KR100531645B1 - Composition comprising the extract of African Phellinus mushroom having human papilloma virus activity - Google Patents
Composition comprising the extract of African Phellinus mushroom having human papilloma virus activity Download PDFInfo
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- KR100531645B1 KR100531645B1 KR10-2003-0071687A KR20030071687A KR100531645B1 KR 100531645 B1 KR100531645 B1 KR 100531645B1 KR 20030071687 A KR20030071687 A KR 20030071687A KR 100531645 B1 KR100531645 B1 KR 100531645B1
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- extract
- african
- mushroom
- mushrooms
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/208—Fungi extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
본 발명은 인유두종 바이러스(HPV) 억제능을 갖는 아프리카산 상황버섯(Phellinus genus) 추출물을 함유하는 조성물에 관한 것으로, 본 발명의 아프리카산 상황버섯 추출물은 항 바이러스 효과에 의해 인유두종 바이러스의 증식을 억제하므로, 인유두종 바이러스로 인한 질환의 치료 및 예방을 위한 의약품 또는 건강기능식품에 유용하게 이용할 수 있다.The present invention relates to a composition containing an African Phellinus genus extract having the ability to inhibit human papillomavirus (HPV), and the African situation S. mushroom extract of the present invention inhibits the growth of the human papilloma virus by an antiviral effect, It can be usefully used for medicines or functional foods for the treatment and prevention of diseases caused by HPV.
Description
본 발명은 인유두종 바이러스 억제능을 갖는 아프리카산 상황버섯 추출물을 함유하는 인유두종 바이러스로 인한 질환의 치료 및 예방을 위한 조성물에 관한 것이다.The present invention relates to a composition for the treatment and prevention of diseases caused by human papilloma virus containing an African situation mushroom extract having the ability to inhibit human papilloma virus.
인유두종 바이러스(Human Papilloma Virus; 이하 HPV)는 환상의 이중나선 DNA와 정 20 면체의 뉴클레오캡시드를 가지는 외피비보유 바이러스로서, 자궁경부암 환자의 80 %에서 HPV가 분리되었다는 보고가 있으며, 국내에서는 가정주부의 30 %, 직업여성의 50 %가 HPV에 감염되었다는 보고도 있다. HPV 감염은 대개 임상적 증상으로 진단되며, 혈청학적 검사는 시행되지 않는데, HPV는 시험관내에서 증식이 어려우며, 현재 바이러스를 검출할 수 있는 검체는 자궁경부암 조직에서 얻을 수 있다. HPV DNA의 존재를 검출하기 위한 DNA 교잡시험이 유용하게 이용된다. HPV의 감염은 성적접촉을 피함으로써 예방될 수 있으며, 아직 이용할 수 있는 백신은 없는 실정이고, 인정된 항바이러스제도 개발되지 않은 상태이다. Human Papilloma Virus (HPV) is a non-enveloped virus with cyclic double-stranded DNA and icosahedral nucleocapsids, which reported HPV isolation in 80% of patients with cervical cancer. It is reported that 30% of housewives and 50% of working women are infected with HPV. HPV infections are usually diagnosed with clinical symptoms and serological tests are not performed. HPV is difficult to proliferate in vitro, and samples capable of detecting the virus can now be obtained from cervical cancer tissue. DNA hybridization tests to detect the presence of HPV DNA are useful. Infection with HPV can be prevented by avoiding sexual contact, no vaccine is available yet, and no approved antiviral agents have been developed.
상황(桑黃)버섯은 옛날부터 중국에서는 상이(桑耳)라고도 하여 뽕나무, 느릅나무, 사시나무, 오리나무 등의 활엽수의 심재부에 자생하는 버섯으로 소나무비늘버섯과(Hymenochaetaceae)의 진흙버섯속(Phellinus Ouel. Em.lmaz)에 속하는 백색부후균이다. 우리나라에서 상황버섯이라 함은 목질 진흙버섯(Phellinus Linteus)을 지칭한다. 상황버섯은 옛날부터 자궁출혈, 월경불순 등의 부인병치료제로 사용되어 왔으며 최근에는 높은 항암율 때문에 각광을 받고 있다. 상황버섯의 약리작용에 대한 약학계 및 의학계의 연구를 통해 지금까지 알려진 바로는 소화기 계통의 암인 위암, 식도암, 십이지장암, 결장암, 직장암을 비롯한 간암 수술 후 화학요법을 병행할 때 면역기능을 활성화한다는 것이다. 상황버섯의 효능이 다당류 베타글루칸에 의한 것으로 밝혀진 이후 다당류에 대한 연구가 활발히 이루어지고 있다. 일반적으로 버섯류는 칼륨, 칼슘, 마그네슘, 비타민 B2, B3, C, 섬유질아미노산을 함유하고 있는데, 상황버섯은 이런 성분 외에 다당류 베타글루칸(polysaccharide β-glucan)을 함유하고 있다. 베타글루칸은 인체의 면역력을 높임으로써 몸안에 침입한 세균이나 이물질을 격퇴하고, 감염되어도 발병을 억제시키는 역할을 한다.Sichuan mushrooms, which are known in China since ancient times, are native to the heart of hardwoods such as mulberry, elm, aspen and alder. ( Phellinus Ouel. Em.lmaz ) is a white fungus belonging to. In Korea, the situation mushroom refers to the woody mud mushroom ( Phellinus Linteus ). Situation mushrooms have been used for the treatment of gynecological diseases such as uterine bleeding and menstrual irregularities. Pharmacological and medical researches on the pharmacological action of situational mushrooms have been known to activate immune function following chemotherapy following liver cancer surgery, including gastric cancer, esophageal cancer, duodenal cancer, colon cancer and rectal cancer, which are cancers of the digestive system . Since the efficacy of the situation mushroom was found to be caused by polysaccharide beta glucan, research on polysaccharides has been actively conducted. In general, mushrooms contain potassium, calcium, magnesium, vitamins B2, B3, C, and fibrous amino acids. In addition to these ingredients, mushrooms contain polysaccharide β-glucan. Beta-glucan reinforces the body's immunity, repels bacteria and foreign substances that have invaded the body, and acts as a suppressor of infection.
상황버섯과 관련한 종래의 발명으로, 한국특허출원 제 2001-0003264호는 펠리누스 린테우스 균주 및 이로부터 분리정제된 항암 면역활성 다당류에 대해 개시하고 있으며, 한국특허출원 제1998-0015617호는 펠리누스속 균주로부터 균사체 또는 자실체를 대량 배양하여 그로부터 면역증강활성을 나타내는 신규한 다당류물질의 분리 정제에 대해 개시하고 있다. 한국특허등록 제 10-0174433호는 펠리누스 린테우스 균주의 균사체로부터 분리 정제된 항암활성 다당류, 이의 제조 방법 및 이를 포함하는 약학적 조성물에 대해 개시하고 있으며, 한국특허출원 제2000-0054319호는 금사상황버섯 추출물의 항암제로서의 용도에 대해 개시하고 있으며, 한국특허등록 제 10-0348115호는 자연산 상황버섯 추출물을 함유하는 발모제 조성물 및 이의 제조방법에 대해 개시하고 있다. 이외에도 상황버섯 및 그 추출물을 이용한 의약품 및 건강식품들에 대한 많은 연구들이 국내외적으로 이루어지고 있다.As a conventional invention related to a situation mushroom, Korean Patent Application No. 2001-0003264 discloses a Pelinus linteus strain and an anticancer immunoactive polysaccharide isolated and purified therefrom, and Korean Patent Application No. 1998-0015617 discloses a genus of Pelinus. Disclosed is a novel separation and purification of a novel polysaccharide that exhibits an immunostimulating activity by mass culturing mycelium or fruiting body from a strain. Korean Patent Registration No. 10-0174433 discloses an anticancer active polysaccharide isolated from mycelium of Felinus linteus strains, a method for preparing the same, and a pharmaceutical composition comprising the same, and Korean Patent Application No. 2000-0054319 describes The use of a mushroom extract as an anticancer agent is disclosed, and Korean Patent Registration No. 10-0348115 discloses a hair regrowth composition containing a natural situation mushroom extract and a method of preparing the same. In addition, many researches on medicines and health foods using situation mushrooms and their extracts have been made at home and abroad.
그러나, 진흙버섯에 속하는 모든 버섯이 이 같은 약효가 있는 것은 아니며 현재까지 밝혀진 바에 의하면 목질진흙버섯, 말똥진흙버섯, 전나무진흙버섯 정도이다.However, not all mushrooms belonging to mud mushrooms have this effect, and to date, it has been found to be wood mud mushrooms, horse mud mushrooms and fir mud mushrooms.
본 발명의 아프리카산 상황버섯(Phellinus genus)은 소나무비늘버섯과(Hymenochaetaceae) 진흙버섯속(Phellinus)에 속하며, 아프리카 케냐의 고산 밀립지대에서 야생상태로 자생하는 뽕나무과에 속하는 활엽수 등에 기생하여 자라고 있는 버섯이다. 이곳의 생태적 특징은 적도부근의 열대 지역임에도 불구하고 해발 5000 m급의 고산지대로서 버섯의 생육에 최적환경을 갖추고 있다. 다년생이며, 표면은 회갈색 또는 암회색을 띄고 딱딱한 외피를 가져 매끈하다. 하면은 움푹하고 담갈색을 띠며 다공성이다. 포자는 작고 유구형이다. 해마다 층이 증가하며, 증가한 층이 뚜렷이 구분되어 쉽게 셀 수 있기에 이로부터 나이를 정확히 측정할 수 있다. 자실체는 다년생 경재이다. 일반적으로 갓은 말굽형이며 높이 11 cm, 폭 20 cm, 두께 8 cm에 이른다. 자라면 윗부분은 검게 갈라지며, 암회색을 띄고 표면이 거칠어진다. 완전히 자라면 갓은 그을 듯한 색을 띄게 되며, 평평해진다. 아프리카산 상황버섯은 살아있거나 죽은 목재를 부패시켜 백목질 및 심재부에 백색 부패물을 생성한다.The African situation mushroom ( Phellinus genus ) of the present invention belongs to the Hymenochaetaceae mud mushroom ( Phellinus ), a mushroom that grows on parasites belonging to wild leaves, etc., which grow wild in the alpine area of Kenya, Africa. to be. The ecological feature of this area is the alpine region of 5000m above sea level, although it is tropical near the equator, it has the best environment for mushroom growth. Perennial, the surface is grayish brown or dark gray, and has a hard shell and smooth. The underside is hollow, light brown, and porous. Spores are small and globose. Each year there is an increase in layers, and the increased layers are clearly distinguished and can be easily counted so that age can be accurately measured. Fruiting bodies are perennial hardwoods. The lampshade is generally horseshoe-shaped, 11 cm high, 20 cm wide, and 8 cm thick. When grown, the upper part is blackened, dark gray, and the surface is rough. When fully grown, the lampshade will be tanned and flattened. African situation mushrooms decay live or dead wood, producing white decay in white matter and heartwood.
질병치료와 관련한 의학적 효능은 이미 오래 전부터 동양의학에서 잘 알려져 있다. 항암효과를 나타내며, 추출물 역시 간암, 폐암 및 기타 여러 암들에 대한 우수한 치료 및 예방효과를 가짐이 임상실험에 의해 입증되었다. The medical efficacy of treating diseases is well known in oriental medicine for a long time. It has anti-cancer effects, and the extract has also been proved by clinical trials as having an excellent therapeutic and preventive effect against liver cancer, lung cancer and many other cancers.
그러나, 지금까지 상기 아프리카산 상황버섯의 항 HPV효과에 대해서는 연구되어진 바 없다.However, the anti-HPV effect of the African situation mushroom has not been studied until now.
본 발명자는 각종 바이러스 질환으로 고생하던 특정지역 주민들이 상황버섯의 끓인 물을 복용하던 중 탁월한 치료효과를 보인 것에 착안하여, 아프리카산 상황버섯 추출물 및 세부 분획물의 HPV 억제능을 실험한 결과, 그 탁월한 효능을 확인하여 본 발명을 완성하였다.The inventors pay attention to the excellent treatment effect of the local residents suffering from various viral diseases while taking boiled water of the situation mushroom. It was confirmed to complete the present invention.
본 발명은 아프리카산 상황버섯 추출물을 함유하는 인유두종 바이러스로 인한 질환의 예방 및 치료용 조성물로써 의약품 및 건강기능식품을 제공하는 것이다. The present invention is to provide a pharmaceutical and nutraceutical as a composition for the prevention and treatment of diseases caused by human papilloma virus containing African situation mushroom extract.
상기 목적을 달성하기 위하여, 본 발명은 아프리카산 상황버섯 (Phellinus genus) 추출물을 함유하는 인유두종 바이러스로 인한 질환의 예방 및 치료용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for the prevention and treatment of diseases caused by human papillomavirus containing African Phellinus genus extract.
상기 상황버섯은 아프리카 케냐, 우간다, 탄자니아 등의 동남부 아프리카 지역, 바람직하게는 케냐에 자생하는 상황버섯을 포함한다.The situation mushroom includes a situation mushroom native to Southeast Africa, preferably Kenya, such as Africa Kenya, Uganda and Tanzania.
상기 인유두종 바이러스로 인한 질환으로는 자궁경부암, 콘딜로마(곤지름) 등이 있다.Diseases caused by the human papilloma virus include cervical cancer, condyloma (gon diameter), and the like.
상기 상황버섯 추출물은 조추출물 또는 비극성 용매 가용성 추출물로써, 조추출물은 물, 에탄올, 메탄올, 부탄올과 같은 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물 또는 메탄올에 가용한 추출물을 의미하며, 비극성 용매 가용추출물은 헥산, 디클로로메탄 또는 에틸 아세테이트와 같은 비극성 용매, 바람직하게는 헥산 또는 에틸 아세테이트에 가용한 추출물을 포함한다.The situation mushroom extract is a crude extract or a non-polar solvent soluble extract, the crude extract means a lower alcohol such as water, ethanol, methanol, butanol or a mixed solvent thereof, preferably an extract available in water or methanol, non-polar solvent Soluble extracts include extracts soluble in nonpolar solvents such as hexane, dichloromethane or ethyl acetate, preferably hexane or ethyl acetate.
또한, 상기 아프리카산 상황버섯 추출물에는 고분자 다당체(분자량 12,000이상) 분획을 갖는 아프리카산 상황버섯 열수 추출물 및 고분자 단백질(분자량 12,000이상) 분획을 갖는 아프리카산 상황버섯 냉침 추출물을 포함한다. In addition, the African situation mushroom extract includes an African situation mushroom hydrothermal extract having a polymer polysaccharide (molecular weight 12,000 or more) fraction and an African situation mushroom cold extract having a polymer protein (molecular weight 12,000 or more) fraction.
이하, 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명의 상황버섯 조추출물, 비극성용매 가용추출물, 고분자 다당체 분획을 갖는 열수 추출물 및 고분자 단백질 분획을 갖는 냉침 추출물은 하기와 같이 수득될 수 있다.The crude mushroom extract of the present invention, the nonpolar solvent soluble extract, the hydrothermal extract having the polymer polysaccharide fraction and the cold needle extract having the polymer protein fraction can be obtained as follows.
본 발명의 상황버섯을 채취하여 물로 깨끗이 수세하고 건조 후 균질기 등을 이용하여 마쇄하여 분말화 한 후, 상황버섯 건조중량의 약 2 내지 30 배, 바람직하게는 약 15 내지 30 배에 달하는 부피의 물, 메탄올, 에탄올 및 부탄올과 같은 저급 알콜 또는 이들의 약 1:0.1 내지 1:10의 혼합비를 갖는 혼합용매로, 바람직하게는 메탄올로 20 내지 100 ℃, 바람직하게는 50 내지 70 ℃의 추출 온도에서 약 0.5시간 내지 2일, 바람직하게는 1 시간 내지 1일 동안 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등의 추출방법, 바람직하게는 열수 추출로 1회 내지 5회, 바람직하게는 3회 연속 추출하여 감압 여과하고 여과추출물을 진공회전농축기로 20 내지 100 ℃, 바람직하게는 20 내지 70 ℃에서 감압 농축하여 물, 저급알콜 또는 이들의 혼합용매에, 바람직하게는 메탄올에 가용한 추출물로써 상황버섯 조추출물을 수득할 수 있다.After collecting the situation mushroom of the present invention, washed with water and dried, pulverized by using a homogenizer and the like, and then powdered, the volume of the mushroom mushroom is about 2 to 30 times the dry weight, preferably about 15 to 30 times Lower alcohols such as water, methanol, ethanol and butanol or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably with an extraction temperature of 20 to 100 캜, preferably 50 to 70 캜 with methanol Extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for about 0.5 hours to 2 days, preferably 1 hour to 1 day, preferably 1 to 5 times, preferably 3 Continuous extraction and filtration under reduced pressure, and the filtrate was concentrated under reduced pressure at 20 to 100 ℃, preferably 20 to 70 ℃ with a vacuum rotary concentrator to water, lower alcohol or a mixed solvent thereof, preferably It can be obtained as a crude extract of Phellinus extracts available to all.
본 발명의 비극성용매 가용 추출물은 상기 상황버섯 조추출물을 물에 현탁한 후, 헥산, 에틸아세테이트, 디클로로메탄, 클로로포름과 같은 비극성 용매를 이용하여 추출하여 본 발명의 상황버섯 비극성용매 가용 추출물을 수득할 수 있다. 좀 더 구체적으로는 상황버섯 조추출물, 바람직하게는 상황버섯 메탄올 추출물에 상황버섯 건조중량의 약 2 내지 30 배, 바람직하게는 약 15 내지 30 배에 달하는 부피의 물을 넣고 잘 저으면서 녹인 후, 여기에 일정량의 헥산 또는 에틸아세테이트를 혼합한 후, 3∼4 차례 반복, 분획하여 각각의 헥산 가용성 추출물 및 에틸아세테이트 가용성 추출물과 같은 비극성 용매 가용 추출물을 수득할 수 있다. The non-polar solvent soluble extract of the present invention is obtained by suspending the crude mushroom crude extract in water and then extracting it using a non-polar solvent such as hexane, ethyl acetate, dichloromethane and chloroform to obtain the non-polar solvent soluble extract of the present invention. Can be. More specifically, the crude mushroom extract, preferably the mushroom mushroom methanol extract in a volume of water of about 2 to 30 times the dry weight of the mushroom, preferably about 15 to 30 times, and then stir well to dissolve After mixing a certain amount of hexane or ethyl acetate, and repeated three to four times, fractionation can be obtained non-polar solvent soluble extract, such as each hexane soluble extract and ethyl acetate soluble extract.
또한, 아프리카산 상황버섯 열수 추출물은 아프리카산 상황버섯을 물로 깨끗이 수세하고 건조시킨 다음, 건조상태의 상황버섯을 균질기를 이용하여 잘게 분쇄한 후, 상황버섯 건조 중량의 약 2 내지 30 배, 바람직하게는 약 3 내지 10 배에 달하는 부피의 물을 넣고 가열 멘틀을 이용하여 약 1 내지 24 시간, 바람직하게는 6 시간 동안 열수 추출한 후, 여지로 여과하여 수득한 열수 추출액에 1 내지 5 배, 바람직하게는 3 배의 저급알콜, 바람직하게는 냉(冷) 에탄올을 첨가하여 약 0 내지 10 ℃, 바람직하게는 4 ℃에서 약 12 내지 48 시간, 바람직하게는 24 시간 동안 침전시킨 다음, 상층액을 제거하고 원심분리하여 침전물을 모아 용매를 증발제거시킨 다음, 약 50 내지 100 ℃, 바람직하게는 60 ℃에서 증류수로 펠렛(pellet)을 재용해시켜 원심분리하는 과정을 2 내지 5 회 반복수행하여 수득된 상층액을 0 내지 10 ℃, 바람직하게는 4 ℃에서 약 12 내지 72 시간, 바람직하게는 48 시간 동안 증류수를 이용하여 투석한 후 동결건조하여 고분자 다당체(분자량 12,000 이상) 분획을 갖는 본 발명의 아프리카산 상황버섯 열수 추출물을 수득할 수 있다.In addition, the African mushroom mushroom hot water extract is washed with water and dried African mushroom mushrooms with water, and then finely pulverized the dried mushroom mushrooms using a homogenizer, about 2 to 30 times the dry weight of the mushroom mushrooms, preferably Is about 3 to 10 times the volume of water and hydrothermal extraction for about 1 to 24 hours, preferably 6 hours using a heating mantle, and then filtrate 1 to 5 times, preferably in the hydrothermal extract obtained by filtration Is precipitated at about 0 to 10 ° C., preferably 4 ° C. for about 12 to 48 hours, preferably 24 hours by adding 3 times lower alcohol, preferably cold ethanol, and then removing the supernatant. And centrifugation to collect the precipitate to evaporate the solvent, and then re-dissolve the pellet in distilled water at about 50 to 100 ℃, preferably 60 ℃ centrifugation process 2 to 5 The supernatant obtained by the repeated repetition was dialyzed with distilled water at 0 to 10 ° C., preferably at 4 ° C. for about 12 to 72 hours, preferably 48 hours, and then lyophilized to obtain a polymer polysaccharide (molecular weight of 12,000 or more). African situation mushroom hot water extract of the present invention can be obtained.
또한, 본 발명의 아프리카산 상황버섯 냉침 추출물은 아프리카산 상황버섯을 채취하여 물로 깨끗이 수세하고 건조시킨 다음, 건조상태의 상황버섯을 균질기를 이용하여 잘게 분쇄한 후, 완충용액을 가하고 약 0 내지 10 ℃, 바람직하게는 4 ℃에서 약 12 내지 24 시간 방치하여 냉침 추출한 후, 추출액을 여과하여 단백질 침전제로 에탄올, 아세톤 또는 황상암모늄(Ammonium sulfate)과 같은 염, 바람직하게는 황산암모늄을 가하고 포화상태로 한 후, 원심분리하여 상층액을 제거하고 수득한 침전물을 완충용액으로 재용해시킨 다음, 재용해된 추출물을 완충용액을 이용하여 0 내지 10 ℃, 바람직하게는 4 ℃에서 50 내지 100 시간 동안, 바람직하게는 74 시간 동안 투석한 후, 단백질 양을 정량하여 고분자 단백질(분자량 12,000이상)분획을 갖는 아프리카산 상황버섯 냉침 추출물을 수득할 수 있다. In addition, the African mushroom mushroom extract of the present invention, the African mushroom mushroom extract, washed with water and dried thoroughly with water, and after grinding the dried mushrooms in a fine state using a homogenizer, adding a buffer solution about 0 to 10 After cold extraction with standing at 12 DEG C., preferably at 4 DEG C for about 12 to 24 hours, the extract was filtered and added with a protein precipitant to a salt such as ethanol, acetone or ammonium sulfate, preferably ammonium sulfate, and saturated. After centrifugation, the supernatant was removed, and the precipitate obtained was redissolved with a buffer solution. Preferably, after dialysis for 74 hours, the amount of protein quantitatively determines the amount of protein protein (molecular weight 12,000 or more) of African situation mushroom cold It is possible to obtain an extract.
본 발명은 상기 제조방법으로 얻어지는 인유두종 바이러스로 인한 질환의 예방 및 치료에 효과적인 아프리카산 상황버섯 추출물을 제공한다.The present invention provides an African situation mushroom extract effective in the prevention and treatment of diseases caused by human papilloma virus obtained by the above method.
또한, 본 발명의 아프리카산 상황버섯 추출물을 유효성분으로 함유하는 인유두종 바이러스로 인한 질환의 예방 및 치료에 효과적인 조성물을 제공한다.In addition, the present invention provides a composition effective in the prevention and treatment of diseases caused by human papillomavirus containing the African situation mushroom extract of the present invention as an active ingredient.
본 발명의 인유두종 바이러스로 인한 질환의 예방 및 치료용 조성물은, 조성물 총 중량에 대하여 상기 추출물을 0.1 내지 80 중량 %, 바람직하게는 1.0 내지 50 중량 %를 포함한다.The composition for the prevention and treatment of diseases caused by human papilloma virus of the present invention, the total weight of the composition comprises 0.1 to 80% by weight, preferably 1.0 to 50% by weight.
본 발명의 추출물의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 분획물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다. Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active fractions, as well as in any suitable collection.
본 발명의 아프리카산 상황버섯 추출물을 포함하는 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다. 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. The composition comprising the African situation mushroom extract of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of a pharmaceutical composition. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명에 따른 추출물을 포함하는 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상세하게는, 제제화할 경우 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 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. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 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.Compositions comprising extracts according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used. Specifically, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used when formulated. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
아프리카산 상황버섯 추출물의 사용량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으나, 일반적으로 0.01 내지 500 mg/㎏의 양, 바람직하게는 0.1 내지 100∼200 mg/㎏의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다. 또한 추출물의 투여량은 투여경로, 질병의 종류 및 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The amount of African situation mushroom extract may vary depending on the age, sex and weight of the patient, but in general, the amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 to 200 mg / kg Can be administered in several divided doses. In addition, the dosage of the extract may be increased or decreased depending on the route of administration, the type and severity of the disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 (intracerebroventricular) 주사에 의해 투여될 수 있다. The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
본 발명의 아프리카산 상황버섯 추출물 자체는 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용 시에도 안심하고 사용할 수 있다. African situation mushroom extract itself of the present invention has little toxicity and side effects, so can be used with confidence even for long-term administration for the purpose of prevention.
본 발명은 상기에 기재된 인유두종 바이러스로 인한 질환의 예방 및 개선 효과를 나타내는 아프리카산 상황버섯 추출물 외에 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 건강기능식품을 제공한다.The present invention provides a health functional food comprising a food supplement acceptable food additives in addition to African situation mushroom extract exhibiting the effect of preventing and improving the disease caused by the human papillomavirus described above.
본 발명의 추출물들을 포함하는 조성물은 인유두종 바이러스로 인한 질환의 예방 및 개선을 위한 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 아프리카산 상황버섯 추출물을 첨가할 수 있는 식품으로는, 각종 식품류, 예를 들어, 캔디, 초콜릿, 음료, 껌, 차, 비타민 복합제, 건강 기능 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.Compositions comprising the extracts of the present invention can be used in a variety of food and beverages for the prevention and improvement of diseases caused by human papilloma virus. Foods to which the African situation mushroom extract of the present invention can be added include various foods, for example, candy, chocolate, beverages, gums, teas, vitamin complexes, health functional foods, and the like, powders, granules, tablets, It can be used in the form of a capsule or a beverage.
이때, 식품 또는 음료 중의 상기 추출물의 양은, 일반적으로 본 발명의 건강 기능 식품 조성물의 경우 전체 식품 중량의 0.01 내지 50 중량 %, 바람직하게는 0.1 내지 20 중량 %로 가할 수 있으며, 건강 기능 음료 조성물의 경우 100 ㎖를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. At this time, the amount of the extract in the food or beverage, in the case of the health functional food composition of the present invention can generally be added to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, In the case of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.
본 발명의 건강 기능 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에는 액체성분에는 특별한 제한점은 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등;과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and sugar alcohols such as xylitol, sorbitol, erythritol and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
이 외 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어, 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 또한, 본 발명의 조성물은 여러가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그다지 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.Other flavors may be advantageously used natural flavors (tautin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.). The composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. Other compositions of the present invention may be used in natural fruit juices and fruit juice beverages and vegetable beverages. The components may be used independently or in combination The ratio of such additives is not critical but is zero per 100 parts by weight of the composition of the present invention. It is generally selected from the range of about 20 parts by weight.
본 발명은 다음의 실시예 및 실험예에 의거하여 더욱 상세히 설명되나, 본 발명이 실시예 또는 실험예에 의해 제한되지는 않는다.The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited to the examples or experimental examples.
실시예 1. 아프리카산 상황버섯 열수 추출물의 제조Example 1. Preparation of hydrothermal extract of African situation mushroom
아프리카산 상황버섯(아프리카 케냐에서 채집)을 10 ㎏ 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조상태의 상황버섯 200 g 을 균질기(homogenizer)를 이용하여 잘게 분쇄한 후, 가열 멘틀(heating mantle)에 상황버섯 20 g 당 물 1 ℓ를 넣고 100 ℃에서 6 시간동안 열수 추출한 후, 여지(filter paper)로 여과(filtering)하여 수득한 열수 추출액 0.8 ℓ에 3배의 냉(cold) 95 % 에탄올을 첨가하여 4 ℃에서 24시간동안 침전시킨 다음, 상층액을 제거하고 2800 rpm, 4 ℃, 20 분의 조건으로 원심분리하여 침전물만을 수집(harves0ting)한 후, 침전물의 에탄올을 증발시킨 후, 60 ℃의 고온에서 증류수로 펠렛(pellet)을 재용해시켜 원심분리하는 과정을 3회 반복수행하여 수득된 상층액을 4 ℃에서 48 시간동안 증류수를 이용하여 투석(투석막 Mw 12,000 이상)하고, 투석이 끝난 후 동결건조하여 고분자 다당체(분자량 12,000 이상) 분획을 갖는 아프리카산 상황버섯 열수 추출물 1 g을 수득하였다.Take 10 kg of African mushrooms (collected from Kenya, Africa), wash them with water, dry them (dried in shaded shade), and then crush 200 g of dried mushrooms using a homogenizer. 1 liter of water per 20 g of mushrooms in a heating mantle, followed by hot water extraction at 100 ° C. for 6 hours, followed by three times cold in 0.8 L of hot water extract obtained by filtering with filter paper. (cold) 95% ethanol was added and precipitated at 4 ° C. for 24 hours, the supernatant was removed, centrifuged at 2800 rpm, 4 ° C. for 20 minutes, and only precipitate was collected (harves0ting). After evaporation of the pellet, the pellet was re-dissolved with distilled water at a high temperature of 60 ° C., and centrifuged three times. The supernatant obtained was dialyzed using distilled water at 4 ° C. for 48 hours (dialysis membrane Mw 12,000 Over) and dialysis After freeze-drying to give the African Phellinus hot water extract 1 g polysaccharide having a high molecular fraction (molecular weight above 12,000).
실시예 2. 아프리카산 상황버섯 냉침 추출물의 제조Example 2 Preparation of Extract from African Mushrooms
아프리카산 상황버섯(아프리카 케냐에서 채집)을 10 ㎏ 을 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조 상태의 상황버섯 200g 을 균질기(homogenizer)를 이용하여 잘게 분쇄한 후, 상황버섯 20 g 당 50 mM 트리스-염산(Tris-HCl, pH 8.0) 완충용액 0.2 ℓ를 가하고 4 ℃에서 하룻밤동안 방치하여 냉침 추출한 후에, 추출액을 여과하여 황산암모늄(Ammonium Sulfate)를 가하고 100 % 포화상태로 한 후 원심분리(8000 g, 20 분)하여 상층액을 제거하고 수득한 침전물을 50 mM 트리스-염산(pH 8.0)과 50 mM 염화나트륨 완충용액으로 재용해 시킨 다음, 재용해된 추출물을 50 mM 트리스-염산(pH 8.0), 50 mM 염화나트륨 완충용액을 이용하여 4 ℃에서 72시간동안 투석(투석막 Mw 12,000 이상)하고, 투석이 끝난 후 단백질 양을 정량하여 고분자 단백질(분자량 12,000 이상) 분획을 갖는 아프리카산 상황버섯 냉침 추출물 15 ㎖ (농도 250㎍/㎖)를 수득하였다.Take 10 kg of African mushrooms (collected from Kenya, Africa), wash them with water, dry them (dried in shaded shade), and then crush 200 g of dried mushrooms using a homogenizer. , 0.2 liter of 50 mM Tris-HCl (pH 8.0) buffer solution per 20 g of the situation mushroom was left at 4 ° C. overnight, followed by cold extraction. The extract was filtered and added with ammonium sulfate (100%). After saturation, the supernatant was removed by centrifugation (8000 g, 20 minutes), and the obtained precipitate was redissolved with 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer, and the redissolved extract was Dialysis (dialysis membrane Mw 12,000 or more) at 4 ° C. using 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer solution for 72 hours, and after the dialysis, the amount of protein was determined to determine the amount of high molecular protein (molecular weight 12,000 or more). 15 ml (concentration 250 μg / ml) of African situation mushroom cold extract having a stroke were obtained.
실시예 3. 아프리카산 상황버섯 조추출물의 제조Example 3. Preparation of African Mushroom Extract
아프리카산 상황버섯(아프리카 케냐에서 채집)을 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조상태의 상황버섯 200 g을 균질기(homogenizer)를 이용하여 잘게 분쇄한 후, 가열 멘틀(heating mantle)에 넣고 메탄올 4 ℓ를 넣은 후, 79 ℃에서 일정시간(12 h) 간격으로 3회 반복 추출하여 얻은 추출액을 여지로 여과한 다음, 회전감압농축기(rotary vacuum evaporator)를 이용하여 감압 농축하여 상황버섯 조추출물 7 g을 수득하였다. Take African situation mushrooms (gathered from Kenya, Africa), wash them with water, dry them (dried in shaded shades), and crush 200 g of dried mushrooms using a homogenizer and heat them. Into a mantle (heating mantle) and 4 liters of methanol, the extract obtained by extracting three times at regular intervals (12 h) at 79 ℃ repeatedly filtered and then filtered using a rotary vacuum evaporator (rotary vacuum evaporator) Concentration under reduced pressure yielded 7 g of crude mushroom extract.
실시예 4. 아프리카산 상황버섯 헥산 가용성 분획물의 제조Example 4 Preparation of Hepatic Mushroom Hexane Fractions from Africa
상기 실시예 3에서 얻은 상황버섯 조추출물 즉, 상황버섯 메탄올 추출물 6.5 g을 300 ㎖ 3차 증류수에 넣고 1시간 가량 저으면서 녹인 후, 헥산 1.5 ℓ를 가하여 혼합하고 3차례 반복 분획하여 헥산 가용성 분획물을 얻은 후, 이 헥산 가용성 분획물을 여과 후 감압 농축시켜 상황버섯 헥산 가용성 추출물 0.5 g을 수득하였고, 4 ℃ 냉장고에 보관하면서 시료로 사용하였다.The crude mushroom extract obtained in Example 3, that is, 6.5 g of methanol extract of mushroom mushrooms was dissolved in 300 ml of tertiary distilled water, and stirred for about 1 hour. Then, 1.5 L of hexane was added, mixed and repeated three times to obtain a hexane-soluble fraction. Thereafter, the hexane soluble fraction was concentrated under reduced pressure after filtration to obtain 0.5 g of a mushroom hexane soluble extract, which was used as a sample while being stored in a 4 ° C. refrigerator.
실시예 5. 아프리카산 상황버섯 에틸아세테이트 가용성 분획물의 제조Example 5 Preparation of Ethyl Acetate Soluble Fraction from African Mushrooms
상기 실시예 3에서 얻은 상황버섯 조추출물 즉, 상황버섯 메탄올 추출물 6.5 g을 300 ㎖ 3차 증류수에 넣고 1시간 가량 저으면서 녹인 후, 에틸아세테이트 1.5 ℓ를 가하여 혼합한 후 3차례 반복, 분획하여 에틸아세테이트 가용성 분획물을 얻은 후, 이 에틸 아세테이트 가용성 분획물을 여과 후 감압 농축시켜 상황버섯 에틸 아세테이트 가용성 추출물 2.5 g을 수득하여 시료로 사용하였고, 4 ℃ 냉장고에 보관하면서 시료로 사용하였다. The crude mushroom extract obtained in Example 3, that is, 6.5 g of methanol extract of mushroom mushrooms was dissolved in 300 ml of tertiary distilled water, and stirred for about 1 hour. Then, 1.5 L of ethyl acetate was added to the mixture, and the mixture was repeatedly mixed and fractionated three times. After obtaining the soluble fraction, the ethyl acetate soluble fraction was concentrated under reduced pressure after filtration to obtain 2.5 g of the ethyl acetate soluble extract of the situation mushroom, which was used as a sample, and used as a sample while being stored in a 4 ° C. refrigerator.
실험예 1. 아프리카산 상황버섯 추출물의 항 HBV 효과Experimental Example 1.Anti-HBV effect of African situation mushroom extract
1-1. 검체 및 시료의 준비1-1. Sample and Sample Preparation
본 실험에 사용한 검체는 자궁경부암에 걸린 환자의 가검물에서 검출한 것으로 HPV 16, 18, 33번 타입(Type)으로, 검체 및 모든 시약은 냉장고에서 꺼내어 실온화 시켰고, 항온수저와 마이크로 플레이트(microplate heater) 온도는 미리 65 ℃로 맞추어 놓았으며, 음성 검량기(Negative cal; 이하 NC), 검량기 A(Cal A) 및 검량기 B(Cal B)는 각각 3개씩 준비하였다. The samples used in this experiment were detected in the specimens of patients with cervical cancer. HPV 16, 18, and 33 types were used. The samples and all reagents were removed from the refrigerator and allowed to room temperature. A thermostat and a microplate heater were used. ), The temperature was previously set at 65 ° C., and three negative checkers (Negative cal; NC), checker A (Cal A), and checker B (Cal B) were prepared.
본 실험에 사용하는 시료는 아프리카산 상황버섯 열수 추출물(상기 실시예 1), 냉침 추출물(상기 실시예 2), 조추출물(상기 실시예 3), 헥산 추출물(상기 실시예 4) 및 에틸 아세테이트 추출물(상기 실시예 5)이며, 모든 시료는 혈청 50 ㎕에 시료 50 ㎕의 비율로 반응시켰으며, 각 시료는 혈청과 2시간, 8시간 동안 37 ℃에서 반응시킨 뒤 냉장 보관 후 계속 실험에 사용하였고, 각 시료의 농도는 아프리카산 상황버섯 1 mg/㎖, 대조군은 혈청 50 ㎕ + PBS(완충액) 50 ㎕였다.Samples used in this experiment are the African situation mushroom hot water extract (Example 1), cold extract (Example 2), crude extract (Example 3), hexane extract (Example 4) and ethyl acetate extract (Example 5), and all samples were reacted with 50 μl of serum at a rate of 50 μl, and each sample was reacted with serum at 37 ° C. for 2 hours and 8 hours, and then used in experiments after refrigerated storage. The concentration of each sample was 1 mg / mL of African situation mushroom, and 50 μl of serum + 50 μl of PBS (buffer).
1-2. 각 추출물의 항 HPV 효과 측정1-2. Determination of the anti-HPV effect of each extract
각 추출물의 항 HPV 효과를 측정하기 위하여 HPV 검사 키트(Digene Hybrid Capture Ⅱ HPV DNA Test Kit 2480, Digene사)를 사용하여 하기와 같은 단계로 실험을 수행하였다.In order to measure the anti-HPV effect of each extract, the experiment was performed using the HPV test kit (Digene Hybrid Capture II HPV DNA Test Kit 2480, Digene) as follows.
1-2-1. 변성(Denaturation)1-2-1. Denaturation
변성 시약 용기(Denaturation Reagent bottle)에 지시약 염료(Indicator dye) 5방울을 넣고 혼합하여 짙은 자색을 띄게 한 후, 변성 시약(Denaturation Reagent)을 검체와 양성 검량기 튜브(positive calibrator tube)에 500 ㎕씩 분주하고, 음성 검량기(negative calibratior)에는 1 ㎖를 분주한 다음, 5초 동안 잘 혼합해 주어 검량기와 검체가 자색을 띄게 하고, 65 ±0 ℃ 수욕상(water bath)에서 45 ±5분 동안 배양(이때 튜브속의 검체가 완전히 물에 잠기어야 함)하며, 배양하는 동안 탐색자 혼합물(probe mixture)을 준비한다.Add 5 drops of indicator dye to the Denaturation Reagent bottle to make it dark purple, and add 500 ml of Denaturation Reagent to the sample and positive calibrator tube. Dispense 1 ml into a negative calibratior and mix well for 5 seconds to make the calibrator and the sample purple, 45 ± 5 minutes in a 65 ± 0 ° C water bath Incubate (the sample in the tube must be completely submerged) and prepare a probe mixture during the incubation.
1-2-2. 탐색자 혼합물(Probe mixture) 준비1-2-2. Probe mixture preparation
HPV 탐색자 B 용기(HPV probe B vial)를 3-5초간 원심 분리한 후, 가볍게 바닥에 두드리고, HPV 탐색자 B를 탐색자 희석기(probe diluent)에 1:25로 희석(희석액 1.75 ㎖ + 탐색자 70 ㎕)하여, 최대속도에서 적어도 5초동안 혼합했다.Centrifuge the HPV probe B vial for 3-5 seconds, then tap on the bottom lightly and dilute HPV probe B to 1:25 in the probe diluent (1.75 mL of diluent + 70 μL of probe). At least for 5 seconds at maximum speed.
1-2-3. 이종교배(HYBRIDIZATION)1-2-3. HYBRIDIZATION
조제된 HPV 탐색자 A 또는 B를 각각의 평판에 25 ㎕씩 분주하고, 수욕상에서 검체를 꺼내 5초 동안 혼합한 후, 각각의 검량기(calibrator)와 검체를 미리 HPV 프로버크(proberk)가 첨가된 평판에 75 ㎕씩 분주(이 때 검체 간 오염에 주의함)한 다음, 평판 덮개(plate sealer)를 덮은 후, 1100 ±100 rpm에서 3 ±2분 동안 흔들어주어(이때 각 검량기와 검체는 노란색을 나타냄) 노란색이 나타나지 않으면 탐색자를 25 ㎕ 더 첨가하고, 평판 덮개를 덮은 후, 65 ±2 ℃ 히터(heater)에서 60 ±5분 동안 배양했다.Dispense 25 μl of the prepared HPV probe A or B into each plate, remove the sample from the water bath, mix for 5 seconds, and then add each of the calibrator and the sample to which HPV proberk was added in advance. Dispense 75 μl onto the plate (note the contamination between samples), cover the plate sealer, and shake for 3 ± 2 minutes at 1100 ± 100 rpm. If no yellow color appears, add 25 μl of probe, cover the plate, and incubate for 60 ± 5 minutes on a 65 ± 2 ° C heater.
1-2-4. 이종 포착(HYBRID CAPTURE) 및 이종 검출(HYBRID DETECTION)1-2-4. HYBRID CAPTURE and HYBRID DETECTION
이종 포착을 위해 히터에서 평판을 꺼낸 후, 포착 마이크로플레이트(Capture Microplate)로 모두 옮기고, 덮개를 이용하여 모두 덮은 뒤 1100 ±100 rpm, 상온에서 1시간동안 배양한 후, 반응이 끝나면 포착 마이크로플레이트의 여액을 피펫을 이용하여 제거한 후 2-3회 흡수지에 두들겨서 여액을 완전 제거하였다.Remove the plate from the heater for heterogeneous capturing, transfer it all to the Capture Microplate, cover everything with a cover, incubate for 1 hour at 1100 ± 100 rpm, room temperature, and then The filtrate was removed using a pipette and then beaten on blotter paper 2-3 times to completely remove the filtrate.
이종 검출을 위해 각각의 포착 마이크로플레이트 평판에 75 ㎕의 검출 시약(Detection Reagent) 1을 분주하고(분주시 웰과 접촉을 피하여 분주함), 덮개를 이용하여 포착 마이크로플레이트를 덮고, 20-25 ℃에서 30분 동안 배양하였다.Dispense 75 μl of Detection Reagent 1 into each capture microplate plate for heterologous detection (dividing avoiding contact with the wells at the time of dispensing), cover the capture microplate with a cover, and at 20-25 ° C. Incubate for 30 minutes.
1-2-5. 세척(WASHING)1-2-5. WASHING
세척용 완충용액(Washing buffer) 100 ㎖를 2.9 ℓ의 증류수에 희석하여 사용하며(총 3 ℓ) 포착 마이크로플레이트 웰(Capture Microplate well)의 여액을 완전히 제거(6회 세척실시)한 다음, 세척한 포착 마이크로플레이트 웰을 깨끗한 흡수지 위에서 뒤집어 10-15분간 방치하였고, 이 때 오염 방지를 위해 검출 시약(Detection Reagent) 2를 분주하기 전까지 뒤집어 놓았다.100 ml of washing buffer is diluted in 2.9 L of distilled water (3 L total), and the filtrate of the Capture Microplate well is completely removed (six times), followed by washing. The capture microplate wells were turned upside down on a clean blotter paper and left for 10-15 minutes at which point they were turned upside down before dispensing Detection Reagent 2 to prevent contamination.
1-2-6. 복제(AMPLIFICATION)1-2-6. Replication (AMPLIFICATION)
각각의 포착 마이크로플레이트 웰에 검출 시약 2를 75 ㎕씩 분주하고, 덮개로 덮은 뒤 실온에서 15분간 직사광선을 피해 배양하였고, 반응이 끝난 뒤 15분 이내에 DML 2000 루미노메터(Luminometer) 장비(Digene사)를 이용하여 측정하였다.75 μl of Detection Reagent 2 was dispensed into each capture microplate well, covered and incubated for 15 minutes in direct sunlight at room temperature, and within 15 minutes after the reaction was completed, a DML 2000 Luminometer instrument (Digene) ) Was measured.
그 결과, 하기 표 1에서 알 수 있는 것처럼, 아프리카 상황버섯 조추출물에서 2시간 후 27 %, 8시간 후 28 %의 DNA 양이 감소함을 확인하였다(도 1a 및 1b 참조).As a result, as can be seen in Table 1, it was confirmed that the amount of DNA decreases in 27% after 2 hours and 28% after 8 hours in the African situation mushroom crude extract (see FIGS.
실험예 2. 아프리카 상황버섯 추출물의 독성 측정 Experimental Example 2. Determination of Toxicity of African Situation Mushroom
2-1. 아프리카 상황버섯 추출물의 세포 독성2-1. Cytotoxicity of African Situation Mushroom Extracts
아프리카산 상황버섯 추출물의 세포 독성을 측정하기 위하여, 장내바이러스에 민감한 포유동물 유래의 세포주인 RD(Rabdomyosarcoma, 6×104/세포)를 사용하여 실험실 내에서 아프리카산 상황버섯 각각의 추출물, 즉 열수 추출물, 조추출물, 헥산 추출물, 에틸 아세테이트 추출물의 세포독성을 MTT 분석법(MTT assay)으로 측정하였으며, 이때 각 추출물의 농도는 1 mg/㎖로 하였다.In order to measure the cytotoxicity of the African mushroom extract, the extract of each of the African mushroom mushrooms, ie, hot water, was used in the laboratory using RD (Rabdomyosarcoma, 6 × 10 4 / cell), a mammalian-derived cell line sensitive to enterovirus Cytotoxicity of the extract, crude extract, hexane extract, ethyl acetate extract was measured by MTT assay (MTT assay), wherein the concentration of each extract was 1 mg / ㎖.
먼저, 세포 단분자층(6×104/세포)을 37 ℃에서 하룻밤 동안 배양한 후, 아프리카산 상황버섯 시료를 10 배 희석하고, 희석된 시료를 100 ㎕가한 다음, 37 ℃에서 3일 동안 배양한 후, MTT 염색약(MTT staining) 50 ㎕를 가하고, 37 ℃에서 2시간 동안 배양한 다음, 배지를 버리고 DMSO 150 ㎕를 가하고, 1 시간동안 휘저은 후 540 nm에서 흡광도를 측정하였다. 그리고 각 추출물의 세포 대조군(PBS) 평균에 대비한 OD 값을 비교하였으며, 세포 대조군의 평균을 일정한 선으로 나타내어 이를 기준으로 이 선보다 높은 것은 세포독성이 없고, 이 선보다 낮은 것은 세포독성이 있는 것으로 판정하였다.First, the cell monolayers (6 × 10 4 / cells) were incubated overnight at 37 ° C., and then the samples of African mushrooms were diluted 10-fold, 100 μl of the diluted samples were incubated at 37 ° C. for 3 days. Then, 50 μl of MTT staining (MTT staining) was added, incubated at 37 ° C. for 2 hours, and then the medium was discarded and 150 μl of DMSO was added, stirred for 1 hour, and the absorbance was measured at 540 nm. And the OD value compared to the average of the cell control (PBS) of each extract was compared, and the average of the cell control is represented by a constant line, based on which the higher than this line is not cytotoxic, the lower than this line is determined to be cytotoxic It was.
그 결과, 아프리카산 상황버섯의 열수 추출물에 대한 세포 독성을 나타낸 하기 표 2 및 그 외 조추출물, 헥산 추출물, 에틸 아세테이트 추출물에 대한 세포 독성을 나타낸 하기 표 3에서 알 수 있는 것처럼, 아프리카산 상황버섯 각각의 추출물들의 유의한 세포독성을 찾을 수 없었다(도 2 및 3 참조).As a result, as shown in Table 2 showing the cytotoxicity of the hydrothermal extracts of African situation mushrooms and the following Table 3 showing the cytotoxicity of the crude extract, hexane extracts, ethyl acetate extract, African situation mushrooms No significant cytotoxicity of each extract was found (see Figures 2 and 3).
2-2. 상황버섯 추출물의 조직 독성2-2. Histotoxicity of Situation Mushroom Extract
아프리카산 상황버섯 추출물의 조직 독성을 측정하기 위하여, 수술시 적출되는 조직중 염색소견상 정상조직으로 판정된 포유동물 유래의 조직인 대장 조직(Nude mouse : normal colon)을 사용하여 아프리카산 상황버섯 조추출물, 헥산 추출물, 에틸 아세테이트 추출물의 조직 독성 실험을 측정하기 위하여 MTT 분석법을 실시하여, 여러 가지 오차를 고려하여 저해율(IR)이 30 % 이상인 경우만 그 추출물에 대하여 독성이 있다고 판단하였다. 저해율은 하기 수학식 1로 계산하였다.In order to measure the histotoxicity of the African situation mushroom extract, the crude extract of African situation mushroom was extracted using a colon mouse (Nude mouse: normal colon), which was found to be normal tissue by staining. In order to measure the tissue toxicity experiments of hexane extracts and ethyl acetate extracts, MTT assays were performed, and it was determined that the extracts were toxic only when the inhibition rate (IR) was 30% or more in consideration of various errors. Inhibition rate was calculated by the following equation (1).
그 결과, 아프리카산 상황버섯 조추출물의 조직 독성은 하기 표 4a 및 도 4a에, 헥산 추출물의 조직독성은 표 4b 및 도 4b에, 에틸 아세테이트 추출물의 조직독성은 표 4c 및 도 4c에 나타내었다. 이 때, 도에는 저해율(IR) 30 %일 때를 일정한 선으로 표시하여, 이 선 이상 막대가 올라간 추출물은 독성이 있다고 판정하였다. 하기 표 4a 내지 4 c(도 4a 내지 4c 참조)에서 볼 수 있는 것처럼, 아프리카산 상황버섯의 각각의 추출물들은 에틸 아세테이트 10 ㎍ 처리농도만 제외하고, 30 % 이상의 저해율을 나타낸 것이 없으므로 정상조직에서 독성이 없는 것으로 판단되었다.As a result, the histotoxicity of the crude extract of African situation mushroom was shown in Tables 4a and 4a, the histotoxicity of hexane extracts in Tables 4b and 4b, and the histotoxicity of ethyl acetate extracts in Tables 4c and 4c. In this case, the time when the inhibition rate (IR) is 30% is indicated by a constant line, and it was determined that the extract having the rod above this line was toxic. As can be seen in Tables 4a to 4c (see FIGS. 4a to 4c), the extracts of the African situation mushrooms were toxic in normal tissues because they showed no inhibition of more than 30% except for 10 ㎍ of ethyl acetate. It was judged that there was no.
참조예 1. 아프리카산 상황버섯 동정Reference Example 1 Identification of African Mushrooms
본 발명의 아프리카산 상황버섯의 종류를 동정하기 위하여, DNA 염기서열을 (주)마이크로 아이디에 의뢰하여 분석하였으며, ITS rDNA 염기서열분석(sequencing)으로 동정하였다. 이 때 먼저, % 유사성(similarity) 값을 구하고, 키무라스 투-파라메터 모델(Kimuras two-parameter model) (Kimura, M. A., J. Mol. Evol., 16, pp111-120, 1980)에 의하여 진화적인 거리(evolutionary distance)를 계산한 다음, 네이버-조이닝(Neighbor-joining) 방법 (Saitou, N. and Nei, M., Mol. Biol. Evol., 4, pp406-425, 1987)으로 계통수(phylogenetic tree)를 작성하였다. 한 편, 계통수(Tree)의 스케일 바(scale bar)는 0.1 또는 0.01 각 위치 당 치환(substitution per site)를 의미하며, 계통수에서 각 가지(branch) 옆의 숫자는 부트스트랩 퍼센테이지(bootstrap percentage)을 의미한다.In order to identify the kinds of African mushrooms of the present invention, DNA sequences were analyzed by Micro ID Co., Ltd., and identified by ITS rDNA sequencing. In this case, first, the% similarity value is obtained, and the evolution is obtained by Kimuras two-parameter model (Kimura, MA, J. Mol. Evol., 16 , pp111-120, 1980). After calculating the evolutionary distance, the phylogenetic tree is determined by the neighbor-joining method (Saitou, N. and Nei, M., Mol. Biol. Evol., 4 , pp406-425, 1987). tree). On the other hand, the tree's scale bar represents 0.1 or 0.01 substitution per site, and the number next to each branch in the tree represents the bootstrap percentage. it means.
본 균주를 ITS 부분의 염기서열에 의해 NCBI 블라스트 서치(blast search)한 결과, 결정된 염기서열의 개수는 701 bp로 그 염기서열은 도 5 및 서열번호 1에 나타내었다. 본 균주는 여러 펠리너스 속(Phellinus genus)에 속하는 진균으로 판명되었다.As a result of NCBI blast search of the strain by the nucleotide sequence of the ITS portion, the determined number of nucleotide sequences was 701 bp, and the nucleotide sequences thereof are shown in FIG. 5 and SEQ ID NO: 1. This strain is a variety of It was found to be a fungus belonging to the genus Phellinus genus .
본 발명의 아프리카산 상황버섯 추출물은 아래와 같은 제형으로 투여할 수 있으며, 아래의 제제예는 본 발명을 예시하는 것일 뿐, 이에 의해 본 발명의 내용이 제한되는 것은 아니다. African situation mushroom extract of the present invention can be administered in the following formulations, the following formulation examples are merely to illustrate the invention, whereby the content of the present invention is not limited.
제제예 1. 주사제제의 제조Formulation Example 1 Preparation of Injection
실시예 3의 아프리카산 상황버섯 조추출물 100 ㎎100 mg of African situation mushroom extract of Example 3
소디움 메타비설파이트 3.0 ㎎Sodium Metabisulfite 3.0 mg
메틸파라벤 0.8 ㎎Methylparaben 0.8 mg
프로필파라벤 0.1 ㎎Propylparaben 0.1 mg
주사용 멸균증류수 적량Proper sterile distilled water for injection
상기의 성분을 혼합하고 통상의 방법으로 최종 부피가 2 ㎖이 되도록 제조한 후, 2 ㎖용량의 앰플에 충전하고 멸균하여 주사제를 제조한다.The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.
제제예 2. 정제의 제조Formulation Example 2 Preparation of Tablet
실시예 3의 아프리카산 상황버섯 조추출물 200 ㎎African situation mushroom extract 200 mg of Example 3
유당 100 ㎎Lactose 100 mg
전분 100 ㎎Starch 100 mg
스테아린산 마그네슘 적량Magnesium stearate proper amount
통상의 정제 제조방법에 따라 상기의 성분을 혼합하고 타정하여 정제를 제조한다.A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.
제제예 3. 캡슐제의 제조Formulation Example 3 Preparation of Capsule
실시예 3의 아프리카산 상황버섯 조추출물 100 ㎎100 mg of African situation mushroom extract of Example 3
유당 50 ㎎Lactose 50 mg
전분 50 ㎎Starch 50 mg
탈크 2 ㎎Talc 2 mg
스테아린산마그네슘 적량Magnesium stearate appropriate amount
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.
제제예 4. 액제의 제조Formulation Example 4 Preparation of Liquid
실시예 3의 아프리카산 상황버섯 조추출물 1000 ㎎1000 mg of crude situation mushroom extract of Example 3
설탕 20 g20 g of sugar
이성화당 20 g20 g of isomerized sugar
레몬향 적량Lemon flavor
정제수를 가하여 전체 1000 ㎖로 맞추었다. 통상의 액제의 제조방법에 따라 상기의 성분을 혼합한 다음, 갈색병에 충전하고 멸균시켜 액제를 제조하였다.Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.
제제예 5. 건강 식품의 제조Formulation Example 5 Preparation of Healthy Food
실시예 3의 아프리카산 상황버섯 조추출물 1000 ㎎1000 mg of crude situation mushroom extract of Example 3
비타민 혼합물 적량Vitamin Mixture
비타민 A 아세테이트 70 ㎍70 μg of Vitamin A Acetate
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B1 0.13 ㎎Vitamin B 1 0.13 mg
비타민 B2 0.15 ㎎Vitamin B 2 0.15 mg
비타민 B6 0.5 ㎎Vitamin B 6 0.5 mg
비타민 B12 0.2 ㎍0.2 μg of vitamin B 12
비타민 C 10 ㎎Vitamin C 10 mg
비오틴 10 ㎍10 μg biotin
니코틴산아미드 1.7 ㎎Nicotinic Acid 1.7 mg
엽산 50 ㎍Folate 50 ㎍
판토텐산 칼슘 0.5 ㎎Calcium Pantothenate 0.5mg
무기질 혼합물 적량Mineral mixture
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 ㎎Calcium Carbonate 100 mg
염화마그네슘 24.8 ㎎Magnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.
제제예 6. 건강 음료의 제조Formulation Example 6 Preparation of Healthy Drink
실시예 3의 아프리카산 상황버섯 조추출물 1000 ㎎1000 mg of crude situation mushroom extract of Example 3
구연산 1000 ㎎Citric acid 1000 mg
올리고당 100 g100 g oligosaccharides
매실농축액 2 gPlum concentrate 2 g
타우린 1 g1 g of taurine
정제수를 가하여 전체 900 ㎖Add 900 ml of purified water
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 ℓ용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. After mixing the above components in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and stored in the refrigerator and then Used to prepare the healthy beverage composition of the invention.
본 발명의 상황버섯 추출물은 인유두종 바이러스의 증식을 억제하는 효과를 가진다. 또한, 생체 내에서 부작용이 없고 약물 내성도 유발하지 않기 때문에 안전하게 장기간 투여할 수 있으므로, 인유두종 바이러스로 인한질환의 치료 및 예방을 위한 약학조성물로서 유용하게 사용될 수 있다. The situation mushroom extract of the present invention has the effect of inhibiting the proliferation of human papilloma virus. In addition, since it can be safely administered for a long time because there is no side effect in vivo and does not cause drug resistance, it can be usefully used as a pharmaceutical composition for the treatment and prevention of diseases caused by HPV.
도 1a 내지 1b는 아프리카산 상황버섯 추출물의 HPV 억제능을 측정한 것으로, Figure 1a to 1b is to measure the HPV inhibitory activity of African situation mushroom extract,
도 1a는 환자의 가검물에서 검출한 HPV와 각각의 추출물을 2시간 동안 반응시킨 결과도이며, Figure 1a is a result of the reaction of HPV and each extract detected in the patient's specimen for 2 hours,
도 2b는 환자의 가검물에서 검출한 HPV와 각각의 추출물을 8시간 동안 반응시킨 결과도이며, Figure 2b is a result of reacting each extract with HPV detected in the patient's specimen for 8 hours,
도 3은 아프리카산 상황버섯의 염기서열을 나타낸 도이다.Figure 3 shows the nucleotide sequence of the African situation mushroom.
<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Composition comprising the extract of African Phellinus mushroom having human papilloma virus activity <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Phellinus genus <400> 1 gaacctgcgg aaggatcatt atcgagtttt tgaaagcgag gcttgctgct ggcgcagaaa 60 cgcgcatgtg cacggccttc gcgctcaaat ccactcaacc ccctgtgcac ctttatcctg 120 tcggagcgag tagcttgaga cctttttggg acgtagtagc cggtaatagt agaaaggagg 180 gtaacagctc cattcgaaag gcgaaaaggc cctaactcga gcgaaaactt tggcttgtat 240 tttataaacc acatttgttg tcctgtgaat gttaatgctc cttgtgggcg agaataaata 300 caactttcaa caacggatct cttggctctc gcatcgatga agaacgcagc gaaatgcgat 360 aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac cttgcgcccc 420 ttggtattcc gaggggcatg cctgtttgag tgtcatgtta acctcaaatc acacttgtct 480 tttggggctt tgtggtttgg acctggaggt ttctgctggc cctgcggtcg gctcctctta 540 aatgcattag ctgggtttcg gctcgcgttt gtggtgtaat agttaattct ttcgcctgag 600 cgcttgcctg atgggcccgc ttctaattgt ctgcttggtc ggacaaggtc ctttggcctt 660 cttgactctt ttgacctcaa atcaggtagg actacccgct g 701<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Composition comprising the extract of African Phellinus mushroom having human papilloma virus activity <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Phellinus genus <400> 1 gaacctgcgg aaggatcatt atcgagtttt tgaaagcgag gcttgctgct ggcgcagaaa 60 cgcgcatgtg cacggccttc gcgctcaaat ccactcaacc ccctgtgcac ctttatcctg 120 tcggagcgag tagcttgaga cctttttggg acgtagtagc cggtaatagt agaaaggagg 180 gtaacagctc cattcgaaag gcgaaaaggc cctaactcga gcgaaaactt tggcttgtat 240 tttataaacc acatttgttg tcctgtgaat gttaatgctc cttgtgggcg agaataaata 300 caactttcaa caacggatct cttggctctc gcatcgatga agaacgcagc gaaatgcgat 360 aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac cttgcgcccc 420 ttggtattcc gaggggcatg cctgtttgag tgtcatgtta acctcaaatc acacttgtct 480 tttggggctt tgtggtttgg acctggaggt ttctgctggc cctgcggtcg gctcctctta 540 aatgcattag ctgggtttcg gctcgcgttt gtggtgtaat agttaattct ttcgcctgag 600 cgcttgcctg atgggcccgc ttctaattgt ctgcttggtc ggacaaggtc ctttggcctt 660 cttgactctt ttgacctcaa atcaggtagg actacccgct g 701
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