KR101662185B1 - Method for producing chaga mushroom extract containing high concentration of? -glucan - Google Patents
Method for producing chaga mushroom extract containing high concentration of? -glucan Download PDFInfo
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- KR101662185B1 KR101662185B1 KR1020140183354A KR20140183354A KR101662185B1 KR 101662185 B1 KR101662185 B1 KR 101662185B1 KR 1020140183354 A KR1020140183354 A KR 1020140183354A KR 20140183354 A KR20140183354 A KR 20140183354A KR 101662185 B1 KR101662185 B1 KR 101662185B1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- ZYBWTEQKHIADDQ-UHFFFAOYSA-N ethanol;methanol Chemical compound OC.CCO ZYBWTEQKHIADDQ-UHFFFAOYSA-N 0.000 description 1
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- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
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- BSUHUYNJLGMEPD-UHFFFAOYSA-N methanol;propan-1-ol Chemical compound OC.CCCO BSUHUYNJLGMEPD-UHFFFAOYSA-N 0.000 description 1
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- 235000013824 polyphenols Nutrition 0.000 description 1
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- 230000008569 process Effects 0.000 description 1
- YGSFNCRAZOCNDJ-UHFFFAOYSA-N propan-2-one Chemical compound CC(C)=O.CC(C)=O YGSFNCRAZOCNDJ-UHFFFAOYSA-N 0.000 description 1
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
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- 210000002784 stomach Anatomy 0.000 description 1
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 description 1
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
본 발명은 β-글루칸을 고농도로 함유하는 차가버섯 추출물의 제조방법에 관한 것으로, 보다 상세하게는 차가버섯에 추출용매를 가하고 40~100℃에서 4~8시간 동안 환류냉각추출하는 단계를 포함하는 고농도의 β-글루칸을 함유하는 차가버섯 추출물의 제조방법에 관한 것이다. 이러한 본 발명은 차가버섯 유래 β-글루칸을 고농도로 함유하는 차가버섯 추출물을 제공할 수 있다.The present invention relates to a method for preparing an extract of chaga mushroom which contains β-glucan at a high concentration, and more particularly to a method for producing chaga mushroom extract comprising a step of adding an extraction solvent to chaga mushroom and refluxing and extracting the mixture at 40 to 100 ° C. for 4 to 8 hours The present invention relates to a method for producing an extract of chaga mushroom containing high concentration of? -Glucan. The present invention can provide chaga mushroom extract containing a high concentration of mushroom-derived? -Glucan.
Description
본 발명은 β-글루칸을 고농도로 함유하는 차가버섯 추출물의 제조방법에 관한 것으로, 보다 상세하게는 차가버섯에 추출용매를 가하고 40~100℃에서 4~8시간 동안 환류냉각추출하는 단계를 포함하는 고농도의 β-글루칸을 함유하는 차가버섯 추출물의 제조방법에 관한 것이다.The present invention relates to a method for preparing an extract of chaga mushroom which contains β-glucan at a high concentration, and more particularly to a method for producing chaga mushroom extract comprising a step of adding an extraction solvent to chaga mushroom and refluxing and extracting the mixture at 40 to 100 ° C. for 4 to 8 hours The present invention relates to a method for producing an extract of chaga mushroom containing high concentration of? -Glucan.
차가버섯(Inonotus obliquus)은 소나무비늘버섯과(Hymenochaetaceae)에 속하는 다년생의 담자균 버섯으로, 자연상태에서 시베리아, 핀란드, 노르웨이, 우크라이나, 홋카이도 등의 북위 45도 이상의 춥고 습한 북반구에 분포하며, 일반적으로 자작나무, 오리나무, 마가목 등의 줄기나 그루터기에 자생하는 극내한성 버섯이다. 국내에서 자생한다는 보고는 아직 없으며, 백색부후균의 일종으로, 자연상태에서 성장하면 검은색의 균핵덩어리가되어 자작나무 등의 줄기에 기생하는 것으로 알려져 있다. Inonotus obliquus is a perennial bamboo mushroom belonging to the pine scales (Hymenochaetaceae). It is distributed in natural and cold northern hemisphere above 45 degrees north of Siberia, Finland, Norway, Ukraine and Hokkaido, It is extreme cold resistant mushroom which grows in stem and stump of tree, duck, There is no report that it is native in Korea. It is a kind of white rot fungus. It grows in the natural state and becomes black sclerotia, and is known to parasitize in the trunk of birch.
차가버섯은 러시아에서는 예로부터 차, 음료로 음용되어 왔으며, 현재는 위장과 관련된 모든 암치료에 사용되고 있고, 일본에서는 카바노아나타케라고 부르고 있으며 에이즈와 O-157 및 간암의 치료제로 이용되고 있다. 이 버섯은 강력한 제암작용과 강한 면역활성작용(일본생약학회 제41회 연회연구발표)과 항에이즈 활성(제7회 일본에이즈학회)을 보이는 것으로 알려져 있다.Chaga mushrooms have been traditionally used in tea and beverages in Russia and are now used for the treatment of all cancers related to the stomach. In Japan, they are called "cabanoanatake" and are used for the treatment of AIDS, O-157 and liver cancer. This mushroom is known to exhibit strong anti-cancer action and strong immune-activating action (the 41th annual meeting of the Japanese Society of Pharmaceutical Sciences) and anti-AIDS activity (the 7th Japanese Society of AIDS).
차가버섯의 유효성분으로는 강한 항산화 효과와 세포활성 효과가 있는 β-글루칸, 헤테로글루칸 등이 있고(제암성다당류에 관한연구, 일본생약학회 제27보), 이 버섯의 균핵과 균사체로부터 추출한 다당류의 수용성 및 수불용성 다당체는 항종양활성 및 혈당강하작용이 있는 것으로 알려져 있다. 이외에도, 알레르기 피부염, 만성간염, 신염 등의 예방에 효력이 있는 것으로 알려지고 있으나 인체 내에서의 대사기능과 약리작용 등, 아직 밝혀지지 않은 것이 많아 아직 연구단계에 있는 것도 많다. 이러한 효력을 나타내는 차가버섯 속의 유효성분들에는 β-글루칸, 트리체페놀산, 호로마도겐, 폴리페놀, 옥시페놀카르본산, 휘노친, 차가산(60%), 바닐라산, 파라옥시향산, 프테린, 스테롤 등의 많은 생리활성성분이 있는 것으로 알려져 있다.Glucan, and heteroglucan, which have strong antioxidative and cell activating effects (studies on anticarcinogenic polysaccharides, Japanese Pharmacopoeia, 27), polysaccharides extracted from scleroti and mycelium of mushroom Of water-soluble and water-insoluble polysaccharides are known to have antitumor activity and hypoglycemic action. In addition, it is known to be effective in the prevention of allergic dermatitis, chronic hepatitis, nephritis, etc. However, many metabolic functions and pharmacological actions in the body have not yet been clarified. The active ingredients of this mushroom, which exhibits such efficacy, include beta -glucan, trichephenol acid, fomlodogen, polyphenol, oxyphenolcarboxylic acid, phinocin, carboxy acid (60%), vanillic acid, , Sterol, and the like.
그런데 이들 성분들은 물리 화학적 성질의 차이에 의해 용해성이나 휘발성이 다르고, 특히 온도에 대한 용해성이 다르기 때문에 추출온도는 유효성분의 효과적인 용출을 위해서 매우 중요한 요인이라 사료된다. 따라서 차가버섯의 유효성분의 물리적 화학적 특성과 관계없이 가능한 한 많은 유효성분을 섭취하기 위한 추출 가공방법이 중요하다.However, these components differ in their solubility and volatility due to differences in their physico-chemical properties, and especially because their solubility in temperature is different, the extraction temperature is considered to be a very important factor for effective elution of the active ingredient. Therefore, it is important to extract and process as many active ingredients as possible regardless of the physicochemical properties of the active ingredients of the mushroom.
차가버섯의 추출방법에 관한 종래기술로는 단순 열수추출 등의 일반적인 한약방에서 사용하는 방법으로, 차가버섯을 30~100℃에서 5~40배 부피의 온수에 5~10분간 용출시키거나, 8시간 동안 1.2~2.5kg/㎤ 정도의 압을 유지한 상태에서 추출하는 방법 등이 알려져 있다.As a conventional technique for extracting mushroom, it is a method used in a general oriental medicine such as simple hot water extraction. The mushroom is extracted from mushroom at 30 to 100 DEG C for 5 to 10 minutes in hot water of 5 to 40 times volume, A method of extracting while maintaining a pressure of about 1.2 to 2.5 kg / cm 3 is known.
그러나 종래에는 버섯과 같은 인체에 유익한 천연물의 경우, 아직 현대과학으로도 분석해내지 못하는 유효성분이 다수 있으므로 추출공정 중에 이를 파괴하거나 소실되지 않도록 해야한다는 어려움이 있었다. 특히, 고온이나 저온으로 추출할 경우 열에 약하거나 휘발성이 강한 유효성분들이 파괴, 소실되거나, 활성 성분이 효율적으로 추출되지 못한다는 문제가 있었다.However, in the case of natural materials useful for human bodies such as mushrooms, there are many effective ingredients that can not be analyzed by modern science yet. Therefore, it has been difficult to destroy or lose the natural materials during the extraction process. Particularly, when extracted at a high temperature or a low temperature, effective substances having weak heat or volatility are destroyed or lost, or active ingredients can not be efficiently extracted.
따라서, 인체에 유익한 차가버섯으로부터 유용한 활성성분들을 효율적으로 추출할 수 있는 방법에 대한 연구가 더욱 필요한 실정이다.Therefore, there is a need for further studies on a method for efficiently extracting useful active ingredients from the mushroom which is useful for human body.
상기와 같은 종래기술의 문제점을 해결하고자, 본 발명은 환류냉각추출법을 이용한 차가버섯 유래 β-글루칸을 고농도로 함유하는 차가버섯 추출물의 제조방법을 제공하는 것을 목적으로 한다.In order to solve the problems of the prior art as described above, it is an object of the present invention to provide a method for preparing chaga mushroom extract containing a high concentration of chaga mushroom-derived? -Glucan using a reflux cooling extraction method.
상기 목적을 달성하기 위하여, 본 발명은 차가버섯을 1~2㎜의 크기로 분쇄하는 단계; 상기 차가버섯 분쇄물에 추출용매를 가하고 40~100℃에서 4~8시간 동안 환류냉각추출하는 단계; 및 상기 차가버섯 추출물을 감압농축하는 단계;를 포함하는 β-글루칸을 고농도로 함유하는 차가버섯 추출물의 제조방법을 제공한다.In order to attain the above object, the present invention provides a method for producing a fermented mushroom, comprising: crushing a mushroom to a size of 1 to 2 mm; Adding an extraction solvent to the ground mushroom and refluxing and cooling at 40 to 100 ° C for 4 to 8 hours; And concentrating the chaga mushroom extract under reduced pressure. The present invention also provides a method for producing chaga mushroom extract containing β-glucan at a high concentration.
상기 추출용매는 증 증류수, 알칼리 환원수, 에탄올, 메탄올, 2-프로판올 및 아세톤 중 선택된 어느 하나 이상을 사용할 수 있으며, 상기 환류냉각추출은 40℃, 80℃ 또는 100℃에서 4시간 또는 8시간 동안 수행되는 것이 바람직하다.The extraction solvent may be at least one selected from the group consisting of distilled water, alkaline reducing water, ethanol, methanol, 2-propanol and acetone. The reflux cooling extraction is carried out at 40 ° C, 80 ° C or 100 ° C for 4 hours or 8 hours .
특히, 상기 차가버섯 추출물은 추출용매로 에탄올을 사용하고, 100℃에서 8시간 동안 환류냉각추출 시 조성물 건조중량에 대하여 22.1~31.7㎎/g의 β-글루칸을 포함하며, 추출용매로 에탄올이나 메탄올을 사용하여 40℃에서 4시간 환류냉각추출 시 조성물 건조중량에 대하여 6.85~8.43㎎/g의 β-1.3-글루칸을 포함하는 차가버섯 추출물을 제조할 수 있다.In particular, the chaga mushroom extract contains 22.1 to 31.7 mg / g of β-glucan as an extracting solvent, ethanol and ethanol as an extraction solvent and ethanol or methanol Was used to prepare chaga mushroom extract containing beta-1.3-glucan in an amount of 6.85 to 8.43 mg / g based on the dry weight of the composition under reflux and cooling for 4 hours at 40 ° C.
또한 본 발명은 상기 방법으로 제조된 β-글루칸을 고농도로 함유하는 차가버섯 추출물을 제공한다.In addition, the present invention provides a chaga mushroom extract containing β-glucan at a high concentration as prepared by the above method.
본 발명에 따른 환류냉각추출법을 이용하여 차가버섯 유래 β-글루칸을 고농도로 함유하는 차가버섯 추출물을 제조할 수 있으며, 이렇게 제조된 차가버섯 추출물은 β-글루칸을 고농도로 함유하여 적은 양으로도 β-글루칸이 가지는 항암, 면역강화, 항염, 항산화, 세포활성 효과를 최대 발현할 수 있어, 약학, 식품 등의 분야에 유용히 적용할 수 있다. By using the reflux cooling method according to the present invention, a chaga mushroom extract containing chaga mushroom-derived? -Glucan at a high concentration can be produced. The chaga mushroom extract thus prepared contains a high concentration of? -Glucan, - Glucan has maximal expression of anti-cancer, immunity enhancement, anti-inflammation, antioxidant, and cell activation effects, and can be applied to fields of pharmacy and food.
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명자는 차가버섯으로부터 β-글루칸을 고농도로 추출, 분리할 수 있는 방법에 대하여 연구하던 중, 특정 추출용매, 추출온도 및 추출시간에서 환류냉각추출법에 의해 추출 시 고농도의 β-글루칸을 함유하는 차가버섯 추출물을 얻을 수 있음을 확인하고, 이를 토대로 본 발명을 완성하였다.The present inventors have studied a method of extracting and separating? -Glucan from chaga mushrooms at a high concentration, and have found that when extracting by a reflux cooling extraction method at a specific extraction solvent, extraction temperature and extraction time, The present invention has been completed on the basis of this finding.
이러한 본 발명은 차가버섯을 분쇄하는 단계, 상기 차가버섯 분쇄물에 추출용매를 가하고 환류냉각추출하는 단계 및 상기 차가버섯 추출물을 감압농축하는 단계를 포함한다.The present invention includes a step of pulverizing a mushroom, a step of adding an extraction solvent to the mushroom pulverized product, followed by reflux cooling extraction, and a step of concentrating the mushroom extract at a reduced pressure.
이하에서는 본 발명의 고농도의 β-글루칸을 함유하는 차가버섯 추출물의 제조방법을 구체적으로 설명한다. Hereinafter, a method for producing the mushroom extract containing the high concentration of? -Glucan of the present invention will be described in detail.
이하 본 발명에서 설명하는 차가버섯 추출물은 추출, 정제의 단계에서 얻어지는 모든 추출물, 정제물, 그들의 희석액, 농축액 또는 건조물을 모두 포함하는 개념이다.The chaga mushroom extract described in the present invention is a concept including all the extracts obtained in the step of extraction and purification, the purified product thereof, the diluted solution thereof, the concentrate or the dried product.
먼저, 차가버섯은 신선하거나 건조된 상태로 이용될 수 있으나, 건조된 차가버섯을 사용하는 것이 좋다.First, chaga mushrooms can be used fresh or dried, but it is better to use dried chaga mushrooms.
상기 건조된 차가버섯은 통상의 분쇄기를 이용하여 차가버섯의 자실체 또는 균사체 입자가 1~2㎜의 크기가 되도록 분쇄하는 것이 최종 얻어진 차가버섯 추출물 중 β-글루칸의 농도를 증가시킬 수 있어 바람직하다.The dried chaga mushroom is preferably pulverized to have a size of 1 to 2 mm in the fruiting body or the mycelium of chaga mushroom using a conventional grinder to increase the concentration of? -Glucan in the resulting chaga mushroom extract.
상기 차가버섯 분쇄물에는 추출용매를 가하여 추출하는 단계를 수행한다.The extraction of the mushroom pulverized product with the extraction solvent is carried out.
통상 차가버섯 추출물을 제조하기 위하여는 다양한 추출용매와 추출방법이 적용될 수 있으나, 본 발명에서는 고농도의 β-글루칸을 포함하는 차가버섯 추출물을 제조하기 위하여 특정 추출용매를 사용하는 것이 바람직하다. 상기 추출용매로는 증류수, 알칼리 환원수, 에탄올, 메탄올, 2-프로판올 및 아세톤 중 선택된 어느 하나 이상의 용매를 사용하는 것이 바람직하며, 특히 에탄올이나 메탄올을 사용하는 것이 최종 얻어진 차가버섯 추출물 중 β-글루칸의 농도를 최대 증가시킬 수 있어 더욱 바람직하다. 특히, 추출용매로 에탄올을 사용할 경우에는 차가버섯 추출물 중 β-글루칸을 고농도로 함유하게 되며, 메탄올을 사용할 경우에는 차가버섯 추출물 중 β-1,3-글루칸이 고농도로 함유하게 된다.In order to prepare chaga mushroom extract in general, various extraction solvents and extraction methods can be applied. However, in the present invention, it is preferable to use a specific extraction solvent to prepare chaga mushroom extract containing high concentration of β-glucan. It is preferable to use at least one solvent selected from distilled water, alkaline reducing water, ethanol, methanol, 2-propanol and acetone as the extraction solvent. In particular, ethanol or methanol is preferably used as the extraction solvent. It is possible to maximize the concentration. In particular, when ethanol is used as the extraction solvent, β-glucan is contained in a high concentration in the mushroom extract, and when methanol is used, β-1,3-glucan is contained in a high concentration in the mushroom extract.
또한 상기 차가버섯 추출물의 추출방법으로는 환류냉각추출(reflux extraction)법을 사용하는 것이 바람직하다.As the extraction method of the mushroom extract, reflux extraction method is preferably used.
상기 추출온도는 40~100℃에서 4~8시간 동안 수행되는 것이 바람직하며, 더욱 바람직하게는 40℃, 80℃ 또는 100℃에서 4시간 또는 8시간 동안 수행하는 것이며, 가장 바람직하게는 100℃에서 8시간 동안 수행하는 것이다. 상기 추출온도가 40℃ 미만일 경우에는 추출물 수율이 낮게 되고, 100℃를 초과할 경우에는 추출물 중 함유된 유효성분들이 파괴될 수 있다. 또한 상기 추출시간이 4시간 미만일 경우에는 추출물 수율이 낮게 되고,8시간을 초과할 경우에는 시간 증가에 따른 추출물의 양에 큰변화가 없게 된다.The extraction temperature is preferably 4 to 8 hours at 40 to 100 ° C, more preferably 4 hours or 8 hours at 40 ° C, 80 ° C or 100 ° C, most preferably 100 ° C 8 hours. When the extraction temperature is lower than 40 ° C, the yield of the extract is lowered. When the extraction temperature is higher than 100 ° C, the active ingredients contained in the extract may be destroyed. When the extraction time is less than 4 hours, the yield of the extract is lowered. When the extraction time exceeds 8 hours, the amount of the extract is not greatly changed with time.
상기 추출완료 후에는 추출액을 원심분리하여 여과한 다음, 상기 여과액을 감압농축하여 차가버섯 추출물을 얻을 수 있으며, 또한 추가로 용매를 증발, 분무건조 또는 동결건조할 수도 있다.After completion of the extraction, the extract is centrifuged and filtered, and then the filtrate is concentrated under reduced pressure to obtain an extract of Chaga mushroom. Further, the solvent may be further evaporated, spray dried or lyophilized.
상기와 같은 방법으로 제조된 본 발명의 차가버섯 추출물은 전술한 추출조건 하에서 환류냉각추출하는 방법 이외의 통상 알려진 다른 추출방법이나 동일한 환류냉각추출 조건에서 실시하더라도 전술한 본 발명의 추출조건을 충족시키지 않을 경우에는 본 발명에 따라 제조된 차가버섯 추출물과 같이 고농도의 β-글루칸을 포함하지 않게 된다.The chicory mushroom extract of the present invention produced by the above-mentioned method may be subjected to extraction under the above-described extraction conditions and other similar extraction methods other than the reflux cooling extraction method, It does not contain a high concentration of? -Glucan as in the mushroom extract prepared according to the present invention.
즉, 본 발명의 추출조건으로 추출된 차가버섯 추출물은 기존 알려진 차가버섯 추출물 제조방법과는 달리 고농도의 β-글루칸을 포함한다.That is, the chaga mushroom extract extracted under the extraction conditions of the present invention contains a high concentration of? -Glucan, unlike the known chaga mushroom extract preparation method.
특히, 본 발명의 차가버섯 추출물은 추출용매로 에탄올을 사용하여 100℃에서 8시간 동안 환류냉각추출 시 최종 얻어진 차가버섯 추출물 건조중량에 대하여 22.1~31.7㎎/g의 β-글루칸을 포함하며, 추출용매로 메탄올을 사용하여 40℃에서 4시간 환류냉각추출 시 6.85~8.43㎎/g의 β-1,3-글루칸을 포함한다.In particular, the mushroom extract of the present invention contains 22.1 to 31.7 mg / g of β-glucan relative to the dry weight of the final mushroom extract obtained by reflux-cooling extraction at 100 ° C. for 8 hours using ethanol as an extraction solvent, 1,3-glucan of 6.85 to 8.43 mg / g when cooled and refluxed for 4 hours at 40 ° C using methanol as a solvent.
상기와 같이 고농도의 β-글루칸을 포함하는 본 발명의 차가버섯 추출물은 추출단계에서 얻어지는 모든 추출물, 정제물, 그들의 희석액, 농축액 또는 건조물의 형태로 이용될 수도 있고, 필요에 따라 본 발명에 따라 제조된 차가버섯 추출물로부터 고농도의 β-글루칸을 분리하여 사용할 수도 있다. 이때 상기 β-글루칸의 분리는 통상의 분리방법을 통하여 고순도로 분리할 수 있다.The chaga mushroom extract of the present invention containing the high concentration of? -Glucan as described above may be used in the form of all the extracts, purified products, dilutions thereof, concentrates or dried products obtained in the extraction step, and if necessary, It is also possible to use a high concentration of? -Glucan separated from the mushroom extract. At this time, the? -Glucan can be separated with high purity through a conventional separation method.
일반적으로 동일한 성분을 추출한다 하더라도 어떠한 물질로부터 추출해내는지와, 사용되는 추출용매나, 추출온도 및 추출시간, 추출방법에 따라 추출효율이 각각 상이하게 나타나며, 본 발명에서 β-글루칸의 추출에 기존 알려진 추출용매, 추출온도, 추출시간과 추출방법을 적용하였다 하더라도 차가버섯 유래 β-글루칸을 고농도로 함유하는 추출물을 얻기 위한 최적의 조건에 대한 연구는 이전까지는 전무하였으며, 어디에도 찾아볼 수 없는 기술이므로, 본 발명은 전혀 예기치 못한 기술임은 자명한 것이다.Generally, even if the same components are extracted, the extraction efficiency differs depending on the substance to be extracted, the extraction solvent used, the extraction temperature, the extraction time, and the extraction method. In the present invention, Even though the extraction solvent, the extraction temperature, the extraction time and the extraction method are applied, the optimum conditions for obtaining the extract containing the high concentration of the mushroom-derived? -Glucan have not been studied until now, It is clear that the present invention is a completely unexpected technique.
따라서, 상기와 같은 본 발명의 추출방법에 따르면 차가버섯 추출물로부터 고농도의 β-글루칸을 추출할 수 있을 뿐 아니라, 고농도의 β-글루칸을 포함하는 차가버섯 추출물을 간단하고 경제적인 방법으로 단시간에 효율적으로 추출할 수 있다. Thus, according to the above extraction method of the present invention, it is possible not only to extract high concentration of? -Glucan from chicory mushroom extract, but also to extract chicory mushroom extract containing high concentration of? -Glucan in a simple and economical manner in a short time .
뿐만 아니라, 본 발명의 제조방법에 따라 추출된 차가버섯 추출물은 β-글루칸을 고농도로 함유하므로, 적은양의 차가버섯 추출물을 사용하여도 β-글루칸이 가지는 활성을 최대 발현할 수 있어, 항암, 면역강화, 항염, 항산화, 세포활성 효과를 요하는 약학, 식품 등의 분야에 유용히 적용할 수 있다. In addition, since the mushroom extract extracted according to the method of the present invention contains? -Glucan at a high concentration, the activity of? -Glucan can be maximally expressed even with a small amount of mushroom extract, Immunity enhancement, antiinflammation, antioxidation, pharmacy which requires a cell activation effect, food, and the like.
이하에서는 실시예를 들어 본 발명에 관하여 더욱 상세하게 설명할 것이나. 이들 실시예는 단지 설명의 목적을 위한 것으로 본 발명의 보호 범위를 제한하고자 하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.
실시예 1. 차가버섯 추출물 제조Example 1. Preparation of chaga mushroom extract
차가버섯을 Food processor를 이용하여 분쇄한 후, 35메쉬 testing sieve로 거른 후 실험에 사용하였다.The mushroom was crushed using a food processor and then sieved through a 35 mesh testing sieve and used in the experiment.
상기 차가버섯 분쇄물과 용매를 1:10(w/v)의 비율로 혼합하여 용기에 넣은 후 냉각관을 부착하여 40℃의 항온수조에서 4시간 또는 8시간 동안 반복 추출하였다. 이때, 용매로는 증류수(DW), 알칼리 환원수(AW), 25% 에탄올(EtOH), 50% 에탄올(EtOH), 75% 에탄올(EtOH), 100% 에탄올(EtOH), 메탄올(MeOH), 2-프로판올(2-Pro) 및 아세톤(Acetone)을 사용하였다. 추출이 끝난 후 5,000g에서 15분간 원심분리(Mega21R, Hanil Corp., Seoul, Korea)한 후 와트만 #4 여과지(Whatman Co., Maidstone, England)를 이용하여 여과하였다. 상기 여과액은 회전 농축기(N-1000VW, Eyela Co., Tokyo, Japan)를 이용하여 농축한 후 동결건조(NB-504, Ilshin Co., Dongducheon, Korea) 하여 -20℃에서 냉동보관하여 실험에 사용하였다. The chopped mushroom pulverized material and the solvent were mixed at a ratio of 1:10 (w / v), placed in a container, and a cooling tube was attached thereto and repeatedly extracted in a constant temperature water bath at 40 ° C for 4 hours or 8 hours. In this case, distilled water (DW), alkali reduced water (AW), 25% ethanol (EtOH), 50% ethanol (EtOH), 75% ethanol (EtOH), 100% ethanol (EtOH), methanol -Propanol (2-Pro) and acetone (Acetone) were used. After extraction, the mixture was centrifuged at 5,000 g for 15 minutes (Mega21R, Hanil Corp., Seoul, Korea) and then filtered using Wattman # 4 filter paper (Whatman Co., Maidstone, England). The filtrate was concentrated by using a rotary evaporator (N-1000VW, Eyela Co., Tokyo, Japan) and freeze-dried (NB-504, Ilshin Co., Dongducheon, Korea) Respectively.
실시예 2. 차가버섯 추출물 제조Example 2. Preparation of chaga mushroom extract
상기 실시예 1에서 추출온도를 60℃로 달리한 것을 제외하고는 상기 실시예 1과 동일하게 실시하였다.The procedure of Example 1 was repeated except that the extraction temperature was changed to 60 ° C in Example 1.
실시예 3. 차가버섯 추출물 제조Example 3 Preparation of Chaga mushroom extract
상기 실시예 1에서 추출온도를 100℃로 달리한 것을 제외하고는 상기 실시예 1과 동일하게 실시하였다.The procedure of Example 1 was repeated except that the extraction temperature was changed to 100 ° C in Example 1.
실험예 1. β-글루칸 함량Experimental Example 1. [beta] -Glucan content
상기 실시예 1 내지 3에서 제조한 차가버섯 추출물 중 함유된 β-글루칸 함량은 Mushroom β-glucan kit(K-YBGL, Megazyme Int., Wicklow, Ireland)를 사용하여 측정하였다. 총 글루칸 함량에서 α-글루칸 함량을 제한 나머지를 β-글루칸으로 측정하였다. 실험을 위해 상기 실시예 1 내지 3에서 제조한 차가버섯 추출물을 각각 100㎎씩 취하여 시료를 준비하였다. The content of? -Glucan contained in the mushroom extracts prepared in Examples 1 to 3 was measured using a Mushroom? -Glucan kit (K-YBGL, Megazyme Int., Wicklow, Ireland). Limiting the? -Glucan content in the total glucan content The rest was determined by? -Glucan. For the experiment, 100 mg of each of the mushroom extracts prepared in Examples 1 to 3 was taken and prepared.
먼저 총 글루칸 함량을 측정하기 위하여 상기 실시예 1 내지 3에서 제조한 차가버섯 추출물 100㎎의 시료가 담긴 각 튜브에 1.5mL의 37% HCl을 가하고 교반시킨 후 30℃의 욕조(water bath)에서 45분간 반응시켰다. 그 다음 10mL의 증류수를 가하고 혼합한 후 100℃의 욕조에서 2시간 동안 더 가열시켜 준 후 실온에서 방냉 한 후 10mL의 2N KOH(Samchun Pure Chemical)를 가하고 1,500rpm에서 10분간 원심분리하여 상층액을 얻었다. 상기 상층액 0.1mL에 엑소-1,3β-글루카나아제(exo-1,3-β-glucanase) 및 β-글루코시다아제 믹스(β-glucosidase mixture)를 각각 0.1mL씩 각 튜브에 가하여 잘 섞은 후 40℃에서 60분간 반응시켰다. 이어서, 3mL의 glucose oxidase/peroxidase/4-amino-anipyrine 혼합물(GOPOD)을 각 튜브에 가하여 40℃에서 20분간 반응시킨 다음 510㎚에서 흡광도를 측정하였다. 총 α-글루칸의 함량은 100㎎의 시료가 담긴 각 튜브에 2N KOH 2mL씩을 가하여 20분간 얼음 위에서 반응시켰다. 그 다음 1.2M 초산나트륨 버퍼(pH 3.8) 8mL 및 아밀로글루코시드가수분해효소(amyloglucosidase)와 인버테이스(invertase)의 혼합물 0.2mL을 가하여 혼합하고 40℃의 욕조에서 반응시켰다. 이어서, 1,500rpm에서 10분간 원심분리하여 얻은 상층액 0.1mL에 3mL의 GOPOD를 가하고 40℃에서 20분간 배양시킨 후 510㎚에서 흡광도를 측정하고, kit의 계산식에 따라 총 β-글루칸의 함량을 계산하였다. 그 결과는 하기 표 1에 나타내었다.First, in order to measure the total glucan content, 1.5 mL of 37% HCl was added to each tube containing 100 mg of the mushroom extract prepared in Examples 1 to 3, and the mixture was stirred. The mixture was stirred in a water bath at 45 ° C for 45 Lt; / RTI > Then, 10 mL of distilled water was added, and the mixture was further heated in a bath at 100 ° C. for 2 hours. After cooling at room temperature, 10 mL of 2N KOH (Samchun Pure Chemical) was added and centrifuged at 1,500 rpm for 10 minutes. . 0.1 mL of exo-1,3-β-glucanase and β-glucosidase mixture was added to each tube in 0.1 mL of the supernatant, Followed by reaction at 40 ° C for 60 minutes. Then, 3 mL of glucose oxidase / peroxidase / 4-amino-anipyrine mixture (GOPOD) was added to each tube, incubated at 40 ° C for 20 minutes, and absorbance was measured at 510 nm. The total amount of α-glucan was determined by adding 2 mL of 2N KOH to each tube containing 100 mg of the sample and reacting on ice for 20 minutes. Subsequently, 8 mL of 1.2 M sodium acetate buffer (pH 3.8) and 0.2 mL of a mixture of amyloglucosidase and invertase were added and reacted in a bath at 40 ° C. Then, 3 mL of GOPOD was added to 0.1 mL of the supernatant obtained by centrifugation at 1,500 rpm for 10 minutes, and the mixture was incubated at 40 DEG C for 20 minutes. Then, the absorbance was measured at 510 nm and the content of total? -Glucan was calculated Respectively. The results are shown in Table 1 below.
a,b,cDifferent small letters represent significant differences among the values at the same extract conditions(p<0.05; one-way ANOVA and Duncan's multiple range test a, b, c Different small letters represent significant differences among the same extract conditions ( p <0.05; one-way ANOVA and Duncan's multiple range test
상기 표 1에 나타낸 바와 같이, 상기 실시예 1 내지 3에서 제조한 각각의 차가버섯 추출물 중 함유된 β-glucan 함량은 40℃, 4시간의 추출조건 보다 40℃, 8시간과 60℃, 4시간의 추출조건에서 감소하였으나, 60℃, 8시간의 추출조건에서는 다시 β-glucan 함량이 증가하였다. 각 차가버섯 추출물 중 함유된 β-glucan 함량은 40℃와 60℃ 온도 조건에서 차이가 크지 않았으나, 100℃, 4시간의 추출조건에서 75% 이상의 에탄올 용액 추출물이 유의적으로 높게 나타났다. 또한 100℃, 8시간의 추출조건에서는 에탄올 추출물의 β-glucan 함량이 가장 높게 나타났다.As shown in Table 1, the content of β-glucan contained in each of the mushroom extracts prepared in Examples 1 to 3 was 40 ° C., 8 hours and 60 ° C., 4 hours But the β-glucan content was increased again at 60 ℃ for 8 hours. The content of β-glucan in the mushroom extracts was not significantly different between 40 ℃ and 60 ℃, but the extracts of ethanol extracts of more than 75% were significantly higher at 100 ℃ for 4 hours. The β-glucan content of the ethanol extract was the highest at 100 ℃ for 8 hours.
실험예 2. β-1,3-글루칸 함량Experimental Example 2. [beta] -l, 3-glucan content
β-1,3-글루칸의 함량 분석은 β-1,3-글루칸의 β-1,3- 결합에 아닐린 블루(Samchun Pure Chemical) 염색약이 반응하여 형광이 발현되는 현상을 이용한 Ko(1)의 방법을 이용하여 측정하였다. Analysis of β-1,3-glucan content showed that Ko (1), which uses the phenomenon that fluorescence is expressed by the reaction of β-1,3-bond of β-1,3-glucan with an aniline blue dye (Samchun Pure Chemical) Method.
상기 실시예 1 및 3에서 제조한 차가버섯 추출물 각각의 시료를 1N NaOH로 희석하여 300μL를 취한 후 1.5mL 튜브에 넣은 뒤 30μL의 6N NaOH를 가하고 80℃에서 30분간 배양하였다. 배양이 끝나면 즉시 얼음위로 옮겨 방냉한 후 0.1% 아닐린 블루 수용액과 1N HCl과 1M 글리신-NaOH 버퍼(pH 9.5)를 40:21:59의 비율로 혼합한 염색 혼합액 630μL를 가하였다. 그 후 50℃에서 30분간 배양하여 β-1,3-글루칸과 형광 색소 복합체를 형성시킨 후 실온에서 30분간 방냉하였다. 그 후 여기 398㎚와 방출 502㎚에서 형광특성을 측정하였다. 표준물질로 커드란(curdlan, Wako Chemical Co.)을 이용하여 검량선을 그려 β-1,3-글루칸 함량을 구하였다. 그 결과는 하기 표 2에 나타내었다.Samples of each of the mushroom extracts prepared in Examples 1 and 3 were diluted with 1N NaOH and taken in a 1.5-mL tube. Then, 30 μL of 6N NaOH was added thereto and cultured at 80 ° C. for 30 minutes. Immediately after incubation, the mixture was transferred to ice and left to cool. Then, 630 μL of 0.1% aniline blue aqueous solution, 1N HCl and 1 M glycine-NaOH buffer (pH 9.5) were mixed at a ratio of 40:21:59. Thereafter, the mixture was incubated at 50 ° C for 30 minutes to form β-1,3-glucan and a fluorescent dye complex, followed by cooling at room temperature for 30 minutes. The fluorescence properties were then measured at 398 nm excitation and 502 nm emission. The calibration curve was drawn using curdlan (Wako Chemical Co.) as a standard substance to determine the content of? -1,3-glucan. The results are shown in Table 2 below.
상기 표 2에 나타낸 바와 같이, 상기 실시예 1 및 3에서 제조한 각각의 차가버섯 추출물 중 함유된 β-1,3-글루칸 함량은 유기용매를 사용하여 추출한 추출물에서 높게 나타남을 확인할 수 있었다. 특히 유기용매들 중 메탄올 추출물은 각 추출조건에서 가장 높은 β-1,3-글루칸 함량을 보였으나, 추출 온도와 시간이 증가할수록 β-1,3-글루 함량은 감소하였다. 2-propanol 추출물의 β-1,3-글루함량은 추출 온도와는 관계없이 추출 시간이 길어질 때 감소하였다. As shown in Table 2, the content of β-1,3-glucan contained in each of the mushroom extracts prepared in Examples 1 and 3 was found to be high in the extracts extracted using an organic solvent. In particular, the methanol extracts of organic solvents showed the highest content of β-1,3-glucan at each extraction condition, but the content of β-1,3-glue decreased with increasing extraction temperature and time. The β-1,3-glutamate content of 2-propanol extract decreased with increasing extraction time regardless of extraction temperature.
이상과 같은 결과로부터, 본 발명의 차가버섯 추출물은 추출용매로 에탄올을 사용하여 100℃에서 8시간 동안 환류냉각추출 시 차가버섯 추출물 중 함유된 β-글루칸의 함량이 가장 높게 나타났으며, 추출용매로 메탄올을 사용하여 40℃에서 4시간 환류냉각추출 시 β-1,3-글루칸의 함량이 가장 높아 최적의 추출조건임을 확인할 수 있었다.From the above results, the content of? -Glucan contained in the mushroom extract of chaga mushroom extract of the present invention was highest when ethanol was used as the extraction solvent and reflux cooled for 8 hours at 100 ° C. The content of β-1,3-glucan was the highest during reflux cooling extraction at 40 ℃ for 4 hours using methanol.
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한 첨부된 청구 범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.
Claims (6)
상기 추출용매가 100% 에탄올인 경우, 상기 환류냉각추출은 100℃에서 8시간 동안 수행되고, 또는
상기 추출용매가 메탄올인 경우, 상기 환류냉각추출은 40℃에서 4시간 동안 수행되는 것인 차가버섯 추출물의 제조방법.Milling the mushroom to a size of 1 to 2 mm to obtain a mushroom pulverized product; Adding 100% ethanol or methanol to the crushed mushroom as an extraction solvent, and refluxing and cooling to prepare a mushroom extract; And concentrating the mushroom extract at a reduced pressure, wherein the mushroom extract has a high concentration of? -Glucan,
If the extraction solvent is 100% ethanol, the reflux cooling extraction is carried out at 100 DEG C for 8 hours, or
Wherein when the extraction solvent is methanol, the reflux cooling extraction is carried out at 40 DEG C for 4 hours.
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