KR101150226B1 - Antioxidant composition comprising enzymatic extracts of Inonotus Obliquus - Google Patents
Antioxidant composition comprising enzymatic extracts of Inonotus Obliquus Download PDFInfo
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- KR101150226B1 KR101150226B1 KR1020100020426A KR20100020426A KR101150226B1 KR 101150226 B1 KR101150226 B1 KR 101150226B1 KR 1020100020426 A KR1020100020426 A KR 1020100020426A KR 20100020426 A KR20100020426 A KR 20100020426A KR 101150226 B1 KR101150226 B1 KR 101150226B1
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- chaga
- extract
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- hydrolase
- antioxidant composition
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Abstract
본 발명은 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물을 함유하는 항산화 조성물에 관한 것으로, 더욱 상세하게는 차가버섯에 효소를 처리함으로써 단백질 또는 당 가수분해 효소반응을 통해 제조한 차가버섯 가수분해 효소 추출물과, 이를 유효성분으로 함유하는 항산화 조성물 및 상기 차가버섯 가수분해 효소 추출물을 제조하는 방법에 관한 것이다.
본 발명에 따른 항산화 조성물은 천연물 유래의 물질로, 독성 및 부작용이 없으며, 탁월한 항산화 효과를 나타내므로 활성산소에 의해 생성되는 산화물들이 기인하는 노화 및 각종 질환의 억제 또는 치료에 안전하고 효과적으로 이용될 수 있다.The present invention chaga ( Inonotus) Obliquus ) relates to an antioxidant composition containing a hydrolase extract, and more particularly, chaga mushroom hydrolase extract prepared by protein or sugar hydrolase reaction by treating an enzyme to chaga and containing it as an active ingredient It relates to an antioxidant composition and a method for preparing the chaga hydrolase extract.
Antioxidant composition according to the present invention is a substance derived from natural products, has no toxicity and side effects, and exhibits an excellent antioxidant effect, and thus can be safely and effectively used for suppressing or treating aging and various diseases caused by oxides produced by active oxygen. have.
Description
본 발명은 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물을 함유하는 항산화 조성물에 관한 것으로, 더욱 상세하게는 차가버섯에 효소를 처리함으로써 단백질 또는 당 가수분해 효소반응을 통해 제조한 차가버섯 가수분해 효소 추출물과, 이를 유효성분으로 함유하는 항산화 조성물 및 상기 차가버섯 가수분해 효소 추출물을 제조하는 방법에 관한 것이다.The present invention chaga ( Inonotus) Obliquus ) relates to an antioxidant composition containing a hydrolase extract, and more particularly, chaga mushroom hydrolase extract prepared by protein or sugar hydrolase reaction by treating an enzyme to chaga and containing it as an active ingredient It relates to an antioxidant composition and a method for preparing the chaga hydrolase extract.
산소는 지구상에서 가장 많은 원소로서(53.8%) 건조대기 중의 21%를 차지하고 있으며, 모든 생물체들은 산소를 이용하여 생명 유지에 필요한 에너지를 발생하게 된다. 그러나, 이와 같이 생명 유지에 절대적으로 필요한 산소이지만 안정한 분자상태인 기저삼중항산소(ground state triplet oxygen)가 체내 효소계, 환원대사, 화학약품, 공해물질, 광화학반응 등의 각종 물리적, 화학적, 환경적 요인 등에 의하여 수퍼옥사이드 라디칼(superoxide radical, O2?), 하이드록실 라디칼(hydroxyl radical, HO?), 과산화수소(hydrogen peroxide, H2O2), 일중항산소(singlet oxygen, HO?)와 같은 반응성이 매우 큰 활성산소(active oxygen)로 전환되면 생체에 치명적인 산소 독성을 일으키는 양면성을 지니고 있다. 즉, 이들 활성산소는 세포구성 성분들인 지질, 단백질, 당, DNA 등에 대하여 비선택적, 비가역적인 파괴 작용을 함으로써 노화는 물론 암을 비롯하여 뇌졸중, 파킨슨병 등의 뇌혈관 심장질환, 허혈, 동맥경화, 피부질환, 소화기질환, 염증, 류마티스, 자기면역질환 등의 각종 질병을 일으키는 것으로 알려져 있다(Halliwell B. 1991. Drug antioxidant effects. Drugs 42: 596-605). 또한, 이들 활성산소에 의한 지질과산화 결과 생성되는 지질과산화물을 비롯하여 여러 가지 체내 과산화물도 세포에 대한 산화적 파괴로 인한 각종 기능장애를 야기함으로써 노화와 질병의 원인이 되기도 한다.Oxygen is the most abundant element on earth (53.8%), accounting for 21% of the dry air, and all living things use oxygen to generate the energy needed to sustain life. However, the ground state triplet oxygen, an oxygen but stable molecular state that is absolutely necessary to maintain life, has various physical, chemical, and environmental factors such as enzyme system, reduction metabolism, chemicals, pollutants, and photochemical reactions. Reactivity such as superoxide radical (O 2 ?), Hydroxyl radical (HO?), Hydrogen peroxide (H 2 O 2 ), singlet oxygen (HO?) The conversion to this very large active oxygen has a double sided effect that causes fatal oxygen toxicity in the living body. In other words, these free radicals have non-selective and irreversible destruction effects on cell components such as lipids, proteins, sugars, and DNA. It is known to cause various diseases such as skin diseases, digestive diseases, inflammation, rheumatism and autoimmune diseases (Halliwell B. 1991. Drug antioxidant effects. Drugs 42: 596-605). In addition, various peroxides in the body, including lipid peroxides resulting from lipid peroxidation by these free radicals, also cause various functional disorders due to oxidative destruction of cells, thereby causing aging and disease.
한편, 정상적인 세포에서도 대사과정 중 어느 정도의 자유 라디칼(free radical)과 기타 활성산소 및 과산화물이 생성되고 있으나, 생체내에는 이들에 대한 방어기구로서 SOD(superoxide dismutase), 퍼옥시다제(peroxidase), 카탈레이즈(catalase) 등의 항산화효소와 함께 비타민 E, 비타민 C, 글루타티온(glutathione), 유비퀴논(ubiquinone, 코엠자임 Q10), 요산 등과 같은 항산화물질이 존재하여 스스로를 보호하고 있다. 그러나 이와 같은 생체방어기구에 이상이 초래되거나 각종 물리적, 화학적 요인들에 의하여 활성산소의 생성이 생체방어계의 용량을 초과하게 될 경우 산화적 스트레스가 야기된다. 따라서, 이와 같은 자유 라디칼을 소거할 수 있는 화합물 또는 과산화물 생성 억제물질과 같은 항산화제들은 이들 산화물들에 기인하는 노화 및 각종 질환의 억제 또는 치료제로서 기대된다.On the other hand, even in normal cells, some degree of free radicals and other free radicals and peroxides are generated during metabolism, but in vivo, SOD (superoxide dismutase), peroxidase, Along with antioxidant enzymes such as catalase, antioxidants such as vitamin E, vitamin C, glutathione, ubiquinone (ubiquinone, coemzyme Q10), and uric acid are present to protect themselves. However, oxidative stress is caused when abnormality is caused in such a bioprotective device or the generation of active oxygen exceeds the capacity of the bioprotective system by various physical and chemical factors. Accordingly, antioxidants such as compounds capable of scavenging such free radicals or inhibitors of peroxide production are expected as agents for inhibiting or treating aging and various diseases caused by these oxides.
항산화제에 대한 연구는 1969년 McCord와 Fridovich가 수퍼옥사이드 라디칼을 소거하는 효소인 SOD를 발견한 것을 계기로 본격적으로 진행되었으며, 최근에는 노화나 성인병 등의 질환이 활성산소의 존재에 의한 것으로 알려져, 활성산소를 조절할 수 있는 항산화제에 관한 연구가 활발히 진행되고 있다. 항산화제로는 효소계인 SOD, 카탈레이즈 외에 BHT(tert-butylhydroxytoluene), BHA(tert-butylhydroxyanisol) 등과 같은 합성 항산화제 및 토코페롤(tocopherol), 아스코르브산(ascorbic acid), 탄닌, 카로테노이드(carotenoids), 플라보노이드(flavonoids) 등과 같은 일부 천연 항산화제가 있으며, 그 외에도 다양한 종류의 항산화제 개발이 이루어지고 있다(Kitahara K., Matsumoto Y., Ueda H.A. and Ueoka R. 1992. Chem. Pharm. Bull. 40: 2208-2209; Hatano T. 1995. Natural Medicines 49: 357-363). The study of antioxidants began in earnest when McCord and Fridovich discovered SOD, an enzyme that eliminates superoxide radicals, in 1969. Recently, diseases such as aging and adult diseases are known to be due to the presence of free radicals. Research into antioxidants that can regulate free radicals is actively underway. Antioxidants include synthetic antioxidants such as BHT (tert-butylhydroxytoluene) and BHA (tert-butylhydroxyanisol), tocopherol, ascorbic acid, tannin, carotenoids, flavonoids some natural antioxidants, such as flavonoids), and various other antioxidants are being developed (Kitahara K., Matsumoto Y., Ueda HA and Ueoka R. 1992. Chem. Pharm. Bull. 40: 2208-2209 Hatano T. 1995. Natural Medicines 49: 357-363).
그러나, 이들 항산화제는 독성, 낮은 활성 및 용도의 한계성 등의 여러 가지 문제로 인하여 사용에 제한을 받고 있다.However, these antioxidants are limited to use due to various problems such as toxicity, low activity and limit of use.
이에 본 발명자들은 독성 및 부작용이 거의 없는 천연물 유래의 항산화제를 개발하기 위하여 예의 노력한 결과, 차가버섯(Inonotus Obliquus)의 가수분해 효소추출물이 자유라디칼 소거 효능을 측정하여 우수한 항산화 효과를 갖는 것을 확인하고 본 발명을 완성하였다. Therefore, the present inventors have made diligent efforts to develop antioxidants derived from natural products with little toxicity and side effects, and confirmed that the hydrolytic enzyme extract of Chaga ( Inonotus Obliquus ) has an excellent antioxidant effect by measuring free radical scavenging efficacy. The present invention has been completed.
결국, 본 발명의 주된 목적은 차가버섯에 효소를 처리함으로써 단백질 또는 당 가수분해 효소 반응을 통해 제조한 차가버섯 가수분해 효소 추출물과, 이를 유효성분으로 함유하는 항산화 조성물 및 상기 차가버섯 가수분해 효소 추출물을 제조하는 방법을 제공하는데 있다.After all, the main object of the present invention is the chaga mushroom hydrolase extract prepared by the protein or sugar hydrolase reaction by treating the enzyme with chaga, an antioxidant composition containing the same as an active ingredient and chaga hydrolase extract To provide a method for producing a.
상기 목적을 달성하기 위하여, 본 발명은 차가버섯(Inonotus Obliquus)에 효소를 처리함으로써 단백질 또는 당 가수분해 효소 반응을 통해 제조한 차가버섯 가수분해 효소 추출물을 제공한다.In order to achieve the above object, the present invention provides chaga mushroom hydrolase extract prepared by protein or glycolytic enzyme reaction by treating the enzyme to chaga ( Inonotus Obliquus ).
본 발명에서, 상기 차가버섯 가수분해 효소 추출물은 차가버섯을 pH 2.0 내지 8.0의 완충액 중에서 단백질 또는 당 가수분해 효소를 처리하여 제조되는 것을 특징으로 한다.In the present invention, the chaga hydrolase extract is characterized in that the chaga mushroom is prepared by treating the protein or sugar hydrolase in a buffer of pH 2.0 to 8.0.
또한, 본 발명은 상기 차가버섯 가수분해 효소 추출물을 유효성분으로 함유하는 항산화 조성물을 제공한다.The present invention also provides an antioxidant composition containing the chaga hydrolase extract as an active ingredient.
상기 항산화 조성물은 자유라디칼 소거 효능을 가지므로 활성산소에 의해 생성되는 산화물들에 기인하는 노화 및 각종 질환의 치료 및 예방에 우수한 효과를 나타내며, 특히, 노화, 만성알콜중독, 죽상동맥경화증, 암, 관상심장질환, 백내장, 당뇨병, 다운증후군, 간염, 허혈이나 재관류성 손상, 류마티스성 관절염, 관절염, 신부전증, 염증반응, 각종 퇴행성 신경질환, 뇌졸중 발작 및 심근경색 등의 이상을 개선함으로써 상기 질환의 치료 및 예방이 가능하다.Since the antioxidant composition has a free radical scavenging effect, it has an excellent effect on the treatment and prevention of aging and various diseases caused by oxides produced by free radicals, and particularly, aging, chronic alcoholism, atherosclerosis, cancer, Treatment of these diseases by improving abnormalities such as coronary heart disease, cataracts, diabetes, Down's syndrome, hepatitis, ischemia or reperfusion injury, rheumatoid arthritis, arthritis, kidney failure, inflammatory reactions, various degenerative neurological diseases, stroke attacks and myocardial infarction And prevention are possible.
또한, 본 발명은 상기 차가버섯 가수분해 효소 추출물을 제공하는 방법을 제공한다.The present invention also provides a method for providing the chaga hydrolase extract.
본 발명의 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물은 자유 라디칼 소거능이 우수하여 탁월한 항산화 효과를 나타내므로, 본 발명에 따른 추출물을 포함하는 조성물은 활성산소에 의해 생성되는 산화물들에 기인하는 노화 및 각족 질환의 억제 또는 치료에 효과적이다.Chaga mushroom of the present invention ( Inonotus) Since the hydrolase extract has excellent free radical scavenging ability and shows excellent antioxidant effect, the composition comprising the extract according to the present invention is useful for suppressing or treating aging and various diseases caused by oxides produced by free radicals. effective.
또한 본 발명의 차가버섯 가수분해 효소 추출물은 천연물에서 유래한 물질로써 독성 및 부작용 없이 안전하게 사용될 수 있다. In addition, chaga hydrolase extract of the present invention can be used safely without toxicity and side effects as a substance derived from natural products.
도 1은 본 발명의 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물의 제조 공정을 나타낸 모식도이다.
도 2는 본 발명의 차가버섯 펩신(Pepsin) 가수분해 효소 추출물의 활성산소종 생성 억제능을 나타낸 것이다.
도 3은 본 발명의 차가버섯 가수분해 효소 추출물의 산화적 스트레스로 인한 DNA 손실에 대한 DNA 보호능력을 측정한 전기영동 사진이다.1 is chaga mushroom of the present invention ( Inonotus) Obliquus ) is a schematic diagram showing the manufacturing process of the hydrolase extract.
Figure 2 shows the inhibitory ability of reactive oxygen species generation of chaga pepsin (Pepsin) hydrolase extract of the present invention.
Figure 3 is an electrophoresis picture of the DNA protection ability of the chaga mushroom hydrolase extract of the present invention against DNA loss due to oxidative stress.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 항산화 효능을 갖는 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물을 제공한다.The present invention chaga ( Anonotus) having antioxidant efficacy Obliquus ) provides a hydrolase extract.
본 발명의 차가버섯(Inonotus Obliquus)은 대체적으로 검은 색상을 띠며, 길이 40 ㎝, 둘레 15 ㎝, 무게 5 ㎏이 되려면 보통 10~15년이 걸린다. 나무에 붙어있는 접합부분은 부드러운 밝은 빛깔을 가지고 있으며, 쉽게 표면하면 자작나무의 버섯이다. 러시아 캄차카 반도가 주 생산지이고, 효능으로는 당뇨와 교혈압에 효과적으며, 칼슘, 마그네슘, 철 등의 생리학적 요소들이 다량 함유되어 있어 차나 약재로 많이 사용되고 있다.Chaga mushroom ( Inonotus Obliquus ) of the present invention is generally black color, it takes 10-15 years to be 40 cm in length, 15 cm in circumference, 5 kg in weight. The joints attached to the trees have a soft light color and are easily birch mushrooms. The Kamchatka Peninsula in Russia is the main producer, and its efficacy is effective for diabetes and blood pressure, and it is widely used as tea or medicine because it contains large amounts of physiological elements such as calcium, magnesium, and iron.
본 발명의 차가버섯 가수분해 효소 추출물은 상기의 차가버섯을 pH 2.0 내지 8.0의 인산염 완충액, 아세트산 완충액 및 글리신-HCl 중에서 선택된 1종 이상의 용매를 사용하여 용해시킨 후, 여기에 효소 또는 효소 혼합물을 첨가하여 가수분해 반응을 진행시켜 제조되는 것이 바람직하다.Chaga mushroom hydrolase extract of the present invention is dissolved in the chaga mushroom at least one solvent selected from phosphate buffer, acetic acid buffer and glycine-HCl pH 2.0 to 8.0, and then added to the enzyme or enzyme mixture It is preferable to make it by advancing a hydrolysis reaction.
상기 효소는 단백질을 가수분해 하는 단백질 가수분해 효소 또는/및 당을 가수분해 하는 당 가수분해 효소로 이루어진 군에서 선택된 1종 이상이 포함되는 것이 바람직하며, 더욱 바람직하게는 프로타맥스(Protamex), 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 알카라제(Alcalase), 파파인(Papain), 키모트립신(α-caymotrypsin), 트립신(Trypsin), 펩신(Pepsin), 프로모자임(Promozyme), 셀루클라스트(Celluclast), 말토게나제(Maltogenase), 비스코자임(Viscozyme), 테르마밀(Termamyl), 덱스트로자임(Dextrozyme), 아밀로글루코시다제(Amlyroglucosidase), α,β-글루코시다제(α,β-glucoxidase), 펙티나제(Pectinase), 나린지나제(Naringinase), 셀루라제(Cellulase), 헤미셀룰라제(hemicellulase) 등에서 선택되는 1종 이상인 것이 좋다.The enzyme may preferably include one or more selected from the group consisting of proteolytic enzymes for hydrolyzing proteins and / or sugar hydrolases for hydrolyzing sugars, more preferably Protamex, Flavozyme, Neutrase, Alcalase, Papain, α-caymotrypsin, Trypsin, Pepsin, Promozyme, Cellul Celluclast, Maltogenase, Viscozyme, Teramyl, Dextrozyme, Amyloglucosidase, α, β-glucosidase (α) , beta -glucoxidase, pectinase (Pectinase), naringinase (Naringinase), cellulase (Cellulase), hemicellulase (hemicellulase) and the like is one or more selected.
또한, 본 발명에서 상기 "추출물"은 추출액, 정제물, 희석액, 농축액, 및 건조물을 모두 포함한다.In addition, in the present invention, the "extract" includes all of the extract, purified, diluent, concentrate, and dried.
본 발명은 또한 상기 차가버섯(Inonotus Obliquus) 가수분해 효소 추출물을 유효성분으로 하는 항산화 조성물을 제공한다.The present invention also Chaga ( Inonotus) Obliquus ) provides an antioxidant composition comprising the hydrolase extract as an active ingredient.
본 발명의 차가버섯 가수분해 효소 추출물은 DPPH 라디칼, 하이드록실 라디칼, 알킬 라디칼 등의 소거능 실험을 통해 항산화 효과가 우수한 것을 확인할 수 있었다.Chaga hydrolase extract of the present invention was confirmed that the antioxidant effect was excellent through the scavenging experiments such as DPPH radical, hydroxyl radical, alkyl radical.
따라서, 상기 차가버섯 가수분해 효소 추출물을 유효성분으로 하는 항산화 조성물은 활성산소에 의해 생성되는 산화물들에 기인하는 노화 및 각종 질환, 바람직하게는 노화, 만성알콜중독, 죽상동맥경화증, 암, 관상심장질환, 백내장, 당뇨병, 다운증후군, 간염, 허혈이나 재관류성 손상, 류마티스성 관절염, 관절염, 신부전증, 염증반응, 각종 퇴행성 신경질환, 뇌졸중 발작 및 심근경색 등에서 선택되는 1종 이상의 질환을 억제 또는 치료하기 위한 의약품, 건강보조세, 화장료, 식품, 식품 첨가제 등에 유용하게 이용될 수 있으며, 이에 의해 제한되지는 않는다.Therefore, the antioxidant composition using the chaga hydrolase extract as an active ingredient is due to the aging and various diseases, preferably aging, chronic alcoholism, atherosclerosis, cancer, coronary heart caused by the oxides produced by active oxygen To inhibit or treat one or more diseases selected from diseases, cataracts, diabetes, Down syndrome, hepatitis, ischemia or reperfusion injury, rheumatoid arthritis, arthritis, renal failure, inflammatory reactions, various degenerative neurological diseases, stroke attacks and myocardial infarction It can be usefully used for medicines, health supplements, cosmetics, food, food additives, etc., but is not limited thereto.
본 발명의 조성물이 의약품으로 이용될 경우에는 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있으며, 통상적인 방법에 따라 약학적으로 허용되는 제형으로 제조될 수 있다.When the composition of the present invention is used as a medicament, it may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, and may be prepared in pharmaceutically acceptable formulations according to conventional methods. .
상기 담체 또는 부형제로는 물, 덱스트린, 칼슘카보네이느, 락토스, 프로필렌글리콜, 리퀴드, 파라핀, 생리식염수, 덱스트로스, 수크로즈, 솔비통, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸셀룰로즈, 폴리비닐피릴리돈, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테라레이트 및 광물류가 있으며, 이들은 1종 이상 사용될 수 있으나, 이에 한정되는 것은 아니며 통상의 담체 및 부형제는 모두 사용 가능하다.The carrier or excipient includes water, dextrin, calcium carbonane, lactose, propylene glycol, liquid, paraffin, physiological saline, dextrose, sucrose, sorbitan, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, polyvinylpyridone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more, but are not limited thereto. And conventional carriers and excipients can be used.
또한, 항산화 조성물을 약제화 하는 경우, 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등을 더 포함할 수 있다.In addition, when the antioxidant composition is formulated, conventional fillers, extenders, binders, disintegrating agents, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.
본 발명의 조성물이 화장료로 이용될 경우에는 주름개선 및 미백 기능성 화장품, 유연화장수, 영양화장수, 아이크림, 영양크림, 맛사지크림, 클렌징크린, 에센스 등의 제형으로 제조될 수 있으며, 각 제형의 화장료 조성물에 있어서, 차가버섯 가수분해 효소 추출물을 포함하여 주름개선 및 미백 원료 또는 통상의 화장료 원료로서 이외의 성분들을 화장료의 제형 또는 사용목적 등에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있다.When the composition of the present invention is used as a cosmetic, wrinkle improvement and whitening functional cosmetics, flexible cosmetics, nourishing cosmetics, eye cream, nutrition cream, massage cream, cleansing clean, essences, etc. In the composition, ingredients other than wrinkle improvement and whitening raw materials or ordinary cosmetic raw materials including chaga hydrolase extract may be appropriately selected and blended by those skilled in the art without difficulty according to the formulation or purpose of use of cosmetics.
본 발명은 또한, 상기 차가버섯(Inonotus Obliquus) 가수분해 추출물의 제조방법을 제공한다.The present invention, the chaga ( Inonotus) Obliquus ) provides a method for preparing a hydrolyzed extract.
구체적으로, 본 발명의 제조방법은, (1) 차가버섯(Inonotus Obliquus)을 완충액 중에 용해시키는 단계; (2) 상기에 단백질 또는 당 가수분해 효소를 첨가하는 단계; 및 (3) 상기를 30 내지 70℃ 범위의 반응온도에서 6 내지 10시간 가수분해하는 단계;를 포함할 수 있다.Specifically, the manufacturing method of the present invention, (1) dissolving chaga ( Inonotus Obliquus ) in a buffer; (2) adding a protein or a sugar hydrolase to the above; And (3) hydrolyzing the above at a reaction temperature in the range of 30 to 70 ° C. for 6 to 10 hours.
본 발명에서, 상기 완충액은 pH 2.0 내지 8.0의 인산염 완충액, 아세트산 완충액 및 글리신-HCl 중에서 선택되는 1종 이상인 것이 바람직하다. 또한, 상기 단백질 가수분해 효소는 프로타맥스(Protamex), 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 알카라제(Alcalase), 파파인(Papain), 키모트립신(α-Caymotrypsin), 트립신(Trypsin), 펩신(Pepsin) 등에서 선택되는 1종 이상인 것이 바람직하며, 상기 당 가수분해효소는 프로모자임(Promozyme), 셀루클라스트(Celluclast), 말토게나제(Maltogenase), 비스코자임(Viscozyme), 테르마밀(Termamyl), 덱스트로자임(Dextrozyme), 아밀로글루코시다제(Amlyroglucosidase), α,β-글루코시다제(α,β-glucoxidase), 펙티나제(Pectinase), 나린지나제(Naringinase), 셀루라제(Cellulase), 헤미셀룰라제(hemicellulase) 등에서 선택되는 1종 이상인 것이 바람직하다.In the present invention, the buffer is preferably at least one selected from phosphate buffer, acetic acid buffer and glycine-HCl of pH 2.0 to 8.0. In addition, the protease is Protamex, Flavozyme, Neutrase, Alcalase, Papain, Chymotrypsin, Trypsin At least one selected from Trypsin, Pepsin, and the like is preferable. The sugar hydrolase is Promozyme, Celluclast, Maltogenase, Maltogenase, Viscozyme, Ter Termamyl, Dextrozyme, Amyloglucosidase, α, β-glucoxidase, Pectinase, Naringinase, It is preferable that it is 1 or more types chosen from a cellulase, a hemicellulase, etc.
또한, 본 발명에서 가수분해시 pH와 반응온도는, 가수분해 효소의 적정 pH와 적정 온도에 따라 달라질 수 있는데, 바람직하게는 단백질 가수분해 효소를 사용하는 경우, pH 6.0 내지 8.0의 인산염 완충액(Phosphate buffer, PB) 또는 pH 2 내지 3의 글리신-HCl(Glycine-HCl, GH) 중에 용해하여 반응온도 30 내지 60℃에서, 또한 당 가수분해 효소의 경우에는 pH 4 내지 5의 아세트산 완충액(Sodium acetate-acetic acid buffer, SB) 또는 pH 5 내지 7의 인산염 완충액(Phosphate buffer, PB) 중에 용해하여 40 내지 70℃에서 6 내지 10시간, 더욱 바람직하게는 8시간 동안 반응시키는 것이 바람직하다. In addition, in the present invention, the pH and reaction temperature during hydrolysis may vary depending on the proper pH and proper temperature of the hydrolase. Preferably, when using a proteolytic enzyme, a phosphate buffer (Phosphate) having a pH of 6.0 to 8.0 buffer, PB) or glycine-HCl (Glycine-HCl, GH) at pH 2 to 3 at a reaction temperature of 30 to 60 ° C, and in the case of sugar hydrolase, an acetate buffer of pH 4 to 5 (Sodium acetate- It is preferable to dissolve in acetic acid buffer (SB) or phosphate buffer (PB) of pH 5 to 7 and react at 40 to 70 ° C for 6 to 10 hours, more preferably 8 hours.
또한, 가수분해 반응 후에는 100℃에서 5 내지 20분간 가열하여 가수분해 반응을 종결시키는 것이 바람직하다.After the hydrolysis reaction, it is preferable to terminate the hydrolysis reaction by heating at 100 ° C. for 5 to 20 minutes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 차가버섯 단백질 가수분해 효소 추출물 제조Example 1. Preparation of Chaga Mushroom Proteinase Extract
음지에서 건조한 차가버섯(Inonotus Obliquus)을 마쇄기로 분쇄하여 분말화 한 차가버섯 분말에 0.1 M 인산염 완충액(phosphate buffer, PB; pH 7.0)을 차가버섯 분말 : 인산염 완충액 = 1 : 50의 비율(w/w)로 용해한 후, 30분간 150 rpm으로 교반하면서 30~60℃로 예열한 다음, 여기에 단백질 가수분해 효소를 상기 차가버섯 분말과 1 : 50의 비율(w/w)로 첨가하였다.Chaga powder powdered by crushing the dried Chaga ( Inonotus Obliquus ) in the shade with crusher was added 0.1 M phosphate buffer (PB; pH 7.0) to Chaga powder: Phosphate buffer = 1: 50 (w / After dissolving in w), the mixture was preheated to 30 to 60 ° C. with stirring at 150 rpm for 30 minutes, and then proteolytic enzyme was added to the chaga powder at a ratio of 1:50 (w / w).
이때, 상기 단백질 가수분해 효소는 프로타맥스(Protamex), 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 알카라제(Alcalase), 키모트립신(α-Caymotrypsin), 펩신(Pepsin), 트립신(Trypsin), 파파인(Papain)을 사용하였으며, 8시간 동안 180 rmp에서 교반하여 가수분해 후, 가수분해가 완료되면 100℃에서 10분간 가열하여 반응을 종료시켰다.At this time, the proteolytic enzymes are Protamex, Flavozyme, Neutrase, Alcalase, Chymotrypsin, Pepsin, Trypsin (Pepsin) Trypsin) and Papain (Papain) were used, and after the hydrolysis by stirring at 180 rmp for 8 hours, when the hydrolysis was completed, the reaction was terminated by heating at 100 ℃ for 10 minutes.
단, 단백질 가수분해 효소로 펩신을 사용하는 경우에는 0.1 M 글리신-HCl(glycine-HCl, GH; pH 2.2)을 사용하였다.However, when pepsin was used as a proteolytic enzyme, 0.1 M glycine-HCl (glycine-HCl, GH; pH 2.2) was used.
반응액은 감압증발기로 농축시킨 다음 동결건조하여 차가버섯 단백질 가수분해 추출물을 제조하였다.
The reaction solution was concentrated with a reduced pressure evaporator and then lyophilized to prepare a chaga protein hydrolyzed extract.
실시예 2. 차가버섯 당 가수분해 효소 추출물 제조Example 2 Preparation of Chaga Sugar Hydrolase Extract
차가버섯(Inonotus Obliquus) 분말에 0.1M 아세트산 완충액(sodium acetate-acetatic acid buffer, SB; pH 4.5~5.0)을 차가버섯 분말 : 아세트산염 완충액 = 1 : 50의 비율(w/w)로 용해한 후, 30분간 150 rpm으로 교반하면서 50~60℃로 예열한 다음, 여기에 당 가수분해 효소를 상기 차가버섯 분말과 1 : 50의 비율(w/w)로 첨가하였다.After dissolving 0.1 M acetate acetate (acetic acid buffer, SB; pH 4.5 ~ 5.0) in chaga ( Inonotus Obliquus ) powder at the ratio (w / w) of chaga mushroom powder: acetate buffer = 1: 50, After preheating at 50-60 ° C. with stirring at 150 rpm for 30 minutes, sugar hydrolase was added to the chaga powder at a ratio of 1:50 (w / w).
이때, 상기 당 가수분해 효소는 프로모자임(Promozyme), 셀루클라스트(Celluclast), 말토게나제(Maltogenase), 비스코자임(Viscozyme), 테르마밀(Termamyl), 덱스트로자임(Dextrozyme), 아밀로글루코시다제(Amlyroglucosidase, AMG)를 사용하였으며, 8시간 동안 180 rmp에서 교반하여 가수분해 후, 가수분해가 완료되면 100℃에서 10분간 가열하여 반응을 종료시켰다.At this time, the sugar hydrolase is Promozyme, Celluclast, Maltogenase, Maltogenase, Biscozyme, Teramyl, Dextrozyme, Amyloglucose A sidase (Amlyroglucosidase, AMG) was used, and after hydrolysis by stirring at 180 rmp for 8 hours, when the hydrolysis was completed, the reaction was terminated by heating at 100 ° C. for 10 minutes.
단, 당 가수분해 효소로 테르마밀을 사용하는 경우에는 0.1 M 인산염 완충액(pH 6.0)을 사용하였다.However, in the case of using termamyl as a sugar hydrolase, 0.1 M phosphate buffer (pH 6.0) was used.
반응액은 감압증발기로 농축시킨 다음 동결건조하여 차가버섯 당 가수분해 추출물을 제조하였다.
The reaction solution was concentrated with a reduced pressure evaporator and then lyophilized to prepare a hydrolyzed extract of chaga.
본 발명에서 사용된 효소의 조성 및 반응 조건은 하기 표 1에 나타내었다.The composition and reaction conditions of the enzyme used in the present invention are shown in Table 1 below.
완충액a Used
Buffer a
(Protamex)Protamex
(Protamex)
(Flavourzyme)Flavozyme
(Flavourzyme)
(Neutrase)Neutraze
(Neutrase)
(Alcalase)Alcalase
(Alcalase)
(Promozyme)Promozyme
(Promozyme)
(Celluclast) Cellulast
(Celluclast)
(Maltogenase)Maltogenase
(Maltogenase)
(Viscozyme)Biscozyme
(Viscozyme)
(Termamyl)Termamil
(Termamyl)
(Dextrozyme)Dextrozyme
(Dextrozyme)
(Amyloglucosidase)Amyloglucosidase
(Amyloglucosidase)
(Papain)Papain
(Papain)
(Pepsin)pepsin
(Pepsin)
(α-chymotrypsin)Chymotrypsin
(α-chymotrypsin)
(tripsing form porcine pancreas)Trypsin
(tripsing form porcine pancreas)
단, 상기에서 a는 효소 가수분해(in Ezymatic hydrolysis), b는 인산염 완충액(phosphate buffer), c는 아세트산 완충액(sodium acetate-acetic acid) 및 d는 글리신-HCl(glycine-HCl)을 의미한다.
However, a denotes enzymatic hydrolysis, b denotes phosphate buffer, c denotes sodium acetate-acetic acid, and d denotes glycine-HCl.
실험예 1. DPPH 라디칼 소거능을 이용한 항산화 효과 확인Experimental Example 1. Confirmation of antioxidant effect using DPPH radical scavenging ability
본 발명에 따른 차가버섯 가수분해 효소 추출물의 항산화 효과를 확인하기 위하여, 전자스핀공명기기(ESR spectrometer, JEX-PX 2000-300 모델, JEOL, 일본)를 이용하여 DPPH(1,1-다이페닐-2-피크릴하이드라질) 라디칼의 소거능을 측정하였다.In order to confirm the antioxidant effect of chaga hydrolase extract according to the present invention, using an electronic spin resonance (ESR spectrometer, JEX-PX 2000-300 model, JEOL, Japan) DPPH (1,1-diphenyl- Scavenging ability of the 2-picrylhydrazyl) radical was measured.
DPPH는 짙은 자주색을 나타내며, 그 자체가 질소 중심의 라디칼로서, 라디칼 전자의 비편재화에 의해 안정화된 상태로 존재하는데, ESR을 이용한 DPPH 라디칼 소거능 측정방법을 다음과 같다.DPPH is dark purple and is itself a nitrogen-centered radical, which exists in a stabilized state by delocalization of radical electrons. DPPH radical scavenging ability measurement using ESR is as follows.
구체적으로, 메탄올에 용해시킨 30 μM의 DPPH 30 ㎕와 초순 증류수에 용해시킨 실시예 1 및 실시예 2에서 제조한 농도별 차가버섯 가수분해 추출물 30 ㎕를 혼합한 후 10초간 강하게 볼텍스(vortex)하여 2분간 실온에서 방치한 다음 모세관 튜브(capillary tube)에 옮겨 ESR 스펙트로미터에서 측정하였다[측정조건 - central field: 3475G, modulation frequency: 100 ㎑, modulation amplitude: 2G, micro power: 5 ㎽, gain: 6.3×105, 온도: 298K].Specifically, 30 μl of DPPH dissolved in methanol and 30 μl of chaga-hydrolyzed extracts of different concentrations prepared in Examples 1 and 2 dissolved in ultrapure distilled water were mixed and strongly vortexed for 10 seconds. After standing at room temperature for 2 minutes, the solution was transferred to a capillary tube and measured on an ESR spectrometer [measurement condition-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, micro power: 5 Hz, gain: 6.3]. X10 5 , temperature: 298 K].
본 발명의 8종의 단백질 가수분해 효소 추출물과 7종의 당 가수분해 효소 추출물에 대한 결과는 표 2에 나타낸 바와 같다.The results for the eight proteolytic enzyme extracts and seven sugar hydrolase extracts of the present invention are shown in Table 2.
하기 표 2에서도 알 수 있듯이, 본 발명의 차가버섯 단백질 및 당 가수분해 효소 추출물은 농도 의존적으로 DPPH 라디칼을 소거하였으며, 특히 단백질 가수분해 효소 중에서는 프로타맥스(Protamex)의 추출물이 DPPH 라디칼 소거 활성이 가장 높았으며, 이때 IC50값은 150 ㎍/㎖이었다. 또한, 당 가수분해 효소 중에서는 셀루클라스(Celluclast) 추출물이 IC50값 155 ㎍/㎖로 DPPH 라디칼 소거 활성이 가장 높았다.As can be seen in Table 2 below, chaga mushroom protein and sugar hydrolase extract of the present invention was DPPH radical-dependently scavenged concentration-dependently, especially among proteolytic enzymes, the extract of Protamex is DPPH radical scavenging activity Was the highest, with an IC 50 value of 150 μg / ml. Among the hydrolases, Celluclast extract had the highest DPPH radical scavenging activity with an IC 50 value of 155 μg / ml.
가수분해
효소Party
Hydrolysis
enzyme
가수분해
효소protein
Hydrolysis
enzyme
실험예 2. 하이드록실 라디칼 소거능을 이용한 항산화 효과 확인Experimental Example 2. Confirmation of antioxidant effect using hydroxyl radical scavenging ability
본 발명에 따른 차가버섯 가수분해 효소 추출물의 항산화 효과를 확인하기 위하여, 상기 실험예 1에서 사용한 전자스핀공명기(ESR spectrometer)를 이용해 하이드록실 라디칼 소거능을 측정하되, 철(Fe) 이온과 과산화수소가 반응하는 펜톤(Fenton) 반응에 따라 하이드록실 라디칼을 생성시켜 DMPO(5,5-디메틸-1-피롤린 N-옥사이드)-OH의 양을 측정하여 하이드록실 라디칼 소거율을 측정하였다.In order to confirm the antioxidant effect of chaga hydrolase extract according to the present invention, the hydroxyl radical scavenging ability was measured using an electron spin resonance (ESR spectrometer) used in Experimental Example 1, iron (Fe) ion and hydrogen peroxide reaction Hydroxyl radical scavenging rate was measured by generating hydroxyl radicals according to the Fenton reaction to measure the amount of DMPO (5,5-dimethyl-1-pyrroline N-oxide) -OH.
구체적으로, 실시예 1 및 2의 농도별 차가버섯 가수분해 효소 추출물 2 ㎖에 0.3 M의 DMPO 0.2 ㎖, 10 mM의 FeSO4 0.2 ㎖ 및 10 mM의 H2O2 0.2 ㎖를 차례로 첨가한 후, 10초간 강하게 vortex 하여 반응 혼합물을 모세관 튜브로 옮겨 ESR 스펙트로미터에서 하이드록실 라디칼 발생량을 측정하였다[측정조건 - central field: 3475G, modulation frequency: 100 ㎑, modulation amplitude: 2G, micro power: 1 ㎽, gain: 6.3×105, 온도: 298K]. Specifically, 0.2 ml of 0.3 M DMPO, 0.2 ml of 10 mM FeSO 4, and 0.2 ml of 10 mM H 2 O 2 were sequentially added to 2 ml of chaga hydrolase extract according to the concentrations of Examples 1 and 2, respectively. Vigorously vortex for 10 seconds to transfer the reaction mixture to a capillary tube and measure the amount of hydroxyl radicals generated on an ESR spectrometer [measured condition-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, micro power: 1 Hz, gain : 6.3 × 10 5 , temperature: 298 K].
그 결과를 나타낸 표 3에서도 알 수 있듯이, 본 발명의 차가버섯 단백질 및 당 가수분해 효소 추출물은 농도 의존적으로 하이드록실 라디칼을 소거하는 것을 확인할 수 있었으며, 그 중 단백질 가수분해 효소인 펩신(Pepsin) 추출물이 IC50값 847 ㎍/㎖로 하이드록실 라디칼 소거 활성이 높았다.As can be seen in Table 3 showing the results, chaga mushroom protein and sugar hydrolase extract of the present invention was confirmed to eliminate the hydroxyl radical in a concentration-dependent, pepsin (Pepsin) extract of the protease The IC 50 value of 847 µg / ml showed high hydroxyl radical scavenging activity.
가수분해
효소Party
Hydrolysis
enzyme
가수분해
효소protein
Hydrolysis
enzyme
실험예 3. 알킬 라디칼 소거능을 이용한 항산화 효과 확인Experimental Example 3. Confirmation of antioxidant effect using alkyl radical scavenging ability
본 발명에 따른 차가버섯 가수분해 효소 추출물의 항산화 효과를 확인하기 위하여, 상기 실험예 1에서 사용한 전자스핀공명기(ESR spectrometer)를 이용해 알킬 라디칼 소거능을 측정하였다.In order to confirm the antioxidant effect of chaga hydrolase extract according to the present invention, the alkyl radical scavenging ability was measured using an electron spin resonance (ESR spectrometer) used in Experimental Example 1.
알킬 라디칼은 APPH(2,2′-아조비스(2-아미디노프로판) 다이하이드로클로라이드)에 의해 생성되며, 소거능은 POBN(α-(4-프로디닐 1-옥사이드)-N-3가-부틸니트론)과의 반응물의 양으로 측정할 수 있다.Alkyl radicals are produced by APPH (2,2′-azobis (2-amidinopropane) dihydrochloride) and the scavenging ability is POBN (α- (4-prodinyl 1-oxide) -N-3valent-butyl Nitron).
본 발명에서는 PBS(phosphate buffered saline, 인산염 완충액)에 상기 실시예 1 및 2의 농도별 차가버섯 가수분해 효소 추출물 10 ㎕, 40 mM의 AAPH 10 ㎕, 40 mM의 POBN 10 ㎕를 차례로 첨가한 후, 10초간 강하게 vortex 하여 반응 혼합물을 37℃ 수욕조(water bath)에서 30분간 반응시킨 다음, 모세관 튜브로 옮겨 ESR 스펙트로미터에서 하이드록실 라디칼 발생량을 측정하였다[측정조건 - central field: 3475G, modulation frequency: 100 ㎑, modulation amplitude: 2G, micro power: 10 ㎽, gain: 6.3×105, 온도: 298K]. In the present invention, 10 μl of chaga hydrolase extract according to the concentrations of Examples 1 and 2, 10 μl of 40 mM AAPH, and 10 μl of 40 mM POBN are sequentially added to PBS (phosphate buffered saline). After vigorously vortexing for 10 seconds, the reaction mixture was reacted for 30 minutes in a 37 ° C water bath, and then transferred to a capillary tube to measure hydroxyl radical generation on an ESR spectrometer. [Measurement condition-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, micro power: 10 Hz, gain: 6.3 × 10 5 , temperature: 298K].
그 결과, 표 4에 나타난 바와 같이, 본 발명의 차가버섯 단백질 및 당 가수분해 효소 추출물은 농도 의존적으로 알킬 라디칼을 소거하는 것을 확인할 수 있었으며, 단백질 가수분해 효소 중 플라보자임(Flavozyme)의 추출물이 알킬 라디칼 소거 활성이 가장 높았으며, 이때 IC50값은 0.082 ㎎/㎖었다. 또한, 당 가수분해 효소 중에서는 말토게네이즈(Maltogenase) 추출물이 IC50값 0.082 ㎎/㎖로 알킬 라디칼 소거 활성이 가장 높았다.As a result, as shown in Table 4, chaga mushroom protein and sugar hydrolase extract of the present invention was confirmed that the alkyl radical radical scavenging concentration-dependent, Flavozyme extract of proteolytic enzymes Alkyl radical scavenging activity was the highest, with an IC 50 value of 0.082 mg / ml. Among the hydrolase, maltogenase extract had the highest alkyl radical scavenging activity with an IC 50 value of 0.082 mg / ml.
가수분해
효소Party
Hydrolysis
enzyme
가수분해
효소protein
Hydrolysis
enzyme
실험예 4. 펩신 가수분해 추출물의 항산화 효과 확인(1)Experimental Example 4. Confirmation of antioxidant effect of pepsin hydrolysis extract (1)
본 발명의 펩신 가수분해 추출물의 항산화 효과를 확인하기 위하여, 신경세포 내 활성산소종(reactive oxygen species, ROS) 생성에 미치는 영향을 측정하였다.In order to confirm the antioxidant effect of the pepsin hydrolysis extract of the present invention, the effect on the production of reactive oxygen species (ROS) in neurons was measured.
활성산소종(ROS)는 호흡과정에서 몸속으로 들어간 산소가 산화과정에 이용되면서 여러 대사과정에서 생성되어 생체조직을 공격하고, 세포를 손상시키는 산화력이 강한 산소종으로 과잉생성시에는 우리 몸의 산화를 촉진시킨다.Free radical species (ROS) is a highly oxidative oxygen species that is produced by various metabolic processes as oxygen enters the body during the respiration process and attacks biological tissues and damages cells. To promote.
본 발명에서는 6-웰 플레이트에 2.1×105개의 신경세포를 접종하고 24시간 뒤에 1 mM H2O2 또는 1 mM H2O2 + 펩신 가수분해 추출물을 처리한 후 37℃, CO2 인큐베이터에서 24시간 반응시킨 다음, 24시간 경과 후에 각 배지에 5 μM의 2′,7′-dichlorofluoroscein diacetate(DCF-DA)를 첨가하여 30분 동안 반응시키고, 이를 수확하여 유세포 분석기(FACScan, BD Science, 미국)로 활성산소종의 생성량을 측정하였다.In the present invention, 2.1 × 10 5 neurons were inoculated into 6-well plates and treated with 1 mM H 2 O 2 or 1 mM H 2 O 2 + pepsin hydrolysis extract 24 hours later in a 37 ° C., CO 2 incubator. After 24 hours of reaction, 5 μM of 2 ', 7'-dichlorofluoroscein diacetate (DCF-DA) was added to each medium for 30 minutes, followed by harvesting and flow cytometry (FACScan, BD Science, USA). The amount of active oxygen species was measured with).
표 5는 본 발명의 펩신 가수분해 추출물의 활성산소종 생성량을 나타낸 것으로, 펩신 가수분해 추출물 처리군이 비처리군(control)과 비교하여 생성량이 감소한 것으로 볼 때, 본 발명의 펩신 가수분해 추출물은 활성산소종의 생성을 억제시키는 것을 알 수 있었다.Table 5 shows the amount of active oxygen species produced by the pepsin hydrolyzate extract of the present invention, the pepsin hydrolyzed extract treated group is considered to be reduced compared to the untreated group (control), pepsin hydrolyzed extract of the present invention is It was found that the production of reactive oxygen species is suppressed.
+ H2O2 1mM0.125 mg / ml
+ H 2 O 2 1 mM
+ H2O2 1mM0.25 mg / ml
+ H 2 O 2 1 mM
+ H2O2 1mM0.5 mg / ml
+ H 2 O 2 1 mM
+ H2O2 1mM1.0 mg / ml
+ H 2 O 2 1 mM
generationRos
generation
±8.9179.18
± 8.9
±8.9176.04
± 8.9
±8.9* 164.41
± 8.9 *
±8.9* 159.70
± 8.9 *
±8.9** 140.23
± 8.9 **
실험예 5. 펩신 가수분해 추출물의 항산화 효과 확인(2)Experimental Example 5. Confirmation of antioxidant effect of pepsin hydrolysis extract (2)
본 발명의 펩신 가수분해 추출물의 항산화 효과를 확인하기 위하여, 산화스트레스로 인한 신경세포 손상을 억제능을 측정하였다.In order to confirm the antioxidant effect of the pepsin hydrolysis extract of the present invention, the ability to inhibit neuronal damage caused by oxidative stress was measured.
요오드 프로피디움(propidium iodine, PI)은 자동세포사멸인 아폽토시스를 측정하기 위해 자주 사용되는 형광색소로 DNA와 결합하므로 PI로 염색된 DNA 양으로부터 세포사멸과 세포주기를 유추할 수 있다.Iodine propidium (PI) binds DNA with fluorescence pigments, which are often used to measure apoptosis, which is an automatic cell death, and thus can induce apoptosis and cell cycle from the amount of DNA stained with PI.
본 발명에서는 6-웰 플레이트에 2.1×105개의 신경세포를 접종하고 24시간 뒤에 1 mM H2O2 또는 1 mM H2O2 + 펩신 가수분해 추출물을 처리한 후 37℃, CO2 인큐베이터에서 24시간 반응시키고, 반응이 완료되면 수확하여 4℃, 13,000 rpm에서 3분간 원심분리하여 상층액을 버리고 인산염 완충액 300 ㎕를 첨가하여 잘 혼합하고, tween 20 0.5%가 함유된 차가운 에탄올 1 ㎖을 첨가하여 4℃에서 고정한 다음, 24시간 후 다시 4℃, 13,000 rpm에서 3분간 원심분리하고, 상층액을 버리고 여기에 RNase 10 ㎍/㎖이 포함된 요오드 프로피디움 용액(최종 농도 50 ㎍/㎖) 300 ㎕를 첨가하여 잘 혼합한 다음 37℃에서 30분 염색하여 유세포 분석기(FACScan, BD Science, 미국)에서 세포사멸과 세포주기를 측정하였다.In the present invention, 2.1 × 10 5 neurons were inoculated into 6-well plates and treated with 1 mM H 2 O 2 or 1 mM H 2 O 2 + pepsin hydrolysis extract 24 hours later in a 37 ° C., CO 2 incubator. Reaction was performed for 24 hours, and when the reaction was completed, harvested and centrifuged at 4 ° C. and 13,000 rpm for 3 minutes to discard the supernatant, add 300 µl of phosphate buffer, mix well, and add 1 ml of cold ethanol containing 20% of tween. Fixed at 4 ° C., followed by centrifugation for 3 min at 4 ° C. and 13,000 rpm after 24 hours, and the supernatant was discarded and an iodine propidium solution (final concentration 50 μg / ml) containing 10 μg / ml of RNase was added thereto. After the addition of μl, the mixture was mixed well and stained at 37 ° C. for 30 minutes, and cell death and cell cycle were measured by flow cytometry (FACScan, BD Science, USA).
도 2는 본 발명의 펩신 가수분해 추출물의 산화스트레스로 인한 신경세포 손상정도를 나타낸 것으로, 펩신 가수분해 추출물 처리군이 비처리군(control)에 비해 세포사멸 수가 감소한 것으로 볼 때 본 발명의 펩신 가수분해 추출물은 신경세포 사멸을 억제시키는 것을 알 수 있었다.
Figure 2 shows the degree of neuronal damage caused by the oxidative stress of the pepsin hydrolyzed extract of the present invention, pepsin hydrolyzate extract treated group of pepsin hydrolyzed when the number of apoptosis is reduced compared to the control (control) Decomposed extract was found to inhibit neuronal cell death.
실험예 6. 자유 라디칼로부터의 DNA 보호 효과 확인 Experimental Example 6. Confirmation of DNA Protection Effect from Free Radicals
본 발명에 따른 차가버섯 가수분해 효소 추출물의 자유 라디칼로부터의 DNA 보호 효과를 확인하기 위하여, 플라스미드 DNA를 이용한 전기영동(electrophoresis)을 실시하여 하이드록실 라디칼에 의해 유도된 충격으로부터의 DNA 보호 효과를 측정하였다.In order to confirm the DNA protection effect from free radicals of chaga hydrolase extract according to the present invention, electrophoresis using plasmid DNA was carried out to measure the DNA protection effect from impact induced by hydroxyl radicals. It was.
정상 플라스마드 DNA를 전기영동하면 초나선 DNA(supercoiled DNA), 선형 DNA(linear DNA), 고리열림 DNA(open circular DNA)로 나뉘어지며, 초나선 DNA가 가장 많은 양을 차지한다. 여기에 펜톤(Fenton) 반응을 실시하여 DNA에 충격을 가하면 DNA는 손실을 입고 초나선 DNA가 고리열림 DNA로 변화하는데, 초나선 DNA 양의 감소로 DNA 손실여부를 알 수 있다.Electrophoresis of normal plasma DNA is divided into supercoiled DNA, linear DNA, and open circular DNA, with the highest amount of super helical DNA. When a Fenton reaction is applied to the DNA, the DNA is lost and the ultra helical DNA is changed to the ring-opened DNA, and the decrease in the amount of the super helical DNA indicates the DNA loss.
본 발명에서는 하이드록실 라디칼 소거 효과가 가장 우수한 펩신 가수분해 추출물을 가지고 DNA 보호 효과를 측정하였으며, 실험방법은 하기와 같다.In the present invention, the DNA protection effect was measured with the pepsin hydrolysis extract with the best hydroxyl radical scavenging effect, and the experimental method is as follows.
0.5 ㎍의 pBR 322 DNA, 3 ㎕의 2 mM FeSO4, 2 ㎕의 농도별 차가버섯 가수분해 효소 추출물 및 4 ㎕의 30% H2O2를 혼합하여 37℃에서 1시간 반응시키고, 상기 반응액은 EDTA로 염색된 0.8% 아가로스 겔 전기영동을 실시하여 밴드를 관찰하였다.0.5 μg of pBR 322 DNA, 3 μl of 2 mM FeSO 4 , 2 μl of chaga hydrolase, and 4 μl of 30% H 2 O 2 were mixed and reacted at 37 ° C. for 1 hour. The bands were observed by conducting 0.8% agarose gel electrophoresis stained with silver EDTA.
그 결과를 나타낸 도 3에서 보듯이, 본 발명의 차가버섯 가수분해 효소 추출물에서 정상 플라스미드 DNA(레인 1)와 비교하여 펜톤 반응 하에서 초나선 DNA가 완전히 고리열림 DNA로 전환된 것을 확인할 수 있었다(레인 2).As shown in Figure 3, the chaga mushroom hydrolase extract of the present invention as compared to the normal plasmid DNA (lane 1) it was confirmed that the ultra-helix DNA was completely converted to the ring-open DNA under the Fenton reaction (lane 2).
이는 본 발명의 차가버섯 펩신 가수분해 효소 추출물이 모두 0.125~1 ㎎의 범위에서 농도 의존적으로 DNA 보호 효과를 나타내는 것을 시사한다.
This suggests that chaga pepsin hydrolase extracts of the present invention all exhibit a DNA protection effect in a concentration-dependent manner in the range of 0.125-1 mg.
이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (11)
(2) 상기 (1)단계에 의해 차가버섯이 용해된 완충액에 프로타멕스(protamex), 플라보자임(flavourzyme), 뉴트라제(neutrase), 알카라제(alcalase), 파파인(papain), 키모트립신(α-caymotrypsin), 트립신(trypsin) 및 펩신(pepsin) 중에서 선택되는 1종 이상의 단백질 가수분해 효소를 첨가하는 단계;
(3) 상기 (2)단계에 의해 단백질 가수분해 효소를 첨가한 다음 반응온도 30 내지 70℃, 반응시간 6 내지 10시간 동안 가수분해하는 단계;를 포함하는 차가버섯(Inonotus Obliquus) 가수분해 추출물의 제조방법.
(1) dissolving Chaga ( Inonotus Obliquus ) in buffer;
(2) protamex, flavozyme, neutrase, alcalase, papain, chymo in chaga-dissolved buffer by step (1) Adding at least one proteolytic enzyme selected from trypsin (α-caymotrypsin), trypsin and pepsin;
(3) adding a proteolytic enzyme by the step (2) and then hydrolyzing the reaction temperature for 30 to 70 ° C. and the reaction time for 6 to 10 hours; a chaga ( Inonotus Obliquus ) hydrolysis extract comprising Manufacturing method.
상기 (1)단계에서 사용되는 완충액은 pH 2.0 내지 8.0의 인산염 완충액, 아세트산 완충액 및 글리신-HCl 완충액 중에서 선택되는 1종 이상인 것을 특징으로 하는 차가버섯 가수분해 추출물의 제조방법.
The method of claim 7, wherein
The buffer used in the step (1) is a method of producing a chaga mushroom hydrolysis extract, characterized in that at least one selected from phosphate buffer, acetic acid buffer and glycine-HCl buffer of pH 2.0 to 8.0.
Chaga hydrolyzed extract prepared by the method of claim 7.
Antioxidant composition comprising the chaga mushroom hydrolysis extract of claim 9 as an active ingredient.
상기 항산화 조성물은 의약품, 건강보조제, 화장료, 식품 또는 식품첨가제로 사용되는 것을 특징으로 하는 항산화 조성물.
11. The method of claim 10,
The antioxidant composition is an antioxidant composition, characterized in that used as medicines, health supplements, cosmetics, food or food additives.
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