KR102117297B1 - Ellagic acid glycoside and manufacturing method thereof - Google Patents
Ellagic acid glycoside and manufacturing method thereof Download PDFInfo
- Publication number
- KR102117297B1 KR102117297B1 KR1020180149803A KR20180149803A KR102117297B1 KR 102117297 B1 KR102117297 B1 KR 102117297B1 KR 1020180149803 A KR1020180149803 A KR 1020180149803A KR 20180149803 A KR20180149803 A KR 20180149803A KR 102117297 B1 KR102117297 B1 KR 102117297B1
- Authority
- KR
- South Korea
- Prior art keywords
- ellagic acid
- glycoside
- sucrose
- glycosides
- ellagic
- Prior art date
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- 235000004132 ellagic acid Nutrition 0.000 title claims abstract description 158
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Abstract
Description
본 발명은 스트레스 질환 및 퇴행성 뇌질환의 예방, 개선 또는 치료효과를 가지는 엘라그산 배당체 및 이의 제조방법에 관한 것이다. The present invention relates to an ellagic acid glycoside having a prophylactic, ameliorating or therapeutic effect on stress diseases and degenerative brain diseases and a method for manufacturing the same.
엘라그산(Ellagic acid)은 폴리페놀 4개의 링으로 구성되어 있으며, 식물체 내에서는 전구물질인 엘라기탄닌(ellagitannin) 형태로 존재하며, 포도, 딸기, 석류, 나무딸기, 땅콩류 및 녹차 등에 존재하는 식물성 페놀물질이다. Ellagic acid is composed of four rings of polyphenols, and is present in the form of precursor ellagitannin in plants, and is present in grapes, strawberries, pomegranates, raspberries, peanuts, and green tea. It is a phenolic substance.
엘라그산은 강력한 항산화제로 피부 미백과 주름 생성 억제에 효과가 있다고 보고되어 있는데, 이 물질은 멜라닌 생성효소인 티로시나아제(tyrosinase) 활성의 중요한 요소인 구리에 강력히 부착하여 멜라닌 생성을 억제한다고 한다. 또한, 사람 피부 세포와 생쥐를 이용한 실험을 통해 엘라그산이 콜라겐 파괴를 일으키는 효소(MMP)가 생산되는 것을 막고, 염증반응 관련 물질(ICAM)의 발현을 감소시킴으로써 피부를 보호한다는 사실이 알려져 있으며, 엘라그산 또는 엘라그산 함유 석류 추출물을 피부에 바르거나, 섭취할 시에도 자외선(UV)에 의한 피부 주름 방지 및 색소침착을 막아준다는 사실이 보고되고 있다. It is reported that ellagic acid is a powerful antioxidant that is effective in inhibiting skin whitening and wrinkle formation, and it is said that this substance strongly inhibits melanin production by strongly attaching to copper, an important element of the tyrosinase, a melanin-producing enzyme. In addition, it is known through experiments with human skin cells and mice that ellagic acid protects the skin by preventing the production of collagen breakdown enzyme (MMP) and reducing the expression of inflammatory reaction-related substances (ICAM). It has been reported that when ellagic acid or pomegranate extract containing ellagic acid is applied to the skin or ingested, it prevents skin wrinkles and pigmentation caused by ultraviolet (UV) rays.
엘라그산은 피부보호 효과 외에도, 항산화, 항바이러스, 항돌연변이, 항암기능을 가지고 있다고 알려져 있다. 엘라그산은 암세포의 세포자연사를 유도하며, 특히 유방, 식도, 피부, 결장, 전립선과 췌장에서의 암세포 활동을 억제한다고 알려져 있으며, 결장 내의 비정상 세포의 성장을 감소시키고, 자궁경부암을 유발하는 HPV 바이러스에 감염된 세포의 성장을 저지하는 것이 확인되었다. Ellagic acid is known to have anti-oxidant, anti-viral, anti-mutagenic and anti-cancer functions, in addition to its skin-protective effect. Ellagic acid is known to induce apoptosis of cancer cells, and in particular, it is known to inhibit cancer cell activity in the breast, esophagus, skin, colon, prostate and pancreas, and decreases the growth of abnormal cells in the colon and causes HPV virus to cause cervical cancer It was confirmed to inhibit the growth of cells infected with.
또한, 엘라그산의 효능 중 스트레스에 대한 뇌세포 보호 및 인지능력 개선, 알츠하이머 예방 및 치료에 대한 연구도 진행되고 있으며, 뇌에 발생되는 외상성 뇌손상(traumatic brain injury, TBI)과 이에 따른 인지능력 손상, 기억력감퇴에 대해 보호 효과 및 치유효과가 보고되어 있다. In addition, among the efficacy of ellagic acid, studies on brain cell protection against stress, improvement of cognitive ability, and prevention and treatment of Alzheimer's have been conducted, and traumatic brain injury (TBI) occurring in the brain and impaired cognitive ability , Protective and healing effects against memory loss have been reported.
한편, 글루칸수크라제(Glucansucrases)는 mono-, di-, 또는 그 이상의 슈크로오스 단위를 다른 탄수화물 수여체(carbohydrate acceptors)에 글리코시드 결합(glycosidic linkages)을 통해 전달하는 효소로, 효소에 의한 배당체의 전달이 몇몇 바이오 활성 물질에 작용하여 그 기능적 특성을 변화시키려는 시도가 되어 왔다. Meanwhile, glucansucrases are enzymes that deliver mono-, di-, or more sucrose units to other carbohydrate acceptors through glycosidic linkages. It has been attempted that the delivery of glycosides acts on several bioactive substances to change their functional properties.
엘라그산은 물에 전혀 녹지 않는 특성이 있어, 산업적 활용이 제한되고 있다. 현재까지 글루칸수크라제를 이용하여 엘라그산의 물리적 특성을 변화시키고 개선시킨 종래기술은 없는 실정이다. 따라서, 엘라그산의 생물학적 전환을 통해 생합성된 엘라그산 배당체를 통해 엘라그산의 물성을 개선하고, 효능을 증대시켜, 식품, 화장품 및 약학분야를 포함하는 다양한 분야에서 사용을 증대시키고자 하는 노력이 필요하다. Ellagic acid has properties that are not soluble in water at all, and thus industrial use is limited. To date, there has been no prior art that changes and improves the physical properties of ellagic acid using glucan sucrase. Therefore, it is necessary to improve the physical properties of ellagic acid through the biosynthetic ellagic acid glycoside through biological conversion of ellagic acid, increase the efficacy, and increase the use in various fields including food, cosmetics and pharmaceutical fields. Do.
한편, 한국등록특허 제1091641호에는 석류로부터 엘라그산을 효율적으로 분리하는 방법 및 이를 함유한 미백, 주름개선 기능성 화장품 조성물이 개시되어 있지만, 본 발명의 엘라그산 배당체 및 이의 제조방법에 관해 개시된 바 없다. On the other hand, Korean Patent No. 1016441 discloses a method for effectively separating ellagic acid from pomegranate and a whitening and wrinkle-improving functional cosmetic composition containing the same, but has not been disclosed about the ellagic acid glycoside of the present invention and a method for manufacturing the same .
본 발명은 상기와 같은 요구에 의해 도출된 것으로, 엘라그산 및 수크로스 혼합액에 당전이 효소를 첨가하여 형성된 엘라그산 배당체 및 이의 제조방법을 제공하고, 상기 방법에 의해 제조된 엘라그산 배당체의 용해도, 뇌 신경세포 보호능, 글루타메이트(glutamate) 자극에 의해 증가된 코티졸(cortisol)의 발현 저해효과 및 글루타메이트(glutamate) 자극에 의해 증가된 아세틸콜린 분해효소(acetylcholineesterase)의 활성 증가효과를 확인함으로써, 본 발명을 완성하였다. The present invention was derived by the above-mentioned needs, provides an ellagic acid glycoside formed by adding a sugar transfer enzyme to a mixture of ellagic acid and sucrose, and a method for preparing the same, and a solubility of the ellagic acid glycoside prepared by the method, Brain neuron protection ability, by inhibiting the expression of cortisol (glutamate) increased inhibitory effect of cortisol (glutamate) stimulation and increased activity of acetylcholine degrading enzyme (acetylcholineesterase) increased by stimulation of glutamate (glutamate), the present invention Was completed.
상기 과제를 해결하기 위하여, 본 발명은 수크로스 및 엘라그산의 혼합액에 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 유래의 글루칸수크라제를 첨가한 후 25~30℃에서 6~36시간 동안 반응하는 단계를 포함하는 하기 화학식 1로 표시되는 엘라그산 배당체의 제조방법을 제공한다.In order to solve the above problems, the present invention is a mixture of sucrose and ellagic acid leuconostoc mesentheroid mesenteroides ) provides a method for producing ellagic acid glycosides represented by the following Chemical Formula 1, which comprises the steps of reacting at 25 to 30 ° C. for 6 to 36 hours after adding glucan sucrase.
또한, 본 발명은 상기 제조방법으로 제조된 화학식 1로 표시되는 엘라그산 배당체를 제공한다. In addition, the present invention provides an ellagic acid glycoside represented by Formula 1 prepared by the above production method.
또한, 본 발명은 엘라그산 배당체 또는 약학적으로 허용 가능한 이의 염을 유효성분으로 함유하는 스트레스성 질환 및 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of stressful diseases and degenerative brain diseases containing ellagic acid glycosides or pharmaceutically acceptable salts thereof as an active ingredient.
또한, 본 발명은 상기 엘라그산 배당체 또는 식품학적으로 허용 가능한 이의 염을 유효성분으로 함유하는 스트레스 및 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for the prevention or improvement of stress and degenerative brain disease, which contains the ellagic acid glycoside or a salt thereof as a food acceptable ingredient.
본 발명은 엘라그산 배당체 및 이의 제조방법에 관한 것으로, 엘라그산 및 수크로스 혼합액에 당전이 효소를 첨가하여 형성된 엘라그산 배당체는 엘라그산에 비해 수용성이 개선되고, 글루타메이트(glutamate) 자극으로부터 신경아세포종인 SH-SY5Y 세포의 생존율을 엘라그산에 비해 향상시키고, 글루타메이트(glutamate) 자극에 의해 증가된 코티졸(cortisol)의 발현을 엘라그산에 비해 감소시키며, 글루타메이트(glutamate) 자극에 의해 증가된 아세틸콜린 분해효소(acetylcholineesterase)의 활성을 감소시키는 효과가 있으므로, 스트레스성 질환 및 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물, 스트레스성 질환 및 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물로 사용할 수 있다. The present invention relates to an ellagic acid glycoside and a method for manufacturing the same, and the ellagic acid glycoside formed by adding a sugar transfer enzyme to a mixture of ellagic acid and sucrose has improved water solubility compared to ellagic acid, and is a neuroblastoma from glutamate stimulation. It improves the survival rate of SH-SY5Y cells compared to ellagic acid, decreases cortisol expression increased by glutamate stimulation compared to ellagic acid, and increases acetylcholine degrading enzyme by glutamate stimulation. Since it has an effect of reducing the activity of (acetylcholineesterase), it can be used as a pharmaceutical composition for preventing or treating stressful diseases and degenerative brain diseases, and as a health functional food composition for preventing or improving stressful diseases and degenerative brain diseases.
도 1은 용매에 따른 엘라그산의 용해도를 확인한 결과이다.
도 2는 본 발명의 제조방법에 의해 엘라그산으로부터 당전이 반응 물질이 생성되는 기작을 도식화한 것이다.
도 3은 엘라그산을 12~24시간 동안 당전이 반응시킨 반응물의 TLC 분석결과이다. 화살표는 당전이 반응 물질(엘라그산 배당체)을 표시한다. EA는 엘라그산, S는 수크로스, G는 글루코오스, F는 프룩토오스, -E는 수크로스 및 효소만 첨가된 반응물, 12h는 엘라그산, 수크로스 및 효소가 첨가된 12시간 반응물 및 24h는 엘라그산, 수크로스 및 효소가 첨가된 24시간 반응물을 로딩한 것으로, 황산발색을 통해서 확인한 것이다. 좌측 EA는 UV 245nm 파장에서 확인한 것이다.
도 4는 엘라그산, 수크로스 및 효소가 첨가된 반응물로부터 엘라그산 배당체를 분리하여 TLC 분석한 결과(A) 및 HPLC로 분리한 크로마토그램(B)이다. (A)의 상단은 UV 245nm 파장에서 엘라그산(EA)을 확인한 것이고, 하단은 황산발색을 통해 엘라그산 배당체(EAG)를 확인한 결과이다. 반응물은 엘라그산, 수크로스 및 효소 첨가 후 24시간 동안 반응시킨 것이다.
도 5는 ESI(-)-MS/MS 분석에 의해 엘라그산 배당체의 분자량을 동정한 결과이다. 엘라그산 배당체의 분자량은 485.1이다. 분자량 531.1은 엘라그산 배당체에 Na 이온 3개가 붙은 [M+3Na]-물질이다.
도 6은 중심합성계획법에 의해 반응한 엘라그산 배당체 생성물의 반응표면분석 3차원 모식도이다. A는 효소(glucanase) 및 수크로스(sucrose)의 양과 엘라그산 배당체 생성물의 관계를 나타낸 것이고, B는 엘라그산(ellagic acid) 및 수크로스(sucrose)의 양과 엘라그산 배당체 생성물의 관계를 나타낸 것이며, C는 효소(glucanase) 및 엘라그산(ellagic acid)의 양과 엘라그산 배당체 생성물의 관계를 나타낸 것이다.
도 7은 엘라그산 및 엘라그산 배당체의 물에 대한 용해도를 확인한 결과이다.
도 8은 본 발명의 엘라그산 배당체에 의한 뇌신경 세포 보호효과를 MTT 분석(A) 및 현미경(B)으로 확인한 결과이다. 글루타메이트는 세포 스트레스 유발 물질이다. Buffer는 화합물의 용매만 처리한 군이고, 테아닌(theanine)은 양성 대조군이고, EA는 엘라그산 농도별 처리군이며, EAG는 엘라그산 배당체의 농도별 처리군이다. *, **은 엘라그산에 비해 엘라그산 배당체의 세포 생존율이 유의미하게 증가하는 것을 의미하며, *은 p<0.05, **은 p<0.01이다.
도 9는 본 발명의 엘라그산 배당체에 의한 코티졸 분비 억제효과를 확인한 것으로, 글루타메이트는 코티졸 분비 유도 물질이다. Buffer는 화합물의 용매만 처리한 군이고, 테아닌(theanine)은 양성 대조군이고, EA는 엘라그산 농도별 처리군이고, EAG는 엘라그산 배당체의 농도별 처리군이다. **은 엘라그산에 비해 엘라그산 배당체의 코티졸 분비량이 유의미하게 감소하는 것을 의미하며, p<0.01이다.
도 10은 본 발명의 엘라그산 배당체에 의한 아세틸콜린에스테라아제 활성 억제효과를 확인한 것으로, 글루타메이트는 아세틸콜린에스테라아제 활성 유도 물질이다. 타크린(tacrine)은 양성 대조군이고, EA는 엘라그산 농도별 처리군이고, EAG는 엘라그산 배당체의 농도별 처리군이다. ***은 엘라그산에 비해 엘라그산 배당체의 아세틸콜린에스테라아제 저해 활성이 유의미하게 증가하는 것을 의미하며, p<0.001이다. 1 is a result confirming the solubility of ellagic acid according to the solvent.
Figure 2 is a schematic diagram showing the mechanism by which a sugar transfer reaction substance is generated from ellagic acid by the production method of the present invention.
3 is a TLC analysis result of the reactant reacted with ellagic acid for 12 to 24 hours. The arrow indicates the sugar transfer reaction material (elagic acid glycoside). EA is ellagic acid, S is sucrose, G is glucose, F is fructose, -E is sucrose and an enzyme-only reactant, 12h is ellagic acid, sucrose and an enzyme-added 12-hour reactant, and 24h is Elagic acid, sucrose, and enzyme-loaded reactants were loaded for 24 hours, and confirmed by sulfuric acid coloring. The left EA is confirmed at UV 245nm wavelength.
4 is a result of TLC analysis by separating ellagic acid glycosides from reactants to which ellagic acid, sucrose and enzymes were added (A) and chromatogram (B) separated by HPLC. The upper part of (A) was confirmed by ellagic acid (EA) at UV 245nm wavelength, and the lower part was the result of confirming ellagic acid glycoside (EAG) through sulfuric acid coloration. The reactants were reacted for 24 hours after the addition of ellagic acid, sucrose and enzymes.
5 is a result of identifying the molecular weight of the ellagic acid glycosides by ESI (-)-MS / MS analysis. The molecular weight of the ellagic acid glycoside is 485.1. Molecular weight 531.1 is [M + 3Na] -substance with three Na ions attached to the ellagic acid glycoside.
6 is a three-dimensional schematic diagram of the reaction surface analysis of the ellagic acid glycoside product reacted by the central synthesis planning method. A represents the relationship between the amount of the enzyme (glucanase) and sucrose (sucrose) and the ellagic acid glycoside product, B represents the relationship between the amount of ellagic acid and sucrose (sucrose) and the ellagic acid glycoside product, C shows the relationship between the amount of enzyme (glucanase) and ellagic acid and the ellagic acid glycoside product.
7 is a result confirming the solubility of ellagic acid and ellagic acid glycosides in water.
8 is a result confirming the neuroprotective effect of the brain cells by ellagic acid glycosides of the present invention by MTT analysis (A) and microscope (B). Glutamate is a cell stress inducer. Buffer is a group treated with only the solvent of the compound, theanine is a positive control group, EA is a treatment group according to the concentration of ellagic acid, and EAG is a treatment group according to the concentration of ellagic acid glycosides. *, ** means that the cell viability of ellagic acid glycosides is significantly increased compared to ellagic acid, * is p <0.05, ** is p <0.01.
Figure 9 confirms the inhibitory effect of cortisol secretion by the ellagic acid glycoside of the present invention, glutamate is a cortisol secretion inducer. Buffer is a group treated with only the solvent of the compound, theanine is a positive control group, EA is a treatment group according to the concentration of ellagic acid, and EAG is a treatment group according to the concentration of ellagic acid glycosides. ** means that the amount of cortisol secretion of ellagic acid glycosides is significantly reduced compared to ellagic acid, and p <0.01.
Figure 10 confirms the inhibitory effect of acetylcholinesterase activity by the ellagic acid glycoside of the present invention, glutamate is an acetylcholinesterase activity inducer. Tacrine is a positive control group, EA is a treatment group according to the concentration of ellagic acid, and EAG is a treatment group according to the concentration of ellagic acid glycosides. *** means that the acetylcholinesterase inhibitory activity of the ellagic acid glycoside is significantly increased compared to ellagic acid, and p <0.001.
본 발명은 수크로스 및 엘라그산의 혼합액에 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 유래의 글루칸수크라제를 첨가한 후 25~30℃에서 6~36시간 동안 반응하는 단계를 포함하는 하기 화학식 1로 표시되는 엘라그산 배당체의 제조방법에 관한 것이다. The present invention is a mixture of sucrose and ellagic acid leuconostoc mesenteroid ( Leuconostoc mesenteroides ) and a method for preparing ellagic acid glycosides represented by the following Chemical Formula 1, which comprises reacting at 25 to 30 ° C. for 6 to 36 hours after adding glucan sucrase.
[화학식 1][Formula 1]
상기 방법은 바람직하게는 140~160mM 수크로스 및 4~6mM 엘라그산의 혼합액에 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 유래의 글루칸수크라제 280~320mU/mL를 첨가한 후 소듐 아세테이트 버퍼에서 26~30℃에서 20~28시간 동안 반응하는 단계를 포함할 수 있으며, 더욱 바람직하게는 150mM 수크로스 및 5mM 엘라그산의 혼합액에 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 유래의 글루칸수크라제 300mU/mL를 첨가한 후 20mM 소듐 아세테이트 버퍼에서 28℃에서 24시간 동안 반응하는 단계를 포함할 수 있다.The method is preferably added to a mixture of 140-160mM sucrose and 4-6mM ellagic acid, followed by addition of 280-320mU / mL of glucan sucrease derived from Leuconostoc mesenteroides , 26-in sodium acetate buffer. It may include a step of reacting for 20 to 28 hours at 30 ℃, more preferably 150mM sucrose and 5mM ellagic acid in a mixture of leuconostoc mesenteroid ( Leuconostoc mesenteroides ) may include a step of reacting for 24 hours at 28 ° C. in 20 mM sodium acetate buffer after adding 300 mU / mL of glucan sucrase.
상기 당전이 효소는 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 유래의 글루칸수크라제를 사용할 수 있으며, 바람직하게는 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 512 FMCM 유래의 글루칸수크라제를 사용하는 것이지만, 이에 한정하는 것은 아니다. The sugar transfer enzyme is Leuconostoc mesenteroides ) -derived glucan sucrease may be used, and preferably, the glucan sucrase derived from Leuconostoc mesenteroides 512 FMCM is used, but is not limited thereto.
또한, 본 발명은 상기 제조방법으로 제조된 화학식 1로 표시되는 엘라그산 배당체에 관한 것이다. In addition, the present invention relates to an ellagic acid glycoside represented by Formula 1 prepared by the above production method.
또한, 본 발명은 상기 엘라그산 배당체 또는 약학적으로 허용 가능한 이의 염을 유효성분으로 함유하는 스트레스성 질환 및 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물에 관한 것이다.In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of stressful diseases and degenerative brain diseases containing the ellagic acid glycoside or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 퇴행성 뇌질환은 아세틸콜린 분해에 의해 유발되는 것으로, 치매(dementia), 알츠하이머 질환(Alzheimer's disease) 및 파킨슨 질환(Parkinson disease)으로 이루어진 군으로부터 선택된 어느 하나인 것일 수 있고, 바람직하게는 치매 또는 알츠하이머 질환일 수 있지만, 이에 한정하는 것은 아니다. The degenerative brain disease is caused by acetylcholine decomposition, and may be any one selected from the group consisting of dementia, Alzheimer's disease and Parkinson's disease, preferably dementia or Alzheimer's It may be a disease, but is not limited thereto.
상기 스트레스성 질환은 코티졸 분비의 증가에 의해 발생하는 우울증(depressive disorder), 수면장애(sleep disturbance) 및 불안장애(anxiety disorder)로 이루어진 군으로부터 선택되는 하나 이상인 것일 수 있으나. 이에 제한되는 것은 아니다. The stressful disease may be one or more selected from the group consisting of depressive disorder, sleep disturbance and anxiety disorder caused by an increase in cortisol secretion. It is not limited thereto.
상기 엘라그산 배당체는 글루타메이트(glutamate) 자극으로부터 뇌신경 세포를 보호하며, 글루타메이트(glutamate) 자극에 의해 증가된 코티졸(cortisol)의 분비량을 감소시키며, 글루타메이트(glutamate) 자극에 의해 증가된 아세틸콜린 분해효소(acetylcholineesterase)의 활성을 저해한다. The ellagic acid glycoside protects cranial nerve cells from glutamate stimulation, reduces the amount of cortisol secreted by glutamate stimulation, and increases acetylcholine degrading enzyme by glutamate stimulation ( acetylcholineesterase).
상기 엘라그산 배당체는 엘라그산에 비해 수용성이 증가한다. The ellagic acid glycoside has increased water solubility compared to ellagic acid.
본 발명에 따른 상기 약학 조성물은 각각 통상의 방법에 따라 캡슐제, 산제, 과립제, 정제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. The pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be.
본 발명에 따른 상기 약학 조성물은 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있다. The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.
본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 약학 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당하는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the pharmaceutical composition, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다.Suitable dosages of the pharmaceutical compositions of the invention may be variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response sensitivity. You can.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있으며, 비경구 투여의 경우, 피부에 국소적으로 도포, 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, topically applied to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.
또한, 본 발명은 상기 엘라그산 배당체 또는 식품학적으로 허용 가능한 이의 염을 유효성분으로 함유하는 스트레스 및 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다. In addition, the present invention relates to a health functional food composition for the prevention or improvement of stress and degenerative brain disease, which contains the ellagic acid glycoside or a salt thereof as an active ingredient.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 양은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 총 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 하는 장기간의 섭취인 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient can be appropriately used depending on the purpose of use (prevention or improvement). In general, the health functional food composition of the present invention in the manufacture of a food or beverage is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the total raw material. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There are no particular restrictions on the type of the health functional food. Examples of foods to which the health functional food composition can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, tea Drinks, alcoholic beverages, and vitamin complexes are included, and all of the health foods in the ordinary sense are included.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients that are conventionally added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink agent, natural carbohydrate or flavoring agent may be included as an additional component in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.) or sugar alcohols ( Xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (eg, tau martin, stevia extract, etc.) and a synthetic flavoring agent (eg, saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.
Various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may further contain a carbonation agent used in beverages. The proportion of the components to be added is not particularly important, but it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다.
Hereinafter, the present invention will be described in more detail using examples. It is obvious to those skilled in the art that these examples are only intended to illustrate the present invention in more detail and that the scope of the present invention is not limited by them.
재료 및 방법Materials and methods
1. 실험재료1. Experimental materials
엘라그산(ellagic acid), 실리카겔(silica gel), 수크로스(sucrose), 글루코오스(glucose), 말토오스(maltose), 나프틸에틸렌-디아민 디하이드로클로라이드(naphthylethylene-diamine dihydrochloride), MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolimbromide], DPPH(1,1-diphenyl-2-picrylhydrazyl) 시약은 시그마(Sigma)에서 구입하였다.
Ellagic acid, silica gel, sucrose, glucose, maltose, naphthylethylene-diamine dihydrochloride, MTT [3- (4 , 5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolimbromide] and DPPH (1,1-diphenyl-2-picrylhydrazyl) reagent were purchased from Sigma.
2. 2. 엘라그산Ellagsan 배당체 생성 Glycoside production
당전이를 위한 글루칸수크라제 효소는 L. mesenteroides B-512 FMCM을 발효 배양한 후, 30K 차단중공사형(cutoff hollow fiber)을 이용하여 정제한 후, Q-세파로오스 컬럼(sepharose column)을 이용하여 정제된 것을 사용하였다. 효소 활성은 28℃에서 기질로 100mM 수크로스와 20mM 소듐 아세테이트(pH 5.2) 완충액을 이용해 다양한 반응시간을 두고 측정하였다. 글루칸수크라제 활성 단위인 1unit은 20mM 소듐 아세테이트 완충용액(pH 5.2)을 사용하였을 때, 28℃에서 분당 1μmol의 프락토스를 분해하는 효소의 활성을 말한다. The glucan sucrase enzyme for sugar transfer is fermented and cultured in L. mesenteroides B-512 FMCM, purified using 30K cutoff hollow fiber, and then Q-sepharose column. The purified one was used. Enzyme activity was measured over a variety of reaction times using 28 mM sucrose and 20 mM sodium acetate (pH 5.2) buffer as a substrate at 28 ° C. 1 unit, the glucan sucrase activity unit, refers to the activity of an enzyme that decomposes 1 μmol of fructose per minute at 28 ° C. when a 20 mM sodium acetate buffer solution (pH 5.2) is used.
엘라그산 배당체 생성조건은 총 250mL에 10mM 엘라그산(75.3mg), 355mM 수크로스(15.9g) 및 상기 분리 정제한 글루칸수크라제(0.65units/mL)를 혼합하고, 반응액을 28℃에서 2~24시간 동안, 설탕이 없어질 때까지 반응시켰다. 그 후 효소를 불활성화시키기 위해 반응액을 5분 동안 100℃에서 끓였다. The conditions for ellagic acid glycoside production were mixed with 10 mM ellagic acid (75.3 mg), 355 mM sucrose (15.9 g) and the separated and purified glucan sucrase (0.65 units / mL) in a total of 250 mL, and the reaction solution was 2 at 28 ° C. For ~ 24 hours, reaction was carried out until sugar disappeared. Then the reaction solution was boiled at 100 ° C. for 5 minutes to inactivate the enzyme.
TLC(Thin-Layer Chromatography)는 실리카 겔 60 F254 TLC 플레이트(Merck Co.)를 이용해 분석하였다. 각 반응액을 실온에서 1㎕씩 실리카 겔 플레이트에 점적한 후 전개 용매 에틸아세테이트/아세트산/물(ethyl acetate/acetic acid/water (3:1:1, v/v/v))을 이용하여 TLC 챔버에서 전개한 후, UV 254nm에서 검출하거나, 황산 발색 용매(0.3%(v/v) N-1-나프틸-에틸렌디아민 및 5%(v/v) 황산이 함유된 메탄올)에 5초 동안 담근후, 110℃ 건조 오븐에서 10분 동안 발색하였다.
TLC (Thin-Layer Chromatography) was analyzed using
3. 3. 엘라그산Ellagsan 배당체의 용매 추출 및 분리 Solvent extraction and separation of glycosides
반응액에 존재하는 당(dextran, fructose 및 glucose) 및 효소 등을 없애기 위해, 비극성 용매인 초산에틸을 사용해 상기 배당체 형성 반응액과 1 대 1로 혼합한 후 200×g에서 30분 동안 쉐이킹(shaking)한 후, 원심분리(10,000×g, 20분, 4℃)한 다음 상등액을 취하였다. 상기 상등액은 47℃ 회전증발농축기를 이용하여 용매를 증발시킨 후 50%(v/v) 메탄올에 용해한 후 C18 컬럼(50×500mm)에 로딩하였다. 그 후 컬럼을 1ml/min의 유속으로 총 300ml의 증류수를 이용하여 1차 세척한 다음, 총 200ml 증류수를 이용하여 2차 세척하였다. 그 후 10~100%(v/v) 메탄올을 이용하여 농도구배하여 용출하였고, 50~60%(v/v) 메탄올을 사용하여 엘라그산 배당체가 포함된 추출액을 획득하였다. In order to remove sugars (dextran, fructose and glucose) and enzymes present in the reaction solution, the mixture is reacted with the glycoside-forming reaction solution in a 1: 1 ratio using ethyl acetate, a non-polar solvent, and shaken at 200 × g for 30 minutes. ), Followed by centrifugation (10,000 × g, 20 minutes, 4 ° C.) and then the supernatant. The supernatant was evaporated with a solvent using a 47 ° C. rotary evaporator, dissolved in 50% (v / v) methanol, and loaded onto a C18 column (50 × 500 mm). Thereafter, the column was first washed with a total of 300 ml of distilled water at a flow rate of 1 ml / min, followed by a second washing with a total of 200 ml of distilled water. Then, it was eluted by concentration gradient using 10-100% (v / v) methanol, and an extract containing ellagic acid glycosides was obtained using 50-60% (v / v) methanol.
배당체 포함 여부를 확인하기 위해 엘라그산 배당체를 함유하고 있는 추출액을 47℃ 회전증발농축기를 이용하여 농축한 후, PDA-MD2015 장비의 HPLC(JASCO, Kyoto, Japan)를 이용하여 배당체를 분리하였다. HPLC 분석에 사용된 컬럼은 u-Bondapak C18 역상 컬럼(300×19mm i.d., Waters)이고, A 용매(0.1%(v/v) 포름산이 함유된 물)와 B 용매(0.1%(v/v) 포름산이 함유된 메탄올)를 사용하여 B 용매 대비 A 용매를 초기 5분 동안은 5%~20%(v/v)까지, 그 후 10분 동안 20%~50%(v/v)까지, 그 후 10분 동안 50%~10%(v/v)까지의 농도로 흘려보냈고, 유속은 0.9ml/min, 컬럼 오븐 온도는 40℃였다. 회수된 추출액은 254nm에서 MD2015 모델 PDA 검출기(JASCO)를 사용해서 분석하였다.
To confirm the presence of glycosides, the extract containing ellagic acid glycosides was concentrated using a 47 ° C rotary evaporator, and then glycosides were separated using HPLC (JASCO, Kyoto, Japan) of PDA-MD2015 equipment. The columns used for HPLC analysis were u-Bondapak C18 reverse phase column (300 × 19 mm id, Waters), solvent A (0.1% (v / v) formic acid water) and solvent B (0.1% (v / v)) Using solvent formic acid), solvent A compared to solvent B is 5% to 20% (v / v) for the first 5 minutes, and then 20% to 50% (v / v) for 10 minutes. After flowing for 10 minutes at a concentration of 50% to 10% (v / v), the flow rate was 0.9 ml / min, and the column oven temperature was 40 ° C. The recovered extract was analyzed using an MD2015 model PDA detector (JASCO) at 254 nm.
4. 4. 엘라그산Ellagsan 배당체 구조 동정 Identification of glycoside structure
본 발명의 엘라그산 배당체는 LC/MS/MS 분석을 통해 분자량 및 분자식을 확인하였다. 배당체는 ESI-MS/MS(electrospray ionization tandem mass spectrometry)를 사용하여 분석하였으며, Positive ESI-MS 분석은 Synapt HDMS system(Waters)을 사용하여 로크 스프레이 인터페에스가 있는 전기 스프레이 이온화 소스(electrospray ionization source with lock spray interface)로 분석하였다.
The ellagic acid glycoside of the present invention confirmed the molecular weight and molecular formula through LC / MS / MS analysis. Glycosides were analyzed using ESI-MS / MS (electrospray ionization tandem mass spectrometry), and positive ESI-MS analysis using an Synapt HDMS system (Waters) with an electrospray ionization source with a lock spray interface. lock spray interface).
5. 5. 엘라그산Ellagsan 배당체 생성 최적화 (중심합성계획법과 반응표면분석법) Optimization of glycoside production (Central Synthesis Planning and Response Surface Analysis)
엘라그산의 배당체는 355mM 수크로스, 650mU/ml 효소 및 10mM 엘라그산을 12시간 반응시켜 얻었다. 엘라그산의 배당체 생성 최적조건을 얻기 위해서 반응표면분석법(RSM)을 이용하였으며, 엘라그산의 배당체 생성에 대한 실험계획은 중심합성계획법을 실시하여 생성 조건에 대한 중요한 독립변수로(Xi) 고려되는 인자, 즉 기질농도 (X1), 효소농도(X2) 및 수용체 농도(엘라그산, X3)에 대한 실험범위를 설정하여 각 5단계로 부호화하여(표1) 17군으로 구분하였다(표 2). 그리고 이들 독립변수에 의해 영향받는 종족변수(Yn), 즉 엘라그산의 배당체의 생성농도를 측정하여 그 값을 회귀분석에 사용하였다.The glycoside of ellagic acid was obtained by reacting 355mM sucrose, 650mU / ml enzyme and 10mM ellagic acid for 12 hours. Response surface analysis (RSM) was used to obtain optimal conditions for the production of glycosides of ellagic acid, and the experimental design for glycoside production of ellagic acid was considered as an important independent variable (X i ) by the central synthesis planning method. The experimental ranges for factors, namely, substrate concentration (X 1 ), enzyme concentration (X 2 ), and receptor concentration (ellagic acid, X 3 ) were set and coded in 5 steps (Table 1) and divided into 17 groups (Table 1). 2). In addition, the concentrations of the race variables (Yn) affected by these independent variables, namely, the glycosides of ellagic acid, were measured and the values were used for regression analysis.
(mM)Sucrose
(mM)
(mU)Glucan sucrase
(mU)
(mM)Ellagsan
(mM)
최적 엘라그산 배당체의 생성조건은 반응표면분석으로 얻어진 수크로스 농도, 글루칸수크라제 농도, 엘라그산 농도 등의 등고선도(contour map)를 슈퍼이미징(superimaging) 했을 때 중복되는 부분의 범위로 예측하였다. 회귀분석 및 그래프를 얻기 위해 Design-Expert 6.0.11 통계 프로그램을 사용하였다.
The optimum conditions for the production of ellagic acid glycosides were predicted as a range of overlapping parts when superimaging contour maps such as sucrose concentration, glucan sucrase concentration, and ellagic acid concentration obtained by reaction surface analysis. . The statistical program of Design-Expert 6.0.11 was used to obtain regression analysis and graphs.
6. 기능성 조사6. Functional investigation
1) 뇌신경 보호 효과 : 1) Neuroprotective effect: MTTMTT 분석 analysis
신경세포 보호 효과를 확인하기 위하여 SH-SY5Y 세포(KCLB 22266, Korean Cell Line Bank, Seoul, Korea)를 실험에 사용하였다. 세포의 생존율은 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 분석을 이용하여 측정하였다. SH-SY5Y cells (KCLB 22266, Korean Cell Line Bank, Seoul, Korea) were used in the experiment to confirm the neuroprotective effect. Cell viability was measured using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis.
뇌신경 세포(SH-SY5Y)(neuroblastoma, human dopaminergic neuronal cell)는 1%(v/v) 항진균제/항생제와 10%(v/v) FBS를 함유한 RPMI-1640 배지에서 배양하였고, 96-웰 플레이트에 세포수가 104~106cell/ml가 되도록 분주한 후, 24시간 후에 엘라그산과 엘라그산 배당체를 농도별로 처리하였다. 그 후 100mM 글루타메이트를 처리해 스트레스를 유발시킨 후 MTT 분석을 이용하여 570nm 파장으로 흡광도를 측정하여 세포독성을 확인하였다. 양성 대조군으로는 뇌 진정작용이 알려진 1~10uM 테아닌(theanine)을 사용하였다.
Neuroblastoma (SH-SY5Y) (neuroblastoma, human dopaminergic neuronal cell) was cultured in RPMI-1640 medium containing 1% (v / v) antifungal / antibiotic and 10% (v / v) FBS, and 96-well plate After dispensing so that the number of cells was 10 4 to 10 6 cells / ml, after 24 hours, ellagic acid and ellagic acid glycosides were treated by concentration. After treatment with 100mM glutamate to induce stress, the MTT assay was used to measure the absorbance at a wavelength of 570nm to confirm cytotoxicity. As a positive control, 1-10 μM theanine, which is known for brain sedation, was used.
2) 2) 항스트레스Antistress 효과 : effect : 코티졸Cortisol 측정( Measure( cortisolcortisol determinationdetermination ))
엘라그산 배당체의 항스트레스 효과를 확인하기 위해 스트레스 호르몬인 코티졸의 함량을 측정하였다. In order to confirm the anti-stress effect of ellagic acid glycosides, the content of the stress hormone cortisol was measured.
세포배양액을 4℃, 10,000×g에서 10분 동안 원심분리한 후 상등액을 취하여 실험에 사용하였다. 상등액의 단백질 함량은 BCA 키트로 조사하였으며, 코티졸 엘라이자 키트(Calbiotech)를 제조사의 프로토콜에 따라 사용하여 코티졸 함량을 분석하였다.
The cell culture solution was centrifuged at 4 ° C and 10,000 × g for 10 minutes, and the supernatant was taken and used in the experiment. The protein content of the supernatant was investigated with a BCA kit, and the cortisol content was analyzed using a cortisol ELISA kit (Calbiotech) according to the manufacturer's protocol.
3) 퇴행성 뇌질환 개선효과 : 3) Degenerative brain disease improvement effect: 아세틸콜린에스테라제Acetylcholinesterase (( AcetylcholinesteraseAcetylcholinesterase , AChE) 억제 분석, AChE) inhibition assay
AChE는 뇌신경 전달물질인 아세틸콜린(Acetylcholin)을 분해하며, 아밀로이드반(amyloid plaque)이나 신경세포섬유매듭(neurofibrillary tangles)의 생성을 유발한다고 알려져 있다. 따라서 뇌신경 세포(SH-SY5Y)에 100mM 글루타메이트를 처리해 스트레스가 유발된 세포에 엘라그산 또는 엘라그산 배당체를 농도별로 처리한 후 세포배양액을 10,000×g에서 10분 동안 원심분리한 다음 상등액을 취하였고, 상등액을 이용하여 AChE 억제 정도를 AChE 억제 분석 키트를 제조사의 프로토콜에 따라 측정하여 항치매 개선효과를 확인하였다.
It is known that AChE breaks down the brain neurotransmitter acetylcholine and causes the production of amyloid plaques or neurofibrillary tangles. Therefore, the brain nerve cells (SH-SY5Y) were treated with 100 mM glutamate to treat stress-induced cells with ellagic acid or ellagic acid glycosides by concentration, and then centrifuged the cell culture solution at 10,000 × g for 10 minutes, and then took the supernatant. The degree of AChE inhibition using the supernatant was measured by measuring the AChE inhibition assay kit according to the manufacturer's protocol to confirm the antidementia improvement effect.
7. 통계처리7. Statistics processing
실험에서 얻어진 결과는 SAS 프로그램(Package relwase 8.01)을 이용하여 평균+표준편차로 표시하였고, 평균값의 통계적 유의성은 p<0.05수준에서 던칸의 다중검정법(multiple range test)에 의해 검정하였다.
The results obtained in the experiment were expressed as the mean + standard deviation using the SAS program (Package relwase 8.01), and the statistical significance of the mean value was tested by Duncan's multiple range test at the p <0.05 level.
실시예Example 1. 용매별 1. By solvent 엘라그산의Ellagic 용해도 Solubility
물에 전혀 녹지 않는 엘라그산을 가용화하기 위해 엘라그산의 농도 및 용매별 용해도를 확인하였다. 그 결과, 도 1에 개시된 바와 같이 25mM의 엘라그산을 50%(v/v) 아세톤을 이용하여 녹였을 때 가장 높은 용해도를 나타냈다. In order to solubilize ellagic acid which is not soluble in water at all, the concentration of ellagic acid and solubility of each solvent were checked. As a result, as shown in FIG. 1, 25 mM ellagic acid showed the highest solubility when dissolved using 50% (v / v) acetone.
따라서 이후 실험에서는 50%(v/v) 아세톤에 용해된 엘라그산을 이용하였다.
Therefore, in the subsequent experiments, ellagic acid dissolved in 50% (v / v) acetone was used.
실시예Example 2. 2. 엘라그산Ellagsan 배당체 생성, 정제 및 동정 Glycoside production, purification and identification
반응액은 50%(v/v) 아세톤에 용해한 10mM의 엘라그산(ellagic acid, EA)(75.3g)에 335mM의 수크로스(15.9g) 및 글루칸수크라제(0.65units/ml)를 혼합한 후 완충용액(0.1mM MgCl2, 40mM 소듐 아세테이트, pH 5.2)을 이용하여 총 250ml 용량으로 제조하였다. 당전이 물질 생성기작은 도 2에 도식화하였다. The reaction solution was mixed with 10 mM ellagic acid (EA) (75.3 g) dissolved in 50% (v / v) acetone, 335 mM sucrose (15.9 g), and glucan sucrase (0.65 units / ml). After the buffer solution (0.1mM MgCl 2 , 40mM sodium acetate, pH 5.2) was prepared in a total capacity of 250ml. The mechanism for generating sugar transfer material is illustrated in FIG. 2.
상기 반응액은 28℃에서 12~24시간 동안 수크로스가 없어질 때까지 반응시켰고, 반응 후 반응액을 TLC(Thin-Layer Chromatography)에 점적하여 전개 용매 에틸아세테이트/아세트산/물(ethyl acetate/acetic acid/water, 3:1:1, v/v/v)을 이용하여 TLC 챔버에서 전개한 후, UV 254nm에서 검출하거나, 황산 발색 용매(0.3%(v/v) N-1-나프틸-에틸렌디아민 및 5%(v/v) 황산이 함유된 메탄올)에 5초 동안 담근 후, 110℃ 건조 오븐에서 10분 동안 발색하였다. The reaction solution was reacted at 28 ° C. for 12 to 24 hours until sucrose disappeared, and after reaction, the reaction solution was added dropwise to TLC (Thin-Layer Chromatography) to develop ethyl acetate / acetic acid / water (ethyl acetate / acetic). After development in a TLC chamber using acid / water, 3: 1: 1, v / v / v), detection at UV 254 nm or sulfuric acid color development solvent (0.3% (v / v) N-1-naphthyl- After soaking for 5 seconds in ethylenediamine and 5% (v / v) sulfuric acid methanol), it was developed for 10 minutes in a 110 ° C. drying oven.
그 결과, 도 3에 개시된 바와 같이 반응 후 당전이 물질이 생성된 것을 확인하였다. 엘라그산은 UV 245nm 파장에서 확인 가능하였고, 엘라그산 배당체는 황산발색을 통해서 확인 가능하였다. As a result, it was confirmed that a sugar transfer material was produced after the reaction as disclosed in FIG. 3. Ellagic acid was confirmed at UV 245nm wavelength, and ellagic acid glycosides were identified through sulfuric acid color development.
또한, 상기 엘라그산 및 엘라그산 배당체가 함유된 효소 반응물을 초산에틸을 이용해 액-액 분리하였으며, C18 칼럼으로 분리 후 TLC 분석한 결과, 도 4A에 개시된 바와 같이 엘라그산 배당체(EAG)는 50%(v/v)~60%(v/v) 메탄올을 이용하여 분리할 수 있었다. 엘라그산은 UV 245nm 파장에서 확인 가능하였고, 엘라그산 배당체는 황산발색을 통해서 확인 가능하였다. In addition, the enzymatic reactants containing ellagic acid and ellagic acid glycosides were liquid-liquid separated using ethyl acetate, and after TLC analysis after separation with a C18 column, as shown in FIG. 4A, ellagic acid glycoside (EAG) was 50%. (v / v) to 60% (v / v) methanol. Ellagic acid was confirmed at UV 245nm wavelength, and ellagic acid glycosides were identified through sulfuric acid color development.
또한, 상기 엘라그산 및 엘라그산 배당체가 함유된 효소 반응물을 초산에틸을 이용해 액-액 분리하였으며, C18 칼럼정제 후 HPLC로 분리한 크로마토그램 결과, 도 4B에 개시된 바와 같이 효소 반응물에서 엘라그산 배당체는 6.7분(70%(v/v) 메탄올), 엘라그산은 9.2분(55%(v/v) 메탄올)에서 추출되었다. In addition, the enzymatic reactant containing the ellagic acid and ellagic acid glycosides was liquid-liquid separated using ethyl acetate, and the chromatogram separated by HPLC after C18 column purification showed that the ellagic acid glycoside in the enzymatic reactant is as shown in FIG. 6.7 minutes (70% (v / v) methanol), ellagic acid was extracted at 9.2 minutes (55% (v / v) methanol).
한편, 엘라그산 배당체는 LC/MS/MS 분석을 통해 분자량 및 분자식을 밝혔으며, 글루칸수크라제 효소 특성에 따라 예상 결합 구조는 α-1,6 결합된 것으로 예측되어, O-α-D-글루코실(1->6) 엘라그산으로 구조를 결정하였다. On the other hand, ellagic acid glycosides revealed molecular weight and molecular formula through LC / MS / MS analysis, and the predicted binding structure was predicted to be α-1,6 bound according to the properties of glucan sucrase, so that O- α-D- The structure was determined with glucosyl (1-> 6) ellagic acid.
LC/MS/MS를 통해 얻어진 엘라그산 분자량은 302.19이고 엘라그산 배당체의 분자량은 485.1로 [M+Na]- 예상구조와 일치하였다. 상기 Na 이온은 효소에서 유래된 것으로, 531.1 분자량은 엘라그산 배당체에 Na 이온 3개가 붙은 [M+3Na]-물질로 판명되었다(도 5).
The molecular weight of ellagic acid obtained through LC / MS / MS was 302.19, and the molecular weight of ellagic acid glycoside was 485.1, which was consistent with [M + Na] -expected structure. The Na ion was derived from an enzyme, and the 531.1 molecular weight was found to be [M + 3Na] -substrate with three Na ions attached to the ellagic acid glycoside (FIG. 5).
실시예Example 3. 3. 엘라그산Ellagsan 배당체 생성 최적화(중심합성계획법 및 반응표면분석법) Optimization of glycoside production (Central Synthesis Planning and Response Surface Analysis)
엘라그산 배당체 생성을 최적화하기 위해 중심합성계획법 및 반응표면분석법을 이용하였다. 중심합성계획법에 의해 수크로스 기질은 10.2~699.8mM, 글루칸수크라제 효소는 61~1238mU/ml, 엘라그산 수용체는 1.5~25mM을 첨가해 총 20가지 조합으로 분석하였다. To optimize the production of ellagic acid glycosides, a central synthesis planning method and a response surface analysis method were used. According to the central synthesis planning method, sucrose substrate was analyzed in a total of 20 combinations by adding 10.2 ~ 699.8mM, glucan sucrase enzyme 61 ~ 1238mU / ml, and ellagic acid receptor 1.5 ~ 25mM.
최적 엘라그산 배당체(EAG) 생성 조건은 글루칸수크라제 효소 300mU/mL, 수크로스 기질 150mM, 수용체 엘라그산 5mM을 첨가해 20mM 소듐 아세테이트 버퍼에서 28℃ 조건에서 24시간 반응시켰을 때, 3.47mM 엘라그산 배당체가 생성된다는 것을 확인하였다. Optimum ellagic acid glycoside (EAG) production conditions are 3.47 mM ellagic acid when reacted for 24 hours at 28 ° C. in 20 mM sodium acetate buffer by adding
반응표면 분석법에 의한 3차원 그래프 3개(도 6)로부터 유출된 공식은 하기 식 1에 표시하였다. The formulas leaked from three 3D graphs (FIG. 6) by the reaction surface analysis method are shown in
[식 1][Equation 1]
Y=-4.630+0.011X1+0.008X2+0.443X3+0.0000004X1 2-0.000008X2 2-0.00004X3 2-0.00001X1X2-0.000004X1X3-0.016X2X3 Y = -4.630 + 0.011X 1 + 0.008X 2 + 0.443X 3 + 0.0000004X 1 2 -0.000008X 2 2 -0.00004X 3 2 -0.00001X 1 X 2 -0.000004X 1 X 3 -0.016X 2 X 3
Y= 엘라그산 배당체 생성양(mM)Y = amount of ellagic acid glycoside production (mM)
X1 수크로스, X2 글루칸수크라제, X3 엘라그산
X 1 sucrose, X 2 glucan sucrase, X 3 ellagic acid
실시예 4. 엘라그산Example 4. Ellagic acid 배당체의 수용성 확인 Checking the acceptability of glycosides
본 발명의 엘라그산 배당체를 물에 용해하여 보았다. 그 결과, 도 7에 개시된 바와 같이 엘라그산 자체는 농도에 상관없이 전혀 물에 녹지 않았으나, 엘라그산 배당체는 50uM 이하 농도에서 물에 녹는 것을 확인하였다.
The ellagic acid glycoside of the present invention was dissolved in water and looked. As a result, as shown in FIG. 7, the ellagic acid itself was not soluble in water at all regardless of the concentration, but it was confirmed that the ellagic acid glycoside was soluble in water at a concentration of 50 uM or less.
실시예Example 5. 5. 엘라그산Ellagsan 배당체의 뇌신경 세포 보호효과 Protective effect of glycosides on brain neurons
본 발명의 엘라그산 배당체의 글루타메이트에 의한 뇌신경 세포 손상에서의 뇌신경 세포 보호효과를 확인하였다. It was confirmed that the protective effect of cranial nerve cells on brain nerve cell damage by glutamate of the ellagic acid glycoside of the present invention.
그 결과, 도 8에 개시된 바와 같이 엘라그산 배당체는 동일 농도에서 엘라그산 대비 20~30% 높은 생존율을 나타냈다. 특히 엘라그산 배당체 처리 농도가 증가할 수록 엘라그산 대비 생존율이 현저하였다.
As a result, as disclosed in FIG. 8, the ellagic acid glycosides exhibited a survival rate of 20-30% higher than that of ellagic acid at the same concentration. In particular, as the concentration of ellagic acid glycoside treatment increased, the survival rate of ellagic acid was remarkable.
실시예Example 6. 6. 글루타메이트에On glutamate 의해 due to 증가된Increased 코티졸의Cortisolic 분비 억제효과 Secretion inhibitory effect
본 발명의 엘라그산 배당체의 글루타메이트에 의한 스트레스 상황에서 코티졸 분비 억제효과를 확인하였다. In the stress situation caused by glutamate of the ellagic acid glycoside of the present invention, the effect of inhibiting cortisol secretion was confirmed.
그 결과, 도 9에 개시된 바와 같이 엘라그산 배당체는 동일 농도에서 엘라그산 대비 코티졸 분비량을 감소시켰으며, 특히 엘라그산 배당체 처리 농도가 증가할 수록 엘라그산 대비 코티졸 분비량의 감소효과가 현저하였다.
As a result, as shown in FIG. 9, the ellagic acid glycosides decreased the amount of cortisol secretion compared to ellagic acid at the same concentration, and in particular, the effect of reducing the cortisol secretion amount relative to ellagic acid was remarkable as the ellagic acid glycoside treatment concentration increased.
실시예 7. 글루타메이트에Example 7. Glutamate 의해 due to 증가된Increased AChEAChE 활성 억제효과 Active inhibitory effect
뇌신경세포(SH-SY5Y)를 이용한 엘라그산 배당체의 퇴행성 뇌질환, 특히 치매 개선 효과는 글루타메이트 처리에 의해 증가된 뇌신경전달물질인 아세틸콜린(acetylcholin)을 분해하는 AChE 효소의 활성을 억제하는 정도로 측정하였다. The degenerative brain disease of ellagic acid glycosides using brain neurons (SH-SY5Y), particularly the dementia improvement effect, was measured to the extent of inhibiting the activity of the AChE enzyme that degrades acetylcholin, a neurotransmitter increased by glutamate treatment. .
그 결과, 도 10에 개시된 바와 같이 AChE 효소억제를 통해 치매와 같은 퇴행성 뇌질환을 치료하는 치료제로 알려진 타크린 대비 엘라그산 배당체는 100uM 농도에서 50% 정도의 AChE 억제효과를 나타냈으며, 특히 100uM 농도는 엘라그산 보다 엘라그산 배당체의 AChE 억제효과가 약 3배 높게 나타났다.As a result, as disclosed in FIG. 10, taculin-glycemic glycosides compared to tacrine, known as a therapeutic agent for treating degenerative brain diseases such as dementia through AChE enzyme inhibition, showed an AChE inhibitory effect of about 50% at a concentration of 100 uM, particularly 100 uM concentration. The AChE inhibitory effect of ellagic acid glycosides was about 3 times higher than that of ellagic acid.
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| Enzyme and Microbial Technology, Vol. 50, pp. 50-56 (2012.) * |
| Frontiers In Microbiology, Vol. 8, pp. 496 (1-12) (2017.03.23.) * |
| IOSR Journal of Pharmacy and Biological Sciences, Vol. 13, pp. 66-70 (2018.04.) * |
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