KR20190081753A - Pharmaceutical composition comprising ishige okamurae extracts for prevention and treatment of neurodegenerative disorders as an active ingredient - Google Patents
Pharmaceutical composition comprising ishige okamurae extracts for prevention and treatment of neurodegenerative disorders as an active ingredient Download PDFInfo
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- KR20190081753A KR20190081753A KR1020170184511A KR20170184511A KR20190081753A KR 20190081753 A KR20190081753 A KR 20190081753A KR 1020170184511 A KR1020170184511 A KR 1020170184511A KR 20170184511 A KR20170184511 A KR 20170184511A KR 20190081753 A KR20190081753 A KR 20190081753A
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Abstract
Description
본 발명은 패(Ishige okamurae) 추출물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 치료용, 및 인지기능 개선용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient, and a composition for improving cognitive function.
패(Ishige okamurae)는 갈조류로서 패과에 속한다. 중국, 일본, 태국 연안에 분포하며, 우리나라에서는 서해안, 남해안, 제주도 해안가의 바위에 붙어 자생한다. 패는 xanthopophyll, fucoxanthin 등을 함유하여 다양한 범위의 생산물에 있어서 alginate 및 항염증, 항산화제의 훌륭한 급원이 되며, 패의 폴리페놀 성분으로 알려져 있는 phlorotannin은 생리활성 물질로 항플라즈민 억제 효과 및 항산화 활성이 있다고 보고되었다. Ishige okamurae is a brown algae belonging to the lacunae. It is distributed in China, Japan, and Thailand coast, and it is attached to the rocks in the west coast, southern coast and Jeju island coast in Korea. Phlorotannin, which is known as a polyphenol component of plaquettes, is an excellent source of alginate, anti-inflammation, and antioxidants in a wide range of products containing xanthopophyll and fucoxanthin. Phlorotannin, a physiologically active substance, .
체내 산소대사의 과정에서 또는 자외선, 약물, 환경오염물질과 같은 여러 가지 외부 요인 및 그 외의 여러 가지 이유에 의해서 몸 안에 활성산소종(ROS)의 양이 크게 늘어날 경우 생명체는 산화적 스트레스를 받아 세포 사멸, 질병 발생 그리고 세포의 노화를 일으키게 된다. 즉, ROS의 생성과 세포의 항산화력 간의 균형이 산화적 스트레스를 결정하게 되고, 산화적 스트레스는 세포내 단백질, 지질 및 DNA에 손상을 입히게 되는 것이다. 이러한 활성산소에 의한 세포 혹은 세포내 물질의 산화적 손상은 알츠하이머 증후군, 파킨슨 증후군, 헌팅턴 증후군과 같은 중추 신경계의 퇴행성 뇌질환의 중요한 요인으로 알려져 있다.When the amount of reactive oxygen species (ROS) in the body is greatly increased in the process of oxygen metabolism in the body or by various external factors such as ultraviolet rays, drugs, environmental pollutants and various other reasons, Death, disease outbreaks and cell aging. That is, the balance between the production of ROS and the antioxidant capacity of cells determines oxidative stress, and oxidative stress damages intracellular proteins, lipids and DNA. Oxidative damage of cellular or intracellular substances by these reactive oxygen species is known to be an important factor in degenerative brain diseases of the central nervous system such as Alzheimer's syndrome, Parkinson's syndrome, and Huntington's syndrome.
세포사멸(apoptosis)은 세포 내에서 일어나는 프로그램 된 사멸 기전으로, 호르몬 또는 화학 물질과 같은 많은 세포사멸(apoptosis) 관련 인자들에 의해 조절되는데, 세포 괴사인 necrosis와는 구별되는 것으로, 암의 치료와도 밀접한 관계가 있기 때문에 다양한 암세포를 대상으로 한 세포사멸(apoptosis) 유도와 관련된 연구가 진행되고 있으며, 특징적으로 핵과 크로마틴의 응축으로 인한 세포의 수축, DNA의 단편화, 단백질의 절단 형성, apoptotic body 등과 같은 현상이 나타난다. 이러한 세포사멸(apoptosis)은 세포 내의 Bcl2 family, caspase의 활성화, poly (ADP-ribose) polymerase (PARP)의 조절에 의해 발생하게 된다. 최근에는 세포사멸(apoptosis)이 reactive oxygen species (ROS)와 많은 관련이 있다고 보고되고 있다. ROS는 세포 대사 과정에서 생성되고 즉시 불활성화되지만 산화 환원의 균형이 무너지게 되면, 세포손상을 가져오게 된다. 이러한 ROS는 스트레스가 없는 상황에서 세포 내 신호전달에서 주요한 역할을 하지만, ROS는 산화적 스트레스를 일으켜 DNA 손상, 노화와 세포사멸(apoptosis) 등을 일으키게 된다.Apoptosis is a programmed death mechanism that occurs in cells and is regulated by a number of apoptosis-related factors such as hormones or chemicals. It is distinct from necrosis, a cell necrosis, Because of its close relationship, studies on the induction of apoptosis in various cancer cells have been carried out. Characteristically, there are contraction of nucleus and chromatin due to condensation, DNA fragmentation, protein cleavage, apoptotic body The same phenomenon appears. This apoptosis is caused by the activation of Bcl2 family, caspase activation and poly (ADP-ribose) polymerase (PARP) in the cells. Recently, apoptosis has been reported to be associated with reactive oxygen species (ROS). ROS is produced in cell metabolism and is inactivated immediately, but when the balance of redox is broken, it causes cell damage. These ROSs play a major role in intracellular signaling in stress-free situations, but ROS causes oxidative stress, resulting in DNA damage, senescence and apoptosis.
본 발명의 목적은 패(Ishige okamurae) 추출물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing Ishige okamurae extract as an active ingredient.
상기 과제를 해결하기 위하여, 본 발명은 패(Ishige okamurae) 추출물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물을 제공한다. In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing and treating degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient.
패 추출물은 glutamate induced 된 HT22 세포를 Akt signaling 조절을 통해 Glutamate 증가에 따른 세포 내 ROS 농도를 감소시켜 cell death를 저해하는 것을 확인하였으며, Aβ peptide가 유도된 PC12 세포에 패 추출물을 처리하였을 때, COX2와 iNOS 같은 pro-imflammatory mediator의 발현이 억제되고, MAPKs signaling pathway 비활성화를 통해 inflammation에 대한 저해 효능을 나타내며, Aβ peptide 증가에 따른 세포 내 ROS 농도를 감소시켜 세포 독성을 감소시키는 것을 확인하였다.The extracts of glutamate-induced HT22 cells inhibited cell death by decreasing the intracellular ROS concentration by increasing glutamate through the control of Akt signaling. When the extract of PC12 cells treated with Aβ peptide, And iNOS, inhibited the inflammation by inactivating the MAPKs signaling pathway, and decreased the cytotoxicity by decreasing intracellular ROS concentration by increasing the Aβ peptide.
도 1은 세포사멸(apoptosis) 신호 경로를 나타내는 도면으로, 단백질 키니 아제 신호 전달 경로와 apoptotic cascade를 도시한다.
도 2는 HT-22 세포에서 glutamate 매개 신경 세포 독성에 대한 패 추출물의 가능한 저해 메커니즘을 도시한다.
도 3은 PC-12 세포에서 glutamate 매개 신경 세포 독성에 대한 패 추출물의 가능한 저해 메커니즘을 도시한다.
도 4는 HT22 세포의 생존율(viability)에 대한 패 추출물의 억제 효과를 나타내는 그래프이다. (a) 세포에 패 추출물을 24시간 동안 0.01 내지 0.7 mg/mL의 농도로 처리하였다. (b) 세포에 24시간 동안 1 mM 내지 10 mM의 상이한 농도의 글루타메이트를 처리하였다. 패 추출물에 대한 세포 생존 능력은 WST-1을 사용하여 평가되었다. 통계 분석은 일원 분산 분석 (One-way ANOVA)을 사용하고 적어도 세 번의 실험을 기반으로 수행되었다(*** : P <0.001).
도 5는 HT22 세포 생존 능력에 대한 패 추출물의 억제 효과를 나타내는 그래프로 패 추출물을 0.05, 0.1, 0.2 mg/mL 각각 처리하였다. 흡광도는 450nm에서 마이크로 플레이트 리더로 측정하였다(## p<0.01, ### p<0.001 versus glutamate alone; ** p<0.01, *** p<0.001 versus control).
도 6은 HT22 cell에 패 추출물 (0.05, 0.1 mg/mL)를 2시간 30분 전 처리하여 5 mM glutamate 14시간 배양한 후 광학 현미경으로 관찰한 사진이다. (a) 대조군; (b) 5 mM 글루타메이트; (c) 패 추출물 0.05 mg/mL 및 글루타메이트 5 mM 처리; (d) 패 추출물 0.1 mg/mL 및 글루타메이트 5 mM 처리.
도 7은 HT22 세포에서 글루타메이트 세포 사멸에 대한 패 추출물의 보호 효과를 나타낸 그래프이다. (a) 대조군; (b) 5 mM 글루타메이트; (c) 패 추출물 0.5 mg/mL 및 글루타메이트 5 mM; (d) 패 추출물 0.1 mg/mL 및 글루타메이트 5 mM.
도 8은 글루타메이트가 처리된 HT22 세포에서의 세포 내 ROS 형성에 대한 패 추출물의 억제 효과를 나타내는 그래프이다. DCF의 형광 강도는 530nm에서 excitation 485nm로 조사하여 측정하였다(*** p <0.001 versus control).
도 9는 글루타메이트가 처리된 HT22 세포에서의 p-Akt 및 Akt 단백질 발현에 대한 패 추출물의 효과를 나타내는 (a)웨스턴 블럿 사진 및 (b)p-Akt의 정량 결과를 나타낸 그래프이다(### p<0.001 versus glutamate alone; *** p<0.001 versus control).
도 10은 글루타메이트가 처리된 HT22 세포에서의 Bcl-2 및 Bax의 발현에 대한 패 추출물의 효과를 나타내는 (a)웨스턴 블럿 사진 및 (b) Bax/Bcl-2의 비(ratio)를 나타낸 그래프이다(### p<0.001 compared to control group; *** p<0.001 compared to glutamate group).
도 11은 글루타메이트가 처리된 HT22 세포에서의 active caspase-3의 발현에 대한 패 추출물의 효과를 나타내는 웨스턴 블럿 사진이다.
도 12는 Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때 HO-1, Nrf2를 mRNA level을 나타내는 그래프이다. HO-1, Nrf2의 mRNA level은 q-PCR을 사용하여 측정되었다(### p<0.001 versus glutamate alone; *** p<0.001 versus control).
도 13은 PC12 세포의 생존율(viability)에 대한 패 추출물의 억제 효과를 나타내는 그래프이다. 통계 분석은 일원 분산 분석 (One-way ANOVA)을 사용하고 적어도 세번의 실험을 기반으로 수행되었다(*** : P <0.001).
도 14는 Aβ peptide가 유도된 PC12 세포에서의 패 추출물의 독성보호 효과를 나타내는 그래프이다. 흡광도는 파장 450nm에서 마이크로 플레이트 리더로 측정하였다(## p<0.01, ### p<0.001 versus glutamate alone; *** p<0.001 versus control).
도 15는 Aβ peptide가 유도된 PC12 cell의 패 추출물의 독성보호효과 및 세포 형태를 나타내는 광학 현미경 사진이다. (a) 대조군; (b) 70 μM aβ 펩티드 처리; (c) 70 μM aβ 펩티드 및 패 추출물 0.125 ㎎/㎖.
도 16은 Aβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때의 ROS 저해 효과를 나타내는 그래프이다. DCF의 형광 강도는 530nm에서 excitation 485nm로 조사하여 측정하였다(*** p <0.001 versus control).
도 17은 Aβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때 MAPKs pathway에 미치는 효과를 나타낸 웨스턴 블럿 결과이다.
도 18은 Aβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때 mRNA level에서 iNOS와 COX2에 미치는 효과를 나타낸 그래프이다. iNOS와 COX2의 mRNA level은 q-PCR을 사용하여 측정되었다(### p<0.001 versus glutamate alone; *** p<0.001 versus control).Figure 1 is a diagram showing the apoptosis signal pathway, showing the protein kinase signaling pathway and the apoptotic cascade.
Figure 2 depicts possible inhibition mechanisms of cardiac extracts on glutamate mediated neurotoxicity in HT-22 cells.
Figure 3 shows the possible inhibition mechanism of plaque extract on glutamate mediated neurotoxicity in PC-12 cells.
FIG. 4 is a graph showing the inhibitory effect of the extract of Lactobacillus on the viability of HT22 cells. (a) Cells were treated with L. extract at a concentration of 0.01 to 0.7 mg / mL for 24 hours. (b) Cells were treated with different concentrations of glutamate ranging from 1 mM to 10 mM for 24 hours. Cell viability for L. extract was evaluated using WST-1. Statistical analysis was performed using at least three experiments using one-way ANOVA (***: P <0.001).
FIG. 5 is a graph showing the inhibitory effect of the extract of Lactobacillus on HT22 cell viability. Lactobacillus extracts were treated at 0.05, 0.1, and 0.2 mg / mL, respectively. Absorbance was measured with a microplate reader at 450 nm (## p <0.01, ### p <0.001 versus glutamate alone; ** p <0.01, *** p <0.001 versus control).
FIG. 6 is a photograph of HT22 cell treated with L-eluate (0.05, 0.1 mg / mL) for 2
FIG. 7 is a graph showing the protective effect of Lactobacillus root extract on glutamate cell death in HT22 cells. (a) control group; (b) 5 mM glutamate; (c) Lactobacillus extract 0.5 mg / mL and
FIG. 8 is a graph showing the inhibitory effect of Lactobacillus root extract on intracellular ROS formation in glutamate-treated HT22 cells. The fluorescence intensity of DCF was measured by irradiating with excitation at 485 nm at 530 nm (*** p < 0.001 versus control).
9 is a graph showing (a) Western blot photograph and (b) p-Akt quantification results showing the effect of a roe extract on p-Akt and Akt protein expression in glutamate-treated HT22 cells p < 0.001 versus glutamate alone; *** p < 0.001 versus control).
10 is a graph showing the ratio of (a) western blot photograph and (b) Bax / Bcl-2 showing the effect of rosacea extract on the expression of Bcl-2 and Bax in glutamate-treated HT22 cells (*** p <0.001 compared to control group; *** p <0.001 compared to glutamate group).
Figure 11 is a western blot photograph showing the effect of Lactobacillus extract on the expression of active caspase-3 in glutamate-treated HT22 cells.
FIG. 12 is a graph showing mRNA levels of HO-1 and Nrf2 when glutamate-induced HT22 cells were treated with L. extract. MRNA levels of HO-1 and Nrf2 were measured using q-PCR (### p <0.001 versus glutamate alone; *** p <0.001 versus control).
FIG. 13 is a graph showing the inhibitory effect of Lactobacillus root extract on the viability of PC12 cells. Statistical analysis was performed using at least three experiments using one-way ANOVA (***: P <0.001).
FIG. 14 is a graph showing the toxic protective effect of the Lactobacillus extract on PC12 cells induced by Aβ peptide. FIG. Absorbance was measured with a microplate reader at a wavelength of 450 nm (## p <0.01, ### p <0.001 versus glutamate alone; *** p <0.001 versus control).
FIG. 15 is an optical microscope photograph showing the cytotoxic effect and cytotoxic effect of A? Peptide-induced PC extract on the cell extract. FIG. (a) control group; (b) 70 [mu] M a [beta] peptide treatment; (c) 70 [mu] M a [beta] peptide and L. extract 0.125 mg / ml.
FIG. 16 is a graph showing the ROS inhibitory effect when A? Peptide-induced PC12 cell was treated with L. extract. The fluorescence intensity of DCF was measured by irradiating with excitation at 485 nm at 530 nm (*** p < 0.001 versus control).
FIG. 17 shows Western blot results showing the effects of Aβ peptide-induced PC12 cells on the MAPKs pathway when treated with L. extract.
FIG. 18 is a graph showing the effect of the extract of Aβ peptide-induced PC12 cells on the iNOS and COX2 levels at the mRNA level. mRNA levels of iNOS and COX2 were measured using q-PCR (### p <0.001 versus glutamate alone; *** p <0.001 versus control).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 패(Ishige okamurae) 추출물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing an extract of Ishige okamurae as an active ingredient.
상기 추출물은 상기 추출물은 C1 내지 C2의 저급 알코올 또는 이들의 혼합물인 용매로 추출한 후, 건조하여 1,3-butylene glycol에 녹인 것일 수 있으나, 이에 한정하지 않는다The extract may be extracted with a solvent which is a lower alcohol of C1 to C2 or a mixture thereof, and then dried to be dissolved in 1,3-butylene glycol, but not limited thereto
상기 저급 알코올은 메탄올 또는 에탄올일 수 있으나, 이에 한정하지 않는다.The lower alcohol may be, but is not limited to, methanol or ethanol.
상기 패 추출물의 추출 방법으로는 여과법, 열수추출, 침지추출, 환류냉각추출 및 초음파추출 등 당업계의 통상적인 방법을 이용할 수 있으며, 열수추출 방법으로 1회 내지 5회 추출하는 것일 수 있고, 보다 구체적으로 3회 반복 추출하는 것일 수 있으나 이에 한정하지 않는다. As the method of extracting the Linalool extract, a conventional method in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction may be used, and extraction may be performed once to five times by hot water extraction method. Specifically, it may be repeated three times, but is not limited thereto.
상기 추출용매는 패에 0.1 내지 10배 첨가할 수 있으며, 0.3 내지 5배 첨가하는 것이 바람직하다. The extraction solvent may be added in an amount of 0.1 to 10 times, preferably 0.3 to 5 times.
추출온도는 20 내지 40인 것일 수 있으나 이에 한정하지 않는다. 또한, 추출시간은 12 내지 48시간인 것일 수 있으나 이에 한정하지 않는다.The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.
상기 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이며, 바람직하게는 동결건조하는 것이나 이에 한정하지 않는다.The drying may be carried out under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, preferably freeze drying.
상기 패 추출물은 AKT를 인산화시켜 글루타메이트 증가에 따른 세포 내 ROS 농도를 감소시키는 것일 수 있으나, 이에 한정하지 않는다. The Lactobacillus root extract may be, but is not limited to, phosphorylating AKT to reduce intracellular ROS concentration with increasing glutamate.
상기 패 추출물은 염증 전 매개체(pro-imflammatory mediator)의 발현을 억제하여 Aβ peptide 증가에 따른 세포 내 ROS 농도를 감소시키는 것일 수 있으나, 이에 한정하지 않는다.The Lactobacillus root extract may inhibit the expression of pro-imflammatory mediator to reduce the intracellular ROS concentration upon increase of A [beta] peptide, but is not limited thereto.
본 발명의 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The compositions of the present invention may be of various oral or parenteral formulations. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다.Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances or preservatives are included .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제 또는 좌제 등이 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations or suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.
본 발명의 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여시 피부외용 또는 복강내, 직장, 정맥,근육, 피하, 자궁내 경막 또는 뇌혈관내 주사 방식을 선택하는 것이 바람직하며, 가장 바람직하게는 피부외용으로 사용한다.The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods for parenteral administration, It is used for external skin.
본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다. The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하며, 일일 투여량은 패 추출물의 양을 기준으로 0.01 내지 1000 ㎎/㎏이고, 바람직하게는 30 내지 500 ㎎/㎏이고, 더욱 바람직하게는 50 내지 300 ㎎/㎏이며, 하루 1 ~ 6 회 투여될 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, The dose is 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and can be administered 1 to 6 times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.
본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
본 발명은 패(Ishige okamurae) 추출물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 및 개선용 건강기능식품을 제공한다.The present invention provides a health functional food for prevention and improvement of degenerative brain diseases containing Ishige okamurae extract as an active ingredient.
상기 식품의 종류에는 특별한 제한은 없다. 상기 추출물을 첨가할 수 있는 식품의 예로는 드링크제, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of the foods to which the extract can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.
본 발명의 패 추출물 또는 이의 분획물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강식품 중의 상기 화합물의 양은 전체 식품 중량의 0.1 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The Lactobacillus extract or fraction thereof of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 화합물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스 파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.
상기 외에 본 발명의 패 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 패 추출물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 패 추출물은 100 중량부 당 0.1 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the Lard extract of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the Lard extract of the present invention can contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of these additives is not so important, it is general that the roots extract of the present invention is selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight.
이하, 실시예 및 실험예를 통해 본 발명을 보다 구체적으로 설명한다. 그러나 이들 예는 본 발명의 이해를 돕기 위한 것일 뿐 어떠한 의미로든 본 발명의 범위가 이들 예로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.
시약 reagent 및 기기And devices
Dulbecco’s modified Eagle’s minimal medium (DMEM), and Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal bovine serum (FBS, corning, USA) and penicillin/streptomycin은 Hyclone에서 구입하였고, FBS는 Corning, HS, PBS, 0.25% Trypsin-EDTA은 Gibco에서 구매하였다. WST-1은 cayman chemical에서 구매하였다. N-Acrtyl-L-cysteine, glutamic acid, 2',7'-Dichlorofluorescin은 Sigma-Aldrich, amyloid β peptide(25-35)는 abcam에서 구매하였으며, TRIzol® Reagent은 ambion을 구매하였다. SYBR® Green Realtime PCR Master Mix, reverTra Ace® qPCR RT Master Mix with gDNA Remover는 TOYOBO에서 구매하였다. Acrylamide solution은 Tech & Innovation에서 구입하였으며, ECL solution은 BIO-RAD에서 구매하였다. (FBS, corning, USA) and penicillin / streptomycin were purchased from Hyclone, FBS was purchased from Corning, HS, PBS, 0.25% Trypsin-EDTA was purchased from Gibco. WST-1 was purchased from cayman chemical. N-Acrtyl-L-cysteine, glutamic acid, 2 ', 7'-Dichlorofluorescin purchased from Sigma-Aldrich, amyloid β peptide (25-35) purchased from abcam and TRIzol ® Reagent purchased ambion. SYBR ® Green Realtime PCR Master Mix, reverTra Ace ® qPCR RT Master Mix with gDNA Remover purchased from TOYOBO. Acrylamide solution was purchased from Tech & Innovation, and ECL solution was purchased from BIO-RAD.
실험에 사용한 모든 Antibody들과 inhibitor들은 아래의 [표 1]에 정리하였다. 본 발명에서 사용된 기기는 ELISA microplate reader (iMark Microplate Reader, Bio-Rad laboratories, Inc.), Real-Time PCR Detection Systems(iMark Microplate Reader, Bio-Rad laboratories, Inc.), Flow cytometry (Beckman Coulter), Nicon Eclipse Ti-S mieroscope (Nikon), Automatic X-RAY filmprocessor (JPI) 등을 사용하였다.All antibiotics and inhibitors used in the experiments are summarized in Table 1 below. The instrument used in the present invention was an ELISA microplate reader (iMark Microplate Reader, Bio-Rad Laboratories, Inc.), Real-Time PCR Detection Systems (iMark Microplate Reader, Bio- Rad Laboratories, Inc.), Flow cytometry (Beckman Coulter) , Nicon Eclipse Ti-S mieroscope (Nikon), and Automatic X-ray film processor (JPI).
실험방법Experimental Method
<Cell culture><Cell culture>
mouse 해마 유래 세포주인 HT22 세포는 한국 세포주은행에서 분양받았으며, DMEM 배지에 100 unit/ml penicillin, 100μg/ml streptomycin 및 10% fatal bovine serum을 첨가하여 사용하였으며, 5% CO2가 포함된 37℃의 배양기 내에서 배양하였다. Rat 크롬친화성 세포주인 PC12 세포는 한국 세포주은행에서 분양받았으며, RPMI 배지에 100 unit/ml penicillin, 100μg/ml streptomycin 및 5% fatal bovine serum, 10% Horse serum을 첨가하여 사용하였으며, 5% CO2가 포함된 37℃의 배양기 내에서 배양하였다. mouse hippocampus-derived cell line, HT22 cells were used to received pre-sale in Korea Cell Line Bank, a 100 unit / ml penicillin, 100μg / ml streptomycin and 10% fatal bovine serum was added in DMEM medium, 5% CO 2 in a 37 ℃ contain And cultured in an incubator. Rat chromium affinity cell line, PC12 cells were used to received pre-sale in Korea Cell Line Bank, it was added to 100 unit / ml penicillin, 100μg / ml streptomycin and 5% fatal bovine serum, 10% Horse serum in RPMI medium, 5% CO 2 Lt; RTI ID = 0.0 > 37 C. < / RTI >
<Cell viability><Cell viability>
HT22 세포를 5×103 cell/well씩 96 well plate에 분주하고, 24시간 배양 후에 serum이 들어있지 않은 배지에 패 추출물을 농도별로 2시간 전 처리하여 다시 18시간 배양하였다. WST-1(Tetrazolium Salts) 10 μl를 각각 첨가하고 2시간 배양하였다. PC12 세포를 1×104 cell/well씩 96 well plate에 분주하고, 24시간 배양 후에 serum이 들어있지 않은 배지에 패 추출물을 농도별로 2시간 전 처리하여 다시 18시간 배양하였다. WST-1 10 μl를 각각 첨가하고 4시간 배양하였다.HT22 cells were plated at a density of 5 × 10 3 cells / well in a 96-well plate. After culturing for 24 hours, the cell extract-free extracts were treated for 2 hours before culturing for 18 hours. 10 μl of WST-1 (Tetrazolium Salts) was added and incubated for 2 hours. PC12 cells were plated at a density of 1 × 10 4 cells / well in a 96-well plate, cultured for 24 hours, and cultured for 2 hours before the treatment for 18 hours. 10 μl of WST-1 was added and incubated for 4 hours.
<< ApoptosisApoptosis assay> assay>
HT22, PC12 세포는 5×106 cell을 10cm cell culture dish에 분주하고 24시간 동안 안정화 시킨 후, serum free배지로 O/N한 다음, 패 추출물을 농도별로 처리하여 24시간 배양하였다. 1×106 cells을 모으고 PBS로 두 번 washing 해준 후, annexin V/PI 시약과 1× binding buffer를 사용하여 암실에서 20분 간 염색 시킨 후, 염색된 세포는 유동세포계수법(flow cytometry)을 이용하여 분석하였다.HT22 and PC12 cells were plated at 5 × 10 6 cells in a 10 cm cell culture dish and stabilized for 24 hours. O / N was performed with serum-free medium, and then L-lysine extract was treated for 24 hours. 1 × 10 6 cells were collected, washed twice with PBS, stained with annexin V / PI reagent and 1 × binding buffer for 20 min in the dark room, and the stained cells were analyzed by flow cytometry Respectively.
<Measurement of <Measurement of ROSROS >>
HT22, PC12 세포는 5×105 cells/well을 6 well에 분주하고 24시간 동안 안정화시킨 후, serum free 배지로 O/N한 다음, 패 추출물을 농도별로 처리하여 24시간 배양하였다. 세포를 분리하여 PBS로 세척한 뒤, 10 μM의 DCFH-DA(dichlorodihydrofluorescein diacetate)를 처리하여 37℃에서 30분간 염색 후 flow cytometry를 사용하여 측정하였다.HT22 and PC12 cells were seeded at 5 × 10 5 cells / well in 6 wells and stabilized for 24 hours. Then, the cells were treated with O / N with serum free medium and cultured for 24 hours. Cells were separated, washed with PBS, and treated with 10 μM DCFH-DA (dichlorodihydrofluorescein diacetate), stained at 37 ° C for 30 min, and then measured using flow cytometry.
<Quantitative real-time <Quantitative real-time PCRPCR >>
HT22, PC12 세포는 5×105 cell을 6 well에 분주하고 24시간 동안 안정화 시킨 후, serum free 배지로 O/N한 다음, 패 추출물을 농도별로 처리하여 24시간 배양하였다. Trizol을 이용하여 RNA를 추출하여, cDNA를 만들어 SYBR Kit로 RT-PCR을 하였다. Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)를 internal standard로 사용하였다.HT22 and PC12 cells were plated in 6 wells at 5 × 10 5 cells and stabilized for 24 hours. Then, O / N was added to serum-free medium, followed by culturing for 24 hours. RNA was extracted using Trizol, cDNA was prepared, and RT-PCR was performed with SYBR kit. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
<Western blotting><Western blotting>
HT22, PC12 세포는 2×105 cells/well을 6 well에 분주하고 24시간 동안 안정화 시킨 후, serum free 배지로 O/N한 다음, 패 추출물을 농도별로 처리하여 24시간 배양하였다. 세포를 분리하여 PBS로 세척한 뒤, 이후 20 mM Tris-HCl, PH7.4, 150 mM NaCl, 5 mM EDTA, 1%(W/V) IGEPAL® CA-630, 5 mM sodium pyrophoahate, 10mM β-glycerophosphate, 1mM phenylmehylsulfonyl fluoride와 protease inhibitor mixture로 구성된 lysis buffer를 처리하여 세포를 분해하였다. 동등한 양의 단백질을 10%의 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)를 이용하여 분리하여, polyvinylidene difluoride membrane으로 transfer하였다. Transfer된 membrane (PVDF)은 5% skim milk를 포함한 20 mM Tris-HCl buffer, pH7.4, 136 mM NaCl, 0.1% Tween 20의 TBS-T buffer로 1시간 blocking 해주었다. 그 후 primary antibody를 4℃에서 overnight 한 뒤 TBS-T buffer로 3회 세척 후, secondary antibody를 실온에서 40분 처리하여 주었다. 다시 TBS-T buuffer로 3회 세척한 후, ECL 시약을 처리하여 Autometic X-RAY film processor를 이용하여 확인하였다. 확인된 protein band는 Image J를 통하여 측정하였다.HT22 and PC12 cells were seeded at 6 × 10 5 cells / well in 6 wells and stabilized for 24 hours. Then, O / N was added to serum-free medium, Cells were separated and washed with PBS. The cells were then washed with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (w / v) IGEPAL ® CA- 630, 5 mM sodium pyrophoahate, glycerophosphate, 1 mM phenylmehylsulfonyl fluoride and a protease inhibitor mixture. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 10% and transferred to a polyvinylidene difluoride membrane. The transferred membrane (PVDF) was blocked with TBS-T buffer (20 mM Tris-HCl buffer, pH 7.4, 136 mM NaCl, 0.1% Tween 20) containing 5% skim milk for 1 hour. The primary antibody was then incubated at 4 ° C, washed three times with TBS-T buffer, and the secondary antibody was treated at room temperature for 40 minutes. After washing three times with TBS-T buuffer, ECL reagent was treated and examined using an Autometic X-ray film processor. Identified protein bands were measured using Image J.
실시예Example : 패 추출물의 제조: Preparation of Lard Extracts
본 실험에서 사용한 패는 제주도 해안에서 재취하여, 세척하고 세절하고 80% 메탄올로 3회 추출하여 여과지 감압 농축을 한 후 동결건조 과정을 거친 후 분말화 하여 -80℃ deep freezer에 보관하였다. 분말로 만든 약재는 85% 1,3-Butylene glycol에 녹인 후, 용액을 원심분리하여, 상층액만 옮겨 사용하였다.The plaques used in this experiment were collected from the shore of Jeju Island, washed, cut into three pieces with 80% methanol, concentrated under reduced pressure, and lyophilized, powdered and stored at -80 ℃ deep freezer. The powdered medicinal material was dissolved in 85% 1,3-butylene glycol, and the solution was centrifuged and only the supernatant was transferred.
1. HT22 cell을 이용한 패 추출물의 glutamate 매개 세포 1. Glutamate-mediated cell of Lactobacillus sp. Extract using HT22 cells 독성에 대한 신경보호작용Neuroprotection against toxicity
실험예Experimental Example 1-1. 패 추출물 처리에 따른 세포 1-1. Cells treated with L. extract 생존률Survival rate (Cell Viability) 변화(Cell Viability) change
먼저 HT22 세포의 cell viability rate를 확인하기 위하여 패 추출물의 농도를 달리하여 WST-1(Tetrazolium Salts) assay를 수행하였다. 패 추출물 농도에 의존적으로 Cell viability가 줄어드는 것을 확인하였고, 대조군과 비교했을 때 HT22 세포에서는 0.5 mg/mL 이상의 농도에서 세포 독성이 나타나며, glutamate를 처리한 경우에도 농도에 의존적으로 cell viability가 줄어드는 것이 확인되었고 대조군과 비교했을 때 1 mM 에서부터 세포독성이 나타남을 확인하였다(도 4).To determine the cell viability rate of HT22 cells, WST-1 (Tetrazolium Salts) assay was performed at different concentrations of Lactobacillus extract. Cell viability was decreased depending on the cell extract concentration, and cell toxicity was observed at a concentration of 0.5 mg / mL or more in HT22 cells compared with the control group, and cell viability was decreased in a concentration-dependent manner even when glutamate was treated And cytotoxicity was observed from 1 mM when compared to the control (Fig. 4).
실험예Experimental Example 1-2. glutamate가 유도된 HT22 cell의 패 추출물의 독성보호 효과 1-2. Toxicity of glutamate-induced HT22 cell line extract
glutamate가 유도된 apoptotic cell death에 대한 패 추출물의 효과를 확인하기 위하여 WST-1을 실행하였다. HT22 세포에 패 추출물(0.05-0.1 mg/mL)을 2시간 30분 전처리를 하고 5 mM glutamate를 14시간 처리하였다. 대조군을 기준으로 glutamate 처리한 cell viability는 약 80% 정도 감소 되었다. 그러나 패 추출물(0.05-0.2 mg/mL) 처리된 cell의 viability가 대조군과 비슷한 비율로 증가 되었다. 패 추출물은 HT22 세포에서 glutamate가 유도되어 독성이 생기는 것을 방지해주며, glutamate가 유도되어 세포가 죽는 것을 보호해주는 효과가 있는 것으로 보인다(도 5).WST-1 was performed to determine the effect of Lactobacillus extract on glutamate-induced apoptotic cell death. HT22 cells were pretreated for 2
실험예Experimental Example 1-3. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때의 세포생존율 확인 1-3. Cell survival rate of Glutamate-induced HT22 cells treated with L. extract
HT22 세포에 패 추출물(0.05, 0.1 mg/mL)을 2시간 30분 전 처리하여 5 mM glutamate 14시간 배양한 후 광학 현미경으로 관찰한 결과, 농도가 높아질수록 세포가 살아나면서 패 추출물이 glutamate 유도된 세포에 보호효과가 있는 것으로 보인다(도 6).HT22 cells were pretreated with 0.05% (0.1 mg / mL) for 2
실험예Experimental Example 1-4. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때의 세포생존율 확인 1-4. Cell survival rate of Glutamate-induced HT22 cells treated with L. extract
HT22 세포에 glutamate를 유도하면 세포사멸(apoptosis)이 일어나게 되며, glutamate 유도된 HT22 세포에 패 추출물이 처리되면 세포사멸(apoptosis) 세포가 줄어들게 된다. Annexin V와 PI의 염색을 이용하여 조사하였다. HT22 세포에 패 추출물(0.05, 0.1 mg/mL)을 전 처리 2시간 30분을 하고 5mM glutamate 14시간 배양한 후, 10 μL의 annexin V-FITC, 5 μL의 PI로 15분간 암반응시켰다. 이후 flow cytometer로 분석하였다.When glutamate is induced in HT22 cells, apoptosis is induced. When glutamate-induced HT22 cells are treated with LAH extract, apoptosis cells are reduced. Annexin V and PI staining. HT22 cells were pretreated for 2
실험결과 패 추출물 농도에 의존적으로 annexin V의 염색량이 감소하였으며 cell death은 대조군은 9.82%였고, glutamate는 34.43%로 증가하는 것을 확인할 수 있었고 패 추출물(0.05, 0.1 mg/mL)은 24.96%, 18.55%로 감소 되었다. 실험결과는 패 추출물로 인해 glutamate 유도된 세포가 독성으로부터 보호 효과를 받는다는 것을 보여준다(도 7).The results showed that the amount of annexin V staining decreased with cell extract concentration, cell death was 9.82%, glutamate increased to 34.43%, cell extract was increased to 24.96%, 18.55% %. Experimental results show that glutamate-induced cells are protected from toxicity due to Lactobacillus extract (Fig. 7).
실험예Experimental Example 1-5. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때의 1-5. When glutamate-induced HT22 cells were treated with Lactobacillus root extract ROSROS 저해 효과 Inhibitory effect
패 추출물을 처리하여 HT22 세포 내 산화적 스트레스 생성량에 대한 저해활성을 DCF-DA assay를 이용하여 측정하였으며 그 결과는 아래와 같다. glutamate로 유도되는 세포 손상 요인 중 산화적 스트레스가 중요한 요소로 작용한다. HT22 세포 내 생성된 산화적 스트레스 생성량은 glutamate만을 처리한 처리 군에서는 대조군 100% 대비 168%의 산화적 스트레스 생성량을 나타냈고 양성군(positive control)인 glutamate와 패 추출물 (0.1 mg/mL)를 동시에 처리한 처리 군에서는 120%로 대조군과 유사한 수준으로 산화적 스트레스 생성량이 감소하는 것으로 나타났다(도 8).The inhibitory activity against the production of oxidative stress in HT22 cells was measured by DCF-DA assay. The results are as follows. Oxidative stress plays an important role in glutamate - induced cell damage. The amount of oxidative stress produced in the HT22 cells was 168% of the oxidative stress in the glutamate-treated group compared to the control group, and the positive control glutamate and L-eluate (0.1 mg / mL) In the treated group, the amount of oxidative stress was decreased to 120%, which was similar to that of the control group (FIG. 8).
실험예Experimental Example 1-6. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때 1-6. When glutamate-induced HT22 cells were treated with L. extract P13KP13K // AktAkt pathway에 미치는 효과 Effect on pathway
glutamate가 유도된 HT22 cell death를 패 추출물이 처리되었을 때 보호효과를 P13K/Akt activation를 통해 확인되었다. 5 mM glutamate 처리한 부분에서 Akt가 phosphorylation이 줄어들었다. 패 추출물 전처리한 부분이 Akt phosphorylation이 증가되었다(도 9). Glutamate - induced HT22 cell death was confirmed by P13K / Akt activation when the extract was treated. The phosphorylation of Akt was reduced in the part treated with 5 mM glutamate. The Akt phosphorylation was increased in the pretreated portion of the extract (Fig. 9).
실험예Experimental Example 1-7. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때 1-7. When glutamate-induced HT22 cells were treated with L. extract BclBcl -2/-2/ Bax의Bax ratio ratio
Bax, Bcl-2의 protein level에서 확인하기 위해 western blotting을 하였다. HT22 세포에 5 mM glutamate를 14h 처리했을 때, pro-apoptotic인 Bax에선 증가하고 anti-apoptotic Bcl-2에선 감소하였다. 그러나 패 추출물과 glutamate를 함께 처리하였을 때, 농도의존적으로 대조군과 비슷해지고 있다. Bax/Bcl-2의 ratio는 glutamate만 처리한 것보다 glutamate와 패 추출물을 같이 처리한 군에서 더 감소하는 것을 확인하였다(도 10).Bax and Bcl-2 protein levels. When HT23 cells were treated with 5 mM glutamate for 14 h, they increased in pro-apoptotic Bax and decreased in anti-apoptotic Bcl-2. However, when Lactobacillus extract and glutamate were treated together, they were similar to the control group in a concentration-dependent manner. The ratio of Bax / Bcl-2 was further reduced in the group treated with glutamate and L-glutamate than in glutamate alone (Fig. 10).
실험예Experimental Example 1-8. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때 1-8. When glutamate-induced HT22 cells were treated with L. extract caspasecaspase -3 활성화 효과-3 activation effect
Caspase-3은 활성화는 세포사멸(apoptosis)에 중요한 역할을 한다. western blotting을 통해 caspase-3활성을 보았다. glutamate 유도된 cell은 패 추출물이 함께 처리되면 cleaved caspase-3 단백질 발현이 감소된다(도 11).Caspase-3 activation plays an important role in apoptosis. Western blotting revealed caspase-3 activity. glutamate-induced cells are reduced when cleaved caspase-3 protein expression is reduced (Fig. 11).
실험예Experimental Example 1-9. Glutamate 유도된 HT22 cell에 패 추출물을 처리하였을 때 HO-1, 1-9. When glutamate-induced HT22 cells were treated with L. extract, HO-1, Nrf2를Nrf2 mRNAmRNA level에서의 효과 Effect at level
HT22 세포에 패 추출물(0, 0.05, 0.1 mg/mL)을 2시간 30분 전 처리하여 5 mM glutamate 14시간 배양하였다. 패 추출물을 처리한 군이 glutamate만 처리한 군보다 HO-1, Nrf2 mRNA level이 증가하는 것을 확인하였다(도 12).HT22 cells were pretreated for 2
2. PC12 cell을 이용한 패 추출물의 2. Cell extracts of PC12 cells AβAβ peptide 매개 세포 peptide-mediated cell 독성에 대한 신경보호 작용Neuroprotection against toxicity
실험예Experimental Example 2-1. 패 추출물 처리에 따른 Cell viability 변화 2-1. Changes in Cell viability by Treatment with Lard Extracts
먼저, PC12 세포의 Cell viability rate를 확인하기 위하여 패 추출물의 농도를 달리하여 WST-1을 수행하였다. 패 추출물 농도에 의존적으로 cell viability가 줄어드는 것을 확인하였고, 대조군과 비교했을 때 PC12 세포에서는 0.15 mg/mL 이상의 농도에서 세포 독성이 나타나며, Aβ peptide에서도 농도에 의존적으로 cell viability가 줄어드는 것이 확인되었고, 대조군과 비교했을 때 20 μM에서부터 세포독성이 나타남을 확인하였다(도 13).First, to determine the cell viability rate of PC12 cells, WST-1 was performed at different concentrations of Lactobacillus extract. The cell viability of PC12 cells was found to be more than 0.15 mg / mL and the cell viability was decreased in Aβ peptide depending on the concentration as compared with the control group. And cytotoxicity was observed from 20 [mu] M (Fig. 13).
실험예Experimental Example 2-2. 2-2. AβAβ peptide가 유도된 PC12 cell의 패 추출물의 독성보호 효과 Toxicity of Peptide-Derived PC12 Cells
Aβ peptide가 유도된 apoptotic cell death에 대한 패 추출물의 효과를 확인하기 위하여 WST-1을 실행하였다. PC12 세포에 패 추출물(0.05-0.125 mg/mL)을 2시간 30분 전처리를 하고 70 μM Aβ peptide를 14시간 처리하였다. 대조군을 기준으로 Aβ peptide를 처리한 cell viability는 약 80% 정도 감소하였다. 그러나 패 추출물(0.05-0.125 mg/mL)이 처리된 세포의 viability가 control group과 비슷한 비율로 증가하였다. 패 추출물은 PC12 세포에서 Aβ peptide가 유도되어 독성이 생기는 것을 방지해주며, Aβ peptide가 유도되어 죽는 것을 보호해주는 효과가 있는 것으로 보여진다(도 14).WST-1 was performed to confirm the effect of extracts on apoptotic cell death induced by Aβ peptide. PC12 cells were pretreated with L. extract (0.05-0.125 mg / mL) for 2
실험예Experimental Example 2-3. 2-3. AβAβ peptide가 유도된 PC12 cell의 패 추출물의 독성보호효과 및 세포 형태 확인 Toxicity and cell morphology of peptide extract induced PC12 cell
PC12 세포에 패 추출물 (0.125 mg/mL)를 2시간 30분 전 처리하여 70 μM Aβ peptide 14시간 배양한 후 광학 현미경으로 관찰한 결과, 농도 높아질수록 세포가 살아나면서 패 추출물의 처리가 Aβ peptide 유도된 세포에 보호효과가 있는 것으로 보여진다(도 15).PC12 cells were treated with L. extract (0.125 mg / mL) for 2
실험예Experimental Example 2-4. 2-4. AβAβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때의 peptide-induced PC12 cells were treated with Lactobacillus casei extract ROSROS 저해 효과 Inhibitory effect
패 추출물을 처리하여 PC12 세포 내 산화적 스트레스 생성량에 대한 저해활성을 DCF-DA assay를 이용하여 측정하였으며 그 결과는 아래와 같다. Aβ peptide에 의해 유도되는 손상 요인 중 산화적 스트레스가 중요한 요소로 작용한다. PC12 세포 내 생성된 산화적 스트레스 생성량은 Aβ peptide만을 처리한 처리 군에서 대조군 100% 대비 250%의 산화적 스트레스 생성량을 나타냈고 양성군(positive control)인 glutamate와 패 추출물(0.125 mg/mL)을 동시에 처리한 처리 군에서는 117%로 대조군과 유사한 수준으로 산화적 스트레스 생성량이 감소하는 것으로 나타났다(도 16). The inhibitory activity against oxidative stress production in PC12 cells was measured by DCF-DA assay. The results were as follows. Oxidative stress plays an important role among Aβ peptide - induced damage factors. The amount of oxidative stress produced in the PC12 cells was 250% of the oxidative stress produced in the Aβ peptide-treated group compared with the control group (100%), and the positive control glutamate and L-eluate (0.125 mg / mL) In the treated group, the amount of oxidative stress was decreased to 117%, similar to that of the control group (FIG. 16).
실험예Experimental Example 2-5. 2-5. AβAβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때 When peptide extracts were treated with peptide-induced PC12 cells MAPKsMAPKs pathway에 미치는 효과 Effect on pathway
Aβ peptide가 유도된 PC12 cell death를 패 추출물이 처리되었을 때 보호효과를 MAPKs phosphorylation이 감소함이 확인되었다. 70 μM Aβ peptide 처리한 부분에서 MAPKs가 phosphorylation이 증가하였다. 패 추출물을 처리한 군에서 MAPKs phosphorylation이 감소되었다(도 17). Aβ peptide - induced PC12 cell death was found to be a protective effect and a decrease in MAPK phosphorylation when the extract was treated. The phosphorylation of MAPKs was increased in the area treated with 70 μM Aβ peptide. MAPKs phosphorylation was reduced in the group treated with Lactobacillus root extract (Fig. 17).
실험예Experimental Example 2-6. 2-6. AβAβ peptide 유도된 PC12 cell에 패 추출물을 처리하였을 때 When peptide extracts were treated with peptide-induced PC12 cells mRNAmRNA level에서 at level iNOS와iNOS and COX2에On COX2 미치는 효과 Effect
PC12 cell에 패 추출물 (0.075, 0.125 mg/mL)를 2시간 30분 전 처리하여 70 μM Aβ peptide 14시간 배양하였다. 패 추출물을 처리한 군이 Aβ peptide만 처리한 군 보다 iNOS, COX2 mRNA level이 증가하는 것을 확인하였다(도 18).PC12 cells were treated with L. extract (0.075, 0.125 mg / mL) for 2
<결과 및 고찰><Results and Discussion>
산화적 스트레스에 의한 뇌 손상의 기전으로 신경흥분 독성이 주요한 기전 중 하나로 알려져 있는데 이는 과다 분비된 glutamate 등 흥분성 아미노산에 의한 수용체들의 과도한 자극으로 인한 신경세포 세포사멸(neuronal cell death)이 일어나게 되는 것이다. 글루타메이트가 유도하는 신경 세포 손상은 글루타메이트 수용체의 과다 흥분에 의한 독성뿐만 아니라 수용체의 매개 없이 산화적 스트레스를 유발해 손상을 일으킬 수도 있다. 따라서 본 실험은 패라는 해양 천연물을 가지고 glutamate-induced된 HT22 세포의 신경독성 보호효과를 보았다.Nerve excitotoxicity is known to be one of the main mechanism of brain damage caused by oxidative stress, which is caused by neuronal cell death due to excessive stimulation of receptors by excitatory amino acids such as excess glutamate. Neuronal damage induced by glutamate may cause damage by inducing oxidative stress without receptor mediation as well as toxicity by excessive excitations of glutamate receptors. Thus, the present experiment demonstrated the neurotoxic protective effect of glutamate-induced HT22 cells in the parasites of marine natural products.
HT22 cell에 패 추출물 0.05, 0.1 mg/mL로 2시간 30분 전 처리하고 14시간 5 mM glutamate 처리하여 농도별로 glutamate-induced 된 HT22 세포의 신경보호 효과를 WST-1으로 확인결과 농도 의존적으로 Cell viability가 증가하는 것으로 보여줬다. AnnexinV와 PI stanning을 통해 농도의존적으로 cell death가 감소하는 것을 확인하였으며, Western blotting을 통해서 Bax/Bcl2 ratio와 Cleaved Caspase-3가 감소하는 것을 통해서 패의 농도 의존적으로 세포독성보호 효과가 일어남을 확인하였다. DCFH-DA 염색을 통해 패의 농도 의존적으로 ROS가 감소하는 것을 확인하였다. Akt/P13K pathway가 활성화되는 것이 glutamate-induce 된 cell death를 줄인다. glutamate-induced 된 cell에 패 추출물을 처리하면 AKT가 인산화된다. 약재를 처리하고 Akt pathway가 활성화되면 Nrf2활성 증가하게 되고 HO-1이 증가하게 되면서 ROS를 저해시켜 cascade 활성을 감소시켜 cell death를 감소시킨다. glutamate-induced된 cell에 패 추출물을 처리하면 Nrf2와 HO-1이 증가하게 되는데 q-PCR로 mRNA level로 확인하였다. Glutamate 증가에 따른 세포 내 ROS 농도를 감소시키는 것으로 판명된다.HT22 cells were pretreated with 0.05% fetal bovine serum (0.05 mg / mL) and 0.1 mg / mL for 2
본 연구는 패 추출물은 glutamate induced 된 HT22 세포를 Akt signaling 조절을 통해 신경보호효과를 나타내며, 산화적 스트레스를 통한 AD의 의약개발에 큰 가능성을 제시한다.The present study demonstrates the neuroprotective effect of glutamate - induced HT22 cells through the regulation of Akt signaling, and suggests great potential for the development of AD medicines through oxidative stress.
Aβ peptide에 의해 유도된 신경염증은 알츠하이머의 중요한 기전이다. PC12 cell에 패 추출물 0.075, 0.125 mg/mL로 2시간 30분 전 처리하고 Aβ peptide 14시간 처리하여 농도별로 Aβ peptide-induced 된 PC12 세포의 신경보호 효과를 WST-1으로 확인결과 농도 의존적으로 Cell viability가 증가하는 것으로 보여줬다. 따라서, Aβ peptide-induced에 패 추출물은 신경독성 보호효과가 있다는 것이 보인다. Aβ peptide가 유도된 PC12 세포에 패 추출물이 처리되었을 때 COX2와 iNOS 같은 pro-imflammatory mediator의 발현이 억제된다. 게다가 패 추출물은 MAPKs signaling pathway 비활성화를 통해 inflammation에 대한 저해 효능이 보인다. Aβ peptide 증가에 따른 세포 내 ROS 농도를 감소시켜 세포 독성을 감소시키는 것으로 판명된다.Aβ peptide-induced neuroinflammation is an important mechanism of Alzheimer's. PC12 cells were pretreated with 0.075 and 0.125 mg / mL of protein extract for 2 hours and 30 minutes and treated with Aβ peptide for 14 hours to determine the neuroprotective effect of Aβ peptide-induced PC12 cells on the concentration by WST-1. Of the total. Thus, it is shown that the L. extract against Aβ peptide-induced has a neurotoxic protective effect. The expression of pro-imflammatory mediators such as COX2 and iNOS is inhibited when PC12 cells induced by Aβ peptide are treated with L. extract. In addition, the extract of Lappa shows inhibitory effect on inflammation through inactivation of MAPKs signaling pathway. Aβ peptide, it is found that it decreases cytotoxicity by decreasing intracellular ROS concentration.
본 연구는 Aβ peptide가 유도된 PC12 세포에 대한 패 추출물 항염증 효과는 향후 AD의 의약개발에 큰 가능성을 제시한다.This study suggests that the anti - inflammatory effect of L - extract on PC12 cells induced Aβ peptide has great potential for development of AD medicines in the future.
Claims (11)
A pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising an extract of Ishige okamurae as an active ingredient.
상기 추출물은 C1 내지 C2의 저급 알코올 또는 이들의 혼합물을 용매로 사용하여 추출한 후, 건조하여 1,3-butylene glycol에 녹인 것을 특징으로 하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물.
The method according to claim 1,
Wherein the extract is extracted with a C1 to C2 lower alcohol or a mixture thereof as a solvent, and then dried to be dissolved in 1,3-butylene glycol.
상기 저급 알코올은 메탄올인 것을 특징으로 하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물.
3. The method of claim 2,
Wherein the lower alcohol is methanol. ≪ RTI ID = 0.0 > 11. < / RTI >
상기 퇴행성 뇌질환은 알츠하이머인 것을 특징으로 퇴행성 뇌질환 예방 및 치료용 약학적 조성물.
The method according to claim 1,
Wherein the degenerative brain disease is Alzheimer's disease, and is a pharmaceutical composition for preventing and treating degenerative brain diseases.
상기 패 추출물은 AKT를 인산화시켜 글루타메이트 증가에 따른 세포 내 ROS 농도를 감소시키는 것을 특징으로 하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물.
The method according to claim 1,
Wherein said extract of Rhodophyll is phosphorylated with AKT to reduce intracellular ROS concentration upon increase of glutamate.
상기 패 추출물은 염증 전 매개체(pro-imflammatory mediator)의 발현을 억제하여 Aβ peptide 증가에 따른 세포 내 ROS 농도를 감소시키는 것을 특징으로 하는 퇴행성 뇌질환 예방 및 치료용 약학적 조성물.
The method according to claim 1,
The pharmaceutical composition for the prevention and treatment of degenerative brain diseases according to claim 1, wherein the extract of Raloxifene inhibits expression of a pro-imflammatory mediator to reduce intracellular ROS concentration upon increase of A? Peptide.
(Ishige okamurae) extract as an active ingredient for the prevention and improvement of degenerative brain diseases.
상기 추출물은 C1 내지 C2의 저급 알코올 또는 이들의 혼합물을 용매로 사용하여 추출한 후, 건조하여 1,3-butylene glycol에 녹인 것을 특징으로 하는 퇴행성 뇌질환 예방 및 개선용 건강기능식품.
8. The method of claim 7,
Wherein the extract is obtained by extracting C1 to C2 lower alcohols or a mixture thereof as a solvent and then drying and dissolving the 1,3-butylene glycol in a 1,3-butylene glycol.
상기 퇴행성 뇌질환은 알츠하이머인 것을 특징으로 퇴행성 뇌질환 예방 및 개선용 건강기능식품.
8. The method of claim 7,
Wherein the degenerative brain disease is Alzheimer's disease, and is a health functional food for preventing and improving degenerative brain diseases.
상기 패 추출물은 AKT를 인산화시켜 글루타메이트 증가에 따른 세포 내 ROS 농도를 감소시키는 것을 특징으로 하는 퇴행성 뇌질환 예방 및 개선용 건강기능식품.
8. The method of claim 7,
Wherein said extract is phosphorylated with AKT to reduce intracellular ROS concentration upon increase of glutamate.
상기 패 추출물은 염증 전 매개체(pro-imflammatory mediator)의 발현을 억제하여 Aβ peptide 증가에 따른 세포 내 ROS 농도를 감소시키는 것을 특징으로 하는 퇴행성 뇌질환 예방 및 개선용 건강기능식품.
8. The method of claim 7,
Wherein the extract of Lahm is a compound for inhibiting the expression of pro-imflammatory mediator and reducing intracellular ROS concentration upon increase of A? Peptide.
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