KR100531644B1 - Composition comprising the extract of African Ganoderma mushroom having human papilloma virus activity - Google Patents
Composition comprising the extract of African Ganoderma mushroom having human papilloma virus activity Download PDFInfo
- Publication number
- KR100531644B1 KR100531644B1 KR10-2003-0071685A KR20030071685A KR100531644B1 KR 100531644 B1 KR100531644 B1 KR 100531644B1 KR 20030071685 A KR20030071685 A KR 20030071685A KR 100531644 B1 KR100531644 B1 KR 100531644B1
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- KR
- South Korea
- Prior art keywords
- extract
- ganoderma lucidum
- african
- sample
- african ganoderma
- Prior art date
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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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- 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
- A61K36/074—Ganoderma
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/208—Fungi extracts
Abstract
본 발명은 인유두종 바이러스(HPV) 억제능을 갖는 아프리카산 영지버섯(Ganoderma genus) 추출물을 함유하는 조성물에 관한 것으로, 본 발명의 아프리카산 영지버섯 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 Ganoderma genus extract having an HPV inhibitory ability, and the African Ganoderma genus extract of the present invention inhibits the growth of HPV by antiviral effects. 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 영지버섯 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 papillomavirus containing an African Ganoderma lucidum extract having the ability to inhibit human papilloma virus.
인유두종 바이러스(Human Papilloma Virus; 이하 HPV)는 환상의 이중나선 DNA와 정 20면체의 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.
본 발명의 아프리카산 영지버섯(Ganoderma genus)은 불로초과(Ganodermataceae), 불로초속(Ganoderma)에 속하며, 아프리카 케냐의 고산 밀림지대에서 야생상태로 자생하는 활엽수 등에 기생하여 자라고 있는 버섯이다. 이곳의 생태적 특징은 적도부근의 열대지역임에도 불구하고 해발 5000 m급의 고산지대로서 버섯의 생육에 최적환경을 갖추고 있다. 목질이 강한 활엽수의 고사목과 그루터기에 자생하며 그루터기에 직각으로 자란다. 그 모양은 말발굽 모양으로써 원형이다. 표면은 매끈하고 콩팥형, 반원형의 형태를 가진다. 앞면은 처음에 황백색을 띠고 있으나 생장하면서 먼저 자란 부분부터 적갈색 내지 자갈색으로 변해간다. 뒷면은 황백색을 띠고 관공이 무수히 나 있다. 대는 갓의 표면과 같은 색으로 약간 굴곡이 생긴다. 가벼운 접촉에 의해 흰색의 관공 표면에 갈색반점이 나타나기 때문에 예술가의 모자(artist conk)라 불리어진다.African Reishi mushroom (Ganoderma genus) of the present invention is a mushroom growing to parasitic bulrochogwa (Ganodermataceae), it belongs to the genus bulrocho (Ganoderma), such as broad-leaved trees growing wild in the wild state in alpine jungle of Kenya. Although the ecological feature of this area is tropical near the equator, it is an alpine area of 5000m above sea level and has the optimal environment for mushroom growth. It grows on dead wood and stump of hardwood hardwood, and grows at right angle to stump. The shape is round in shape of a horseshoe. The surface is smooth and has the shape of a kidney, semicircle. The front is yellowish white at first, but grows from reddish brown to reddish brown. The back side is yellowish white and has a lot of bureaucracy. The stem is slightly curved with the same color as the surface of the lampshade. It is called an artist's hat because light spots show brown spots on the surface of white pores.
기억력 감소 방지, 노화억제, 생리활성 세포활성, 항염, 항균작용, 혈압 강화작용, 항알레르기, 항히스타민, 항남성호르몬작용 등에 유효한 것으로 알려져 있다. 그 외 항암효과를 나타내며, 추출물 역시 간암, 폐암 및 기타 여러 암들에 대한 우수한 치료 및 예방효과를 가짐이 임상실험에 의해 입증되었다. It is known to be effective in preventing memory loss, inhibiting aging, physiologically active cell activity, anti-inflammatory, antibacterial action, blood pressure strengthening action, anti-allergy, antihistamine, anti-male hormone action. In addition, it has anti-cancer effects, and the extract has also been proved by clinical trials to have excellent therapeutic and preventive effects on liver cancer, lung cancer and many other cancers.
그러나, 지금까지 상기 아프리카산 영지버섯의 항 HPV효과에 대해서는 연구되어진 바 없다.However, the anti-HPV effect of the African Ganoderma lucidum has not been studied until now.
본 발명자는 각종 바이러스 질환으로 고생하던 특정지역 주민들이 영지버섯의 끓인 물을 복용하던 중 탁월한 치료효과를 보인 것에 착안하여, 아프리카산 영지버섯 추출물 및 세부분획물의 HPV 억제능을 실험한 결과, 그 탁월한 효능을 확인하여 본 발명을 완성하였다.The present inventors focused on the excellent treatment effect of the residents of a particular region suffering from various viral diseases while taking boiled water of Ganoderma lucidum mushroom, and tested the HPV inhibitory ability of African Ganoderma lucidum mushroom extracts and subfractions, its excellent efficacy It was confirmed to complete the present invention.
본 발명은 아프리카산 영지버섯 추출물을 함유하는 인유두종 바이러스로 인한 질환의 예방 및 치료용 조성물로써 의약품 및 건강기능식품을 제공하는 것이다. The present invention is to provide a pharmaceutical and dietary supplement as a composition for the prevention and treatment of diseases caused by human papillomavirus containing African Ganoderma lucidum extract.
상기 목적을 달성하기 위하여, 본 발명은 아프리카산 영지버섯(Ganoderma 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 an African Ganoderma genus extract.
상기 영지버섯은 아프리카 케냐, 우간다, 탄자니아 등의 동남부 아프리카 지역, 바람직하게는 케냐에 자생하는 영지버섯을 포함한다.The ganoderma lucidum mushroom includes ganoderma lucidum 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 영지버섯 추출물은 조추출물 또는 비극성 용매 가용성 추출물을 포함하며, 본 발명의 조추출물은 물, 에탄올, 메탄올, 부탄올과 같은 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물 또는 메탄올에 가용한 추출물을 포함하며, 비극성 용매 가용추출물은 헥산, 디클로로메탄 또는 에틸 아세테이트와 같은 비극성 용매, 바람직하게는 헥산 또는 에틸 아세테이트에 가용한 추출물을 포함한다.The Ganoderma lucidum extract includes a crude extract or a non-polar solvent soluble extract, and the crude extract of the present invention comprises a lower alcohol such as water, ethanol, methanol, butanol, or a mixed solvent thereof, preferably an extract available in water or methanol. And non-polar solvent soluble extracts include extracts soluble in non-polar solvents such as hexane, dichloromethane or ethyl acetate, preferably hexane or ethyl acetate.
또한, 상기 아프리카산 영지버섯 추출물은 고분자 다당체(분자량 12,000이상)분획을 갖는 아프리카산 영지버섯 열수 추출물 및 고분자 단백질(분자량 12,000이상)분획을 갖는 아프리카산 영지버섯 냉침 추출물을 포함한다. In addition, the African Ganoderma lucidum mushroom extract includes an African Ganoderma lucidum hydrothermal extract having a polymer polysaccharide (molecular weight 12,000 or more) fraction and an African Ganoderma lucidum mushroom extract having a high molecular 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 영지버섯 조추출물, 비극성용매 가용추출물, 고분자 다당체 분획을 갖는 열수 추출물 및 고분자 단백질로서의 냉침 추출물은 하기와 같이 수득될 수 있다.The ganoderma lucidum extract of the present invention, a nonpolar solvent soluble extract, a hydrothermal extract having a polymer polysaccharide fraction, and a cold needle extract as a polymer protein can be obtained as follows.
본 발명의 영지버섯을 채취하여 물로 깨끗이 수세하고 건조 후 균질기 등을 이용하여 마쇄하여 분말화 한 후, 영지버섯 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 영지버섯 조추출물을 수득할 수 있다.After collecting the Ganoderma lucidum mushroom of the present invention, washed with water and dried, pulverized using a homogenizer and the like, and then powdered, Ganoderma lucidum mushroom has a volume of 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 Ganoderma lucidum extract available in coming.
본 발명의 비극성용매 가용 추출물은 상기 영지버섯 조추출물을 물에 현탁한 후, 헥산, 에틸아세테이트, 디클로로메탄, 클로로포름과 같은 비극성 용매를 이용하여 추출하여 본 발명의 영지버섯 비극성용매 가용 추출물을 수득할 수 있다. 좀 더 구체적으로는 영지버섯 조추출물, 바람직하게는 영지버섯 메탄올추출물에 영지버섯 건조중량의 약 2 내지 30 배, 바람직하게는 약 15 내지 30 배에 달하는 부피의 물을 넣고 잘 저으면서 녹인 후, 여기에 일정량의 헥산 또는 에틸 아세테이트를 혼합한 후, 3∼4 차례 반복, 분획하여 각각의 헥산 가용성 추출물 및 에틸 아세테이트 가용성 추출물과 같은 비극성 용매 가용 추출물을 수득할 수 있다.The non-polar solvent soluble extract of the present invention, the suspension of the Ganoderma lucidum crude extract in water, and then extracted using a non-polar solvent such as hexane, ethyl acetate, dichloromethane, chloroform to obtain the Ganoderma lucidum non-polar solvent soluble extract of the present invention. Can be. More specifically, in the Ganoderma lucidum crude extract, preferably Ganoderma lucidum methanol extract, add about 2 to 30 times the dry weight of Ganoderma lucidum mushroom, preferably about 15 to 30 times the volume of water, and then stir well to dissolve it. After mixing a certain amount of hexane or ethyl acetate, it may be repeated 3 to 4 times, and fractionated to obtain a nonpolar solvent soluble extract such as each hexane soluble extract and ethyl acetate soluble extract.
또한, 아프리카산 영지버섯 열수 추출물은 아프리카산 영지버섯을 물로 깨끗이 수세하고 건조시킨 다음, 건조상태의 영지버섯을 균질기를 이용하여 잘게 분쇄한 후, 영지버섯 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 약 1 내지 5 배, 바람직하게는 3 배의 저급알콜, 바람직하게는 냉(冷)에탄올을 첨가하여 약 0 내지 10 ℃, 바람직하게는 4 ℃에서 12 내지 48 시간, 바람직하게는 24 시간 동안 침전시킨 다음, 상층액을 제거하고 원심분리하여 침전물을 모아 용매를 증발제거시킨 후, 약 50 내지 100 ℃, 바람직하게는 60 ℃에서 증류수로 펠렛(pallet)을 재용해시켜 원심분리하는 과정을 2 내지 5 회 반복수행하여 수득된 상층액을 약 0 내지 10 ℃, 바람직하게는 4 ℃에서, 약 12 내지 72 시간, 바람직하게는 48 시간 동안 증류수를 이용하여 투석한 후 동결건조하여 고분자 다당체(분자량 12,000이상) 분획을 갖는 본 발명의 아프리카산 영지버섯 열수 추출물을 수득할 수 있다.In addition, the African Ganoderma lucidum mushroom hydrothermal extract is washed with water and dried African Ganoderma lucidum with water, and then pulverized dried Ganoderma lucidum using a homogenizer, and then about 2 to 30 times the dry weight of Ganoderma lucidum mushroom, preferably Is about 3 to 10 times the volume of water and hot water extraction for about 1 to 24 hours, preferably 6 hours using a heating mantle, and then about 1 to 5 times, preferably in the hydrothermal extract obtained by filtration Preferably, three times lower alcohols, preferably cold ethanol, are added to precipitate at about 0 to 10 DEG C, preferably 4 DEG C for 12 to 48 hours, preferably 24 hours, and then the supernatant is removed. After centrifugation to collect the precipitate to remove the solvent by evaporation, the process of centrifugation by re-dissolving the pellet (pellet) with distilled water at about 50 to 100 ℃, preferably 60 ℃ 2 to 5 times The supernatant obtained by repeating is dialyzed with distilled water at about 0 to 10 ℃, preferably 4 ℃, for about 12 to 72 hours, preferably 48 hours, and then lyophilized to give a polymer polysaccharide (molecular weight of 12,000 or more). African Ganoderma lucidum hydrothermal extract of the present invention having a fraction can be obtained.
또한, 본 발명의 아프리카산 영지버섯 냉침 추출물은 아프리카산 영지버섯을 채취하여 물로 깨끗이 수세하고 건조시킨 다음, 건조상태의 영지버섯을 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 황산암모늄(Ammonium sulfate)과 같은 염, 바람직하게는 황산암모늄을 가하고 포화상태로 한 후, 원심분리하여 상층액을 제거하고 수득한 침전물을 완충용액으로 재용해 시킨 다음, 재용해된 추출물을 완충용액을 이용하여 약 0 내지 10 ℃, 바람직하게는 4 ℃에서 약 50 내지 100 시간동안, 바람직하게는 74 시간 동안 투석한 후, 단백질 양을 정량하여 고분자 단백질(분자량 12,000이상) 분획을 갖는 아프리카산 영지버섯 냉침 추출물을 수득할 수 있다.In addition, the African Ganoderma lucidum mushroom cold sediment extract of the present invention, after collecting the African Ganoderma lucidum mushroom washed with water and dried, and then crushed the dried Ganoderma lucidum finely 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, and the redissolved extract was then buffered at about 0 to 10 ° C., preferably at 4 ° C. for about 50 to 100 hours. During dialysis, preferably for 74 hours, and then quantitate the amount of protein to produce a fraction of high molecular protein (molecular weight of 12,000 or more) Pop tall can be obtained naengchim extract.
본 발명은 상기 제조방법으로 얻어지는 인유두종 바이러스로 인한 질환의 예방 및 치료에 효과적인 아프리카산 영지버섯 추출물을 제공한다.The present invention provides an African Ganoderma lucidum extract effective for the prevention and treatment of diseases caused by human papilloma virus obtained by the above method.
또한, 본 발명의 아프리카산 영지버섯 추출물을 유효성분으로 함유하는 인유두종 바이러스로 인한 질환의 예방 및 치료에 효과적인 조성물을 제공한다.In addition, the present invention provides an effective composition for the prevention and treatment of diseases caused by human papillomavirus containing the African Ganoderma lucidum 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 영지버섯 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 Ganoderma lucidum extract of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. 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 영지버섯 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 Ganoderma lucidum 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 영지버섯 추출물 자체는 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용 시에도 안심하고 사용할 수 있다. African Ganoderma lucidum extract itself of the present invention has little toxicity and side effects, so can be used with confidence even for prolonged administration for prophylactic purposes.
본 발명은 상기에 기재된 인유두종 바이러스 및 인유두종 바이러스로 인한 질환의 예방 및 개선 효과를 나타내는 아프리카산 영지버섯 추출물 외에 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 건강기능식품을 제공한다.The present invention provides a health functional food comprising a food supplement acceptable additive in addition to the African Ganoderma lucidum extract exhibiting the prevention and improvement effect of the above-described human papillomavirus and the disease caused by the human papilloma virus.
본 발명의 추출물들을 포함하는 조성물은 인유두종 바이러스로 인한 질환의 예방 및 개선을 위한 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 아프리카산 영지버섯 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 Ganoderma lucidum 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 영지버섯 열수 추출물의 제조Example 1 Preparation of Hot Water Extract of African Ganoderma Lucidum Mushroom
아프리카산 영지버섯(아프리카 케냐에서 채집)을 10 ㎏을 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조상태의 영지버섯 20 g을 균질기(homogenizer)를 이용하여 잘게 분쇄한 후, 가열 멘틀(heating mantle)에 영지버섯 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 24시간 동안 침전시킨 다음, 상층액을 제거하고 2800 rpm, 4 ℃, 20 분의 조건으로 원심분리하여 침전물만을 수집(harvesting)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 12,000이상)하고, 투석이 끝난 후 동결건조하여 고분자 다당체(분자량 12,000이상) 분획을 갖는 아프리카산 영지버섯 열수 추출물 1 g을 수득하였다.Take 10 kg of African Ganoderma lucidum mushrooms (from Kenya, Africa), wash them with water and dry them (dried in shaded shade), and then 20 g of dried Ganoderma lucidum mushrooms using a homogenizer. Then, 1 liter of water per 20 g of Ganoderma lucidum was added to a heating mantle, followed by hot water extraction at 100 ° C. for 6 hours, followed by three times with 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, then the supernatant was removed and centrifuged at 2800 rpm, 4 ° C. for 20 minutes to collect only the precipitate. After evaporation of ethanol, the supernatant obtained by re-dissolving the pellet (pellet) with distilled water at a high temperature of 60 ° C. and repeating three times was dialyzed using distilled water at 4 ° C. for 48 hours (dialysis membrane Mw). 12,000 or more), and dialysis After freeze-drying to give a 1 g African Ganoderma hot-water extract with the polysaccharide polymer fraction (molecular weight above 12,000).
실시예 2. 아프리카산 영지버섯 냉침 추출물의 제조Example 2 Preparation of African Ganoderma Lucidum Mushroom Extract
아프리카산 영지버섯(아프리카 케냐에서 채집)을 10 ㎏을 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조 상태의 영지버섯 50 g을 균질기(homogenizer)를 이용하여 잘게 분쇄한 후, 영지버섯 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)을 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 72시간 동안 투석(투석막 Mw 12,000 이상)하고, 투석이 끝난 후 단백질 양을 정량하여 고분자 단백질(분자량 12,000이상) 분획을 갖는 아프리카산 영지버섯 냉침 추출물 15 ㎖(농도 300 ㎍/㎖)를 수득하였다.Ten kilograms of African Ganoderma lucidum mushrooms (collected from Kenya, Africa) were collected, washed with water, dried (dried in shaded shades), and then 50 g of dried Ganoderma lucidum was ground finely using a homogenizer. After adding 0.2 L of 50 mM Tris-HCl (pH 8.0) buffer solution per 20 g of Ganoderma lucidum mushroom, it was allowed to stand overnight at 4 ° C, followed by cold extraction. The extract was filtered and added with ammonium sulfate (Ammonium Sulfate). 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 then the redissolved extract. Dialysis (at 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 minute 15 ml (concentration 300 µg / ml) of African Ganoderma lucidum extract with a stroke was obtained.
실시예 3. 아프리카산 영지버섯 조추출물의 제조Example 3. Preparation of Crude Mushroom Extracts from Africa
아프리카산 영지버섯(아프리카 케냐에서 채집)을 채취하여 물로 깨끗이 수세하고 건조(차광된 음지에서 건조)시킨 다음, 건조상태의 영지버섯 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 영지버섯 조추출물 7 g을 수득하였다. Take an African Ganoderma lucidum mushroom (collected from Kenya, Africa), wash it with water, dry it (dried in shaded shade), and 200 g of dried Ganoderma lucidum using a homogenizer, and then heat it. 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 gave 7 g of ganoderma lucidum extract.
실시예 4. 아프리카산 영지버섯 헥산 가용성 분획물의 제조Example 4. Preparation of African Ganoderma lucidum hexane soluble fraction
상기 실시예 3에서 얻은 영지버섯 조추출물 즉, 영지버섯 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 영지버섯 헥산 가용성 추출물 0.5 g을 수득하였고, 4 ℃ 냉장고에 보관하면서 시료로 사용하였다.The Ganoderma lucidum crude extract obtained in Example 3, that is, 6.5 g of Ganoderma lucidum methanol extract was dissolved in 300 ml tertiary distilled water and stirred for about 1 hour, and then mixed with 1.5 L of hexane and repeatedly fractionated three times to obtain a hexane-soluble fraction. Then, this hexane soluble fraction was filtered and concentrated under reduced pressure to obtain 0.5 g of Ganoderma lucidum hexane soluble extract, which was used as a sample while being stored in a 4 ° C refrigerator.
실시예 5. 아프리카산 영지버섯 에틸아세테이트 가용성 분획물의 제조Example 5 Preparation of Ethyl Acetate Soluble Fraction of African Ganoderma Lucidum Mushroom
상기 실시예 3에서 얻은 영지버섯 조추출물 즉, 영지버섯 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 영지버섯 에틸 아세테이트 가용성 추출물 2.5 g을 수득하여 시료로 사용하였고, 4 ℃ 냉장고에 보관하면서 시료로 사용하였다. The Ganoderma lucidum crude extract obtained in Example 3, that is, 6.5 g of Ganoderma lucidum methanol extract was added to 300 ml of tertiary distilled water and stirred for 1 hour, and then dissolved by adding 1.5 L of ethyl acetate, followed by mixing three times, fractionating and ethyl acetate. After the soluble fraction was obtained, the ethyl acetate soluble fraction was concentrated under reduced pressure after filtration to obtain 2.5 g of Ganoderma lucidum ethyl acetate soluble extract, which was used as a sample, and used as a sample while being stored in a 4 ° C. refrigerator.
실험예 1. 아프리카산 영지버섯 추출물의 항 HPV 효과Experimental Example 1.Anti HPV effect of African Ganoderma lucidum extract
1-1. 검체 및 시료의 준비1-1. Sample and Sample Preparation
본 실험에 사용한 검체는 자궁경부암에 걸린 환자의 가검물에서 검출한 것으로 HPV 16, 17번 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, 17 type, and the samples and all reagents were removed from the refrigerator and allowed to room temperature, and the temperature of the thermostat and the microplate heater. 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 영지버섯 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 영지버섯 1 mg/㎖, 대조군은 혈청 50 ㎕ + PBS(완충액) 50 ㎕였다.Samples used in this experiment are African Ganoderma lucidum hot water extract (Example 1), cold sediment 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 Ganoderma lucidum mushroom, and the control group had 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 ±2 분 동안 흔들어 주어(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 영지버섯 헥산 추출물에서 2시간 후 26 %, 8시간 후 30 %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 26% after 2 hours, 30% after 8 hours in the African Ganoderma lucidum hexane extract (see FIGS. 1A and 1B).
실험예 2. 아프리카산 영지버섯 추출물의 독성 측정Experimental Example 2. Determination of Toxicity of African Ganoderma lucidum Extract
2-1. 아프리카산 영지버섯 추출물의 세포 독성2-1. Cytotoxicity of African Ganoderma Lucidum Extract
아프리카산 영지버섯 추출물의 세포 독성을 측정하기 위하여, 장내바이러스에 민감한 포유동물 유래의 세포주인 RD(Rabdomyosaarcoma, 6×104/세포)를 사용하여 실험실 내에서 아프리카산 영지버섯 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.African to measure the cytotoxicity of the acid Ganoderma lucidum extract, a sensitive mammalian origin in enterovirus cell line RD (Rabdomyosaarcoma, 6 × 10 4 / cell) using the in the laboratory African Ganoderma each extract, that is, hot water 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 영지버섯 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 monolayer (6 × 10 4 / cell) was incubated overnight at 37 ° C., and then the African Ganoderma lucidum sample was diluted 10-fold, 100 μl of the diluted sample was added, and then cultured 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 영지버섯의 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 영지버섯 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 extract of African Ganoderma lucidum mushroom and Table 3 below showing the cytotoxicity of the crude extract, hexane extract, ethyl acetate extract, African Ganoderma lucidum No significant cytotoxicity of each extract was found (see Figures 2 and 3).
2-2. 아프리카산 영지버섯 추출물의 조직 독성2-2. Histotoxicity of African Ganoderma Lucidum Extract
아프리카산 영지버섯 추출물의 조직 독성을 측정하기 위하여, 수술시 적출되는 조직중 염색소견상 정상조직으로 판정된 포유동물 유래의 조직인 대장 조직(Nude mouse : normal colon)을 사용하여 아프리카산 영지버섯 조추출물, 헥산 추출물, 에틸 아세테이트 추출물의 조직 독성 실험을 측정하기 위하여 MTT 분석법을 실시하여, 여러 가지 오차를 고려하여 저해율이 30 % 이상인 경우만 그 추출물에 대하여 독성이 있다고 판단하였다. 저해율은 하기 수학식 1로 계산하였다.In order to measure the histotoxicity of African Ganoderma lucidum mushroom extract, crude Ganoderma lucidum extract of the Ganoderma lucidum extract using normal mouse (Nude mouse: normal colon), which was found to be normal tissue by staining of tissues extracted during surgery In order to measure the tissue toxicity experiments of the hexane extract and the ethyl acetate extract, MTT assay was performed, and it was determined that the extract was toxic only when the inhibition rate was 30% or more in consideration of various errors. Inhibition rate was calculated by the following equation (1).
그 결과, 아프리카산 영지버섯 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 영지버섯의 각각의 추출물들은 30 % 이상의 저해율을 나타낸 것이 없으므로 정상조직에서 독성이 없는 것으로 판단되었다.As a result, the histotoxicity of the crude extract of African Ganoderma lucidum is shown in Tables 4a and 4a, histotoxicity of hexane extracts in Tables 4b and 4b, and 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), each extract of the African Ganoderma lucidum mushroom was determined to be non-toxic in normal tissues because it showed no inhibition of more than 30%.
참조예 1. 아프리카산 영지버섯 동정REFERENCE EXAMPLE 1 Identification of African Ganoderma lucidum Mushroom
본 발명의 아프리카산 영지버섯의 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)로 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)을 의미하며, 계통수에서 각 가지(branch) 옆의 숫자는 부트스트랩 비율(bootstrap percentage)을 의미한다. 참고로 가노더마 속의 ITS 염기서열의 경우 ITS1과 ITS2가 독립적으로 데이터베이스에 등록된 경우가 많기 때문에 같은 균주에 대한 두개의 염기서열을 다운로드(download)하여 하나의 염기서열로 합하여 분석하였다. ITS1과 ITS2 사이에 존재하는 5.8S rDNA 염기서열은 분석에서 제외하였다.In order to identify the type of African Ganoderma lucidum mushroom of the present invention, DNA sequencing was analyzed by Micro ID Co., Ltd., and ITS rDNA sequencing was identified. 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 (phylogenetic) method was used with the neighbor-joining method (Saitou, N. and Nei, M., Mol. Biol.Evol ., 4 , pp406-425, 1987). tree). The tree's scale bar, on the other hand, 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. For reference In the case of ITS nucleotide sequences in the genus ITS1 and ITS2 are frequently registered in a database independently, two nucleotide sequences of the same strain were downloaded and analyzed into a single nucleotide sequence. The 5.8S rDNA sequences present between ITS1 and ITS2 were excluded from the analysis.
그 결과, 결정된 염기서열의 개수는 567 bp로 그 염기서열은 도 5 및 서열번호 1에 나타내었다. 본 균주는 가노더마 속(Ganoderma genus)에 속하는 진균으로 판명되었다.As a result, the determined number of nucleotide sequences is 567 bp and the nucleotide sequences are shown in FIG. 5 and SEQ ID NO: 1. This strain is Ganoderma It was found to be a fungus belonging to the genus Ganoderma genus .
본 발명의 아프리카산 영지버섯 추출물은 아래와 같은 제형으로 투여할 수 있으며, 아래의 제제예는 본 발명을 예시하는 것일 뿐, 이에 의해 본 발명의 내용이 제한되는 것은 아니다. African Ganoderma lucidum extract of the present invention can be administered in the following formulations, the following formulation examples are merely to illustrate the present invention, thereby not limiting the contents of the present invention.
제제예 1. 주사제제의 제조Formulation Example 1 Preparation of Injection
실시예 3의 아프리카산 영지버섯 조추출물 100 ㎎100 mg of African Ganoderma lucidum 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 영지버섯 조추출물 200 ㎎African Ganoderma lucidum extract of Example 3 200 mg
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 영지버섯 조추출물 100 ㎎100 mg of African Ganoderma lucidum 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 영지버섯 조추출물 1000 ㎎African Ganoderma lucidum extract of Example 3 1000 mg
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 영지버섯 조추출물 1000 ㎎African Ganoderma lucidum extract of Example 3 1000 mg
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 영지버섯 조추출물 1000 ㎎African Ganoderma lucidum extract of Example 3 1000 mg
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 영지버섯 추출물은 인유두종 바이러스의 증식을 억제하는 효과를 가진다. 또한, 생체내에서 부작용이 없고 약물 내성도 유발하지 않기 때문에 안전하게 장기간 투여할 수 있으므로, 인유두종 바이러스로 인한질환의 치료 및 예방을 위한 약학조성물로서 유용하게 사용될 수 있다. Ganoderma lucidum extract of the present invention has the effect of inhibiting the growth of human papillomavirus. In addition, since it can be safely administered for a long time because there are no side effects and does not cause drug resistance in vivo, it can be usefully used as a pharmaceutical composition for the treatment and prevention of diseases caused by human papilloma virus.
도 1a 내지 1b는 아프리카산 영지버섯 추출물의 HPV 억제능을 측정한 것으로, 1a to 1b is a measure of the HPV inhibitory activity of African Ganoderma lucidum 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 1b는 환자의 가검물에서 검출한 HPV와 각각의 추출물을 8시간 동안 반응시킨 결과도이며, 1B is a result of reacting HPV and each extract detected in the patient's specimen for 8 hours,
도 2는 아프리카산 영지버섯의 염기서열을 나타낸 도이다.Figure 2 is a diagram showing the nucleotide sequence of African Ganoderma lucidum.
<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Composition comprising the extract of African Ganoderma mushroom having human papilloma virus activity <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 567 <212> DNA <213> Ganoderma genus <400> 1 gaaggatcat tatcgaattt ttgaccgggt tgtagctggc cttccgaggc atgtgcacgc 60 cctgctcaat ccactctaca cctgtgcact tactgtgggt gacggatcgc aaagcgggct 120 tcttgtccgt tataaagcgc atctgtggcc tgcgtttatc acaaactctt tgaaagtact 180 agaatgtaat attgggatat aatagatcta tatacaactt tcagcaacgg atctcttggc 240 tctcgcatcg atgaagaacg cagcgaaatg cgataagtaa tgtgaattgc agaattcagt 300 gaatcatcga atctttgaac gcaccttgcg ctccttggta ttccgaggag catgcctgtt 360 tgagtgtcat gaaatcttca acttgcaacc tctttgcgga gtttgtaggc ttggacttgg 420 agggcttgtc ggcctttaac ggtcggctcc tcttaaatgc attagcttga ttccttgcgg 480 atcggctgtc ggtgtgataa aatgtctacg ccgtgaccgt gaagcgtttg gatgagcttc 540 caaccgtctt gcttcaaaga caacttt 567<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Composition comprising the extract of African Ganoderma mushroom having human papilloma virus activity <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 567 <212> DNA <213> Ganoderma genus <400> 1 gaaggatcat tatcgaattt ttgaccgggt tgtagctggc cttccgaggc atgtgcacgc 60 cctgctcaat ccactctaca cctgtgcact tactgtgggt gacggatcgc aaagcgggct 120 tcttgtccgt tataaagcgc atctgtggcc tgcgtttatc acaaactctt tgaaagtact 180 agaatgtaat attgggatat aatagatcta tatacaactt tcagcaacgg atctcttggc 240 tctcgcatcg atgaagaacg cagcgaaatg cgataagtaa tgtgaattgc agaattcagt 300 gaatcatcga atctttgaac gcaccttgcg ctccttggta ttccgaggag catgcctgtt 360 tgagtgtcat gaaatcttca acttgcaacc tctttgcgga gtttgtaggc ttggacttgg 420 agggcttgtc ggcctttaac ggtcggctcc tcttaaatgc attagcttga ttccttgcgg 480 atcggctgtc ggtgtgataa aatgtctacg ccgtgaccgt gaagcgtttg gatgagcttc 540 caaccgtctt gcttcaaaga caacttt 567
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