KR101408101B1 - Food for improving liver function comprising black rice culture of Lentinus edodes mycelia adding Hovenia dulcis extract as effective component - Google Patents
Food for improving liver function comprising black rice culture of Lentinus edodes mycelia adding Hovenia dulcis extract as effective component Download PDFInfo
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- KR101408101B1 KR101408101B1 KR1020120133377A KR20120133377A KR101408101B1 KR 101408101 B1 KR101408101 B1 KR 101408101B1 KR 1020120133377 A KR1020120133377 A KR 1020120133377A KR 20120133377 A KR20120133377 A KR 20120133377A KR 101408101 B1 KR101408101 B1 KR 101408101B1
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
본 발명은 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 식품 및 약학조성물에 관한 것으로, 본 발명의 배양액은 간세포 보호 및 간기능 개선효과가 증진되어 간기능 개선을 위한 식품 및 약학조성물 등에 유용하게 사용할 수 있다.The present invention relates to a food and a pharmaceutical composition for improving liver function, which comprises a culture solution obtained by inoculating mycelia with a mixture of cereals and Hovenia dulcifera, wherein the culture medium contains hepatocyte protection and liver function The improvement effect is improved and the composition can be effectively used for foods and pharmaceutical compositions for improving liver function.
Description
본 발명은 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 식품 및 약학조성물에 관한 것이다.The present invention relates to a food composition for improving liver function and a pharmaceutical composition containing a culture solution obtained by inoculating shiitake mushroom mycelium with a mixture of grains and Hovenia dulcis extract as an active ingredient.
간은 인체에서 혈액 저장 및 순환, 혈액량 조절과 방어해독작용을 하며 정신적 활동과 밀접하게 관련되어 있다고 알려져 있다. 산업화에 따른 공해물질, 유독물질에 우리의 몸은 항상 노출되어 있어 우리의 간은 끊임없이 해독작용에 시달리고 있다.The liver is known to be closely related to mental activities, such as blood storage and circulation, blood volume control and defense detoxification in the human body. Our body is always exposed to pollution and toxic substances by industrialization, and our liver is constantly suffering from detoxification.
간은 완충능력이 큰 기관으로 질환의 초기단계에서는 잘 나타나지 않고 상당히 악화되어서야 발견된다. 간경화, 간암 등은 각종 간질환이 만성적으로 진행될 경우 공통적으로 이르는 마지막 단계이다. 그 원인으로는 알코올, 약물, 화학약품, 바이러스성 간염, 담도질환, 혈색소증(hematochromatosis)과 같은 대사성 질환, 자가 면역성 질환 등이 있으나 원인을 알 수 없는 경우도 많아 간은 초기의 건강관리가 매우 중요한 기관이다.The liver is an organ with a large buffer capacity. It is not found in the early stages of the disease and is not found until it gets worse. Liver cirrhosis, and liver cancer are the last steps that are common when various liver diseases progress chronically. The cause is metabolic diseases such as alcohol, drugs, chemicals, viral hepatitis, biliary disease, hematochromatosis, and autoimmune disease, but there are many cases where the cause is unknown. It is an institution.
최근 한국인의 건강수준에서 간암에 의한 사망률은 10만 명당 23.4명으로 세계 1위이고 만성 간질환의 경우도 28.8명으로 3번째로 조사되었다. 또한 최근 통계청에서 우리나라 40대의 경우 인구 10만 명당 56.1명이 간질환으로 가장 높은 사망원인으로 발표하였다.In Korea, the mortality rate of liver cancer was 23.4 per 100,000 people in the world, the first in the world, and the chronic liver disease was the third in 28.8 cases. In recent years, the National Statistical Office (NSO) has reported that 56 deaths per 100,000 population in Korea are among the highest causes of liver disease.
더욱 심각한 것은 정신적인 스트레스로 인한 간 손상이다. 정신적 휴식을 가질 경우 손상된 간세포는 복구되지만 급박한 현대사회에서 정신적 휴식의 여유를 찾을 수 없어 정신적 스트레스, 과음, 흡연으로 간 손상을 가중시켜 인체가 방어 해독 작용을 하지 못해 면역 체계에 이상을 가져와 다른 질병의 원인이 되기도 한다.More serious is liver damage due to mental stress. If you have a mental break, damaged hepatocytes are restored, but in an imminent modern society, you can not find a room for mental relaxation. Because of mental stress, excessive drinking and smoking, liver damage is increased and the body fails to defend and detoxify. It also causes illness.
헛개나무는 우리나라 특산수종으로 주독 해소, 이뇨 갈증해소 등 각종 해독작용을 하는 것으로 본초학이나 식물도설 등에 기록되어 있다. 헛개나무 열매자루(果柄)가 간경화 방지와 알코올성 간 손상에 보호효능이 뛰어나다는 연구결과에 따라 간기능 개선 관련 상품으로 많은 주목을 받고 있다.It is recorded in the herbal medicine and the phytosanitary, and it plays various detoxification functions such as resolving the poisoning by the special species of Korea, dissolving diuretic thirst, and so on. As a result of research that hunting fruit bag has excellent protective effect against liver cirrhosis and alcoholic liver damage, it has attracted much attention as a product for improvement of liver function.
버섯은 식물성 단백질과 아미노산, 효소, 지방, 철분, 섬유소, 비타민, 미네랄 등과 같이 인체에 중요한 각종 영양성분을 함유하고 있으며 특유의 향과 맛 때문에 예로부터 사람들이 즐겨 먹었고, 지방질이 적고 식이섬유와 단백질이 풍부한 저칼로리 식품으로 알려져 있다. 또한, 여러 가지 건강기능성 물질이 많이 함유되어 있는 식재료로써 노화예방을 위한 항산화활성, 항암, 항당뇨, 체중감량, 면역력증강, 고혈압예방 등의 성인병 예방에 관한 생리활성 효과를 가지고 있어 건강기능식품 및 의약품 원료로 많이 이용되고 있으며 자실체뿐만 아니라 균사체를 이용한 면역력 개선 제품이 시판되고 있다. Mushrooms contain various nutrients important to human body such as vegetable proteins, amino acids, enzymes, fats, iron, fiber, vitamins and minerals. Due to their unique flavor and taste, they have been enjoyed by people for a long time. This is known as a rich low-calorie food. In addition, it has a physiological activity effect on prevention of adult diseases such as antioxidant activity for preventing aging, anticancer, antidiabetic, weight loss, immunity enhancement and prevention of hypertension as a food material containing many health functional substances, It is widely used as a raw material for pharmaceuticals, and a product for improving immunity using mycelium as well as fruiting bodies is on the market.
표고균사체는 식물성 단백질과 아미노산, 효소, 지방, 철분, 섬유소, 비타민, 미네랄 등과 같이 인체에 중요한 각종 영양성분을 함유하고 있으며 특유의 향과 맛 때문에 예로부터 사람들이 즐겨 먹었고, 지방질이 적고 식이섬유와 단백질이 풍부한 저칼로리 식품으로 알려져 있다.Icelandic mycelium contains various essential nutrients such as plant protein, amino acids, enzymes, fats, iron, fiber, vitamins and minerals. Because of its unique flavor and taste, it has been enjoyed by people for a long time. It is known as a protein-rich low-calorie food.
헛개를 이용한 건강기능식품 인정내용은 간 기능개선에 주기능으로 되어있으나 식품학적 가치에서 볼 때 영양성분 공급에 대한 부분은 취약하므로, 한국인의 주식으로 이용하는 쌀을 주원료로 표고버섯 균사체를 배양한 배양미를 활용한다면 기능적 측면뿐만 아니라 영양학적 측면에서도 우수한 제품을 기대할 수 있을 것으로 생각되어, 헛개와 버섯균사체에 대한 간 기능 개선에 관한 연구를 실시하고 이에 대한 과학적 근거를 확보하고 이를 건강식품 소재로 개발하여 산업화함으로써 국민의 건강 증진을 도모하여 삶의 질을 높이는 연구가 필요하다.The recognition of health functional foods using hinoki has been the main function of improving liver function, but the portion of nutritional ingredient supply is weak from the viewpoint of food value. Therefore, it is considered that cultivation of shiitake mushroom mycelium It is believed that if the beauty is utilized, it can expect superior products in terms of nutritional as well as functional aspects. Research on improvement of hepatic function in the horny mushroom and mycelium is carried out and scientific basis is secured and developed as health food material In order to improve the quality of life by promoting the health of the people by industrialization, research is needed.
한국등록특허 제0937781호에는 에나활성미네랄 A 활성수를 유효성분으로 함유하는 간손상 방지 또는 간기능 개선용 약학조성물이 개시되어 있고, 한국공개특허 제2012-0122264호에는 간 기능 개선용 오가피 발효물과 이의 추출물을 유효성분으로 하는 간기능 개선용 제제가 개시되어 있으나, 본 발명의 헛개나무 추출물을 첨가한 표고버섯 균사체 흑미 배양액을 유효성분으로 함유하는 간기능 개선용 식품과는 상이하다.Korean Patent No. 0937781 discloses a pharmaceutical composition for preventing liver damage or improving liver function containing ena-active mineral A-active water as an active ingredient, Korean Patent Publication No. 2012-0122264 discloses a pharmaceutical composition for improving liver function, And an extract thereof are disclosed as active ingredients. However, this preparation is different from a food for improving liver function containing as an active ingredient a black rice culture solution of a shiitake mushroom mycelium supplemented with a Hodgkin's extract of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명의 목적은 헛개나무 어린가지, 잎, 과병 등 부위별 성분분석 및 효능평가를 통한 이용성 확대 및 버섯균사체를 이용하여 간 기능 개선에 대한 효능 연구를 실시하여 과학적 근거를 제시하고 간기능 개선 기능성을 가지는 건강식품 소재의 생산 기술 확립과 안정적인 생산체계를 마련하기 위해, 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종한 후 최적의 배양조건을 확립하여 배양한 배양액을 유효성분으로 함유함으로써, 간세포 및 간기능 개선 효과를 증진시킬 수 있는 조성물을 제공하는 데 있다.SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and an object of the present invention is to provide a novel method for improving liver function using mushroom mycelium, In order to establish the production technology of health food materials with liver function improvement function and to provide a stable production system by presenting the scientific basis by carrying out the research, the mixture of the mixture of grains and Hovenia dulcis was inoculated with the mycelium of mushroom, And to provide a composition capable of enhancing hepatocyte and hepatic function-improving effect by containing a cultured liquid obtained by establishing culture conditions as an effective ingredient.
상기 과제를 해결하기 위해, 본 발명은 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 식품을 제공한다.In order to solve the above problems, the present invention provides a food for improving liver function containing as an active ingredient a culture solution obtained by inoculating shiitake mushroom mycelium into a mixture of cereal and Hovenia dulcis extract.
또한, 본 발명은 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for improving liver function, comprising a culture solution obtained by inoculating shiitake mushroom mycelium with a mixture of grains and Hovenia dulcifera as an active ingredient.
또한, 본 발명은 흑미를 3~5시간 동안 4~6 brix 농도로 조정된 헛개가지 추출물에 수침하고 25~35분간 물을 뺀 후, 110~130℃에서 30~50분간 멸균한 후 표고버섯 균사체를 접종하고 23~27℃에서 배양하여 제조하는 것을 특징으로 하는 간기능 개선효과와 아미노산 함량이 증진된 배양액의 제조방법을 제공한다.In addition, the present invention relates to a method for treating a Japanese black mushroom mycelium, which comprises treating the black rice with water for 4 to 6 brix for 3 to 5 hours, removing the water for 25 to 35 minutes, sterilizing the mixture at 110 to 130 ° C for 30 to 50 minutes, And culturing the cells at 23 to 27 ° C. The present invention also provides a method for producing a culture solution with enhanced amino acid content.
기존 헛개 추출물만을 사용한 건강기능식품의 경우 헛개나무의 소비가 증가함에 따라 국산 헛개나무 수급에 어려움이 따르고 있다. 헛개 과병의 경우 식재 후 7~8년의 긴 기간이 소요되고 이에 따른 중국산 수입이 불가피하므로 이러한 문제점을 해결하고자 헛개나무 어린가지와 한국인의 주식으로 이용하는 쌀을 주원료로 하여 단위시설에서 일정한 생산량과 추출이 가능한 표고균사체를 배양한 표고균사체 배양 헛개나무 어린가지 배양액을 활용하여 건강기능식품 소재로 활용할 수 있다.In the case of health functional foods using only conventional hinoki extract, consumption of hinoki trees is increasing, and thus it is difficult to supply and receive domestic hinoki trees. In order to solve these problems, it is necessary to take a certain amount of production and extraction from the unit facilities as the main raw material for the use of the rice as the stocks of the Japanese hawthorn tree and Korean people. It is possible to utilize this culture as a health functional food material by utilizing the culture medium of Hodgkin's young shoots cultured in a superficial mycelium cultured as a possible upper level mycelium.
또한, 본 발명의 배양액은 간기능 개선뿐만 아니라, 구성아미노산 함량이 증진되어 건강 기능적 측면에서 시너지 효과를 기대할 수 있어, 상기 배양액을 이용하여 제조된 약학 또는 식품 조성물은 간기능 개선 및 인체의 유용성분인 아미노산 섭취를 위해 효과적으로 이용할 수 있다.In addition, the culture solution of the present invention can improve not only the liver function but also the constitutional amino acid content, and the synergistic effect can be expected from the health functional aspect. Thus, the pharmaceutical or food composition prepared using the culture solution can improve the liver function, Can be effectively used for the intake of amino acids.
도 1은 헛개가지 추출물(A), 흑미 추출물(B)과 본 발명의 배양액 추출물(C)의 다양한 농도(0, 10, 50, 100, 500 ㎍/mL)별로 간세포 생존율을 비교한 그래프이다.
도 2는 100 ㎍/mL의 헛개가지 추출물, 100 ㎍/mL의 흑미 추출물과 본 발명의 배양액 추출물(헛개가지 추출물이 첨가된 흑미에 배양한 표고버섯 균사체)을 아세트아미노펜(acetaminophen) 처리하여 간독성을 일으킨 간세포에 처리하여 간세포 보호활성을 비교한 그래프이다.
도 3은 100 ㎍/mL의 헛개가지 추출물, 100 ㎍/mL의 흑미 추출물과 본 발명의 배양액 추출물(헛개가지 추출물이 첨가된 흑미에 배양한 표고버섯 균사체)을 아세트아미노펜(acetaminophen) 처리하여 간독성을 일으킨 간세포에 처리하여 간기능 보호활성을 비교한 그래프이다.FIG. 1 is a graph comparing hepatocyte survival rates at various concentrations (0, 10, 50, 100, 500 / / mL) of the extracts of A, B, and C of the present invention.
Fig. 2 shows the results of acetaminophen treatment of 100 쨉 g / mL skimmed egg extract, 100 쨉 g / mL of black rice extract, and the culture extract of the present invention (black mushroom mycorrhiza cultured on black rice) Fig. 2 is a graph comparing the hepatocyte protective activity with the treated hepatocytes. Fig.
FIG. 3 is a graph showing the results of the acetaminophen treatment of 100 ㎍ / mL of Hovenia dulcis extract, 100 ㎍ / mL of black rice extract, and the culture extract of the present invention (the shiitake mushroom cultured in black rice supplemented with Hovenia dulcis Thunb extract) The results are shown in FIG.
본 발명의 목적을 달성하기 위하여, 본 발명은 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 식품을 제공한다.In order to accomplish the object of the present invention, the present invention provides a food for improving liver function containing as an active ingredient a culture solution obtained by inoculating mycelia of shiitake mushroom into a mixture of cereals and Hovenia dulcis extract.
본 발명의 간기능 개선용 식품에서, 상기 곡물은 흑미, 백미, 적토미, 강낭콩, 검정콩, 녹두, 메주콩 또는 팥일 수 있으며, 바람직하게는 흑미일 수 있으나, 이에 제한되지 않는다.In the food for improving liver function of the present invention, the cereal may be black rice, white rice, red mackerel, kidney bean, black soybean, green bean, mexican or bean, preferably black rice, but is not limited thereto.
또한, 본 발명의 간기능 개선용 식품에서, 상기 헛개나무의 부위는 어린가지, 뿌리, 줄기, 열매 또는 잎일 수 있으며, 바람직하게는 어린가지일 수 있는데, 상기 어린가지는 12개월 미만된 가지일 수 있으나, 이에 제한되지 않는다.In addition, in the food for improving liver function of the present invention, the part of the hornbreeze may be a young branch, a root, a stem, a fruit or a leaf, preferably a young branch, But is not limited thereto.
또한, 본 발명의 간기능 개선용 식품에서, 상기 배양액은 곡물을 3~5시간 동안 4~6 brix 농도로 조정된 헛개가지 추출물에 수침하고 25~35분간 물을 뺀 후, 110~130℃에서 30~50분간 멸균한 후 표고버섯 균사체를 접종하고 23~27℃에서 배양하여 제조될 수 있으며, 더욱 바람직하게는 곡물을 4시간 동안 4 brix 농도로 조정된 헛개가지 추출물에 수침하고 30분간 물을 뺀 후, 121℃에서 40분간 멸균한 후 표고버섯 균사체를 접종하고 25℃에서 배양하여 제조될 수 있다. 상기 방법으로 배양액을 제조하는 것이 헛개나무 추출물 및 흑미 추출물에 비해 간세포 및 간기능 보호활성이 증진될 뿐만 아니라 인체의 유용성분인 아미노산 함량을 최대로 증진시킬 수 있었다.In addition, in the food for improving liver function of the present invention, the culture liquid is prepared by soaking the grains in the horsetail egg extract adjusted to a concentration of 4 to 6 brix for 3 to 5 hours, removing water for 25 to 35 minutes, And then cultured at 23 to 27 ° C. More preferably, the grains are soaked in a 4 brix-adjusted 4-brix concentration of Hovenia dulcis Thunb. Extract, and water is added for 30 minutes. , Sterilized at 121 ° C for 40 minutes, inoculated with mycelium of mushroom, and cultured at 25 ° C. In addition to the extracts of Hovenia dulcis Thunb. And black rice extract, the production of the culture medium by this method not only enhanced the hepatocyte and liver function - protecting activity, but also maximized the amino acid content, which is a useful component of the human body.
상기 식품은 간기능을 개선시키기 위해 섭취할 수 있는 것이면 특별히 제한되지 않는다.The food is not particularly limited as long as it can be ingested to improve liver function.
본 발명의 배양액을 식품첨가물로 사용하는 경우, 상기 배양액을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 배양액은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the culture broth of the present invention is used as a food additive, the culture broth can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the culture medium of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .
상기 식품의 종류에는 특별한 제한은 없다. 상기 배양액을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of the food to which the culture liquid can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.
상기 외에 본 발명의 건강음료 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강음료 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01~0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health beverage composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health beverage composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
본 발명은 또한, 곡물과 헛개나무 추출물을 혼합한 혼합물에 표고버섯 균사체를 접종하여 배양한 배양액을 유효성분으로 함유하는 간기능 개선용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for improving liver function, comprising a culture solution obtained by inoculating mycelia of shiitake mushroom with a mixture of grains and Hovenia dulcifera as an active ingredient.
본 발명의 배양액은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 포함할 수 있다.The culture broth of the present invention may contain suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.
본 발명의 배양액의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage forms of the culture broths 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 compounds as well as in suitable aggregates.
본 발명에 따른 배양액은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 배양액에 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 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The culture solution according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a usual method have. Examples of carriers, excipients and diluents that can be contained in the culture liquid include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Various compounds or mixtures including cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 배양액의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명의 배양액은 1일 0.0001 내지 100 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the culture medium of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the culture solution of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 배양액은 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, 자궁 내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.The culture medium of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
본 발명은 또한, 흑미를 3~5시간 동안 4~6 brix 농도로 조정된 헛개가지 추출물에 수침하고 25~35분간 물을 뺀 후, 110~130℃에서 30~50분간 멸균한 후 표고버섯 균사체를 접종하고 23~27℃에서 배양하여 제조하는 것을 특징으로 하는 간기능 개선효과와 아미노산 함량이 증진된 배양액의 제조방법을 제공한다.The present invention also relates to a method for preparing a mushroom mycelium of the present invention, which comprises immersing the black rice in an extract of Hovenia dulcis which has been adjusted to a concentration of 4 to 6 brix for 3 to 5 hours, removing water for 25 to 35 minutes, sterilizing it at 110 to 130 ° C for 30 to 50 minutes, And culturing the cells at 23 to 27 ° C. The present invention also provides a method for producing a culture solution with enhanced amino acid content.
본 발명의 배양액의 제조방법은 바람직하게는 흑미를 4시간 동안 4 brix 헛개가지 추출물에 침지하고 30분간 물을 뺀 후, 121℃에서 40분간 멸균한 후 표고버섯 균사체를 접종하고 25℃에서 배양하여 제조할 수 있다.
Preferably, the black rice is immersed in a 4-brix Hovenia dulcis extract for 30 minutes, then sterilized at 121 ° C for 40 minutes, inoculated with the mycelium of mushroom, and cultured at 25 ° C. Can be manufactured.
이하, 본 발명의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
1) 원료1) Raw materials
헛개나무 어린가지 농축액은 ㈜PNK에서, 흑미는 장흥농협에서 구입하여 사용하였다.
The juice concentrate was purchased from PNK Co., Ltd. and the black rice was purchased from Jangheung Nonghyup.
2) 표고버섯 균주2) Shiitake mushroom strain
LentinusLentinus
edodesedodes
3) 배양방법3) Culture method
40 brix 헛개가지 농축액에 정제수를 가하여 4 brix 침지액을 만들고 이에 수세한 흑미를 4시간 동안 침지 후 30분간 물빼기를 하였다. 이를 배양봉지에 500 g씩 넣은 후, 121℃에서 40분간 멸균하고 식혀 곡립배지를 제조하고, 곡립배지 중량대비 표고버섯 균사체 곡립 종균을 10% 내외로 접종하고 배양기를 이용하여 25℃에서 40일간 배양하였다.
4 brix immersion liquid was prepared by adding purified water to a 40 brix Houttuyniae japonica concentrate. The black rice washed with water was immersed for 4 hours and then drained for 30 minutes. 500 g of each of them was put into a culture bag, sterilized at 121 ° C for 40 minutes, cooled to prepare a curd medium, and 10% of the mycorrhizal fungi were inoculated to the medium at a rate of about 10% Respectively.
4) 시료제조 방법4) Sample preparation method
분쇄한 각 시료 6 g에 정제수 100 mL을 넣고 60℃에서 4시간 동안 추출하고, Whatman filter paper No. 2로 여과한 후 동결건조하여 실험에 사용하였다.
100 mL of purified water was added to 6 g of each of the pulverized samples, and the mixture was extracted at 60 캜 for 4 hours. 2 and then lyophilized and used in the experiment.
실험방법Experimental Method
1) 구성아미노산 분석1) Analysis of constituent amino acids
추출 후 동결건조한 각 시료를 분쇄하여 0.3 g을 시험관에 넣고 6N-HCl 용액 10 mL를 가한 후 110℃에서 24시간 가수분해해서 얻은 여액을 원심분리하고, 상등액을 50℃에서 농축하여 염산과 물을 완전히 증발시킨 후, 20 mM HCl(pH 2.2)을 사용하여 5 mL로 정용한 다음 0.45 막 필터(membrane filter)로 여과하여 여액을 취하고 AccQ-Tag 시약을 사용하여 유도체화 시킨 후 HPLC용 분석 시료로 사용하였다. 분석조건은 표 1과 같고, 아미노산 함량은 적분기(integrator)에 의한 외부표준법으로 계산하였다.After lyophilization, each lyophilized sample was pulverized and 0.3 g was placed in a test tube, 10 mL of 6N-HCl solution was added, and the filtrate obtained by hydrolysis at 110 ° C. for 24 hours was centrifuged. The supernatant was concentrated at 50 ° C., After completely evaporating, the solution is adjusted to 5 mL with 20 mM HCl (pH 2.2), filtered through a 0.45 membrane filter, and the filtrate is then derivatized with AccQ-Tag reagent and used as an analytical sample for HPLC Respectively. The analysis conditions are shown in Table 1, and the amino acid content was calculated by an external standard method using an integrator.
(Waters Co., 150 mm L × 3.9 mm ID)AccQ-Tag ™
(Waters Co., 150 mm L x 3.9 mm ID)
B : AccQ-Tag Eluent B(60% acetonitrile)
C : DWA: AccQ-Tag Eluent A (acetate-phosphate buffer)
B: AccQ-Tag Eluent B (60% acetonitrile)
C: DW
2) 간보호 및 간기능 개선 실험2) Liver protection and liver function improvement experiment
가) 콜라게나제 주입(Collagenase perfusion)A) Collagenase perfusion.
Sprague-Dawley(SD) 랫트의 간세포는 Berry와 Friend의 방법을 수정한 2단계 콜라게나제 주입(collagenase perfusion) 방법으로 분리하였다. 랫트 간 세포(primary rat hepatocyte)를 얻기 위해 SD 랫트는 에테르로 흡입 마취시키고 70% 에탄올(ethanol)로 복부를 소독한 후, 개복하고 우심방을 절개하여 혈액이 제거될 수 있도록 하고 좌심실에 21 게이지(gauge) 주사기로 HBSS 100 mL을 주입(perfusion)하였다. 혈액을 제거한 후, 100 U/mL 콜라게나제(collagenase)가 포함된 HBSS를 100 mL 재순환시켜 세포(hepatocyte)가 유리화 되도록 하였다. 간 조직을 떼어내어 100 mm 배양 접시(culture dish)에서 HBSS 60 mL를 첨가하고 No. 11 블레이드(blade)로 세절(chopping)하여 단일 세포(single cell)들을 얻을 수 있었다. 이 세포(hepatocyte) 현탁액에 콜라게나제(collagenase)를 제거하기 위해 3회 Waymouth’s MB 752/1 배지(5% 소태아혈청, 2.0 mg/mL 소 혈청 알부민, 10-6 M 덱사메타손, 107 M 인슐린, 5.32 x 10-2 M L-세린, 4.09 x 10-2 M L-알라닌, 2.67 x 10-2 M NaHCO3, 100 IU/mL 페니실린, 100 IU/mL 스트렙토마이신, 50 ㎍/mL 겐타마이신 설페이트)로 헹궈주었다. 이 랫트 간 세포(primary rat hepatocyte)는 1x105 cell/mL로 희석하여 사전에 콜라겐(collagen)으로 코팅되어 있는 96-웰 플레이트에 4시간 동안 부착시킨 후, 각 시료를 처리하였다.
Hepatocytes from Sprague-Dawley (SD) rats were separated by a two-step collagenase perfusion method modified by Berry and Friend's method. To obtain primary rat hepatocytes, SD rats were anesthetized with ether and sterilized with 70% ethanol. The abdomen was opened and the right atrium was opened to allow blood to be removed. The left ventricle was filled with 21 gauge gauge syringes were perfused with 100 mL of HBSS. After removal of the blood, 100 mL of HBSS containing 100 U / mL collagenase was recirculated to allow the hepatocytes to liberate. The liver tissue was removed and 60 mL of HBSS was added in a 100 mm culture dish. 11 chopped with a blade to obtain single cells. In order to remove collagenase from the hepatocyte suspension, the cells were washed three times with Waymouth's MB 752/1 medium (5% fetal bovine serum, 2.0 mg / mL bovine serum albumin, 10 -6 M dexamethasone, 10 7 M insulin , 5.32 x 10 -2 M L-serine, 4.09 x 10 -2 M L-alanine, 2.67 x 10 -2 M NaHCO 3 , 100 IU / mL penicillin, 100 IU / mL streptomycin, 50 μg / mL gentamycin sulfate ). Primary rat hepatocytes were diluted to 1 × 10 5 cells / mL and adhered to a collagen-coated 96-well plate for 4 hours before each sample was treated.
나) 세포독성분석B) Cytotoxicity analysis
랫트 간 세포(rat primary hepatocyte)는 96-웰 플레이트에 1 x 105 cell/mL의 농도로 배양하여 시료를 처리하였다. 24시간 처리 후, 0.5 ㎍/mL tetrazolium-based colorimetric(MTT)가 첨가된 Waymouth’s MB 752/1 배지로 4시간 동안 포르마잔(formazan)을 형성시켰다. 반응이 끝나면 배지를 제거하고, 150 μL DMSO로 포르마잔(formazan)을 용해시켜 540 nm로 multiplate reader(BioTek, USA)를 이용하여 측정하였다. 각 시료의 세포 생존율은 무처리군을 100%로 하여 상대적으로 계상하였다.
Rat primary hepatocytes were cultured in 96-well plates at a concentration of 1 × 10 5 cells / mL. After 24 hours of treatment, formazan was formed for 4 hours with Waymouth's MB 752/1 medium supplemented with 0.5 μg / mL tetrazolium-based colorimetric (MTT). At the end of the reaction, the medium was removed and the formazan was dissolved in 150 μL DMSO and measured using a multiplate reader (BioTek, USA) at 540 nm. The cell survival rate of each sample was calculated relative to the untreated group as 100%.
다) 간기능 검사C) liver function test
간기능 검사를 하기 위해, 랫트 간 세포(Rat primary hepatocyte)는 96-웰 플레이트에 1 x 105 cell/mL의 농도로 배양하여 시료를 처리하였다. 24시간 처리 후, 배지 50 μL를 이용하여 BlueGene Co.(Shanghai, China)에서 구매한 Rat lactate dehydrogenase(LDH), Rat aspartate transaminase(AST), Rat alanine transaminase(ALT) ELISA kit를 사용하여 효소를 정량적으로 측정하였다. 흡광도는 450 nm로 multiplate reader(BioTek, USA)를 이용하여 측정하였다. 각 시료의 LDH, AST, ALT의 함량은 0, 10, 25, 50, 100, 250 ng/mL의 농도로 표준곡선을 만들이 정량하였다.
To the liver function tests, rat liver cells (Rat primary hepatocyte) is treated samples were incubated in a 96-well plate to 1 x 10 5 cell / mL concentration. After 24 hours of treatment, the enzyme was quantitatively analyzed by using Rat lactate dehydrogenase (LDH), Rat aspartate transaminase (AST), and Rat alanine transaminase (ELISA) ELISA kit purchased from BlueGene Co. (Shanghai, China) . Absorbance was measured at 450 nm using a multiplate reader (BioTek, USA). The concentrations of LDH, AST and ALT in each sample were determined as standard curves at concentrations of 0, 10, 25, 50, 100 and 250 ng / mL.
실시예Example 1: 구성아미노산 함량 1: Amount of constituent amino acid
구성아미노산 분석결과 흑미에 헛개가지 추출물 10%를 첨가 후 표고 균사체를 배양한 시료에서 총 구성아미노산 함량은 1693.09 mg%로 흑미 추출물의 총 구성 아미노산 함량보다 높게 검출되었다. 총 아미노산과 필수아미노산의 비율에 있어서는 두 시료가 비슷하게 나타났다.The total amino acid content of the sample was 1693.09 mg%, which was higher than the total amino acid content of black rice extract. The ratio of total amino acids to essential amino acids was similar for both samples.
표고균사체 배양액Black rice + hinoki branch extract 10%
Cultured mycelium
1)TAA: 총 아미노산 1) TAA: total amino acid
2)EAA: 필수아미노산
2) EAA: Essential amino acids
실시예Example 2: 시료에 대한 간세포 독성확인 2: Identification of hepatocellular toxicity against the sample
헛개나무 어린가지 추출물(10%)이 첨가된 흑미에 배양한 표고버섯 균사체 추출물(C)과 흑미 추출물(B) 및 헛개가지 추출물(A)을 다양한 농도(0, 10, 50, 100, 500 μg/mL) 별로 간세포 독성을 확인한 결과, 모든 농도에서 간세포 독성은 보이지 않았다(도 1).
10, 50, 100, and 500 μg of mycelia extract (C), black rice extract (B), and hinoki extract (A) were cultivated in black rice with the addition of 10% / mL), hepatocellular toxicity was not observed at all concentrations (FIG. 1).
실시예Example 3: 3: 아세트아미노펜(acetaminophen)에In acetaminophen 대한 간세포 보호활성 Hepatocyte protective activity
고농도의 아세트아미노펜(acetaminophen)을 처리하여 간독성을 일으키고, 100 μg/mL의 시료들로 간세포 보호효과를 확인하였다. 이때, 간독성을 일으킨 아세트아미노펜(acetaminophen) 처리군에서는 세포생존율이 84.2±3.2%로 나타났으나 각각의 헛개가지 추출물 및 흑미 추출물을 처리한 세포 생존율은 101.8±1.9%, 107.3±3.6%로 나타났으며, 100 μg/mL 헛개나무 어린가지 추출물이 첨가된 흑미에 배양한 표고버섯 균사체 배양액 추출물에서는 116.7±3.1%로 나타났다. 이는, 세 시료 모두 아세트아미노펜(acetaminophen)에 의한 간독성을 완화하는 것으로 확인되었으며, 본 발명의 균사체 배양액 추출물은 헛개나무 추출물에 비해 간세포 보호활성이 증진되는 것을 확인할 수 있었다(도 2).
A high concentration of acetaminophen was treated to induce hepatotoxicity, and hepatocyte protective effects were confirmed with 100 μg / mL samples. At this time, cell survival rate was 84.2 ± 3.2% in acetaminophen treated with hepatotoxicity, but cell survival rate was 101.8 ± 1.9% and 107.3 ± 3.6%, respectively, , And 116.7 ± 3.1% in the culture solution of the mushroom mycelium cultured in the black rice supplemented with 100 μg / mL Hinoki extract. This confirms that all three samples alleviate hepatotoxicity due to acetaminophen, and that the mycelial culture extract of the present invention has an enhanced hepatocyte protective activity as compared to the Hovenia dulcis extract (Fig. 2).
실시예Example 4: 4: 아세트아미노펜(acetaminophen)에In acetaminophen 대한 간기능 보호활성 Liver function-protecting activity
고농도의 아세트아미노펜(acetaminophen)으로 간세포에 독성을 일으켰을 때, 배지로 분비되는 LDH의 함량을 측정한 결과(도 3의 A), 대조구(무처리구, control)보다 31.6 ng/mL이 증가되었는데, 이는 간세포에 염증반응이 발생하여 LDH 함량이 높아졌다. 헛개가지 추출물이 첨가된 흑미를 배양한 표고버섯 균사체 배양액 추출물과 흑미 추출물을 각각 첨가하였을 때, 염증반응이 감소하여 LDH의 함량이 감소되었다. 그중에서도 헛개가지 추출물이 첨가된 흑미를 배양한 표고버섯 균사체 추출물이 49.7 ng/mL이 감소되어 간기능 보호활성이 탁월한 것으로 사료된다.When the hepatic cells were toxicized with acetaminophen at a high concentration, the content of LDH secreted from the medium was measured (FIG. 3 A), which was 31.6 ng / mL higher than that of the control (untreated control) And the LDH content was increased. The content of LDH was decreased due to the decrease of the inflammatory reaction when the culture broth of Lentinus edodes cultivated with black rice added with extracts of Hovenia dulcis Thunb. Among them, 49.7 ng / mL of shiitake mushroom mycorrhizae cultivated with black rice added with extracts of L. japonica extract showed excellent activity for protecting liver function.
배지 내에 AST의 경우에는, 아세트아미노펜(acetaminophen)으로 간독성을 일으켰을 때 대조구(무처리구, control)보다 6.9 ng/mL이 증가되었으나, 헛개가지 추출물이 첨가된 흑미를 배양한 표고버섯 균사체 배양액 추출물에서는 4.3 ng/mL로 현저하게 감소되어 대조군과 비슷한 함량으로 확인되었다. 이는 상기 본 발명의 배양액이 간에 염증반응을 보호하고 간 기능을 높여주는 것으로 사료된다(도 3의 B).In the case of AST in the medium, 6.9 ng / mL of acetaminophen was increased by 6.9 ng / mL than that of the control (control, control), but 4.3 ng / mL was found in the culture of mushroom mycelium / mL and was found to be similar to that of the control group. This suggests that the culture medium of the present invention protects the liver inflammatory response and enhances liver function (FIG. 3B).
ALT의 경우, 헛개가지 추출물이 첨가된 흑미를 배양한 표고버섯 균사체 배양액 추출물이 아세트아미노펜(acetaminophen)으로 간독성을 일으킨 처리구보다 8.6 ng/mL을 감소시켜 간 손상을 줄이고 간 기능을 높이는 것으로 사료된다(도 3의 C).In the case of ALT, the extract of the cultured shiitake mushroom cultured with the black rice added with the extract of Houttuynia cordata reduced 8.6 ng / mL compared to the treatment with acetaminophen for hepatotoxicity, thus reducing liver damage and enhancing liver function 3C).
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