험군은 5마리씩 6군으로 발열 유발 30분 전에 용아초 추출물(25, 50, 100 mg/kg)과 양성대조군(indomethacin, INN, 100 mg/kg)를 경구투여하였다. 발열을 유발하기 전(0 h) 및 유발 6시간 후(6 h)에 직장 내 체온을 측정하여 도 8에 나타내었다.A body temperature increase inducer (lipopolysaccharides, LPS; sigma, USA) was dissolved in physiological saline at 2.5 mg/kg and administered intraperitoneally to the experimental animals to induce fever. The experimental group was divided into 6 groups of 5 animals each, and Yongacho extract (25, 50, 100 mg/kg) and a positive control group (indomethacin, INN, 100 mg/kg) were orally administered 30 minutes before fever induction. The rectal body temperature was measured before induction of fever (0 h) and 6 hours after induction (6 h), and it is shown in FIG. 8 .
체온 상승 유발물질(LPS) 투여군은 투여 6시간 후 투여 전보다 체온이 상승하였다. 그러나 용아초 추출물를 투여한 군은 모두 농도의존적으로 체온 증가를 억제하는 것을 확인하였다. 이러한 결과로부터 용아초 추출물은 발열을 억제하는 해열 효능이 우수한 것을 확인하였다. In the group administered with a body temperature increase inducer (LPS), body temperature increased 6 hours after administration compared to before administration. However, it was confirmed that all of the groups administered the Yongacho extract inhibited the increase in body temperature in a concentration-dependent manner. From these results, it was confirmed that the Yongacho extract has excellent antipyretic efficacy in suppressing fever.
상기 실시예들에 의해 본 발명의 용아초 추출물을 사용하여 SARS-CoV-2 바이러스에 대한 플라크 감소 실험을 진행한 결과 농도의존적으로 유의적인 결과를 확인하였다. 또한, SARS-CoV-2의 spike protein(스파이크 단백질)의 RNA 복제억제 실험을 진행한 결과 매우 우수하게 바이러스의 복제를 억제함을 확인하였다. 따라서, 용아초 추출물은 SARS-CoV-2의 RNA복제를 억제하여 코로나 바이러스를 억제하는 것으로 나타났다. 또한 체온 상승 유발물질로 유도된 고열에서도 용아초 추출물이 체온 상승을 억제하여 바이러스 감염에 의한 발열을 억제할 수 있을 것으로 생각된다. 이를 통하여 고농도에서 독성이 없으면서 치료효과가 우수한 코로나 바이러스 감염의 예방 또는 개선용 조성물의 개발이 가능할 것이며, 이를 통하여 코로나 바이러스의 예방, 개선 또는 치료용 조성물로서 추가 연구를 통해 건강기능식품 또는 의약품으로 개발 가능할 것이다. As a result of performing a plaque reduction experiment on SARS-CoV-2 virus using the Yongacho extract of the present invention according to the above examples, significant results were confirmed in a concentration-dependent manner. In addition, as a result of the RNA replication inhibition experiment of the spike protein of SARS-CoV-2, it was confirmed that it inhibited virus replication very well. Therefore, it was shown that the Yongacho extract inhibits the RNA replication of SARS-CoV-2 to suppress the corona virus. In addition, even in high fever induced by a substance that induces body temperature increase, Yongacho extract suppresses body temperature increase, so it is thought that fever caused by virus infection can be suppressed. Through this, it will be possible to develop a composition for the prevention or improvement of corona virus infection, which is non-toxic at high concentration and has excellent therapeutic effect, and through this, it is developed as a health functional food or drug through additional research as a composition for the prevention, improvement or treatment of corona virus. It will be possible.
이상에서 살펴본 바와 같이, 본 발명의 구체적인 실시예를 상세하게 설명되었으나, 본 발명의 사상을 이해하는 당업자는 동일한 사상의 범위 내에서 다른 구성요소를 추가, 변경, 삭제 등을 통하여, 퇴보적인 다른 발명이나 본 발명 사상의 범위 내에 포함되는 다른 실시예를 용이하게 제안할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상술한 상세한 설명보다는 후술하는 특허청구의 범위에 의하여 나타내어지며, 특허청구의 범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions. However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the claims described later rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as
Claims (11)
상기 용아초 추출물은 물, C1 내지 C4의 알코올, 에틸아세테이트, 클로로포름 및 헥산 중에서 선택된 적어도 어느 하나의 용매로 추출된 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract is water, C 1 to C 4 of alcohol, ethyl acetate, chloroform, and extracted with at least one solvent selected from hexane, a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection.
상기 용아초 추출물은 30 내지 70% 에탄올을 사용하여 추출된 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract is a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection, which is extracted using 30 to 70% ethanol.
상기 코로나 바이러스는 베타-코로나 바이러스인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The corona virus is a beta-coronavirus, a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection.
상기 용아초 추출물은 코로나 바이러스(SARS-CoV-2)에 대한 플라크 감소 효과를 가지는 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract is a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection, which has a plaque reducing effect on the corona virus (SARS-CoV-2).
상기 용아초 추출물은 코로나 바이러스(SARS-CoV-2) 복제를 억제시키는 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract inhibits the replication of the corona virus (SARS-CoV-2), a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection.
상기 용아초 추출물은 우르솔산(ursolic acid) 또는 케르세틴(quercetin) 성분을 포함하는 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract is a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection, which includes a component of ursolic acid or quercetin.
상기 용아초 추출물은 코로나 바이러스로 인한 발열을 억제하는 효과를 가지는 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The Yongacho extract is a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection, which has the effect of suppressing fever due to corona virus.
상기 조성물은 코로나 바이러스 스파이크 단백질의 세포막 결합을 차단하여 감염을 억제하는 것인, 코로나 바이러스(SARS-CoV-2) 감염의 예방 또는 개선용 건강기능식품 조성물.According to claim 1,
The composition is a health functional food composition for preventing or improving corona virus (SARS-CoV-2) infection, which inhibits infection by blocking the cell membrane binding of the coronavirus spike protein.
A pharmaceutical composition for preventing or treating coronavirus (SARS-CoV-2) infection comprising ursolic acid or quercetin as an active ingredient isolated from Yongacho.
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| WO2023008594A1 (en) * | 2021-07-26 | 2023-02-02 | 에이피알지 주식회사 | Composition for prevention or treatment of coronavirus (sars-cov-2) infection, comprising agrimonia pilosa extract as active ingredient |
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| KR20200054862A (en) | 2018-11-09 | 2020-05-20 | 주식회사 레이크머티리얼즈 | Sublimation device for high purity semiconductor |
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| KR20200054862A (en) | 2018-11-09 | 2020-05-20 | 주식회사 레이크머티리얼즈 | Sublimation device for high purity semiconductor |
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| Microbiol. Immunol. 54, 11-19쪽(2010.) 1부.* * |
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| WO2023008594A1 (en) * | 2021-07-26 | 2023-02-02 | 에이피알지 주식회사 | Composition for prevention or treatment of coronavirus (sars-cov-2) infection, comprising agrimonia pilosa extract as active ingredient |
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