KR0143511B1 - Extracting hethods for separating the active comdonents contazning polysaccharive s from ganoderma lucidum - Google Patents
Extracting hethods for separating the active comdonents contazning polysaccharive s from ganoderma lucidumInfo
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- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
본 발명은 톱밥을 주성으로 한 고체 배지내에서 장이버섯과(Polyporaceae)에 속하는 담자균류의 일종인 영지버섯(Ganoderma lucidum, 불로초)을 재배 수확한 후 폐기처리되는 완숙된 균사배양물(이하 완숙 균사체라함)을 재료로 활용하여 영지 균사체의 유효성분을 값싸게 대량으로 생산할 수 있는 추출방법에 관한 것으로 톱밥을 주원료로 한 배지로 부터 영지버섯(Ganoderma lucidum)을 재배하여 자실체를 수확한 후 얻어지는 영지버섯 완숙 균사체를 용매속에서 가온하거나 가압하여 다당체를 함유하는 유효성분을 추출하는 것을 특징으로 한다.The present invention is a mature mycelium culture (hereinafter referred to as a mature mycelium) which is disposed of and discarded after cultivating and harvesting Ganoderma lucidum (Bulocho), which is a type of basidiomycete belonging to the genus Polyporaceae, in a solid medium mainly composed of sawdust. Regarding the extraction method, it is possible to produce a large amount of active ingredients of Ganoderma lucidum mycelium cheaply by using it as a material.The Ganoderma lucidum is grown from sawdust-based medium and Ganoderma lucidum is obtained after harvesting fruiting bodies. It is characterized by extracting the active ingredient containing the polysaccharide by heating or pressing the mature mycelia in a solvent.
Description
본 발명은 톱밥을 주성분으로 한 고체 배지내에서 장이버섯과(Polyporaceae)에 속하는 담자균류의 일종인 영지버섯(Ganoderma lucidum)을 재배 수확한 후 폐기처리되는 완숙된 균사배양물(이하에서는 완숙 균사체라 칭하기로 함)을 재료로 활용하여 영지 균사체의 유효성분을 값싸게 대량으로 생산할 수 있는 독창적이며 진보적인 추출 방법에 관한 것이다.The present invention is a mature mycelium culture (hereinafter referred to as a mature mycelium) which is disposed of and discarded after cultivating and harvesting Ganoderma lucidum, a type of basidiomycete belonging to the genus Polyporaceae, in a solid medium mainly composed of sawdust. The present invention relates to a unique and advanced extraction method that can produce a large amount of active ingredients of Ganoderma lucidum mycelium inexpensively and in large quantities.
영지버섯은 고대로 부터 신비의 상약으로 알려져 각종 질병에 대한 민간요법제(신농본초경 500년경 강홍경 및 본초강목 1552년 이시진)로 이용되어 왔을 뿐만 아니라 최근에는 항암작용(김병각등, 한국균학회지 8권, 107-113쪽, 1980년)을 위시하여 항앨러지작용(Kohda et al., Chem. Pharm. Bull., 33권, 1367-1374쪽, 1985년), 항고혈압작용(Kanmatsuse et al., 약학잡지(일본), 105권, 942-947쪽:kabir et al., J.Nutr. Sci. vitaminol., 34권, 1988년)등 다양한 생리활성이 입증되었다. 특히 영지 등 담자균류가 생산하는 항암성 다당류들은 부작용이 거의 없고 매우 안전한 반면 인체내 면역계의 기능을 강화시켜 탁월한 항암효과가 있는 것으로 알려졌다.Ganoderma lucidum mushroom has been used as a folk medicine for various diseases since ancient times, and has been used as a folk remedy for various diseases (Kang Hong-kyung, 500 years old, and Lee Si-jin, 1552), as well as anti-cancer activity (Kim Byeong-kak, etc. Antiallergic action (Kohda et al., Chem. Pharm. Bull., Vol. 33, 1367-1374, 1985), antihypertensive action (Kanmatsuse et al., Pharmacy Magazine). (Japan), Vol. 105, pp. 942-947: A variety of biological activities have been demonstrated, including kabir et al., J. Nutr. Sci. Vitaminol., 34, 1988). In particular, anticancer polysaccharides produced by basidiomycetes such as Ganoderma lucidum have little side effects and are very safe, but have been known to have excellent anticancer effects by enhancing the function of the immune system in the human body.
영지의 유효성분을 공업적으로 추출하여 식품 또는 의약품으로 이용하는 방법은 크게 두가지가 있는데 그 첫째는 재배된 영지버섯 자실체를 재료로 이용하는 방법이며, 둘째는 균사를 액내 배양하여 그로부터 유효성분을 추출하는 방법이다. 그러나 영지버섯 자실체는 재배기간이 수개월씩 소요됨은 물론 많은 노동력이 투입되고 수확량도 낮기 때문에 영지버섯 자실체를 원료로 사용하는 방법은 경제성이 없다.There are two ways to extract the active ingredient of Ganoderma lucidum industrially and use it as food or medicine. The first method is to use the cultivated Ganoderma lucidum as a material, and the second is to incubate the hyphae in liquid and extract the active ingredient therefrom. to be. However, the Ganoderma lucidum fruiting body takes months of cultivation as well as a lot of labor and low yield. Therefore, it is not economical to use Ganoderma lucidum fruiting body as a raw material.
한편 균사체 액내 배양법은 엄청난 설비 투자가 요구되고 고도의 첨단 기술이 요구될 뿐만 아니라 배양된 균사체는 완숙되지 않아서 2차 대사물 즉 영지버섯 자체의 고유 성분들을 고르게 형성하지 않기 때문에 영지버섯 자체 즉 자실체와는 성분상 차이가 나는 등 많은 문제점이 있다.On the other hand, mycelium liquid culture requires tremendous equipment investment and advanced technology, and since the cultured mycelium is not mature, it does not evenly form the inherent components of secondary metabolites, namely ganoderma lucidum itself, because There are many problems such as differences in composition.
이 때문에 인삼박 등의 특수원료를 주성분으로 한 배지에서 균사체를 얻는 독특한 방안들이 제안된 바 있으나 이들 방법들은 배지를 형성하는 재료를 구하기가 힘들고 비용이 많이들므로 경제성면에 있어서 불리하다. 한편 농가에서는 대량으로 영지버섯을 재배하고 있는바 영지버섯 재배에는 값싸게 얻을 수 있는 톱밥을 부직포 등의 종균캡슐에 담아 배지를 형성하고 균주를 심어 영지버섯을 길러 자실체를 수확한 후 종균캡슐배지는 소각이나 매립처리하여왔다.For this reason, unique methods of obtaining mycelium from a medium containing special raw materials such as ginseng meal have been proposed, but these methods are disadvantageous in terms of economics because it is difficult and expensive to obtain the material forming the medium. On the other hand, farmers are cultivating large amounts of ganoderma lucidum mushrooms. For cultivation of ganoderma lucidum mushrooms, sawdust, which can be obtained cheaply, is placed in spawn capsules such as non-woven fabrics to form a medium, and the strains are planted to cultivate ganoderma lucidum to harvest fruiting bodies. It has been incinerated or landfilled.
그러나 상기와 같이 영지버섯 자실체의 수확 후 발생되는 다량의 종균캡슐배지를 폐기처분하게 되므로써 환경공해를 유발하고 있으나 본 발명자는 각 농가에서 영지버섯 재배과정에서 발생하는 다량의 폐기물인 종균캡슐 톱밥배지내에 균사체가 다량 포함되어 있는점에 착안하여 환경폐기물에 불과한 종균캡슐 톱밥배지를 재활용차원에서 이용하여 다당체를 함유하는 유효성분을 값싸게 대량으로 얻을 수 있음을 발견하게 되었다.However, as described above, a large amount of seed capsule capsules generated after harvesting Ganoderma lucidum fruit body is disposed to cause environmental pollution, but the present inventors have caused a large amount of wastes generated in the process of growing Ganoderma lucidum mushrooms in each farm. Taking into consideration the fact that a large amount of mycelium is contained, it has been found that a large amount of active ingredients containing polysaccharides can be obtained inexpensively using a seed capsule capsule, which is only an environmental waste, in terms of recycling.
이에 따라 본 발명의 목적은 영지버섯 재배 과정에서 대량으로 발생하여 폐기 대상이 되어 왔던 톱밥 배지내 완숙 균사체를 이용하여 영지버섯 자실체에 가까운 유효성분들을 매우 경제적이고 효과적으로 추출하는 방법을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a method of extracting the active ingredient close to the ganoderma lucidum fruit body very economically and effectively using the mature mycelium in the sawdust medium that has been generated in large quantities in the process of cultivating Ganoderma lucidum.
본 발명의 부수적인 목적은 영지버섯 재배를 통해 발생되는 다량의 폐기물인 종균캡슐 톱밥배지를 재활용하여 다당체 함유 유효성분을 추출하므로써 환경오염유발을 최소화하는 데 있다.An additional object of the present invention is to minimize the environmental pollution by recycling the active ingredient containing polysaccharides by recycling the seed capsule capsule spawn medium, which is a large amount of waste generated through cultivation of Ganoderma lucidum.
본 발명은 상기한 목적을 달성하기 위하여 톱밥을 주원료로 한 배지로 부터 영지버섯(Ganoderma lucidum)을 재배하여 자실체를 수확한 후 얻어지는 영지버섯 완숙 균사체를 용매속에서 가온하거나 가압하여 다당체를 함유하는 유효성분을 추출하는 것을 특징으로 하고 있다.In order to achieve the above object, the present invention is effective to contain the polysaccharide by heating or pressurizing the Ganoderma lucidum mature mycelium obtained after harvesting fruiting bodies by cultivating Ganoderma lucidum from a medium containing sawdust as a main ingredient. It is characterized by extracting a component.
이와 같은 추출 방법은 경제성이 높은 방법임은 물론 영지버섯 고유의 고미성분등이 다량 추출되는 등 액내배양법 보다도 우수한 장점을 갖고 있다. 그 내용을 좀더 자세히 설명하면 다음과 같다.This extraction method is not only a high economic method, but also has an advantage over the liquid culture method, such as the extraction of a large amount of gourds unique to Ganoderma lucidum. The details are as follows.
현재 유통되고 있는 영지버섯은 거의가 농가에서 길러낸 재배품이며, 초기에는 주로 원목 재배법이 이용되었으나 현재는 참나무 톱밥을 주원료로 한 인공고체 배지에 재배하는 방법이 주로 이용되고 있다. 즉, 톱밥을 주성분으로 하고 여기에 쌀겨 등을 첨가한 배지를 플라스틱 용기 또는 폴리플로필렌 포대 등에 충전하고, 살균시킨 다음 영지버섯 종균을 접종시켜 균사 성장에 적합한 온도(25-30℃)에서 배양하여 톱밥배지내에 균사를 충만하게 성장시킨 후 버섯 발생에 적합한 온도 및 습도 조건을 맞추어 줌으로써 영지버섯을 발생시킨다.Most of the Ganoderma lucidum in circulation is a cultivated product grown by farmers. In the early days, wood cultivation was mainly used, but now, cultivation is carried out in an artificial solid medium mainly made of oak sawdust. In other words, a medium containing sawdust and rice bran added thereto are filled in a plastic container or polypropylene bag, sterilized, inoculated with Ganoderma lucidum spawn and incubated at a temperature suitable for mycelial growth (25-30 ° C.). After growing mycelia in sawdust medium, Ganoderma lucidum is generated by adjusting temperature and humidity conditions suitable for mushroom generation.
이때 건실한 자실체를 생성시키기 위하여 용기당 통상 1-2개의 영지버섯이 자라도록 나머지 영지버섯 생장점들은 절단 제거한다. 이러한 이유 등으로 인해 영지의 수율은 느타리버섯 등 다른 일반 식용 버섯 등에 비하여 현저히 낮으며 따라서 영지버섯은 매우 고가일 수 밖에 없다. 그럼에도 불구하고 영지버섯 수확 후 톱밥 배지에 완숙하게 성장한 균사체 즉 완숙 균사체는 활용되지 않고 대부분 소각 폐기되고 있다.At this time, the remaining Ganoderma lucidum growth points are cut and removed so that 1-2 ganoderma lucidum usually grows per container to produce a healthy fruiting body. For this reason, the yield of Ganoderma lucidum is significantly lower than other common edible mushrooms, such as oyster mushroom, and thus Ganoderma lucidum is very expensive. Nevertheless, the mycelia grown in the sawdust medium after harvesting ganoderma lucidum, i.e., mature mycelium, are not used and most of them are incinerated.
따라서 재배 농가의 입장에서는 이들을 처분하는 것이 또 하나의 일거리가 되고 있는 실정이다. 한편, 이들 완숙 균사체는 접종한 영지 균사체에 의해 톱밥 성분이 대부분 분해 흡수되어 영지 균사체로 전환된 상태이며 충분한 기간 동안 완숙되는 과정에서 영지의 고유하고 다양한 성분들이 생성됨으로써 영지의 고유한 쓴맛의 향을 그대로 지니게 된다.Therefore, from the standpoint of cultivated farmers, disposing of them becomes another job. On the other hand, these mature mycelium is a state in which the sawdust component is mostly decomposed and absorbed by the inoculated Ganoderma mycelium, which is converted into Ganoderma mycelium. It will remain as it is.
따라서 이들을 활용하여 유효성분을 추출 이용하는 것은 고귀한 자원의 활용은 물론 미완숙 균사체와는 달리 영지버섯 자체에 가까운 맛과 향을 가짐으로써 기호 식품으로서의 가치는 물론 그 유효성분의 약리활성을 기대하는 기호식품 등으로서의 장점도 갖게 된다. 뿐만 아니라 균사체는 영지 버섯 자체보다도 쉽게 고운 세말 상태로 파쇄되며 추출 작업이 용이하므로 유효성분이 대부분 그대로 남아 있는 완숙 균사체로 부터 영지의 유효성분을 추출 생산하는 것이 가능하게 된다.Therefore, extracting active ingredients by using them is not only utilizing noble resources but also having a taste and aroma close to that of Ganoderma lucidum itself, unlike raw mycelium, it is a favorite food that expects not only its value as a food but also its pharmacological activity. It also has advantages as a back light. In addition, the mycelium is crushed into finer finer state than the Ganoderma lucidum itself, and is easy to extract, so that it is possible to extract and produce the active ingredient of Ganoderma lucidum from the mature mycelium, which remains the most active ingredient.
이하, 본 발명을 실시예에 따라 보다 구체적으로 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail with reference to Examples.
즉, 완숙 균사체에 적당량의 물이나 0.1-2.0 N NaOH 등 알칼리 용액 또는 0.1-1.0 N HCl 등 산용액 등을 가하고 즉시 또는 2∼18시간 방치 후 80∼100℃에서 1-2시간씩 2-3회 균사체 성분을 추출한다. 유기용매를 사용할 경우에는 95% 에탄올, 에테르, 클로로포름, 에틸아세테이트 등의 용매를 가하고 30분 내지 3시간 진탕추출한 후 추출물만을 회수한다. 이때 물이나 알칼리, 또는 산 용액으로 추출한 경우는 추출물을 진공농축 한 후 2-5배량의 95% 에탄올을 가하고 2시간 내지 하룻밤 정치한 다음 1000-8000rpm에서 10-30분간 원심분리한다.That is, a suitable amount of water, an alkaline solution such as 0.1-2.0 N NaOH or an acid solution such as 0.1-1.0 N HCl is added to the mature mycelium immediately or left for 2 to 18 hours, followed by 1-2 hours at 80 to 100 ° C for 2-3 hours. Ash mycelium component is extracted. In the case of using an organic solvent, a solvent such as 95% ethanol, ether, chloroform, ethyl acetate and the like is added thereto, followed by shaking for 30 minutes to 3 hours to recover only the extract. At this time, if extracted with water, alkali, or acid solution, the extract was concentrated in vacuo, 2-5 times of 95% ethanol was added and allowed to stand for 2 hours to overnight, then centrifuged at 1000-8000 rpm for 10-30 minutes.
이와 같이 하여 얻은 추출물은 다시 소량의 물에 녹여서 분자량 8000 이하의 물질을 투석할 수 있는 투석막을 이용하여 약 2-7일간 투석 하거나 극초여과(ultrafiltration)한 후 -70℃에서 동결 건조하면 다당체를 함유하는 고분자 추출물을 0.4-5%의 수득률로 얻을 수 있다. 한편, 유기용매를 사용할 경우에는 그 추출물을 감압농축하여 진한 엑기스 상으로 회수한다.The extract obtained in this way was dissolved in a small amount of water again using a dialysis membrane capable of dialysis of a substance having a molecular weight of 8000 or less, and dialyzed for about 2-7 days or ultrafiltration and then freeze-dried at -70 ° C to contain polysaccharides. A polymer extract can be obtained with a yield of 0.4-5%. On the other hand, when an organic solvent is used, the extract is concentrated under reduced pressure and recovered in a concentrated extract phase.
[실시예1]Example 1
영지 완숙 균사체의 고분자 추출물 제조Preparation of Polymer Extracts from Ganoderma lucidum Mycelium
톱밥을 주원료로 한 배지에서 영지버섯을 재배하여 자실체를 수확한 후 얻어진 영지버섯 완숙 균사체 50g을 1리터의 1.0 N NaOH를 가하고 40℃ 오븐내에 4시간 방치후 121℃에서 30분간 추출한다. 균사체 케익은 제거하고 추출물만을 감압여과하여 회수한 후의 부피로 감축되도록 진공 농축 하고 3배량의 95% 에탄올을 가하여 하룻밤을 정치한다. 3000rpm에서 30분간 원심분리 하여 침전물을 분리한 후 이 침전물을 다시 소량의 물에 녹여서 분자량 8000 이하의 물질을 투석할 수 있는 투석막을 이용하여 약 3-7일간 투석한 다음 다시 ½∼⅓ 의 부피로 감축되도록 감압 농축하여 동결건조하면 다당체를 함유하는 고분자 추출물 0.8-1.5g 가량(수득률 0.8-3.0%)을 얻을 수 있다.50 g of the ripened mycelium obtained from the growth of ganoderma lucidum by harvesting fruiting bodies in a medium containing sawdust as a main ingredient is added with 1 liter of 1.0 N NaOH and left for 4 hours in an oven at 40 ° C., followed by extraction at 121 ° C. for 30 minutes. The mycelium cake is removed, concentrated only in vacuo to reduce the volume to the volume after recovery of the extract by filtration under reduced pressure. The precipitate was separated by centrifugation at 3000 rpm for 30 minutes, and then the precipitate was dialyzed for about 3-7 days using a dialysis membrane capable of dissolving it again in a small amount of water and dialysis material having a molecular weight of 8000 or less, and then returning to a volume of ½ to ⅓. Concentration under reduced pressure to reduce the lyophilization yields about 0.8-1.5 g of the polymer extract containing polysaccharides (yield 0.8-3.0%).
[실시예2]Example 2
면역 활성 효과Immune activity effect
상기 실시예 1에서 얻어진 완숙 균사체 추출물을 아이씨알 마우스(ICR-mouse)에 투여하고 면역활성을 조사하였다. 즉, 실험약물 100㎎을 10㎖의 생리식 염수에 용해시켜 고압멸균하여 매일 1회 0.1㎖씩(50㎎/㎏) 10회 아이씨알 마우스의 복강내에 투여하였다. 10일후에 비장을 적출하여 비장내 면역 세포들의 조성을 보고한 방법(정 경수, 신약개발의 실제와 문제(한국응용약품학회 추계 심포지움 초록집, 7-12쪽, 1993년)에 따라 면역형광염색(immunofluorescence stating)및 유세포분석법(flow cytometry)으로 분석하였다. 그 결과 표 1과 같이 완숙 균사 추출물을 투여함으로써 비장내 B 임파구(sIg+)및 대식세포(macrophage, Mac-1+)가 증가하였으며 CD4+T임파구에 대한 CD8+T임파구 비율도 증가하였다. 이들 대식세포, B임파구, CD8+T임파구 등이 CD4+T임파구와 달리 주로 면역효능세포(effector cell)임을 감안할때 영지 완숙 균사체 추출물이 면역 기능을 효과적으로 강화함을 알 수 있다.The mature mycelia extract obtained in Example 1 was administered to ICR-mouse and examined for immunological activity. That is, 100 mg of the experimental drug was dissolved in 10 ml of physiological saline and sterilized by autoclaving, and 0.1 ml (50 mg / kg) was administered intraperitoneally once a day. After 10 days, the spleen was extracted and the composition of immune cells in the spleen was reported (Jung, Kyung-Soo, Practice and Development of New Drugs, Abstracts Symposium Abstract, 7-12, 1993). The results were as follows: stating) and flow cytometry, which resulted in an increase in splenic B lymphocytes (sIg + ) and macrophages (Mac-1 + ) and CD4 + T by the administration of mature mycelia extracts as shown in Table 1. The ratio of CD8 + T lymphocytes to lymphocytes also increased, and given that these macrophages, B lymphocytes, and CD8 + T lymphocytes are mainly effector cells, unlike CD4 + T lymphocytes, Ganoderma lucidum mycelium extracts showed immune function. It can be seen that it strengthens effectively.
이상과 같은 본 발명에 의하면 농가에서 영지버섯 재배의 부산물로 발생되어 폐기처리되는 종균캡슐 속의 톱밥배지를 회수한 후, 그 톱밥배지 속의 완숙균사체를 이용하여 유효성분을 추출하게 되므로 종래에 비해 비용이 크게 절감되고 재활용에 의한 환경오염이 감소될 뿐만 아니라 유효성분 추출을 위해 별도로 배지를 배양하지 아니하고 농가로부터 버려지는 영지버섯 재배후의 톱밥배지를 회수하여 이용하므로 유효성분의 추출시간이 크게 단축되는 효과가 있다.According to the present invention as described above, after recovering the sawdust medium in the spawn capsule which is generated as a by-product of Ganoderma lucidum cultivation in the farmhouse, the active ingredient is extracted using the mature mycelium in the sawdust medium, the cost is higher than in the prior art. Not only does it significantly reduce the environmental pollution caused by recycling, but also extracts the sawdust medium after cultivation of the Ganoderma lucidum, which is discarded from the farm, without culturing the medium separately for the extraction of the active ingredient. have.
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