KR102578902B1 - Composition for preventing, improving and treating age-related macular degeneration comprising Eruca sativa Mill extracts - Google Patents
Composition for preventing, improving and treating age-related macular degeneration comprising Eruca sativa Mill extracts Download PDFInfo
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- KR102578902B1 KR102578902B1 KR1020210051266A KR20210051266A KR102578902B1 KR 102578902 B1 KR102578902 B1 KR 102578902B1 KR 1020210051266 A KR1020210051266 A KR 1020210051266A KR 20210051266 A KR20210051266 A KR 20210051266A KR 102578902 B1 KR102578902 B1 KR 102578902B1
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- arugula
- extract
- retinal
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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Abstract
본 발명은 루꼴라 추출물을 유효성분으로 함유하는 약학적 조성물, 식품 조성물에 관한 것으로서 세포독성이 없으면서도 항염증, 소포체 스트레스 억제, VEGF 억제 효과가 탁월하여 AMD 예방, 치료, 개선용 약학적 조성물, 식품 조성물로 유용하게 사용될 수 있다.The present invention relates to a pharmaceutical composition and food composition containing arugula extract as an active ingredient. It is non-cytotoxic and has excellent anti-inflammatory, endoplasmic reticulum stress inhibition, and VEGF inhibitory effects, making it a pharmaceutical composition and food for preventing, treating, and improving AMD. It can be usefully used as a composition.
Description
본 발명은 루꼴라 추출물을 함유하는 약학적 조성물, 식품 조성물에 관한 것으로서, 더욱 상세하게는 루꼴라 추출물을 함유하여 망막 세포의 항염증, 소포체 스트레스 억제 및 VEGF 억제 효과가 우수한 노인성 황반 변성 예방, 개선 및 치료용 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition and a food composition containing arugula extract, and more specifically, to a pharmaceutical composition and food composition containing arugula extract for the prevention, improvement and treatment of age-related macular degeneration, which has excellent anti-inflammatory, endoplasmic reticulum stress inhibition and VEGF inhibitory effects on retinal cells. It relates to a composition for use.
노인성 황반 변성(age-related macular degeneration, AMD) 환자는 2019년에 약 20만 명으로 조사되어 2014년을 기준으로 매년 약 20% 증가하는 추세이다(Health Insurance Review & Assessment Service, 2020). 황반변성은 크게 망막의 광수용체와 세포들이 죽는 건성과 황반 아래 맥락막에서 새 혈관이 자라는 습성으로 나뉜다. 습식 노인성 황반변성은 비정상적으로 생성된 맥락막신생혈관(Choroidal Neovascularization, CNV)이 특징이며(Ambati 등, 2012) 생성된 혈관에서의 출혈 및 부종으로 인한 시력의 손실 및 망막의 기능손실을 유발하여 망막 박리(Retinal detachment)를 유발할 수 있다(Do 등, 2020). The number of age-related macular degeneration (AMD) patients was estimated to be approximately 200,000 in 2019, and is increasing by approximately 20% every year as of 2014 (Health Insurance Review & Assessment Service, 2020). Macular degeneration is largely divided into dry type, where photoreceptors and cells in the retina die, and new type, where new blood vessels grow in the choroid under the macula. Wet age-related macular degeneration is characterized by abnormally generated choroidal neovascularization (CNV) (Ambati et al., 2012), which causes loss of vision and loss of retinal function due to hemorrhage and edema in the generated blood vessels, leading to retinal detachment (Ambati et al., 2012). Retinal detachment) (Do et al., 2020).
ARPE-19 세포는 사람 눈의 맥락막과 광수용체 사이에 있는 시각기능을 위한 세포층을 나타내는 망막 상피세포로 AMD, 당뇨망막병증(diabetic retinopathy) 등의 안구질환에 관여하기 때문에 안구질환 연구에서 주요한 망막 세포주로 사용된다(Boulton 등, 2001). ARPE-19 cells are retinal epithelial cells that represent the cell layer for visual function between the choroid and photoreceptors of the human eye. They are involved in eye diseases such as AMD and diabetic retinopathy, so they are a major retinal cell line in eye disease research. (Boulton et al., 2001).
소포체 스트레스는 ARPE-19 세포의 손상 및 VEGF(vascular endothelial growth factor)를 과발현하여 CNV 형성에 기여한다(Chen 등, 2014; Salminen 등, 2010). VEGF는 혈관 형성을 자극하는 세포에서 생성되는 신호 단백질로 혈관의 새로운 형성과 관련된 중요한 단백질이며 ARPE-19 세포에서의 VEGF의 과발현은 망막 상피세포의 CNV 형성에 중요한 역할을 하여 AMD의 주요 원인이다(Jager 등, 2008; Lopez 등, 1996; Datta 등, 2017). 또한, 안구에서 염증은 안구 건조증(dry eyes)이나 포도막염(uveitis)과 당뇨망막병증(diabetic retinopathy)을 유발하는 원인으로 작용한다(Hong 등, 2020; Qin 등, 2014). 안구염증에 의하여 발병된 망막병증은 CNV 형성에 관여할 수 있으며 ARPE-19 세포에서의 염증관련 사이토카인의 발현이 CNV 형성을 증가켜 염증은 AMD 발병의 원인으로 제시되었다(Izumi 등, 2007). 즉, ARPE-19 세포에서 염증과 소포체 스트레스의 억제 및 VEGF의 억제는 AMD 발병 억제의 가능성을 의미한다. Endoplasmic reticulum stress contributes to CNV formation by damaging ARPE-19 cells and overexpressing vascular endothelial growth factor (VEGF) (Chen et al., 2014; Salminen et al., 2010). VEGF is a signaling protein produced by cells that stimulate angiogenesis and is an important protein related to the formation of new blood vessels. Overexpression of VEGF in ARPE-19 cells plays an important role in the formation of CNV in retinal epithelial cells and is a major cause of AMD ( Jager et al., 2008; Lopez et al., 1996; Datta et al., 2017). Additionally, inflammation in the eye acts as a cause of dry eyes, uveitis, and diabetic retinopathy (Hong et al., 2020; Qin et al., 2014). Retinopathy caused by ocular inflammation may be involved in CNV formation, and the expression of inflammation-related cytokines in ARPE-19 cells increases CNV formation, suggesting that inflammation is a cause of AMD (Izumi et al., 2007). In other words, inhibition of inflammation and endoplasmic reticulum stress and VEGF in ARPE-19 cells imply the possibility of inhibiting AMD development.
AMD의 치료를 위해 주로 사용하는 방법은 베르테포핀(Verteporfin)으로 명명되는 물질을 사용하여 AMD에 의하여 생성된 신생 혈관을 특정적으로 염색하여 제거하는 것이다. 베르테포핀을 사용한 치료법은 비교적 안전하지만 병태의 원인을 제거하는 치료가 아니기 때문에 치료를 반복해야 하는 단점이 있으며(Bressler 등, 2001) 자각 증상이 거의 없어 약물의 처방이 어렵다(Sin 등, 2013). 또한, AMD 발병자는 지속해서 증가하고 있으며 자각 증상이 늦게 나타나 조기 치료가 힘들어 의학적 치료의 한계를 보이며, 치료 후에도 재발 가능성이 높다. 따라서, 망막 병태의 진행을 예방하거나 지연시키는 것은 AMD 예방, 치료에 합리적인 전략이 될 수 있으며 의학적 치료의 대안으로 비의료적 치료법인 기능성 식품 섭취를 통한 AMD의 예방이 될 수 있다. 또한, 천연물에서 추출한 기능성 식품은 일반적으로 부작용이 적은 것으로 보고되어(Granato 등, 2020) 장기간 섭취에 대한 안전성을 기대할 수 있다.The method mainly used to treat AMD is to specifically stain and remove new blood vessels created by AMD using a substance called Verteporfin. Treatment using verteporpine is relatively safe, but it has the disadvantage of having to repeat treatment because it does not remove the cause of the condition (Bressler et al., 2001), and it is difficult to prescribe the drug because there are almost no subjective symptoms (Sin et al., 2013). In addition, the number of patients with AMD continues to increase, and symptoms appear late, making early treatment difficult, showing limitations in medical treatment, and the possibility of recurrence is high even after treatment. Therefore, preventing or delaying the progression of retinal pathology may be a reasonable strategy for the prevention and treatment of AMD, and prevention of AMD through the consumption of functional foods, a non-medical treatment alternative to medical treatment, may be possible. In addition, functional foods extracted from natural products are generally reported to have few side effects (Granato et al., 2020), so they can be expected to be safe for long-term consumption.
한편, 루꼴라는 십자화과(Brassicaceae)에 속하는 식물로 항비만 및 혈당 저하 효과(Piragine 등, 2020), 항산화 효과(Son 등, 2020; Taviano 등, 2017; Heimler 등, 2007), 항혈전 형성(Fuentes 등, 2014), 항균 활성과 피부장벽 개선 효과(Kim 등, 2014), 간 보호 효과(Alqasoumi, 2010) 및 위 병변에 대한 항궤양 활성(Alqasoumi 등, 2009) 등의 다양한 생리활성이 보고된 바 있다. 하지만 루꼴라의 망막 질환의 예방, 치료 효과에 대해서는 아직 알려진 바 없다. Meanwhile, arugula is a plant belonging to the Brassicaceae family and has anti-obesity and blood sugar-lowering effects (Piragine et al., 2020), antioxidant effects (Son et al., 2020; Taviano et al., 2017; Heimler et al., 2007), and anti-thrombosis formation (Fuentes et al. , 2014), various physiological activities have been reported, including antibacterial activity and skin barrier improvement effect (Kim et al., 2014), liver protection effect (Alqasoumi, 2010), and anti-ulcer activity against gastric lesions (Alqasoumi et al., 2009). . However, nothing is yet known about the effectiveness of arugula in preventing and treating retinal diseases.
이에 본 발명자들은 안전성이 입증된 천연물 소재인 루꼴라로부터 루꼴라 추출물의 망막 세포에 대한 항염증, 소포체 스트레스 억제 및 VEGF 억제 효과를 확인하여 AMD 예방, 개선 및 치료 용도로 사용될 수 있음을 확인하였다. 또한, 루꼴라 열수 추출물에 비해 루꼴라 에탄올 추출물은 다양한 농도에서 우수한 항염증, 소포체 스트레스 억제 및 VEGF 억제 효과를 나타내었고, 루꼴라 동결 건조물에 비해 열풍 건조물에 대한 에탄올 추출물에서 더욱 뛰어난 망막 세포의 항염증, 소포체 스트레스 억제 및 VEGF 억제 효과를 확인하여, 본 발명을 완성하게 되었다. Accordingly, the present inventors confirmed that the anti-inflammatory, endoplasmic reticulum stress inhibition, and VEGF inhibitory effects of arugula extract on retinal cells from arugula, a natural material with proven safety, can be used for the prevention, improvement, and treatment of AMD. In addition, compared to arugula hot water extract, arugula ethanol extract showed superior anti-inflammatory, endoplasmic reticulum stress inhibition, and VEGF inhibitory effects at various concentrations, and compared to arugula freeze-dried product, the ethanol extract for hot air dried product showed superior anti-inflammatory and endoplasmic reticulum effects on retinal cells. By confirming the stress inhibition and VEGF inhibition effects, the present invention was completed.
따라서, 본 발명이 해결하고자 하는 과제는 루꼴라 추출물을 유효성분으로 포함하는 망막 질환의 예방, 치료용 약학 조성물을 제공하는 것이다. Therefore, the problem to be solved by the present invention is to provide a pharmaceutical composition for preventing and treating retinal diseases containing arugula extract as an active ingredient.
본 발명이 해결하고자 하는 다른 과제는 루꼴라 추출물을 유효성분으로 함유하는 망막 질환의 예방, 개선용 식품 조성물을 제공하는 것이다.Another problem to be solved by the present invention is to provide a food composition for preventing and improving retinal diseases containing arugula extract as an active ingredient.
본 발명이 해결하고자 하는 과제들은 이상에서 언급된 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.
상기 목적을 달성하기 위하여, 본 발명에서는 유효성분으로 루꼴라 추출물을 함유하는 약학적 조성물을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition containing arugula extract as an active ingredient.
본 발명에 있어서, 상기 "추출"은 당업계에 공지된 추출, 분리 및 분획하는 방법을 사용하여 천연으로부터 추출, 분리 및 분획하여 수득한 것을 사용할 수 있다. 본 발명에서 정의된 "추출물"은 적적한 용매를 이용하여 루꼴라로부터 추출 처리에 의해 얻어지는 것이며, 예를 들어 루꼴라 줄기, 잎, 뿌리 또는 이들의 혼합의 조추출물, 극성용매 가용 추출물 또는 비극성용매 가용 추출물을 모두 포함한다. In the present invention, the “extraction” may be obtained by extraction, separation, and fractionation from nature using extraction, separation, and fractionation methods known in the art. “Extract” as defined in the present invention is obtained by extraction treatment from arugula using an appropriate solvent, for example, crude extract, polar solvent-soluble extract, or non-polar solvent-soluble extract of arugula stem, leaf, root, or a mixture thereof. Includes all.
본 발명에 있어서, 상기 루꼴라 추출물은 다양한 추출 용매와 추출 방법에 따라 추출될 수 있으며, 루꼴라로부터 추출물을 추출하기 위한 적절한 용매로는 예를 들어, 상기 용매로는 물, 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등의 C1-C4 알코올 또는 이의 혼합용매를 사용할 수 있다. 본 발명의 추출물의 추출 용매로 에탄올, 50 내지 90% (v/v) 에탄올 또는 80% (v/v) 에탄올을 사용할 수 있으나 이에 제한되는 것은 아니다. 에탄올을 사용하여 루꼴라 추출물을 제조하는 경우 루꼴라 추출물 내 지용성 및 수용성 활성성분이 모두 충분히 녹아들 수 있고, 이에 최적 효과를 나타내는 추출물을 제조할 수 있다. 본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 탄소수 1 내지 4의 알코올로 추출된다. 본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 70 내지 90% (v/v) 에탄올을 추출용매로 하여 추출된 것을 특징으로 한다. In the present invention, the arugula extract can be extracted according to various extraction solvents and extraction methods, and suitable solvents for extracting the extract from arugula include, for example, water, methanol, ethanol, propanol, and isopropanol. , C1-C4 alcohols such as butanol, or mixed solvents thereof can be used. Ethanol, 50 to 90% (v/v) ethanol, or 80% (v/v) ethanol may be used as an extraction solvent for the extract of the present invention, but is not limited thereto. When preparing an arugula extract using ethanol, both fat-soluble and water-soluble active ingredients in the arugula extract can be sufficiently dissolved, and an extract showing optimal effects can be prepared. According to one embodiment of the present invention, the arugula extract is extracted with alcohol having 1 to 4 carbon atoms. According to one embodiment of the present invention, the arugula extract is characterized in that it is extracted using 70 to 90% (v/v) ethanol as an extraction solvent.
상기 추출 온도는 상온인 것이 바람직하며, 이에 제한되지 않는다. 또한 추출방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법이 사용될 수 있다.The extraction temperature is preferably room temperature, but is not limited thereto. In addition, extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression can be used.
본 발명에 있어서, 상기 루꼴라 추출물은 루꼴라를 동결 건조, 열풍 건조 또는 이들의 조합으로 건조시킨 후 추출하는 것이다. 구체적으로, 루꼴라를 동결 건조시킨 후 추출한 루꼴라 동결건조 추출물, 루꼴라를 열풍 건조시킨 후 추출한 루꼴라 열풍건조 추출물일 수 있다. In the present invention, the arugula extract is extracted after drying arugula by freeze-drying, hot air drying, or a combination thereof. Specifically, it may be an arugula freeze-dried extract extracted after freeze-drying arugula, or an arugula hot-air-dried extract extracted after arugula is hot-air dried.
본 발명의 일 실시예에서는 건조 방법에 따른 루꼴라 추출물의 LPS에 의해 염증이 유도된 망막 세포주에서 염증관련 사이토카인(TNFα, IL-1β)의 생성에 미치는 영향을 실험한 결과 모든 루꼴라 추출물에서 대조군과 비교하여 발현이 감소함을 확인하였다. 동결건조 루꼴라 추출물은 TNFα, IL-1β를 높은 비율로 감소시켰으며, 열풍건조 루꼴라 추출물은 TNFα 발현을 감소시켰다.In one embodiment of the present invention, the effect of drying arugula extract on the production of inflammation-related cytokines (TNFα, IL-1β) in retinal cell lines in which inflammation was induced by LPS was tested. As a result, all arugula extracts were compared to the control group. By comparison, it was confirmed that expression decreased. Freeze-dried arugula extract reduced TNFα and IL-1β at a high rate, and hot air-dried arugula extract reduced TNFα expression.
본 발명의 일 실시예에서는 건조 방법에 따른 루꼴라 추출물의 LPS에 의해 염증이 유도된 망막 세포주에서 염증관련 단백질(p-NF-κB, p-IκBα) 인산화에 미치는 영향을 실험한 결과, 동결건조 루꼴라 추출물은 NF-κB의 인산화를, 열풍건조 루꼴라 추출물은 NF-κB와 IκBα의 인산화를 억제시켰다.In one embodiment of the present invention, as a result of testing the effect of the arugula extract according to the drying method on phosphorylation of inflammation-related proteins (p-NF-κB, p-IκBα) in retinal cell lines in which inflammation was induced by LPS, the freeze-dried arugula extract was tested. The extract inhibited the phosphorylation of NF-κB, and the hot-air dried arugula extract inhibited the phosphorylation of NF-κB and IκBα.
본 발명의 일 실시예에서는 건조 방법에 따른 루꼴라 추출물의 LPS에 의해 염증이 유도된 망막 세포주에서 염증 경로를 유발하는 MAPK 단백질(p-JNK) 발현에 미치는 영향을 실험한 결과, 열풍건조 루꼴라 추출물은 p-JNK의 발현을 감소시켰다.In one embodiment of the present invention, as a result of testing the effect of the arugula extract according to the drying method on the expression of MAPK protein (p-JNK), which induces the inflammatory pathway, in a retinal cell line in which inflammation was induced by LPS, the hot air dried arugula extract Reduced expression of p-JNK.
본 발명의 실시예에서는 건조 방법에 따른 루꼴라 추출물의 타프시가진에 의해 소포체 스트레스가 유도된 망막 세포주에 대한 항소포체 스트레스 효과를 확인하기 위해 소포체 스트레스 단백질(CHOP, XBP-1) 발현에 미치는 영향을 실험한 결과 열풍건조 루꼴라 추출물은 CHOP, XBP-1 발현을 감소시켰다.In an example of the present invention, the effect on the expression of endoplasmic reticulum stress protein (CHOP, As a result of the experiment, hot air dried arugula extract decreased the expression of CHOP and XBP-1.
본 발명의 일 실시예에서는 건조 방법에 따른 루꼴라 추출물의 타프시가진에 의해 소포체 스트레스가 유도된 망막 세포주의 세포사멸을 확인하기 위한 cleaved-caspase 9, cleaved-caspase 3 단백질 발현에 미치는 영향을 실험한 결과, 열풍건조 루꼴라 에탄올 추출물은 cleaved-caspase 9, cleaved-caspase 3 발현을 감소시켰다.In one embodiment of the present invention, the effect on cleaved-caspase 9 and cleaved-caspase 3 protein expression was tested to confirm apoptosis of retinal cell lines in which endoplasmic reticulum stress was induced by tapsigazine of the arugula extract according to the drying method. As a result, hot air dried arugula ethanol extract decreased the expression of cleaved-caspase 9 and cleaved-caspase 3.
본 발명의 일 실시예에서는 루꼴라 추출물이 타프시가진에 의해 소포체 스트레스가 유도된 망막 세포주에서 VEGF 생산 및 칼슘 수준에 미치는 영향을 실험한 결과, 루꼴라 추출물은 VEGF 억제 및 세포질 내 칼슘 수준을 감소시켰다. In one embodiment of the present invention, the effect of arugula extract on VEGF production and calcium levels was tested in a retinal cell line in which endoplasmic reticulum stress was induced by tapsigazine. As a result, the arugula extract inhibited VEGF and reduced cytoplasmic calcium levels.
루꼴라 추출물을 얻은 이후에는 당업계에서 알려진 통상의 방법으로 상온에서 냉침, 가열 및 여과하여 액상물을 얻을 수 있으며, 또는 추가로 용매를 증발, 분무건조 또는 동결건조할 수도 있다.After obtaining the arugula extract, a liquid product can be obtained by immersing, heating, and filtering at room temperature by conventional methods known in the art, or the solvent can be further evaporated, spray-dried, or freeze-dried.
본 발명에 있어서, 상기 제조한 루꼴라 추출물은 추가적으로 증류한 후 분획하여 얻을 수 있으며, 구체적으로는 실리카겔 컬럼 크로마토그래피(silica gel column chromatography), 박층크로마토그래피(thin layer chromatography), 고성능 액체 크로마토그래피(high performance liquid chromatography) 등과 같은 다양한 크로마토그래피를 이용하여 추가로 정제된 분획으로도 얻을 수 있으며, 바람직하게는 박층크로마토그래피를 이용하여 분획할 수 있으나 이에 본 발명이 제한되는 것은 아니고, 적절한 용매를 이용하여 추가 분획물을 얻을 수 있는 방법이라면 어느 것이든지 이용할 수 있다.In the present invention, the prepared arugula extract can be obtained by additional distillation and then fractionation, specifically, silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. It can be obtained as an additional purified fraction using various chromatographies such as performance liquid chromatography, and preferably fractionated using thin layer chromatography, but the present invention is not limited thereto, and the fraction can be obtained by using an appropriate solvent. Any method by which additional fractions can be obtained can be used.
본 발명에 있어서, 상기 분획물을 얻기 위해 사용하는 용매는 클로로포름, 에틸 아세트산, 부탄올 및 물로 이루어진 군으로부터 선택된 어느 하나 이상일 수 있으며, 클로로포름, 에틸 아세트산, 부탄올을 사용할 수 있으며, 에틸 아세트산을 사용할 수 있으나 이에 제한되는 것은 아니다.In the present invention, the solvent used to obtain the fraction may be any one or more selected from the group consisting of chloroform, ethyl acetic acid, butanol, and water. Chloroform, ethyl acetic acid, and butanol may be used, and ethyl acetic acid may be used. It is not limited.
본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 건조시킨 루꼴라를 에탄올을 첨가한 후 60 ~ 80℃에서 2 ~ 4 시간 환류 냉각한 후 여과하여 감압 농축하는 것을 포함하는 방법에 의하여 제조된다. According to one embodiment of the present invention, the arugula extract is prepared by a method comprising adding ethanol to dried arugula, cooling it under reflux at 60 to 80°C for 2 to 4 hours, then filtering and concentrating under reduced pressure.
본 발명에 따른 치료용 약학적 조성물은 루꼴라 에탄올 추출물을 유효성분으로 포함하는 망막 질환의 예방, 치료용 식의약학적 조성물이다. The pharmaceutical composition for treatment according to the present invention is a food and pharmaceutical composition for the prevention and treatment of retinal diseases containing arugula ethanol extract as an active ingredient.
본 발명의 실시예에서 LPS가 유도하는 ARPE-19 세포의 염증 반응에서 염증관련 사이토카인(TNFα, IL-1β)의 mRNA 발현 수준 및 염증관련 단백질(NF-κB와 IκBα, JNK)의 인산화와 타프시가진이 유도하는 ARPE-19 세포의 소포체 스트레스 단백질(CHOP, XBP-1, cleaved-caspase 9, cleaved-caspase 3), VEGF 및 세포질 내 칼슘 수준을 분석하여, 루꼴라 추출물은 항염증, 소포체 스트레스, VEGF를 억제, 세포질 내 칼슘 수준을 감소시킴을 확인하여 이와 관련된 망막 질환의 예방, 치료에 사용될 수 있다. In an example of the present invention, the mRNA expression level of inflammation-related cytokines (TNFα, IL-1β) and the phosphorylation and TARP of inflammation-related proteins (NF-κB, IκBα, JNK) in the inflammatory response of ARPE-19 cells induced by LPS By analyzing the endoplasmic reticulum stress proteins (CHOP, It has been confirmed that it inhibits VEGF and reduces calcium levels in the cytoplasm, so it can be used to prevent and treat retinal diseases related to this.
본 발명에 있어서, 상기 망막 질환은 노인성 황반 변성, 습식 노인성 황반 변성, 당뇨병 황반 부종, 당뇨병 망막증(당뇨병 황반 부종 제외), 포도막염, 중심성 삼출성 맥락망막증, 망막색소선조증, 망막 색소 상피 박리, 다발성 맥락막염, 신생혈관 황반증(고도 근시, 경사 유두 증후군 또는 맥락막 골종을 원인으로 하는 사례에 한함), 미숙아 망막증, 망막 색소변성증, 레베르병(Leber's disease), 망막 동맥 폐색증, 망막 정맥 폐색증, 중심성 장액성 맥락망막증, 망막 동맥류, 망막 박리, 증식성 유리체망막증, 스타가르트병(Stargardt's disease), 맥락막 경화증, 전맥락막위축증, 난황상 황반 이영양증, 오구치병, 안저백점증, 백점상 망막염 및 뇌회전형망맥락막위축증으로 이루어지는 군에서 선택되는 1종 이상이다. In the present invention, the retinal disease includes age-related macular degeneration, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy (excluding diabetic macular edema), uveitis, central exudative chorioretinopathy, retinitis pigmentosa, retinal pigment epithelial detachment, and multiple choroids. Retinitis, neovascular maculopathy (limited to cases caused by high myopia, oblique papillary syndrome, or choroidal osteoma), retinopathy of prematurity, retinitis pigmentosa, Leber's disease, retinal artery occlusion, retinal vein occlusion, and central intestine. Aqueous chorioretinopathy, retinal aneurysm, retinal detachment, proliferative vitreoretinopathy, Stargardt's disease, choroidal sclerosis, panchoroidal atrophy, vitelline macular dystrophy, coracoid disease, fundus white spot, white-spot retinitis, and gyroscopic reticulum. It is one or more types selected from the group consisting of choroidal atrophy.
상기에 언급한 바와 같이 본 발명을 의약으로 사용하는 경우 본 발명의 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바납, 합성규산 알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당 등이 사용될 수 있다.As mentioned above, when the present invention is used as a medicine, the composition of the present invention may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, Lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnaubanap, synthetic Aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. can be used.
본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 내지 90 중량부 포함되는 것이 바람직하나 이에 한정되는 것은 아니다.The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
본 발명에 있어서 상기 조성물은 실제 임상 투여시에 경구 또는 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있으며, 당해 기술 분야에 알려진 적합한 제제는 문헌 (Remington's Pharmaceutical Science, 최근, Mack Publishing Company, Easton PA)에 개시되어 있는 것을 이용하는 것이 바람직하다. 상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유 등이 있다.In the present invention, the composition can be administered in various oral or parenteral formulations during actual clinical administration. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It can be prepared using, and suitable preparations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently published by Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, These include cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil.
상기 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 카보네이트(Calcium carbonate), 수크로스 (Sucrose) 또는 락토오스(Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 또한, 상기 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. In addition, the liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. This may be included.
상기 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 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, 글리세로제라틴 등이 사용될 수 있다. 상기 비경구투여는 피부 외용 또는 복강 내 주사, 직장 내 주사, 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 사용하여 이루어질 수 있다.The preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used. The parenteral administration may be performed externally on the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
상기 본 발명의 조성물은 약학적으로 유효한 양으로 투여될 수 있다. 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 감염된 바이러스 종류, 약물의 활성약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 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 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, type of virus infected, and the amount of drug administered. It can be determined based on factors including sensitivity to the active drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.
본 발명에 있어서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.The term “administration” as used in the present invention means providing a given composition of the present invention to an individual by any suitable method.
본 발명에 있어서 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다.In the present invention, the pharmaceutical composition can be administered to an individual through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection.
본 발명의 다른 구현예에 의하면 상기 약학적 조성물을 대상에게 투여하는 단계를 포함하는 망막 질환의 예방 및 치료방법을 제공한다. According to another embodiment of the present invention, a method for preventing and treating retinal diseases is provided, comprising administering the pharmaceutical composition to a subject.
본 발명에 따른 식품 조성물은 루꼴라 추출물을 유효성분으로 포함하는, 망막 질환의 예방, 개선용 건강기능식품을 제공하는 것이다. The food composition according to the present invention provides a health functional food for preventing and improving retinal disease, containing arugula extract as an active ingredient.
본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 C1-C4 알코올로 추출된다. According to one embodiment of the present invention, the arugula extract is extracted with C1-C4 alcohol.
본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 50 내지 90%(v/v) 에탄올을 추출용매로 하여 추출된다. According to one embodiment of the present invention, the arugula extract is extracted using 50 to 90% (v/v) ethanol as an extraction solvent.
본 발명의 일 구현예에 따르면, 상기 루꼴라 추출물은 루꼴라를 동결 건조 또는 열풍 건조한 것을 추출하는 것이다. According to one embodiment of the present invention, the arugula extract is extracted from freeze-dried or hot-air dried arugula.
본 발명에 따른 건강기능식품은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강기능식품은 유효성분인 본 발명에 따른 루꼴라 추출물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합 양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다. The health functional food according to the present invention may be provided in the form of powder, granules, tablets, capsules, syrup or beverage, and the health functional food is used with other foods or food additives in addition to the arugula extract according to the present invention, which is an active ingredient. , can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, such as prevention, health, or therapeutic treatment.
상기 건강기능식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 초코렛, 캔디류, 스넥류, 과자류, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다. There are no particular restrictions on the types of health functional foods, and examples include meat, sausages, bread, chocolate, candy, snacks, confectionery, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drink preparations. , alcoholic beverages, and vitamin complexes.
상술한 본 발명의 조성물에 대한 설명은 중복된 내용의 기재에 의한 과도한 복잡성을 피하기 위하여 그 기재를 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다. The description of the composition of the present invention described above is omitted to avoid excessive complexity due to duplicate description, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention pertains. It is to have.
본 발명에 의한 루꼴라 추출물을 함유하는 약학적 조성물 및 식품 조성물은 세포독성이 없으면서도 항염증 효과, 소포체 스트레스 및 VEGF 억제 효과가 탁월하여 노인성 황반변성 예방, 개선 및 치료용 조성물로 유용하게 사용될 수 있다.The pharmaceutical composition and food composition containing the arugula extract according to the present invention are non-cytotoxic and have excellent anti-inflammatory effects, endoplasmic reticulum stress, and VEGF inhibitory effects, and can be usefully used as compositions for preventing, improving, and treating age-related macular degeneration. .
도 1은 루꼴라 추출물(ESE) 및 LPS가 세포 생존력에 미치는 영향을 MTT 분석을 통해 실험한 결과이다. (A) 세포를 루꼴라 추출물 (25 ~ 1,000 μg/mL)로 3 시간 동안 전처리하고 LPS가 없는 상태에서 24 시간 동안 배양한 결과. (B) 세포를 LPS (0.25 ~ 4 μg/mL)로 처리하고 24 시간 동안 배양한 결과. (C) 세포를 루꼴라 추출물(100 ~ 1,000 μg/mL)로 3 시간 동안 전처리하고 LPS가 있는 상태에서 24 시간 동안 배양한 결과 (유의성 수준은 LPS 0 μg/mL 대비 ***P<0.001, 각 값은 평균 ± SD, n = 3)
도 2는 루꼴라 추출물(ESE) 및 타프시가진(thapsigargin)이 세포 생존력에 미치는 효과를 MTT 분석을 통해 실험한 결과이다. (A) 세포를 타프시가진(0.5 ~ 10 μM)으로 처리하고 24 시간 동안 배양한 결과. (B) 세포를 루꼴라 추출물(100 ~ 1,000 μg/mL)로 3 시간 동안 전처리한 후 타프시가진 존재하에 24 시간 동안 배양한 결과 (유의성 수준은 P < 0.05, 각 값은 평균± SD, n = 3)
도 3은 루꼴라 추출물(ESE)이 LPS 유도 ARPE-19 세포에서 TNFα, IL-1β 발현에 미치는 영향을 정량적 실시간 PCR 분석으로 실험한 결과이다. (A),(B)는 열풍 건조 루꼴라 추출물(ESH) (A) 및 동결 건조 루꼴라 추출물(ESF) (B)에서 TNFα의 상대적 mRNA 수준을 나타낸 결과. (C), (D)는 열풍 건조 루꼴라 추출물(ESH) (C) 및 동결 건조 루꼴라 추출물(ESF) (D)에서 IL-1β의 상대적 mRNA 수준을 나타낸 결과. (GAPDH는 내부 표준으로 사용, 유의성 수준 P < 0.05, 각 값은 평균± SD, n = 3).
도 4는 루꼴라 추출물(ESE)이 LPS에 의해 염증이 유도된 ARPE-19 세포에서 p-NF-κB, p-IκBα 및 p-JNK 발현에 미치는 영향을 실험한 결과이다. (A), (B)는 p-NF-κB, (C), (D)는 p-IκBα, (E), (F)는 p-JNK의 수준을 각 대조군의 값에 따라 추정한 결과. 표시된 평균은 LPS 존재 또는 부재 그룹간에 차이가 있었다(유의성 수준 각 *P<0.05, **P<0.01 and ***P<0.001, 각 값은 평균± SD, n = 3).
도 5는 루꼴라 추출물이(ESE) 타프시가진에 의해 소포체 스트레스가 유도된 ARPE-19 세포에서 CHOP, XBP-1, cleaved-caspase 9 및 cleaved-caspase 3 발현에 미치는 영향을 실험한 결과이다. (A), (B)는 CHOP, (C), (D)는 XBP-1, (E), (F)는 cleaved-caspase 9, (G), (H)는 cleaved-caspase 3의 수준을 측정한 결과. 타프시가진의 유무에 따라 유의하게 차이가 있었다 (유의성 수준 각 P<0.05, ##P<0.01, ###P<0.001, 각 값은 평균 ± SD, n = 3).
도 6은 루꼴라 추출물(ESE)이 타프시가진에 의해 소포체 스트레스가 유도된 ARPE-19 세포에서 VEGF 생산 및 세포 내 칼슘 수준에 미치는 영향을 실험한 결과이다. (A)는 루꼴라 추출물 및 타프시가진 처리 후 상층액을 수집하여 VEGF 생산을 측정한 결과. (B)는 세포 내 칼슘 수준을 측정한 결과.(유의성 수준 각 P <0.05, 각 값은 평균 ± SD, n = 3). Figure 1 shows the results of testing the effects of arugula extract (ESE) and LPS on cell viability through MTT analysis. (A) Results of cells pretreated with arugula extract (25 to 1,000 μg/mL) for 3 hours and cultured for 24 hours in the absence of LPS. (B) Results of cells treated with LPS (0.25 to 4 μg/mL) and cultured for 24 hours. (C) Cells were pretreated with arugula extract (100 to 1,000 μg/mL) for 3 hours and cultured in the presence of LPS for 24 hours (significance level: ***P<0.001 compared to LPS 0 μg/mL, each Values are mean ± SD, n = 3)
Figure 2 shows the results of testing the effects of arugula extract (ESE) and thapsigargin on cell viability through MTT analysis. (A) Results of cells treated with tapsigazine (0.5 to 10 μM) and cultured for 24 hours. (B) Cells were pretreated with arugula extract (100 to 1,000 μg/mL) for 3 hours and then cultured in the presence of tapsigazine for 24 hours (significance level is P < 0.05, each value is mean ± SD, n = 3)
Figure 3 shows the results of quantitative real-time PCR analysis of the effect of arugula extract (ESE) on TNFα and IL-1β expression in LPS-induced ARPE-19 cells. (A) and (B) show the relative mRNA levels of TNFα in hot air dried arugula extract (ESH) (A) and freeze dried arugula extract (ESF) (B). (C) and (D) show the relative mRNA levels of IL-1β in hot air dried arugula extract (ESH) (C) and freeze dried arugula extract (ESF) (D). (GAPDH was used as an internal standard, significance level P < 0.05, each value is mean ± SD, n = 3).
Figure 4 shows the results of testing the effect of arugula extract (ESE) on the expression of p-NF-κB, p-IκBα, and p-JNK in ARPE-19 cells in which inflammation was induced by LPS. The levels of p-NF-κB in (A) and (B), p-IκBα in (C) and (D), and p-JNK in (E) and (F) are estimated based on the values of each control group. The displayed means differed between groups with or without LPS (significance levels of *P<0.05, **P<0.01 and ***P<0.001, respectively; each value is mean ± SD, n = 3).
Figure 5 shows the results of testing the effect of arugula extract (ESE) on the expression of CHOP, The levels of CHOP in (A), (B), XBP-1 in (C), (D), cleaved-caspase 9 in (E), (F), and cleaved-caspase 3 in (G), (H). Measured results. There was a significant difference depending on the presence or absence of tapsigazine (significance level: P<0.05, ##P<0.01, ###P<0.001, each value is mean ± SD, n = 3).
Figure 6 shows the results of testing the effects of arugula extract (ESE) on VEGF production and intracellular calcium levels in ARPE-19 cells in which endoplasmic reticulum stress was induced by tapsigazine. (A) shows the results of measuring VEGF production by collecting the supernatant after treatment with arugula extract and tapsigin. (B) Results of measuring intracellular calcium levels. (Significance level: P <0.05, each value is mean ± SD, n = 3).
이하, 본 발명을 제조예, 실시예 및 실험예에 의하여 상세히 설명한다. Hereinafter, the present invention will be described in detail through preparation examples, examples and experimental examples.
단, 하기 제조예, 실시예 및 실험예는 본 발명을 구체적으로 예시하는 것일 뿐, 본 발명의 내용이 제조예, 실시예 및 실험예에 의해 한정되는 것은 아니다. However, the following production examples, examples, and experimental examples only specifically illustrate the present invention, and the content of the present invention is not limited by the production examples, examples, and experimental examples.
실시예 1 : 루꼴라 추출물의 제조 Example 1: Preparation of arugula extract
루꼴라는 2018년 5월 그림팜(Seoul, Korea)에서 구입하여 사용하였다. 루꼴라의 이물질 제거를 위해 흐르는 물에 세척하였고 열풍 건조 및 동결 건조로 시료를 건조하였다. Arugula was purchased and used from Grim Farm (Seoul, Korea) in May 2018. To remove foreign substances from the arugula, it was washed under running water, and the sample was dried by hot air drying and freeze drying.
시료의 동결 건조는 -70 ℃ 딥 프리저(MDF-U52 V, Sanyo, Osaka, Japan)에서 냉동시켜 동결 건조기(ED 8512, Ilshin, Yangju, Korea)에서 72 시간 동안 건조시켰다. 시료의 열풍 건조는 열풍 건조기(HJ120, Hanil, Jangseong, Korea)를 이용하여 60 ℃에서 40 시간 건조시켰다. 열풍건조 및 동결건조된 시료는 분쇄기(SMX-M41KP, Shinil, Cheonan, Korea)를 사용하여 80 mesh로 분쇄하여 -70 ℃에 보관하여 사용하였다. The samples were frozen in a deep freezer (MDF-U52 V, Sanyo, Osaka, Japan) at -70°C and dried in a freeze dryer (ED 8512, Ilshin, Yangju, Korea) for 72 hours. The sample was dried at 60°C for 40 hours using a hot air dryer (HJ120, Hanil, Jangseong, Korea). The hot air dried and freeze dried samples were ground to 80 mesh using a grinder (SMX-M41KP, Shinil, Cheonan, Korea) and stored at -70°C.
루꼴라 추출물은 열풍 건조 및 동결 건조 분말 각 100 g에 80 % 에탄올 1.5 L를 첨가한 후 65 ℃에서 3시간 환류 냉각을 3회 반복하였다. 추출물은 여과지(Whatman No.2, GE Healthcare, Buckinghamshire, UK)로 여과하여 로터리 진공 증발기(N-1110S-W, EYELA, Tokyo, Japan)로 감압농축한 후 동결건조 처리하였다. 건조된 추출물은 -70 ℃에 냉동 보관하여 사용하였다. For the arugula extract, 1.5 L of 80% ethanol was added to each 100 g of hot air-dried and freeze-dried powder, and then reflux cooling at 65°C for 3 hours was repeated three times. The extract was filtered through filter paper (Whatman No.2, GE Healthcare, Buckinghamshire, UK), concentrated under reduced pressure with a rotary vacuum evaporator (N-1110S-W, EYELA, Tokyo, Japan), and then freeze-dried. The dried extract was stored frozen at -70°C before use.
실시예 2 : ARPE-19 (retinal pigment epithelial cell line) 세포 배양Example 2: ARPE-19 (retinal pigment epithelial cell line) cell culture
ARPE-19 세포는 ATCC(American Type Culture Collection, Manassas, VA, USA)에서 구입하여 사용하였다. ARPE-19 세포는 10 % FBS(fetal bovine serum; Gibco BRL, Grand Island, NY, USA)과 100 U/mL 페니실린, 100 μg/mL 스트렙토마이신이 혼합된 항생제(Thermo Scientific Inc., MA, USA)를 첨가한 DMEM/F12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12; Gibco)를 사용하여 37 ℃, 5 % CO₂의 인큐베이터(VS-2180CW, Vision Scientific Co. LTD., Daejeon, Korea)에서 배양하였다.ARPE-19 cells were purchased and used from ATCC (American Type Culture Collection, Manassas, VA, USA). ARPE-19 cells were treated with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and antibiotics mixed with 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Scientific Inc., MA, USA). DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12; Gibco) supplemented with was used and cultured in an incubator (VS-2180CW, Vision Scientific Co. LTD., Daejeon, Korea) at 37°C and 5% CO₂. .
세포 생존율을 측정하기 위해 96-well plate에 ARPE-19 세포를 2.0 x 104 cell/well 농도로 분주 후 24 시간 동안 37 ℃, 5 % CO₂의 인큐베이터에서 배양하였다. qRT-PCR 및 웨스턴 블롯 실험에서는 6-well plate에 5.0 x 105 cell/well 농도로 분주 후 37 ℃, 5 % CO₂의 인큐베이터에서 48 시간 동안 배양하였다.To measure cell viability, ARPE-19 cells were distributed in a 96-well plate at a concentration of 2.0 x 10 4 cells/well and cultured in an incubator at 37°C and 5% CO₂ for 24 hours. In qRT-PCR and Western blot experiments, the cells were dispensed into 6 -well plates at a concentration of 5.0
실시예 3 : ARPE-19 세포 생존력 분석 Example 3: ARPE-19 cell viability analysis
루꼴라 추출물, LPS(lipopolysaccharides) 및 타프시가진(thapsigargin)이 ARPE-19 세포 생존율에 미치는 영향은 MTT 분석(Mosmann, 1983)을 통하여 측정하였다. 96-well plate에 ARPE-19 세포를 배양한 후 1 % FBS 조건 하에 루꼴라 추출물, LPS, 타프시가진, LPS + 루꼴라 추출물, 타프시가진 + 루꼴라 추출물을 처리하여 37 ℃, 5 % CO₂조건으로 24 시간 동안 배양하였다. The effects of arugula extract, lipopolysaccharides (LPS), and thapsigargin on ARPE-19 cell viability were measured through MTT assay (Mosmann, 1983). After culturing ARPE-19 cells in a 96-well plate, they were treated with arugula extract, LPS, taphxigin, LPS + arugula extract, and taphxigin + arugula extract under 1% FBS conditions and incubated for 24 hours at 37°C and 5% CO₂. It was cultured for some time.
LPS 및 타프시가진의 각 농도에서 ARPE-19 세포의 세포 생존율을 확인할 때는 루꼴라 추출물 대신 1 % FBS 배지를 사용하였으며 루꼴라 추출물의 세포 생존율을 확인할 때는 LPS 및 타프시가진 대신 1 % FBS 배지를 사용하였다. 루꼴라 추출물(ESE)의 경우 동결 건조물 추출물(ESF) 과 열풍 건조물 추출물(ESH)에 대한 실험을 구분하여 진행하였다. When checking the cell viability of ARPE-19 cells at each concentration of LPS and Tapsigazine, 1% FBS medium was used instead of arugula extract. When checking the cell viability of arugula extract, 1% FBS medium was used instead of LPS and Tapsigazine. . In the case of arugula extract (ESE), experiments were conducted separately on freeze-dried extract (ESF) and hot-air dried extract (ESH).
MTT(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide; Biosesang, Seoul, Korea)는 5.5 mg/mL(in 1 x PBS)로 20 μL를 처리하여 4 시간 동안 배양하고 웰의 배지를 모두 제거한 후 DMSO를 100 μL 넣어 포르마잔(formazan)을 용해하여 1 시간 상온에서 반응을 기다린 후 540 nm 흡광도에서 측정하였으며 아래와 같이 계산하였다.MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide; Biosesang, Seoul, Korea) was treated with 20 μL at 5.5 mg/mL (in 1 x PBS). After culturing for 4 hours and removing all of the medium in the wells, 100 μL of DMSO was added to dissolve formazan. After waiting for the reaction at room temperature for 1 hour, the absorbance was measured at 540 nm and calculated as follows.
세포 생존률 (%) = (시료 흡광도 / 대조군 흡광도) x 100Cell viability (%) = (sample absorbance / control absorbance) x 100
실험 결과, ARPE-19 세포에서 루꼴라 열풍 건조 추출물(ESH) 및 루꼴라 동결 건조 추출물(ESF) 처리군 모두 25 ~ 1,000 μg/mL 농도에서 유의적인 차이가 없었다(도 1 A).As a result of the experiment, there was no significant difference between the arugula hot air dried extract (ESH) and arugula freeze dried extract (ESF) treatment groups at concentrations of 25 to 1,000 μg/mL in ARPE-19 cells (Figure 1 A).
ARPE-19 세포에서 LPS(from Escherichia coli O55:B5, Sigma)의 처리 시 LPS 농도 2 μg/mL까지 유의적인 차이를 보이지 않았으며, 4 μg/mL에서 12.58% 감소하였다(도 1 B).When ARPE-19 cells were treated with LPS (from Escherichia coli O55:B5, Sigma), there was no significant difference in LPS concentration up to 2 μg/mL, and it decreased by 12.58% at 4 μg/mL (Figure 1 B).
LPS 1 μg/mL가 처리된 ARPE-19 세포에서 루꼴라 추출물(ESE)을 처리한 세포 생존율은 LPS 1 μg/mL 처리한 대조군과 LPS 1 μg/mL 및 ESE를 각 100, 250, 500, 1,000 μg/mL 처리하였을 때의 실험군 간 세포 생존율에 유의적인 차이가 없었다(도 1 C). The cell survival rate in ARPE-19 cells treated with LPS 1 μg/mL and arugula extract (ESE) was compared to the control group treated with LPS 1 μg/mL and 100, 250, 500, and 1,000 μg of LPS 1 μg/mL and ESE, respectively. There was no significant difference in cell survival rate between experimental groups when treated with /mL (Figure 1 C).
ARPE-19 세포에서 타프시가진의 처리 시 타프시가진은 0.5, 1, 2.5, 5, 10 μM 농도로 24 시간 동안 처리하였을 때 각 100, 91.67, 84.17, 64.27, 49.31, 18.70 %의 세포 생존율이 나타나 농도 의존적인 감소를 확인할 수 있었고 5 μM 농도에서 49.31%로 반수 치사량에 가까운 세포사멸률을 확인하였다(도 2 A). When ARPE-19 cells were treated with Tapsigazine at concentrations of 0.5, 1, 2.5, 5, and 10 μM for 24 hours, cell survival rates were 100, 91.67, 84.17, 64.27, 49.31, and 18.70%, respectively. A concentration-dependent decrease was confirmed, and a cell death rate close to the half-lethal dose was confirmed at 49.31% at a concentration of 5 μM (Figure 2 A).
타프시가진 5 μM가 처리된 ARPE-19 세포에서 루꼴라 추출물(ESE)의 세포 생존율은 타프시가진 5 μM이 처리된 대조군은 무 처리 세포에 비하여 세포 생존율이 49.16% 감소하였고 타프시가진 5 μM 및 ESE를 각 100, 250, 500, 1,000 μg/mL 처리하였을 때 세포 생존율의 추가적인 감소는 없었다(도 2 B).The cell viability of arugula extract (ESE) in ARPE-19 cells treated with 5 μM of Taphxigin was decreased by 49.16% in the control group treated with 5 μM of Taphxigin, compared to untreated cells. There was no additional decrease in cell viability when treated with ESE at 100, 250, 500, and 1,000 μg/mL, respectively (Figure 2 B).
루꼴라 추출물은 모든 처리 농도에서 세포 생존률에 영향을 미치지 않음을 확인하였다.It was confirmed that arugula extract did not affect cell viability at all treatment concentrations.
실시예 4: 페놀 및 플라보노이드 함량 측정 Example 4: Determination of phenol and flavonoid content
루꼴라 동결 건조 추출물, 루꼴라 열풍 건조 추출물의 페놀 및 플라보노이드 함량을 측정하였다. 페놀 함량은 Gutfinger 방법(Gutfinger T., Journal of the American Oil Chemists Society, Vol. 58, No.11, 1981)을 응용하여 측정하였다. 추출 시료용액 1㎖에 50% Foiln-Ciocalteu's phenol 시약 0.5 ㎖를 가하여 실온에서 3분간 반응시켰다. 반응용액에 Na2CO3 포화용액 1 ㎖와 7.5 ㎖ 증류수를 차례로 혼합하여 30 분간 방치시킨 뒤, 12,000 rpm에서 10 분간 원심분리한 후 상청액을 취해 760 nm에서 흡광도를 측정하였다. 총 폴리페놀 함량은 갈릭산(gallic acid)을 표준물질로 이용하여 작성한 검량선에 따라 함량을 구하였으며 측정단위로는 GAE(Gallic acid equivalent)/g을 사용하였다The phenol and flavonoid contents of arugula freeze-dried extract and arugula hot air-dried extract were measured. Phenol content was measured using the Gutfinger method (Gutfinger T., Journal of the American Oil Chemists Society, Vol. 58, No. 11, 1981). To 1 ml of the extraction sample solution, 0.5 ml of 50% Foiln-Ciocalteu's phenol reagent was added and reacted at room temperature for 3 minutes. In the reaction solution, 1 ml of saturated Na 2 CO 3 solution and 7.5 ml of distilled water were sequentially mixed and left for 30 minutes. Then, centrifuged at 12,000 rpm for 10 minutes, the supernatant was taken, and the absorbance was measured at 760 nm. The total polyphenol content was calculated according to a calibration curve prepared using gallic acid as a standard material, and GAE (Gallic acid equivalent)/g was used as the unit of measurement.
각각의 추출물에 존재하는 총 페놀 함량은 3회에 거쳐 실험한 후 나온 값의 평균값과 표준 편차값을 계산하였고 갈릭산 표준용액의 표준검정곡선을 통해 추출물 및 용매 분획물의 중량 1g당 함유하고 있는 갈릭산의 양(GAE; gallic acid equivalent)으로 환산하여 측정하였다. 갈릭산을 표준물질로 하여 측정한 결과, 하기 표 1에 나타냈다. The total phenol content present in each extract was tested three times, and the average and standard deviation values were calculated, and the gallic content per gram of weight of the extract and solvent fraction was calculated through the standard calibration curve of the gallic acid standard solution. It was measured by converting it to the amount of acid (GAE; gallic acid equivalent). The results of measurement using gallic acid as a standard material are shown in Table 1 below.
플라보노이드 함량은 Nieva Moreno 방법(김미혜, 남부대학교 박사학위논문, 2012)을 응용하여 측정하였다. 각 샘플 0.1 ㎖와 80 % 에탄올 0.9 ㎖을 혼합한 혼합물 0.5 ㎖에 10 % 질산 알루미늄과 1M 아세트산 칼륨 0.1 ㎖ 그리고 80% 에탄올 4.3㎖을 가하여 실온에 40 분 방치한 뒤 415 nm에서 흡광도를 측정하였으며, 케르세틴(quercetin)을 이용하여 작성한 표준곡선으로부터 함량을 구하였다.Flavonoid content was measured using the Nieva Moreno method (Mihye Kim, Nambu University doctoral thesis, 2012). To 0.5 ml of a mixture of 0.1 ml of each sample and 0.9 ml of 80% ethanol, 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1M potassium acetate, and 4.3 ml of 80% ethanol were added, left at room temperature for 40 minutes, and the absorbance was measured at 415 nm. The content was obtained from a standard curve prepared using quercetin.
각각의 추출물에 존재하는 총 플라보노이드 함량은 3회에 거쳐 실험한 후 나온 값의 평균값과 표준 편차값을 계산하였고 케르세틴 표준용액의 표준검정곡선을 통해 추출물 및 용매 분획물의 중량 1g당 함유하고 있는 케르세틴의 양(QE; quercetin equivalent)으로 환산하여 측정되었다. 케르세틴을 표준물질로 하여 측정한 결과를 표 1에 나타냈다. The total flavonoid content present in each extract was tested three times, and the average and standard deviation values were calculated, and the quercetin content per 1g weight of the extract and solvent fraction was calculated through the standard calibration curve of the quercetin standard solution. It was measured by converting it to quantity (QE; quercetin equivalent). The results of measurement using quercetin as a standard material are shown in Table 1.
루꼴라 동결 건조 추출물은 페놀 163.43±1.75 GAE mg/g을, 루꼴라 열풍 건조 추출물은 페놀 150.52 ± 2.00 mg/g을 함유하며, 루꼴라 동결 건조 추출물은 플라보노이드 317.88 ± 5.40 QE mg/g를, 루꼴라 열풍 건조 추출물은 플라보노이드 301.99 ± 6.14 QE mg/g를 함유함을 확인하였다. Arugula freeze-dried extract contains 163.43 ± 1.75 GAE mg/g of phenol, arugula hot-air dried extract contains phenol 150.52 ± 2.00 mg/g, arugula freeze-dried extract contains flavonoids 317.88 ± 5.40 QE mg/g, arugula hot-air dried extract contains flavonoids 317.88 ± 5.40 QE mg/g. It was confirmed that it contained 301.99 ± 6.14 QE mg/g of flavonoid.
실시예 5: 총 RNA 분리 및 실시간 중합효소 연쇄 반응(PCR)Example 5: Total RNA Isolation and Real-Time Polymerase Chain Reaction (PCR)
루꼴라 추출물이 LPS에 의해 염증이 유도된 ARPE-19 세포에서 염증관련 사이토카인(TNFα, IL-1β) 발현에 미치는 영향을 확인하기 위해 총 RNA 분리 및 실시간 중합효소 연쇄 반응(PCR)을 수행하였다.To confirm the effect of arugula extract on the expression of inflammation-related cytokines (TNFα, IL-1β) in ARPE-19 cells where inflammation was induced by LPS, total RNA isolation and real-time polymerase chain reaction (PCR) were performed.
ARPE-19 세포에 루꼴라 추출물 0.1 ~ 100 mg/mL 처리하여 최종농도 1 ~ 1000μg/mL이 되도록 한 후 3 시간 동안 배양하였다(이 때, 대조군과 LPS군은 루꼴라 추출물의 용매인 DMSO를 넣어준다). 이후 염증을 유도하는 LPS 500 μg/mL를 3 μL 넣어 최종농도 1 μg/mL가 되도록 하여 2 시간 동안 배양하였다.ARPE-19 cells were treated with 0.1 to 100 mg/mL of arugula extract to reach a final concentration of 1 to 1000 μg/mL and then cultured for 3 hours (at this time, DMSO, a solvent for arugula extract, was added to the control and LPS groups). . Afterwards, 3 μL of 500 μg/mL of LPS, which induces inflammation, was added to a final concentration of 1 μg/mL and cultured for 2 hours.
위와 같이 처리된 ARPE-19 세포는 NucleoZOL(Macherey-Nagel, Duren, Germany)을 사용하여 제조업체 지침에 따라 총 RNA를 추출하고 DNA-free™ Kit(Thermo Fisher)를 제조업체 지침에 따라 처리하였다. 추출한 RNA는 SpectraDrop™ Micro-Volume Microplate(SpectraMax iD3, Molecular Devices, San Jose, CA, USA)를 사용하여 정량하였다. 그리고 iScript™ cDNA Synthesis Kit(Bio-Rad Laboratories, Hercules, CA, USA) 지침에 따라 RNA 1 ug을 cDNA로 역전사시켰다. cDNA 합성은 T100 Thermal Cycler(Bio-Rad)를 사용하여 Priming(25℃ 5분), Reverse transcription(46℃, 20분), RT inactivation(95℃, 1분) 단계로 진행하였다.ARPE-19 cells treated as above were extracted for total RNA using NucleoZOL (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions, and treated with the DNA-free™ Kit (Thermo Fisher) according to the manufacturer's instructions. The extracted RNA was quantified using SpectraDrop™ Micro-Volume Microplate (SpectraMax iD3, Molecular Devices, San Jose, CA, USA). Then, 1 ug of RNA was reverse transcribed into cDNA according to the instructions of the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). cDNA synthesis was performed using a T100 Thermal Cycler (Bio-Rad) in the following steps: priming (25°C, 5 minutes), reverse transcription (46°C, 20 minutes), and RT inactivation (95°C, 1 minute).
qRT-PCR은 CFX Connect Real-Time PCR Detection System(BIo-Rad)을 사용하여 수행하였고 사용한 프라이머는 표 2와 같다.qRT-PCR was performed using the CFX Connect Real-Time PCR Detection System (BIo-Rad), and the primers used are listed in Table 2.
dehydrogenaseGlyceraldehyde 3-phosphate
dehydrogenase
Multiplate™ 96-Well PCR Plates(Bio-Rad)의 각 웰에 cDNA 2 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, Nuclease-free water 5 μL, SsoAdvanced Universal SYBR Green Supermix(Bio-Rad) 11 μL로 총 부피 20 μL를 넣었고 Microseal 'B' PCR Plate Sealing Film(Bio-Rad)을 덮어 반응시켰다. Thermal cycling은 polymerase activation, DNA Denaturation(95℃ 30초)가 선행되었고 Denaturation(95℃,10초)와 Annealing/Extension and Plate Read(55℃, 30초)가 49회 반복 진행되어 Melt Curve Analysis(65℃, 5초; 95℃, 5초)로 마무리되었다. 정량화는 Bio-Rad CFX Maestro로 진행하였다. House keeping gene인 GAPDH를 내부 대조군으로 사용하였으며 상대적 mRNA 수준(fold change)은 아래의 식과 같이 계산하여 표기하였다. In each well of Multiplate™ 96-Well PCR Plates (Bio-Rad), 2 μL of cDNA, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 5 μL of Nuclease-free water, and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) ) A total volume of 20 μL was added to 11 μL, and the reaction was performed by covering it with Microseal 'B' PCR Plate Sealing Film (Bio-Rad). Thermal cycling was preceded by polymerase activation and DNA denaturation (95°C, 30 seconds), and denaturation (95°C, 10 seconds) and Annealing/Extension and Plate Read (55°C, 30 seconds) were repeated 49 times, leading to Melt Curve Analysis (65°C). °C, 5 seconds; 95°C, 5 seconds). Quantification was performed with Bio-Rad CFX Maestro. GAPDH, a house keeping gene, was used as an internal control, and the relative mRNA level (fold change) was calculated and expressed according to the formula below.
Fold change = 2ESE 값 (Primer qv - GPADH qv) - 대조군 값 (Primer qv - GPADH qv) (qv : quantification value)Fold change = 2ESE value (Primer qv - GPADH qv) - Control value (Primer qv - GPADH qv) (qv: quantification value)
LPS로 유도된 ARPE-19 세포에서의 염증관련 사이토카인(TNFα, IL-1β)의 mRNA 발현 정도(fold change)는 도 3에 나타냈다. ESH와 ESF를 전처리한 ARPE-19 세포에서 LPS로 유도된 TNFα의 mRNA 발현은 LPS에 의해 유의적으로 증가하였으며 ESH의 500, 1,000 μg/mL 농도에서 20.15, 25.59% 감소하였고 ESF의 100, 500, 1,000 μg/mL 농도에서 40.76, 45.69, 51.19% 감소하였다(도 3 A, B). IL-1β의 mRNA 발현 정도는 LPS에 의하여 유의적으로 증가하였고 ESH의 100, 500, 1,000 μg/mL 농도에서는 감소하지 않았으나 ESF는 100, 500, 1,000μg/mL 농도에서 각각 98.70%, 75.83%, 59.87% mRNA 발현이 감소하였다.The mRNA expression level (fold change) of inflammation-related cytokines (TNFα, IL-1β) in ARPE-19 cells induced by LPS is shown in Figure 3. In ARPE-19 cells pretreated with ESH and ESF, LPS-induced mRNA expression of TNFα was significantly increased by LPS and decreased by 20.15 and 25.59% at concentrations of 500 and 1,000 μg/mL of ESH, and decreased by 20.15 and 25.59% at concentrations of 500 and 1,000 μg/mL of ESF, respectively. At a concentration of 1,000 μg/mL, it decreased by 40.76, 45.69, and 51.19% (Figure 3 A, B). The level of IL-1β mRNA expression was significantly increased by LPS and did not decrease at concentrations of 100, 500, and 1,000 μg/mL for ESH, but was 98.70%, 75.83%, and 75.83% for ESF at concentrations of 100, 500, and 1,000 μg/mL, respectively. 59.87% mRNA expression decreased.
ESH 1,000 μg/mL에서 TNFα를 ESF 1,000 μg/mL에서 TNFα와 IL-1β의 mRNA 발현 억제를 확인하여, 루꼴라 추출물은 염증관련 사이토카인의 mRNA 발현 수준을 낮추는 것을 확인하였다. By confirming the inhibition of mRNA expression of TNFα and IL-1β at 1,000 μg/mL of ESH and 1,000 μg/mL of ESF, it was confirmed that arugula extract lowers the mRNA expression level of inflammation-related cytokines.
실시예 6: 웨스턴 블롯Example 6: Western Blot
ESE와 LPS의 처리는 실시예 5와 동일하게 진행하였으며 ESE와 타프시가진의 처리는 ARPE-19 세포에 루꼴라 추출물 0.1 ~ 100 mg/mL 처리하여 최종농도 1 ~ 1000 μg/mL이 되도록 한 후 3 시간 배양한다(이때 대조군과 타프시가진 처리군은 루꼴라 추출물의 용매인 DMSO를 넣어준다). 이후 소포체 스트레스를 유도하는 타프시가진 5 mM을 1.5 μL 넣어 최종농도 5μM이 되도록 하여 24 시간 동안 배양하였다. The treatment of ESE and LPS was carried out in the same manner as in Example 5, and the treatment of ESE and tapsigazine was performed by treating ARPE-19 cells with 0.1 to 100 mg/mL of arugula extract to reach a final concentration of 1 to 1000 μg/mL, and then 3 Incubate for time (at this time, DMSO, a solvent for arugula extract, is added to the control group and the Tapsigin treatment group). Afterwards, 1.5 μL of 5 mM tapsigazine, which induces endoplasmic reticulum stress, was added to a final concentration of 5 μM and cultured for 24 hours.
ESE, LPS 및 타프시가진이 처리된 ARPE-19 세포는 Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail_100X(Thermo fisher) 및 radioimmunoprecipitation assay(RIPA) lysis buffer(ATTO, MA, USA)가 1 : 99 비율로 혼합된 용해 버퍼(lysis buffer)에서 추출되었다. digital sonifier(BRANSON, Danbury, CT, USA)를 사용하여 초음파 처리하고 12,000g, 4 ℃에서 20 분간 원심분리하여 상층액을 얻었다. 단백질 함량은 Pierce™ BCA Protein Assay Kit(Thermo fisher)을 사용하여 측정하였으며, 단백질 30 μg(SDS loading buffer x5 포함) 10% 혹은 15% 아크릴아미드(30% Acrylamide-Bis Solution) 젤에서 PowerPac™ HC Power Supply(Bio Rad)를 사용하여 전기영동하였다. 전기영동에 의하여 분자량대로 분리된 겔은 PVDF(polyvinylidene fluoride) 멤브레인(Millipore, Temecula, CA, USA)으로 트랜스퍼를 120 volt에서 1시간 30분 진행하였다. 그리고 5% 스킴 밀크(BD biosciences, Bedford, MA, USA)함유된 TBST(tris buffered saline with tween 20Sigma-Aldrich, St. Louis, USA)으로 1시간 동안 교반 하여 블로킹하였다. 블로킹 후 멤브레인은 TBST로 3회 세척하여 1차 항체(표 3)를 4℃에서 9~18 시간 반응시켰다. 1차 항체와 반응한 멤브레인은 TBST로 3회 세척한 후, 상온에서 2차항체로 1시간 반응시켰다. ARPE-19 cells treated with ESE, LPS, and tapsigazine were incubated with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail_100X (Thermo fisher) and radioimmunoprecipitation assay (RIPA) lysis buffer (ATTO, MA, USA) at a ratio of 1:99. Extracted from mixed lysis buffer. The supernatant was obtained by sonicating using a digital sonifier (BRANSON, Danbury, CT, USA) and centrifuging at 12,000g for 20 minutes at 4°C. Protein content was measured using the Pierce™ BCA Protein Assay Kit (Thermo fisher), and 30 μg of protein (including SDS loading buffer x5) was assayed on a 10% or 15% acrylamide (30% Acrylamide-Bis Solution) gel using PowerPac™ HC Power. Electrophoresis was performed using Supply (Bio Rad). The gel separated by molecular weight by electrophoresis was transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore, Temecula, CA, USA) at 120 volts for 1 hour and 30 minutes. Then, it was blocked by stirring for 1 hour with TBST (tris buffered saline with tween 20Sigma-Aldrich, St. Louis, USA) containing 5% skim milk (BD biosciences, Bedford, MA, USA). After blocking, the membrane was washed three times with TBST and reacted with primary antibody (Table 3) at 4°C for 9 to 18 hours. The membrane reacted with the primary antibody was washed three times with TBST and then reacted with the secondary antibody for 1 hour at room temperature.
이후 멤브레인을 TBST로 3회 세척하고 super signal west pico plus(Thermo-fisher)를 사용하여 chemidoc(Davinch western system, Seoul, Korea)으로 스캔하여 Image J software(ver 1.8, National Institutes of Health, MD, USA)를 사용하여 정량화하였다. 단백질 발현 정도는 하우스 키핑 단백질 인 α-tublin 또는 β-actin을 내부 대조군으로 사용하여 각 단백질 발현의 정량 값을 대조군에서의 정량 값에 대한 fold 값으로 나타내어 결과의 평균은 도 4 내지 5에 나타냈다. Afterwards, the membrane was washed three times with TBST, scanned with chemidoc (Davinch western system, Seoul, Korea) using super signal west pico plus (Thermo-fisher), and imaged with Image J software (ver 1.8, National Institutes of Health, MD, USA. ) was quantified using . The level of protein expression was determined by using the housekeeping protein α-tublin or β-actin as an internal control, and the quantitative value of each protein expression was expressed as a fold value with respect to the quantitative value in the control group. The average of the results is shown in Figures 4 and 5.
NF-κB 관련 단백질 발현 분석NF-κB-related protein expression analysis
인간 망막 상피세포에서 ESE의 항염증 효과를 확인하기 위해 주요 염증 신호전달 경로 단백질인 NF-κB(Nuclear Factor Kappa light chain enhancer of activatedB cells)와 IκBα(nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)의 인산화 정도를 확인하였다. To confirm the anti-inflammatory effect of ESE in human retinal epithelial cells, the major inflammatory signaling pathway proteins, NF-κB (Nuclear Factor Kappa light chain enhancer of activatedB cells) and IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells) were examined. The degree of phosphorylation of inhibitor, alpha) was confirmed.
ARPE-19 세포를 ESH 또는 ESF (0, 100, 500 또는 1,000 μg/mL)로 3 시간 동안 전처리 한 다음 LPS (1 μg/mL)로 2 시간 동안 전처리하였다. 웨스턴 블롯 분석을 위해 ARPE-19 전체 세포 용해물을 준비했으며, α-tublin은 내부 표준으로 사용하였다. 3 개의 독립적인 실험의 정량적 데이터가 각 패널에 표시되며 대표적인 웨스턴 블롯이 삽입으로 표시된다. 각 단백질의 상대적인 양은 밀도 분석에 의해 결정되었다. ARPE-19 cells were pretreated with ESH or ESF (0, 100, 500, or 1,000 μg/mL) for 3 h and then with LPS (1 μg/mL) for 2 h. ARPE-19 whole cell lysates were prepared for Western blot analysis, and α-tublin was used as an internal standard. Quantitative data from three independent experiments are shown in each panel and representative Western blots are shown as inserts. The relative amount of each protein was determined by density analysis.
ESH와 ESF를 전처리한 ARPE-19 세포에서 LPS로 유도된 p-NF-κB와 p-IκBα의 단백질 발현 정도는 도 4 A-D에 나타냈다. p-NF-κB와 p-IκBα는 모두 LPS 처리 하에 유의적으로 증가되는 것을 확인할 수 있었다. ESH가 처리된 ARPE-19 세포의 p-NF-κB는 1,000 μg/mL에서 53.53% 감소하였다. p-IκBα 또한 1,000 μg/mL에서 52.87% 감소하는 것을 확인하였다. ESF 처리된 ARPE-19 세포의 p-NF-κB 단백질 발현 정도는 100, 500, 1,000 μg/mL에서 각각 34.92, 51.38, 74.25%의 농도 의존적인 감소가 나타났다. 그 결과 ESF와 ESH 모두 NF-κB의 인산화를 억제하고 ESH는 IκBα의 인산화를 억제하여, 루꼴라 추출물의 항염증 효과를 확인하였다. The protein expression levels of p-NF-κB and p-IκBα induced by LPS in ARPE-19 cells pretreated with ESH and ESF are shown in Figure 4 A-D. Both p-NF-κB and p-IκBα were confirmed to be significantly increased under LPS treatment. p-NF-κB in ARPE-19 cells treated with ESH decreased by 53.53% at 1,000 μg/mL. p-IκBα was also confirmed to decrease by 52.87% at 1,000 μg/mL. The level of p-NF-κB protein expression in ESF-treated ARPE-19 cells showed a concentration-dependent decrease of 34.92, 51.38, and 74.25% at 100, 500, and 1,000 μg/mL, respectively. As a result, both ESF and ESH inhibited phosphorylation of NF-κB, and ESH inhibited phosphorylation of IκBα, confirming the anti-inflammatory effect of arugula extract.
MAPK 관련 단백질 발현 분석MAPK-related protein expression analysis
MAPK(Microtubule Associated Protein Kinase) familly 단백질은 LPS에 의해 자극되어 NF-κB를 활성화하거나 다른 염증 경로를 유발하기 때문에 MAPK familly 단백질의 발현을 확인하여 항염증 효과를 확인하였다. Since the MAPK (Microtubule Associated Protein Kinase) family protein is stimulated by LPS to activate NF-κB or induce other inflammatory pathways, the expression of the MAPK family protein was confirmed to confirm the anti-inflammatory effect.
ESH, ESF 전처리 및 LPS 처리된 ARPE-19 세포의 MAPK 단백질(p-JNK)의 발현 정도는 도 4 E, F에 나타냈다. p-JNK는 LPS 처리 하에 유의적으로 증가되는 것을 확인할 수 있었다. ESH가 처리된 ARPE-19 세포의 p-JNK 단백질 발현은 1,000 μg/mL에서 67.47% 유의적으로 감소하였다. The expression levels of MAPK protein (p-JNK) in ARPE-19 cells pretreated with ESH, ESF, and LPS were shown in Figure 4 E,F. p-JNK was confirmed to be significantly increased under LPS treatment. The p-JNK protein expression of ESH-treated ARPE-19 cells was significantly reduced by 67.47% at 1,000 μg/mL.
그 결과, 루꼴라 추출물은 NF-κB의 인산화를 억제하는 것과 함께 MAPK의 인산화를 억제하여 항염증 효과를 기대할 수 있다. As a result, arugula extract can be expected to have an anti-inflammatory effect by inhibiting phosphorylation of NF-κB and MAPK.
소포체 스트레스 관련 단백질 발현 분석Endoplasmic reticulum stress-related protein expression analysis
루꼴라 추출물(ESE)의 소포체 스트레스의 억제 효과를 확인하기 위해 소포체 스트레스 관련 단백질(CHOP, BiP, XBP-1)의 발현 수준을 확인하였다. 또한, 소포체 스트레스에 의한 ARPE-19의 세포사멸을 확인하기 위해 cleaved-caspase 9, cleaved-caspase 3 단백질의 발현 수준을 확인하였다. To confirm the inhibitory effect of arugula extract (ESE) on endoplasmic reticulum stress, the expression levels of endoplasmic reticulum stress-related proteins (CHOP, BiP, XBP-1) were checked. Additionally, to confirm apoptosis of ARPE-19 caused by endoplasmic reticulum stress, the expression levels of cleaved-caspase 9 and cleaved-caspase 3 proteins were checked.
ARPE-19 세포를 ESH 또는 ESF(0, 100, 500 또는 1,000 μg/mL)로 3 시간 동안 전처리 한 다음, 타프시가진(5 μM)으로 24 시간 동안 전처리하였다. ARPE-19 세포를 성장시킨 다음 웨스턴 블롯 분석을 위해 전체 세포 용해물을 준비하였다. β-actin은 내부 표준으로 사용하였다. 3 개의 독립적인 실험에서 얻은 정량 데이터가 각 패널에 표시되었으며 대표적인 웨스턴 블롯이 삽입으로 표시되었다. 각 단백질의 상대적인 양은 밀도 분석에 의해 결정되었다.ARPE-19 cells were pretreated with ESH or ESF (0, 100, 500, or 1,000 μg/mL) for 3 hours and then with tapsigazine (5 μM) for 24 hours. ARPE-19 cells were grown and whole cell lysates were prepared for Western blot analysis. β-actin was used as an internal standard. Quantitative data from three independent experiments are shown in each panel and representative Western blots are shown as inserts. The relative amount of each protein was determined by density analysis.
타프시가진 처리된 ARPE-19 세포에서 ESH, ESF 전처리에 의한 CHOP, XBP-1 단백질의 발현 정도는 그림 A 내지 F에 나타냈다. CHOP, XBP-1 모두 타프시가진 처리하에 유의적으로 증가하는 것을 확인할 수 있었다. ESH가 처리된 ARPE-19 세포에서 CHOP은 1,000 μg/mL에서 51.91%로 유의적으로 감소하였다. XBP-1 또한 1,000 μg/mL에서 40.59%로 유의적으로 감소하는 것을 확인하였다. The expression levels of CHOP and Both CHOP and XBP-1 were confirmed to significantly increase under treatment with Tapsiazine. In ARPE-19 cells treated with ESH, CHOP was significantly reduced to 51.91% at 1,000 μg/mL. XBP-1 was also confirmed to significantly decrease to 40.59% at 1,000 μg/mL.
ER 스트레스에 의하여 발생하는 ARPE-19 세포 사멸에서 ESH, ESF에 의한 cleaved-caspase 9, cleaved caspase-3 단백질의 발현 정도는 도 5 E 내지 H에 나타냈다. cleaved-caspase 9, cleaved-caspase 3는 타프시가진 처리하에 유의적으로 증가하는 것을 확인할 수 있었다. ESH가 처리된 ARPE-19 세포에서 cleaved-caspase 9은 1,000 μg/mL에서 41.20 %로 단백질 발현이 감소하였다. Cleaved-caspase 3는 1,000 μg/mL에서 48.67%로 유의적인 감소를 확인하였다. The expression levels of cleaved-caspase 9 and cleaved caspase-3 proteins by ESH and ESF in ARPE-19 cell death caused by ER stress are shown in Figure 5 E to H. cleaved-caspase 9 and cleaved-caspase 3 were confirmed to significantly increase under tapsigazine treatment. In ARPE-19 cells treated with ESH, the protein expression of cleaved-caspase 9 was reduced to 41.20% at 1,000 μg/mL. Cleaved-caspase 3 was confirmed to be significantly reduced to 48.67% at 1,000 μg/mL.
활성화된 IRE1은 XBP-1을 스플라이싱시킬 수 있고 후속적으로 발현하는 BiP(Hirota 등, 2006)과 CHOP은 소포체 스트레스를 확인하는 지표로 사용될 수 있다. CHOP에 의하여 나타나는 세포사멸에서 cleaved-caspase 9,3(활성화된 caspase)는 세포사멸을 확인할 수 있는 단백질의 발현 정도를 확인하였다. ESH가 1,000 μg/mL 농도에서 CHOP, XBP-1, cleaved-caspase 9, cleaved-caspase 3의 단백질 발현을 억제하는 것을 확인하여, ESH는 XBP-1 단백질의 발현을 억제하여 CHOP에 의하여 유도되는 세포사멸 단백질의 발현을 낮춘다고 추측된다. Activated IRE1 can splice XBP-1, and the subsequent expression of BiP (Hirota et al., 2006) and CHOP can be used as indicators to identify endoplasmic reticulum stress. In the cell death caused by CHOP, the expression level of cleaved-caspase 9,3 (activated caspase), a protein that can confirm cell death, was confirmed. It was confirmed that ESH suppresses the protein expression of CHOP, It is assumed that it lowers the expression of death proteins.
실시예 7: VEGF 억제 및 세포질 내 칼슘 수준 분석Example 7: VEGF inhibition and analysis of intracytoplasmic calcium levels
타프시가진으로 소포체 스트레스가 유도된 ARPE-19 세포에서 루꼴라 추출물(ESE)의 전처리에 의해 VEGF의 억제를 확인함으로 소포체 스트레스의 기능적인 감소를 확인하여 AMD의 예방 효과를 확인하는 실험을 수행하였다. 또한, 타프시가진으로 인하여 소포체 내 칼슘 저장의 항상성이 파괴된 세포는 세포질 내로 칼슘을 방출하기 때문에 세포질 내의 칼슘의 감소를 확인함으로 타프시가진로 인한 소포체 스트레스의 억제를 확인하여 AMD의 예방 효과를 확인해 볼 수 있다. An experiment was performed to confirm the preventive effect of AMD by confirming the functional reduction of endoplasmic reticulum stress by confirming the inhibition of VEGF by pretreatment with arugula extract (ESE) in ARPE-19 cells in which endoplasmic reticulum stress was induced with tapsigazine. In addition, cells in which the homeostasis of calcium storage in the endoplasmic reticulum is disrupted due to tapsigazine release calcium into the cytoplasm. By confirming the decrease in calcium in the cytoplasm, the inhibition of endoplasmic reticulum stress caused by tapsigazine was confirmed, confirming the preventive effect of AMD. You can check it.
ARPE-19 세포를 ESH, ESF 또는 U0126 (0, 100, 500 또는 1,000 μg/mL)으로 3 시간 동안 전처리 한 다음 타프시가진 5 μM로 24 시간 동안 전처리했다. ARPE-19 cells were pretreated with ESH, ESF, or U0126 (0, 100, 500, or 1,000 μg/mL) for 3 h and then with tapsigazine 5 μM for 24 h.
VEGF는 abcam에서 제조한 Human VEGF ELISA Kit (ab222510)를 사용하여 측정하였으며 해당 제품에서 제공하는 프로토콜에 의거하여 실험을 진행하였다. 세포질 내 칼슘수준은 Thermo Fisher Scientifi에서 제조한 Fluo-4 NW Calcium Assay Kit (F36206)을 사용하여 측정하였으며 제품에서 제공하는 프로토콜에 의거하여 실험을 진행하였다. VEGF was measured using the Human VEGF ELISA Kit (ab222510) manufactured by abcam, and the experiment was conducted according to the protocol provided by the product. The calcium level in the cytoplasm was measured using the Fluo-4 NW Calcium Assay Kit (F36206) manufactured by Thermo Fisher Scientific, and the experiment was conducted according to the protocol provided by the product.
그 결과, 타프시가진 처리된 ARPE-19 세포에서 ESH, ESF 전처리에 의한 VEGF 발생 정도는 도 6 A에 나타냈다. VEGF는 타프시가진 처리하에 유의적으로 증가하는 것을 확인할 수 있었다. VEGF는 ESH가 500, 1,000 μg/mL 농도로 처리된 ARPE-19 세포에서 농도 의존적으로 감소하였다. VEGF는 ESF가 500, 1,000 μg/mL 농도로 처리된 ARPE-19 세포에서 농도 의존적으로 감소하였다. MAPK 억제제인 U0126을 전처리하였을 때 타프시가진으로 증가된 VEGF를 억제하는 것을 확인할 수 있었으며 ESH와 ESF 1,000 μg/mL에서와 비슷한 억제 효과를 나타내었다.As a result, the degree of VEGF generation by ESH and ESF pretreatment in ARPE-19 cells treated with tapsigazine is shown in Figure 6 A. VEGF was confirmed to significantly increase under tapsigazine treatment. VEGF decreased in a concentration-dependent manner in ARPE-19 cells treated with ESH at concentrations of 500 and 1,000 μg/mL. VEGF decreased in a concentration-dependent manner in ARPE-19 cells treated with ESF at concentrations of 500 and 1,000 μg/mL. When pretreated with U0126, a MAPK inhibitor, it was confirmed that VEGF increased by tapsigazine was suppressed, and it showed a similar inhibitory effect as that of ESH and ESF at 1,000 μg/mL.
타프시가진 처리된 ARPE-19 세포에서 ESH, ESF 전처리에 의한 세포질 내 칼슘 농도는 도 6 B에 나타냈다. 세포질 내의 칼슘은 타프시가진 처리하에 유의적으로 증가하는 것을 확인할 수 있었다. ESH가 처리된 ARPE-19 세포에서 세포질 내의 칼슘은 1,000 μg/mL 농도에서 유의적으로 감소하였다.The cytoplasmic calcium concentration by ESH and ESF pretreatment in ARPE-19 cells treated with tapsigazine is shown in Figure 6B. Calcium in the cytoplasm was confirmed to significantly increase under tapsigazine treatment. In ARPE-19 cells treated with ESH, calcium in the cytoplasm was significantly decreased at a concentration of 1,000 μg/mL.
모든 실험은 3회 실시하였고 각 실험의 결과값을 평균 ± 표준편차로 표현하였다. SPSS 25.0(Statistical Package for the Social Science, SPSS Inc., Chicago, IL, USA)를 사용하여 독립표본 T 검정, one-way ANOVA를 이용하여 분석하고, P<0.05 수준에서 Tukey's post-hoc test를 실시하여 각 시료 간의 유의성을 검증하였다. All experiments were performed three times, and the results of each experiment were expressed as mean ± standard deviation. SPSS 25.0 (Statistical Package for the Social Science, SPSS Inc., Chicago, IL, USA) was used for analysis using an independent sample T test and one-way ANOVA, and Tukey's post-hoc test was performed at the P<0.05 level. The significance between each sample was verified.
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