KR102296419B1 - Pharmaceutical composition for prevention or treatment inflammatory diseases comprising comprising root extract Hovenia dulcis, or fractions thereof, or compounds isolated from therefrom - Google Patents
Pharmaceutical composition for prevention or treatment inflammatory diseases comprising comprising root extract Hovenia dulcis, or fractions thereof, or compounds isolated from therefrom Download PDFInfo
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- KR102296419B1 KR102296419B1 KR1020200152718A KR20200152718A KR102296419B1 KR 102296419 B1 KR102296419 B1 KR 102296419B1 KR 1020200152718 A KR1020200152718 A KR 1020200152718A KR 20200152718 A KR20200152718 A KR 20200152718A KR 102296419 B1 KR102296419 B1 KR 102296419B1
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- Prior art keywords
- inflammatory
- extract
- root extract
- prevention
- inflammatory diseases
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Abstract
본 발명은 헛개나무 뿌리 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 관한 것으로, 구체적으로 헛개나무 뿌리 추출물, 이의 분획물 또는 이로부터 분리된 화합물을 포함하는 예방 또는 치료용 약학적 조성물 및 예방 또는 개선용 식품 조성물에 관한 것이다.
본 발명의 헛개나무 뿌리 추출물, 이의 분획물 및 이로부터 분리한 화합물은 염증 매개인자인 염증성 사이토카인, 프로스타글란딘 E2 및 산화질소의 생성을 억제하므로 염증성 질환의 예방, 치료 또는 개선에 유용하게 사용될 수 있다. 또한, 본 발명의 헛개나무 뿌리 추출물, 이의 분획물 및 이로부터 분리한 화합물은 천연물로부터 유래한 것이므로, 염증성 질환의 예방 또는 치료 및 개선에 부작용 없이 안전하게 사용할 수 있다.The present invention relates to a composition for the prevention or treatment of inflammatory diseases comprising, as an active ingredient, an extract of the rhododendron tree root, a fraction thereof, or a compound isolated therefrom, and specifically, the extract, a fraction thereof, or a compound isolated therefrom. It relates to a pharmaceutical composition for prevention or treatment, and a food composition for prevention or improvement, including.
Since the extract of Heotgae tree root of the present invention, a fraction thereof, and a compound isolated therefrom inhibit the production of inflammatory cytokines, prostaglandin E2, and nitric oxide, which are inflammatory mediators, they can be usefully used for the prevention, treatment or improvement of inflammatory diseases. In addition, since the root extract, fractions thereof, and compounds isolated therefrom of the present invention are derived from natural products, they can be safely used for preventing or treating and improving inflammatory diseases without side effects.
Description
본 발명은 헛개나무 뿌리 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 관한 것으로, 구체적으로 헛개나무 뿌리 추출물, 이의 분획물 또는 이로부터 분리된 화합물을 포함하는 예방 또는 치료용 약학적 조성물 및 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a composition for the prevention or treatment of inflammatory diseases comprising, as an active ingredient, an extract of the rhododendron tree root, a fraction thereof, or a compound isolated therefrom, and specifically, the extract, a fraction thereof, or a compound isolated therefrom. It relates to a pharmaceutical composition for prevention or treatment, and a food composition for prevention or improvement, including.
염증 반응은 생체나 조직에 물리적 작용이나 화학적 물질, 세균감염 등의 어떠한 기질적 변화를 가져오는 침습이 가해질 때 그 손상부위를 수복 재생하려는 기전을 의미한다.Inflammatory reaction refers to a mechanism that attempts to repair and regenerate damaged areas when an invasion that causes any organic changes such as physical action, chemical substances, or bacterial infection is applied to a living body or tissue.
대식세포는 염증반응과 면역기능을 조절하며, 항상성을 유지하는데 중요한 역할을 한다. 그람 음성균의 외막성분인 리포폴리사카라이드(lipopolysaccharide, LPS)는 대식세포의 감염초기에 반응하고 숙주 방어에 중추적인 역할을 하나, 과도한 리포폴리사카라이드 자극에 의해 활성화된 대식세포는 종양 괴사 인자(tumor necrosis factor, TNF)-α, 인터류킨(interleukin, IL)-1β 및 IL-6와 같은 전염증성 사이토카인을 분비시키며, 산화질소(NO), 프로스타글란딘 E2(PGE2) 등의 염증 매개 물질을 다량 분비하게 된다(Jenog et al., 2014). NO는 대식세포가 활성화되면 유도형 산화질소 생성 효소(inducible NO synthase, iNOS)로부터 생산되며, 과도한 NO의 생성은 염증을 유발시키게 되며 병리학적 혈관 확장, 세포독성, 조직 손상 등 유해 작용을 일으키는 것으로 알려져 있다(Forstermann and Sessa, 2012). TNF-α, IL-1b 및 IL-6 와 같은 전염증성 사이토카인 및 단백질의 유전자 발현은 유사분열 활성화 단백질 인산화효소 (Mitogen-activated protein kinases, MAPKs)와 핵 인자 카파 B(nuclear factor kappa B, NF-kB)에 의해 조절된다. 면역과 염증 반응에 관계된 유전자의 발현에 있어서 NF-kB가 활성화되면 NF-kB와 결합해 있던 IkBα(inhibitory kappa B α)가 분해되면서 NF-kB가 세포 원형질에서 핵으로 들어가게 되며 이후 TNF-α, IL-6와 같은 사이토카인 발현의 전사 인자로서 중요한 역할을 한다(Jang et al., 2005, Majdalawieh and Ro, 2010).Macrophages regulate inflammatory response and immune function, and play an important role in maintaining homeostasis. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, responds to macrophages in the early stage of infection and plays a pivotal role in host defense. It secretes pro-inflammatory cytokines such as tumor necrosis factor, TNF)-α, interleukin (IL)-1β and IL-6, and secretes large amounts of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). (Jenog et al., 2014). NO is produced from inducible NO synthase (iNOS) when macrophages are activated, and excessive NO production causes inflammation and causes harmful effects such as pathological vasodilation, cytotoxicity and tissue damage known (Forstermann and Sessa, 2012). Gene expression of pro-inflammatory cytokines and proteins such as TNF-α, IL-1b and IL-6 is dependent on mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF). -kB). When NF-kB is activated in the expression of genes related to immune and inflammatory responses, IkBα (inhibitory kappa B α) bound to NF-kB is degraded, NF-kB enters the nucleus from the cell protoplasm, and then TNF-α, It plays an important role as a transcription factor in the expression of cytokines such as IL-6 (Jang et al., 2005, Majdalawieh and Ro, 2010).
힘 옥시게나제(Heme oxygenase, HO)의 대사를 조절하는 효소인 HO-1은 세포의 힘(heme)을 분해하여 일산화탄소(CO), 제1철(ferrous iron) 및 빌리버딘(biliverdin)으로 전환하게 되고, 빌리버딘은 다시 환원효소에 의해 빌리루빈(bilirubin)으로 환원된다(Elbirt and Bonkovsky, 1999). HO-1과 생성된 부산물들은 항염증 및 항세포사멸에 관련된 유전자 발현과 효소의 활성에 영향과 산화적 스트레스로부터 세포와 조직을 보호하는 효소로 인식되고 있다(Gozzelino et al., 2010, Tsan et al., 1989). 빌리루빈은 iNOS와 COX-2의 발현 및 NO의 생성을 억제하며 빌리버딘은 전염증성 사이토카인인 IL-6와 IL-1β의 생성을 억제시키는 것으로 보고 되었다. 특히 리포폴리사카라이드를 처리한 RAW 264.7 대식세포에서 HO-1은 NO의 생성과 iNOS 유전자 발현을 억제함으로써 항염증 작용을 통하여 세포 손상을 억제한다는 연구 결과가 보고 되었다. HO-1, an enzyme that regulates the metabolism of heme oxygenase (HO), breaks down cellular heme into carbon monoxide (CO), ferrous iron and biliverdin. bilirubin, which is then reduced to bilirubin by reductase (Elbirt and Bonkovsky, 1999). HO-1 and its by-products are recognized as enzymes that protect cells and tissues from oxidative stress and affect the activity of enzymes and gene expression related to anti-inflammatory and anti-apoptosis (Gozzelino et al., 2010, Tsan et al.) al., 1989). It has been reported that bilirubin inhibits the expression of iNOS and COX-2 and the production of NO, and biliburdin inhibits the production of pro-inflammatory cytokines IL-6 and IL-1β. In particular, it has been reported that HO-1 inhibits NO production and iNOS gene expression in RAW 264.7 macrophages treated with lipopolysaccharide, thereby inhibiting cell damage through anti-inflammatory action.
헛개나무(Hovenia dulcis Thunb.)는 갈매나무과의 납엽활엽교목으로 내한성과 내음성이 강하고 맹아력이 강한 수종으로, 우리나라에서는 설악산, 오대산, 지리산 및 한라산 등에 주로 자라며, 중북부 지방보다는 남쪽지방에서 잘 생육한다. 헛개나무의 많은 화학성분이 알려졌으며 주요 활성물질로는 사포닌 계열의 호베노사이드(Hovenoside) I-VII, 호바세르보사이드(Hovacerboside) A1, 호베니둘시오사이드(Hovenidulcioside) A1, A2, B1, B2, 호둘로사이드(Hoduloside) III 등과 플라보노이드 (Flavonoid) 계열의 퀘르세틴(Quercetin), 캠페롤(Kaempferol), (+)암펠로신(Ampelosin), 호베니틴(Hovenitin) I~III, 호베노둘리놀(Hovenodulinol) 등이 분리되어 구조가 연구되었다(Park et al., 2015). 지금까지 보고된 약리 활성으로 단맛 억제 효과, 간세포 보호 효과, 근육 이완 억제 효과, 이뇨작용, 항산화 작용, 항암작용 및 당뇨 치료 효과 등이 보고되고 있다(Yand et al., 2013). 최근 연구에서는 헛개나무 열매 추출물의 항염증 효능 및 성분 분석에 대한 문헌이 보고되었다(Park et al., 2016).Hovenia dulcis Thunb. do. Numerous chemical components of the sycamore tree are known, and the main active substances include saponin-type Hovenoside I-VII, Hovacerboside A1, Hovenidulcioside A1, A2, B1, B2, Hoduloside III and other flavonoids Quercetin, Kaempferol, (+) Ampelosin, Hovenitin I~III, Hobenoduli Hovenodulinol and others have been isolated and studied (Park et al., 2015). The pharmacological activities reported so far include a sweetening inhibitory effect, hepatocellular protective effect, muscle relaxation inhibitory effect, diuretic, antioxidant, anticancer, and diabetes treatment effect (Yand et al., 2013). In a recent study, the literature on the anti-inflammatory efficacy and compositional analysis of extracts from sycamore trees was reported (Park et al., 2016).
헛개나무 열매 추출물이 항염증 효과를 나타낸 보고가 있으나 부위별(잎, 줄기, 뿌리) 추출물의 항염증 효과에 대한 연구는 전무하다.Although there have been reports of anti-inflammatory effects of the fruit extract of serrata, there has been no study on the anti-inflammatory effects of extracts for each part (leaf, stem, root).
그러므로 헛개나무 뿌리 추출물을 활용하여 염증성 질환 치료 및 예방에 뛰어난 효능을 보이고 천연 원료를 사용하여 부작용과 약물 내성을 줄일 수 있는 염증성 질환 예방 또는 치료용 조성물의 개발이 필요한 실정이다.Therefore, there is a need for the development of a composition for preventing or treating inflammatory diseases that can reduce side effects and drug resistance by using natural raw materials and show excellent efficacy in the treatment and prevention of inflammatory diseases by using the root extract of Heotgae tree.
이에 본 발명에서는 헛개나무 부위별(잎, 줄기, 뿌리) 추출물의 NO 생성 억제효과에서 탁월한 뿌리추출물의 항염증 효과를 확인하기 위해 NO, 프로스타글란딘 E2의 생성량과 전염증성 사이토카인 (IL-1β, IL-6, TNF-α) 분비를 측정하였고, 그 조절기전으로 HO-1, MAPKs 및 Ik-Ba를 조사하였다. 또한, 헛개나무 뿌리 추출물로부터 유효성분 분리 및 정제하여 분광학적 방법으로 화학 구조를 동정하였다.Accordingly, in the present invention, in order to confirm the anti-inflammatory effect of the root extract, which is excellent in the NO production inhibitory effect of the extracts of each part (leaf, stem, and root) of the H. -6, TNF-α) secretion was measured, and HO-1, MAPKs and Ik-Ba were investigated as regulatory mechanisms. In addition, the chemical structure was identified by spectroscopic method by separating and purifying the active ingredient from the root extract of Heotgae tree.
본 발명의 하나의 목적은 헛개나무 뿌리 추출물 또는 이의 분획물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising an extract of Heotgae tree root or a fraction thereof as an active ingredient.
본 발명의 다른 하나의 목적은 헛개나무 뿌리 추출물 또는 이의 분획물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for the prevention or improvement of inflammatory diseases, comprising the Heotgae root extract or a fraction thereof as an active ingredient.
본 발명의 또 다른 하나의 목적은 하기 화학식 Ⅰ의 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising a compound of Formula I as an active ingredient.
[화학식 Ⅰ][Formula Ⅰ]
본 발명의 또 다른 하나의 목적은 하기 화학식 Ⅰ의 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for the prevention or improvement of inflammatory diseases comprising the compound of Formula I as an active ingredient.
[화학식 Ⅰ][Formula Ⅰ]
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 헛개나무 뿌리 추출물 또는 이의 분획물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising the Heotgae tree root extract or a fraction thereof as an active ingredient.
본 발명에서는, 헛개나무 뿌리 추출물은 염증성 사이토카인, 프로스타글란딘 E2 및 산화질소와 같은 염증 매개인자의 생성을 억제하는 효과를 나타냄을 확인함으로써, 상기 추출물은 염증성 질환 치료제의 유효성분으로 사용될 수 있음을 확인하였다.In the present invention, it was confirmed that the extract of Heotgae tree root exhibits an effect of inhibiting the production of inflammatory mediators such as inflammatory cytokines, prostaglandin E2 and nitric oxide, thereby confirming that the extract can be used as an active ingredient for treating inflammatory diseases. did.
본 발명에 있어서, 헛개나무는 시장으로부터 구입하거나 직접 채취한 것일 수 있다. 본 발명의 헛개나무는 한국산 (Hovenia dulcis var. Korean Nakai.), 일본산 (Hovenia dulcis var. tomentella Makino) 및 중국산 (Hovenia acerba Lindl., Hoveniaacerba var. kiukiangensis C. Y. WU, Hovenia trichocarpa Chun et Tsiang, Hovenia trichocarpa var. fulvotomemtosa) 헛개나무를 모두 포함한다.In the present invention, Heotgae tree may be purchased from the market or directly collected. Hovenia dulcis var. Korean Nakai.), Japanese (Hovenia dulcis var. tomentella Makino) and Chinese (Hovenia acerba Lindl., Hoveniaacerba var. kiukiangensis CY WU, Hovenia trichocarpa Chun et Tsiang, Hovenia trichocarpa Chun et Tsiang) var.
발명의 용어, "추출물"은 상기 헛개나무 뿌리를 추출 처리하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다.As used herein, the term "extract" refers to an extract obtained by extracting the roots of H. serrata, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as an extract It includes extracts of all formulations that can be formed by themselves and using extracts.
상기 헛개나무 뿌리를 추출하는 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 열수 추출법, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다.The method of extracting the root of Heotgae tree is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, and these may be performed alone or in combination of two or more methods.
본 발명에서 상기 헛개나무 뿌리를 추출하는데 사용되는 추출 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다.In the present invention, the type of the extraction solvent used for extracting the root of Heotgae tree is not particularly limited, and any solvent known in the art may be used.
상기 추출 용매의 비제한적인 예로는 물, 탄소수 1 내지 4의 알코올 또는 이들의 혼합 용매 등을 들 수 있으며, 이들은 단독으로 사용되거나 1종 이상 혼합하여 사용될 수 있다. 구체적으로 에탄올이 사용될 수 있지만, 이에 제한되는 것은 아니며, 상기 에탄올은 0.1 내지 99.99%(v/v), 구체적으로 30 내지 90%(v/v), 더욱 구체적으로 80%(v/v) 메탄올일 수 있지만, 이에 제한되는 것은 아니다.Non-limiting examples of the extraction solvent include water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and these may be used alone or in combination of one or more. Specifically, ethanol may be used, but is not limited thereto, and the ethanol is 0.1 to 99.99% (v/v), specifically 30 to 90% (v/v), more specifically 80% (v/v) methanol may be, but is not limited thereto.
본 발명에서, 상기 헛개나무 뿌리 추출물은 헛개나무 뿌리 80%(v/v) 메탄올 추출물 또는 물 추출물일 수 있다.In the present invention, the Heotgae root extract may be an 80% (v/v) methanol extract or water extract of Heotgae root.
상기 헛개나무 뿌리 에탄올 추출물을 제조하는 일 예로, 헛개나무 뿌리 분쇄물을 메탄올로 추출한 뒤, 감압농축하고 동결건조하여 헛개나무 뿌리 에탄올 추출물을 제조할 수 있다.As an example of preparing the Heotgae root ethanol extract, after extracting the pulverized Heotgae root with methanol, concentrated under reduced pressure and freeze-dried, the Heotgae root ethanol extract can be prepared.
본 발명의 용어, "분획물"은 여러 다양한 구성 성분들을 포함하는 혼합물로부터 특정 성분 또는 특정 성분 그룹을 분리하기 위하여 분획을 수행하여 얻어진 결과물을 의미한다. As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific group of components from a mixture including various components.
본 발명에서 상기 분획물을 얻는 분획 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 수행될 수 있다. 상기 분획 방법의 비제한적인 예로는, 다양한 용매를 처리하여 수행하는 용매 분획법, 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 수행하는 한외여과 분획법, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)를 수행하는 크로마토그래피 분획법, 및 이들의 조합 등이 있다. 구체적으로, 본 발명의 헛개나무 뿌리를 추출하여 얻은 추출물에 소정의 용매를 처리하여 상기 추출물로부터 분획물을 얻는 방법을 들 수 있다.The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity) or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof. Specifically, there may be mentioned a method of obtaining a fraction from the extract by treating the extract obtained by extracting the root of the present invention with a predetermined solvent.
본 발명에서 상기 분획물을 얻는 데 사용되는 분획 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 분획 용매의 비제한적인 예로는 물, 탄소수 1 내지 4의 알코올 등의 극성 용매; 헥산(Hexan), 에틸 아세테이트(Ethyl acetate), 클로로포름(Chloroform), 디클로로메탄(Dichloromethane) 등의 비극성 용매; 또는 이들의 혼합용매 등을 들 수 있다. 이들은 단독으로 사용되거나 1종 이상 혼합하여 사용될 수 있지만, 이에 제한되는 것은 아니며, 구체적으로, 헥산, 에틸 아세테이트, 부탄올(butanol) 또는 물이 단독으로 사용되거나 1종 이상 혼합하여 사용될 수 있지만, 이에 제한되는 것은 아니다. In the present invention, the type of the fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, a polar solvent such as an alcohol having 1 to 4 carbon atoms; hexane (Hexan), ethyl acetate (Ethyl acetate), chloroform (Chloroform), a non-polar solvent such as dichloromethane (Dichloromethane); or a mixed solvent thereof. These may be used alone or in combination of one or more, but are not limited thereto, and specifically, hexane, ethyl acetate, butanol, or water may be used alone or in combination of one or more, but limited thereto it's not going to be
또한, 상기 추출물 또는 분획물은 추출 후 건조 분말 형태로 제조되어 사용될 수 있지만, 이에 제한되는 것은 아니다.In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.
본 발명의 헛개나무 뿌리 추출물 및 이의 분획물은 염증성 질환의 예방 또는 치료에 효과적으로 사용될 수 있다.The Heotgae root extract and its fractions of the present invention can be effectively used for the prevention or treatment of inflammatory diseases.
본 발명에서 용어 “염증성 질환”은 아토피 피부염, 부종, 피부염, 알레르기, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome) 및 다발성 경화증으로 이루어지는 군으로부터 선택되는 어느 하나 이상인 것을 포함하나, 이에 제한되지 않는다.As used herein, the term “inflammatory disease” refers to atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn’s disease, colitis, hemorrhoids, gout, ankylosing spondylitis, Any one or more selected from the group consisting of rheumatoid lupus lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, peri-shoulder arthritis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, and multiple sclerosis including, but not limited to.
본 발명에 있어서, 용어 “예방”은 상기 조성물의 투여에 의해 염증성 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.In the present invention, the term “prevention” refers to any action that suppresses or delays the onset of an inflammatory disease by administration of the composition.
본 발명에 있어서, 용어 “치료”는 상기 조성물의 투여에 의해 염증성 질환을 낫게 하는 행위를 모두 의미하는 것으로, 질환, 질환의 증상, 질병 또는 질환의 2차 질환, 또는 이에 대한 소인(predisposition)을 치료, 경감, 완화, 요법(remedy), 또는 향상하기 위한 목적과 함께 질환, 질환의 증상, 질병 또는 질환의 2차 질환, 또는 이에 대한 소인을 갖는 피험자(인간 또는 동물)에게 헛개나무 뿌리 추출물을 포함하는 조성물의 적용 또는 투여로 정의된다.In the present invention, the term “treatment” refers to all acts of curing an inflammatory disease by administration of the composition, and a disease, a symptom of a disease, a secondary disease of the disease or disease, or a predisposition thereof To a subject (human or animal) having a disease, a symptom of a disease, a secondary disease of the disease or disorder, or a predisposition therefor, for the purpose of treatment, alleviation, alleviation, remedy, or amelioration It is defined as the application or administration of a composition comprising
본 발명의 약학적 조성물은 총 조성물의 중량 대비 상기 헛개나무 뿌리 추출물을 0.0001 내지 80 중량%로 포함할 수 있으며, 구체적으로 0.01 중량% 내지 40 중량%로 포함할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may include 0.0001 to 80% by weight of the H. root extract relative to the total weight of the composition, specifically 0.01 to 40% by weight, but is not limited thereto.
본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 약학적으로 허용가능한 담체, 부형제 또는 희석제를 추가로 포함할 수 있고, 상기 담체는 비자연적 담체(non-naturally occuring carrier)를 포함할 수 있다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, wherein the carrier includes a non-naturally occurring carrier. can do.
상기 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. .
상기 "약학적으로 허용가능한"은 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다.The "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.
구체적으로, 상기 담체의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용되고 약학적으로 허용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다. 또한, 필요한 경우 항산화제, 완충액 및/또는 정균제 등 다른 통상의 첨가제를 첨가하여 사용할 수 있으며, 희석제, 분산제, 계면 활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제 등으로 제제화하여 사용할 수 있다.Specifically, the type of the carrier is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in mixture of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostats can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. It can be used by formulating it into a dosage form, pill, capsule, granule, or tablet.
본 발명에 따른 염증성 질환의 예방 또는 치료용 약학적 조성물의 투여 방식은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방식에 따를 수 있다. 상기 투여 방식의 비제한적인 예로, 조성물을 경구 투여 또는 비경구 투여 방식으로 투여할 수 있다. 또한, 본 발명의 염증성 질환의 예방, 개선 또는 치료용 조성물은 목적하는 투여 방식에 따라 다양한 제형으로 제조될 수 있다.The administration method of the pharmaceutical composition for the prevention or treatment of inflammatory diseases according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. In addition, the composition for preventing, improving or treating an inflammatory disease of the present invention can be prepared in various formulations according to the desired administration method.
본 발명의 또 다른 하나의 양태는 헛개나무 뿌리 추출물 또는 이의 분획물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for the prevention or improvement of inflammatory diseases, comprising the Heotgae root extract or a fraction thereof as an active ingredient.
이때, 상기 헛개나무 뿌리, 추출물, 분획물, 염증성 질환 및 예방의 정의는 상기에서 설명한 바와 같다.In this case, the definitions of the hutgae tree root, extract, fraction, inflammatory disease and prevention are the same as described above.
본 발명의 용어, "개선"은 본 발명의 조성물의 투여로 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter, eg, a symptom, associated with a condition to be treated by administration of the composition of the present invention.
본 발명의 용어, "식품"은 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료, 비타민 복합제, 건강 기능 식품 및 건강 식품 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.
본 발명의 식품 조성물은 일상적으로 섭취하는 것이 가능하기 때문에 높은 염증성 질환 개선 효과를 기대할 수 있으므로, 건강 증진 목적으로 매우 유용하게 사용될 수 있다.Since the food composition of the present invention can be consumed on a daily basis, a high inflammatory disease improvement effect can be expected, and thus it can be very usefully used for health promotion purposes.
상기 건강 기능(성) 식품(functional food)이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 '기능(성)'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한, 상기 식품의 제형은 식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품용 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 천연물을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나므로, 본 발명의 식품은 염증성 질환의 개선 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using a natural product as a raw material, and it is excellent in portability. The food of the present invention can be ingested as an adjuvant for enhancing the improvement effect of inflammatory diseases.
상기 건강 식품(health food)은 일반식품에 비해 적극적인 건강유지나 증진 효과를 가지는 식품을 의미하고, 건강보조식품(health supplement food)은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강 기능 식품, 건강식품, 건강보조식품의 용어는 호용된다.The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are preferred.
구체적으로, 상기 건강 기능 식품은 본 발명의 추출물을 음료, 차류, 향신료, 껌, 과자류 등의 식품 소재에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용이 없는 장점이 있다. Specifically, the health functional food is a food prepared by adding the extract of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc., and when ingested, It means to bring an effect, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.
상기 식품 조성물은 생리학적으로 허용 가능한 담체를 추가로 포함할 수 있는데, 담체의 종류는 특별히 제한되지 않으며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다. The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
또한, 상기 식품 조성물은 식품 조성물에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu), 크륨(Cr) 등의 미네랄; 및 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다.In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.
또한, 상기 식품 조성물은 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 데히드로초산나트륨 등), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨류엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 글루타민산나트륨 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(food additives)을 포함할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, improvers, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 식품 조성물의 일 예로 건강음료 조성물로 사용될 수 있으며, 이 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 슈크로스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 건강음료 조성물 100 mL 당 일반적으로 약 0.001 ~ 0.4 g, 구체적으로 약 0.002 ~ 0.3 g이 될 수 있다.As an example of the food composition of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatin, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.001 to 0.4 g, specifically about 0.002 to 0.3 g per 100 mL of the health beverage composition of the present invention.
상기 외에 건강음료 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일주스, 과일주스 음료, 또는 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 건강음료 조성물 100 중량부당 0.001 ~ 0.10 중량부의 범위에서 선택되는 것이 일반적이다. In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.001 to 0.10 parts by weight per 100 parts by weight of the health beverage composition of the present invention.
본 발명의 또 다른 하나의 양태는 하기 화학식 Ⅰ의 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising a compound of Formula I as an active ingredient.
[화학식 Ⅰ][Formula Ⅰ]
이때, 상기 헛개나무 뿌리, 추출물, 염증성 질환, 예방, 치료 및 약학적 조성물의 정의는 상기에서 설명한 바와 같다.At this time, the definitions of the root, extract, inflammatory disease, prophylaxis, treatment and pharmaceutical composition of the wormwood tree are as described above.
본 발명에서 “화학식 Ⅰ”은 하기 화학식으로 표시되는 화합물을 의미한다.In the present invention, "Formula I" means a compound represented by the following formula.
본 발명의 상기 화학식 Ⅰ로 표시되는 화합물은 27-O-프로토카테쿠오일베툴린산(27-O-protocatechuoylbetulinic acid)으로 명명된다.The compound represented by Formula I of the present invention is named 27-O-protocatechuoylbetulinic acid.
상기 27-O-프로토카테쿠오일베툴린산은 구체적으로 헛개나무 뿌리로부터 분리된 것일 수 있으나, 이에 특별히 제한되는 것은 아니며, 천연물에서 분리된 것 또는 상업적으로 판매하는 것을 구입하여 사용할 수 있다.The 27-O-protocatequoyl betulinic acid may be specifically isolated from the root of the serrata, but is not particularly limited thereto, and may be purchased and used either isolated from natural products or commercially available.
본 발명에서, 상기 화학식 Ⅰ로 표시되는 화합물을 천연으로부터 분리 및 정제하여 사용할 경우에는, 종래 물질을 추출하고 분리하는 방법을 이용하여 천연재료로부터 수득할 수 있으며, 상기 화학식 Ⅰ로 표시되는 화합물을 천연으로부터 분리 및 정제하기 위한 추출방법은 물 또는 유기용매를 통한 용매추출법을 사용할 수 있다. 이때, 상기 유기용매로는 이에 제한되지는 않으나, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol), 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다.In the present invention, when the compound represented by Formula I is separated and purified from nature, it can be obtained from natural materials by using a conventional method for extracting and separating a substance, and the compound represented by Formula I can be obtained from natural sources. As an extraction method for separation and purification from , a solvent extraction method through water or an organic solvent may be used. At this time, the organic solvent is not limited thereto, but methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene ( Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.
또한, 상기 방법으로부터 수득한 추출물은 이후 당업계에 공지된 분리 및 정제 방법을 사용하여 상기 추출물로부터 활성 성분을 수득할 수 있는데, 일반적으로 실리카겔(silica gel)이나 활성 알루미나(alumina)등의 각종 합성수지를 충진한 컬럼 크로마토그래피(column chromatography) 및 고속액체크로마토그라피(HPLC) 등을 단독으로 혹은 병행하여 사용할 수 있다. 활성성분의 추출 및 분리정제 방법은 반드시 상기한 방법에 한정되는 것은 아니다.In addition, the extract obtained from the above method can then obtain an active ingredient from the extract using a separation and purification method known in the art. In general, various synthetic resins such as silica gel or activated alumina Column chromatography and high-performance liquid chromatography (HPLC) may be used alone or in combination. The extraction and separation and purification methods of the active ingredients are not necessarily limited to the above methods.
본 발명의 또 다른 하나의 양태는 하기 화학식 Ⅰ의 화합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for preventing or improving inflammatory diseases comprising a compound of Formula I as an active ingredient.
[화학식 Ⅰ][Formula Ⅰ]
이때, 상기 헛개나무 뿌리, 추출물, 분리 및 정제 과정, 염증성 질환, 예방, 개선, 식품 조성물 및 화학식 Ⅰ로 표시되는 화합물인 27-O-프로토카테쿠오일툴베린산의 정의는 상기에서 설명한 바와 같다.At this time, the definition of 27-O-protocatequoyl tulberic acid, which is the compound represented by formula I, inflammatory disease, prevention, improvement, food composition, and inflammatory disease, is the same as described above. .
중복되는 내용은 본 명세서의 복잡성을 고려하여 생락하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술 분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the technical field to which the present invention pertains.
본 발명의 헛개나무 뿌리 추출물, 이의 분획물 및 이로부터 분리한 화합물은 염증 매개인자인 염증성 사이토카인, 프로스타글란딘 E2 및 산화질소의 생성을 억제하므로 염증성 질환의 예방, 치료 또는 개선에 유용하게 사용될 수 있다. 또한, 본 발명의 헛개나무 뿌리 추출물, 이의 분획물 및 이로부터 분리한 화합물은 천연물로부터 유래한 것이므로, 염증성 질환의 예방 또는 치료 및 개선에 부작용 없이 안전하게 사용할 수 있다.Since the extract of Heotgae tree root of the present invention, a fraction thereof, and a compound isolated therefrom inhibit the production of inflammatory cytokines, prostaglandin E2, and nitric oxide, which are inflammatory mediators, they can be usefully used for the prevention, treatment or improvement of inflammatory diseases. In addition, since the root extract, fractions thereof, and compounds isolated therefrom of the present invention are derived from natural products, they can be safely used for preventing or treating and improving inflammatory diseases without side effects.
도 1a, 도 1b, 도 1c 및 도 1d는 헛개나무 뿌리 추출물이 리포폴리사카라이드로 자극한 RAW 264.7 세포 내에서 산화질소 및 프로스타글란딘 E2 생산, iNOS mRNA 및 COX-2 mRNA 발현에 대해 미치는 영향을 확인하고자 RAW 264.7 세포를 LPS (1 μg/ml)의 존재 또는 부재하에 24 시간 동안 표시된 수준의 RE로 자극시켰을 때의 결과를 나타낸 것으로, 도 1a는 배양 배지 내 그리스 반응을 사용하여 NO의 양을 결정한 것이며, 도 1b는 세포 배양 배지 내 PGE2 수준을 ELISA 분석으로 검출한 것이며, 도 1c 및 도 1d는 LPS 자극으로부터 6시간 후 각각 iNOS 또는 COX-2 단백질 수준을 분석한 것이다.
도 2a, 도 2b, 도 2c, 도 2d, 도 2e, 도 2f 및 도 2g는 RAW 264.7 세포 내에서 헛개나무 뿌리 추출물이 전염증성 사이토카인의 발현에 대해 미치는 영향을 확인하고자 뿌리 추출물 및/또는 LPS(1 μg/ml)를 24시간 동안 표시된 농도로 처리하였을 때의 결과를 나타낸 것으로, 도 2a, 도 2b 및 도 2c는 각각 ELISA 분석법을 사용하여 배양 배지 내 각각 IL-6, IL-1β 또는 TNF-α의 단백질 수준을 측정한 것이며, 도 2d, 도 2e 및 도 2f는 특이적 프라이머를 이용하여 실시간-PCR 분석으로 각각 IL-6, IL-1β 또는 TNF-α의 mRNA 수준을 측정한 것이며, 도 2g는 뿌리 추출물로 24시간 동안 처리한 후 세포 생존력을 세포 카운팅 키트-8로 평가하고 그 결과를 비처리 대조군과 비교하여 백분율로 나타낸 것이다.
도 3a 및 도 3b는 리포폴리사카라이드-자극 RAW 264.7 대식 세포 내 NF-kB 경로 활성화에 대한 헛개나무 뿌리 추출물이 주는 영향을 확인하고자 세포를 5분간 리포폴리사카라이드(1 mg/ml) 처리 전에 표시된 농도로 헛개나무 뿌리 추출물로 30분간 전처리한 결과를 나타낸 것으로, 도 3a는 핵 및 시토졸 단백질을 표시된 항체로 웨스턴 블롯 분석한 결과를 나타낸 것이며, 도 3b는 상기 실험의 결과를 상대밀도 그래프로 나타낸 것이다.
도 4a는 RAW 264.7 세포 내 리포폴리사카라이드-자극 MAPKs 활성화에 대한 헛개나무 뿌리 추출물의 영향을 확인하고자 ERK, p38 및 JNK의 인산화 및 총 단백질 발현을 리포폴리사카라이드로-자극 헛개나무 뿌리 추출물 세포로부터 추출하여 웨스턴 블롯으로 분석한 것이며, 도 4b는 상기 실험의 결과를 상대밀도 그래프로 나타낸 것이다.
도 5a, 도 5b 및 도 5c는 LPS 자극 RAW 264.7 세포 내 시토졸 Nrf2, 핵 Nrf2 및 HO-1 발현에 대한 헛개나무 뿌리 추출물의 효과를 확인하고자, 세포를 다양한 농도의 헛개나무 뿌리 추출물(10 내지 40 μg/ml)로 1시간 동안 전처리하고 리포폴리사카라이드(1 μg/ml)로 24시간 동안 자극한 결과를 나타낸 것으로, 도 5a는 시토졸 Nrf2, 핵 Nrf2 및 HO-1 단백질 발현을 웨스턴 블랏으로 분석한 결과를 나타낸 것이며, 도 5b는 상기 실험의 결과를 상대밀도 그래프로 나타낸 것이며, 도 5c는 헛개나무 뿌리 추출물 투여 농도에 따른 HO-1의 mRNA 발현 정도를 그래프로 나타낸 것이다.
도 6a 및 도 6b는 LPS 자극 RAW 264.7 세포 내 iNOS, COX-2, HO-1 및 Nrf2 발현에 대한 헛개나무 뿌리 추출물의 효과를 확인하고자, 세포를 다양한 농도의 헛개나무 뿌리 추출물(10 내지 40 μg/ml)을 SnPP(tin protoporphyrin IX) 10 μM로 1시간 동안 먼저 전처리하였고 리포폴리사카라이드(1 μg/ml)로 24시간 동안 자극한 결과를 나타낸 것으로, 도 6a는 NOS, COX-2, HO-1 및 Nrf2 단백질 발현을 웨스턴 블랏으로 분석한 결과를 나타낸 것이며, 도 6b는 상기 실험의 결과를 상대밀도 그래프로 나타낸 것이다.
도 7a는 254nm에서 헛개나무 부위에 따른(뿌리, 줄기, 잎) 추출물의 대표 HPLC 크로마토그램을 나타낸 것이며, 도 7b는 헛개나무 뿌리 추출물의 주요 화합물의 구조인 27-O-프로토카테쿠오일베툴린산을 나타낸 것이다.1a, 1b, 1c, and 1d show the effect of the H. root extract on nitric oxide and prostaglandin E2 production, iNOS mRNA and COX-2 mRNA expression in lipopolysaccharide-stimulated RAW 264.7 cells. To show the results when RAW 264.7 cells were stimulated with the indicated levels of RE for 24 hours in the presence or absence of LPS (1 μg/ml), Figure 1a shows the determination of the amount of NO using a grease reaction in the culture medium 1b shows the detection of PGE2 levels in the cell culture medium by ELISA analysis, and FIGS. 1c and 1d show iNOS or COX-2 protein levels analyzed after 6 hours from LPS stimulation, respectively.
2a, 2b, 2c, 2d, 2e, 2f and 2g show the effect of H. root extracts on the expression of pro-inflammatory cytokines in RAW 264.7 cells. Root extracts and/or LPS (1 μg/ml) shows the results when treated at the indicated concentrations for 24 hours, FIGS. 2a, 2b and 2c are respectively IL-6, IL-1β or TNF in the culture medium using an ELISA assay. The protein level of -α was measured, and FIGS. 2d, 2e and 2f are real-time-PCR analysis using specific primers to measure the mRNA level of IL-6, IL-1β or TNF-α, respectively, Figure 2g shows the cell viability was evaluated with the cell counting kit-8 after treatment with the root extract for 24 hours, and the results are expressed as a percentage compared to the untreated control.
Figures 3a and 3b are lipopolysaccharide-stimulated RAW 264.7 macrophage NF-kB pathway activation in macrophages in order to determine the effect of the extract of the
Figure 4a shows the phosphorylation of ERK, p38 and JNK and total protein expression of ERK, p38, and JNK to lipopolysaccharide-stimulated H. root extract cells to confirm the effect of lipopolysaccharide-stimulated MAPKs activation in RAW 264.7 cells. was extracted and analyzed by Western blot, and FIG. 4b shows the results of the experiment as a graph of relative density.
5a, 5b, and 5c are diagrams showing the effect of H. serratus root extract on LPS-stimulated RAW 264.7 intracellular cytosolic Nrf2, nuclear Nrf2 and HO-1 expression, cells were treated with various concentrations of H. 40 μg/ml) for 1 hour and stimulation with lipopolysaccharide (1 μg/ml) for 24 hours. 5b is a graph showing the results of the above experiment, and FIG. 5c is a graph showing the mRNA expression level of HO-1 according to the concentration of Heotgae root extract administered.
6a and 6b show the effect of H. rhododendron root extract on the expression of iNOS, COX-2, HO-1 and Nrf2 in LPS-stimulated RAW 264.7 cells. /ml) was first pretreated with 10 μM of SnPP (tin protoporphyrin IX) for 1 hour and stimulation with lipopolysaccharide (1 μg/ml) for 24 hours, Figure 6a shows NOS, COX-2, HO Shows the results of Western blot analysis of -1 and Nrf2 protein expression, and FIG. 6b shows the results of the experiment as a graph of relative density.
Figure 7a shows a representative HPLC chromatogram of the extract according to the part of H. serrata at 254nm, and Figure 7b is the structure of the main compound of the H. root extract, 27-O-protocatequoylbetulinic acid. is shown.
이하, 본 발명의 이해를 돕기 위하여 실시예 및 실험예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예 및 실험예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예 및 실험예에 한정되는 것은 아니다. 본 발명의 실시예 및 실험예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples and experimental examples will be described in detail to help the understanding of the present invention. However, the following Examples and Experimental Examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following Examples and Experimental Examples. Examples and experimental examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
실시예Example
이하, 실시예를 통해 본 발명을 더욱 상세히 설명한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명의 범위가 실시예에 의해 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only provided for easier understanding of the present invention, and the scope of the present invention is not limited by the examples.
재료ingredient
본 실험에 사용한 헛개나무(Hovenia dulcis) 시료는 2017년 8월에 경남 고성군 일대의 야산에서 채취하였으며, 정확히 감정한 후에 음건·세절하여 실험에 사용하였으며, 표본은 국립백두대간수목원 자원식물산업실에 보관하였다(E01708130066). 마쇄된 헛개나무 줄기, 잎, 뿌리 건조 시료 1.5 kg을 취하여 10L 메탄올을 가하여 30℃에서 24시간 추출한 후 원심분리하여 상등액을 1차적으로 회수하고 다시 침전물을 재추출하여 원심분리하여 상등액을 회수하여 1차 상등액과 혼합한 후 감압 농축하여, -20℃의 냉동고에 보관하면서 항염증 효과 및 HPLC 분석을 위한 시료로 사용하였다. The Hovenia dulcis sample used in this experiment was collected in August 2017 from the hillside in Goseong-gun, Gyeongsangnam-do. Stored (E01708130066). After taking 1.5 kg of ground dried horseradish stem, leaf, and root sample, adding 10L methanol, extracting it at 30°C for 24 hours, centrifuging to recover the supernatant, and re-extracting the precipitate again to centrifuge to recover the supernatant. After mixing with the tea supernatant, it was concentrated under reduced pressure and stored in a freezer at -20°C, and used as a sample for anti-inflammatory effect and HPLC analysis.
시약reagent
Dulbecco’s modified Eagle’s medium(DMEM)과 우태아혈청(fetal bovine serum, FBS) 등의 세포배양용 시약들은 Gibco BRL사(Grand Island, NY, USA)에서 구입하였다. 리포폴리사카라이드(LPS)와 3’-(4,5-디메틸티아졸-2-일)-2,5-디페닐테트라졸륨 브로마이드(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, MTT)는 Sigma사(Louis, MO, USA)에서 구입하였다. 96-well 세포 배양 플라스크(tissue culture plates)와 기타 세포 배양 디쉬 (tissue culture dishes)는 Falcon사(Corning, NY, USA) 제품을 이용하였다. 실험에 사용된 일차 항체인 iNOS, COX-2 그리고 라민(Lamin) B는 Santa Cruz Biotechnology사 (SantaCruz, CA, USA)에서 구입하였고, p65, phosphor-p65, IκBα, HO-1, Nrf2, phospho-ERK, ERK, phospho-p38, p38, phosphor-JNK, JNK (Cell Signaling, Beverly, MA, USA)에서 구입하였다. 2차 항체인 호스래디쉬 퍼옥시다제가 결합된 항 토끼-염소 항체(Horseradish peroxidase, HRP-conjugated anti-rabbit or goat antibodies)는 Jackson ImmunoResearch사(West Grove, PA, USA)에서 구입하였다. 그리스 시약 시스템(Griess Reagent System)은 Promega사(Madison, WI, USA)에서 구입하였고, 효소 결합 면역 흡착검사 키트는 R&D 시스템(Minneapolis, MN, USA)에서 구입하였다.Cell culture reagents such as Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS) and 3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2 ,5-Diphenyltetrazolium Bromide, MTT) was purchased from Sigma (Louis, MO, USA). 96-well cell culture plates and other cell culture dishes were manufactured by Falcon (Corning, NY, USA). Primary antibodies iNOS, COX-2 and Lamin B used in the experiment were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA), and p65, phosphor-p65, IκBα, HO-1, Nrf2, phospho- ERK, ERK, phospho-p38, p38, phosphor-JNK, and JNK were purchased from (Cell Signaling, Beverly, MA, USA). Secondary antibody, Horseradish peroxidase-conjugated anti-rabbit-goat antibody (HRP-conjugated anti-rabbit or goat antibodies) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The Griess Reagent System was purchased from Promega (Madison, WI, USA), and the enzyme-linked immunosorbent assay kit was purchased from R&D Systems (Minneapolis, MN, USA).
세포배양cell culture
마우스 대식세포주인 RAW 264.7 세포는 American Type Culture Collection (ATCC, Manassas, VA, USA)에서 동결상태로 구입하여 10% 우태아혈청(FBS)과 1% 항생제(100 U/ml 페니실린 G, 100 μg/ml 스트렙토마이신)을 첨가한 DMEM 배지로 37℃, 5% CO2 환경에서 배양하였다.RAW 264.7 cells, a mouse macrophage cell line, were purchased frozen from the American Type Culture Collection (ATCC, Manassas, VA, USA) in 10% fetal bovine serum (FBS) and 1% antibiotic (100 U/ml penicillin G, 100 μg/ ml streptomycin) was added to the DMEM medium and cultured at 37° C., 5% CO 2 environment.
세포 생존율 측정Cell viability measurement
뿌리 추출물(Root Extract, RE)의 세포독성 유무를 알아보기 위하여 세포 카운팅 키트(Cell counting kit)-8 (Dojindo, Japan)을 이용하여 측정하였다. RAW 264.7 세포를 96 well 플레이트에 2x104 cells/ml로 분주하고 24시간 배양한 다음, 뿌리 추출물(RE)을 농도별(0, 10, 20 및 40 μg/ml)로 처리하여 24시간 배양하였다. 각 well당 10 μl의 CCK-8 용액을 첨가하여 37 ℃, 5% CO2 조건에서 2시간 반응시킨 후 마이크로플레이트 리더(BioTek, Winooski, VT, USA)를 이용하여 450 nm 에서 흡광도를 측정하였다. In order to determine the presence or absence of cytotoxicity of the root extract (RE), it was measured using a cell counting kit-8 (Dojindo, Japan). RAW 264.7 cells were seeded in a 96-well plate at 2x10 4 cells/ml and cultured for 24 hours. 10 μl of CCK-8 solution was added to each well and reacted at 37 ° C., 5% CO 2 condition for 2 hours, and then absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
산화질소(NO) 및 프로스타글란딘 E2(PGE2)의 측정Determination of nitric oxide (NO) and prostaglandin E2 (PGE2)
리포폴리사카라이드 자극에 의해 RAW 264.7세포에서 유도된 염증반응에 대한 각 부위별 추출물의 항염증 효과를 측정하기 위한 일환으로, 세포 배양액 중의 NO를 정량하여 비교하였다. NO 소거 활성을 측정하기 위하여 세포를 96-well 플레이트에 2x104 cells/ml로 분주 하여 24시간 배양한 후 뿌리 추출물을 0, 10, 20, 40 μg/ml의 농도로 전처리하여 1시간 동안 배양한 후, 리포폴리사카라이드를 1 μg/ml 의 농도로 처리하여 24시간 동안 배양하였다. 배양 상층액 50 μl와 동량의 그리스 시약(Promega, Madison, WI, USA)를 혼합하여 반응시킨 뒤 540 nm에서 마이크로플레이트 리더(BioTek, Winooski, VT, USA)를 사용하여 흡광도 값을 측정하였다. 아질산나트륨(NaNO2)의 농도별 표준곡선을 이용하여 NO값을 산출하였다. 그리고 세포배양액 내의 PGE2 수준은 PGE2 효소 결합 면역 흡착검사 분석 키트(R&D System, Minneapolis, MN, USA)를 사용하여 측정하였다. As part of measuring the anti-inflammatory effect of the extracts for each site on the inflammatory response induced in RAW 264.7 cells by lipopolysaccharide stimulation, NO in the cell culture was quantified and compared. To measure NO scavenging activity, cells were aliquoted in a 96-well plate at 2x10 4 cells/ml and cultured for 24 hours. Then, lipopolysaccharide was treated at a concentration of 1 μg/ml and cultured for 24 hours. 50 μl of the culture supernatant and the same amount of grease reagent (Promega, Madison, WI, USA) were mixed and reacted, and the absorbance value was measured at 540 nm using a microplate reader (BioTek, Winooski, VT, USA). The NO value was calculated using a standard curve for each concentration of sodium nitrite (NaNO 2 ). And the level of PGE2 in the cell culture was measured using a PGE2 enzyme-linked immunosorbent assay kit (R&D System, Minneapolis, MN, USA).
사이토카인(IL-1β, IL-6, TNF-α) 측정Cytokine (IL-1β, IL-6, TNF-α) measurement
NO 측정과 같은 방법으로 세포를 배양한 후, 각 well에서 세포 배양액을 회수하였다. 세포배양 상층액 내의 TNF-α, IL-1β, IL-6 농도는 효소 결합 면역 흡착검사(enzyme linked immunosorbent assay, Elisa) 키트(R&D system, MN, USA)를 이용하여 측정하였다.After culturing the cells in the same way as for NO measurement, the cell culture solution was recovered from each well. TNF-α, IL-1β, and IL-6 concentrations in the cell culture supernatant were measured using an enzyme linked immunosorbent assay (ELIsa) kit (R&D system, MN, USA).
정량 중합 효소 반응 실시간 중합효소연쇄반응(real-time reverse transcription polymerase chain reaction, RT-PCR)Quantitative polymerase reaction real-time reverse transcription polymerase chain reaction (RT-PCR)
RAW 264.7 세포를 6-well 플레이트에 6x105 cells/ml로 분주하여 24시간 배양한 뒤 뿌리 추출물을 1시간 동안 처리한 후, 리포폴리사카라이드를 1 μg/ml의 농도로 24시간 동안 반응하였다. 이후 Trizol(Invitrogen, USA) 시약을 이용하여 모든 RNA를 분리하였다. 총 RNA를 정량하고, RT-PreMix(Promega, Madison, WI, USA)를 이용하여 1 μg의 각각의 모든 RNA로부터 각각의 cDNA를 합성하였다. 각 cDNA의 주형(template)과 iNOS, COX-2, IL-6, IL-1β, TNF-α 및 β-액틴의 프라이머를 이용하여 mRNA 발현을 확인하였다. 각 유전자의 프라이머 서열을 표 1에 나타내었다.RAW 264.7 cells were seeded in a 6-well plate at 6x10 5 cells/ml, cultured for 24 hours, treated with the root extract for 1 hour, and then reacted with lipopolysaccharide at a concentration of 1 μg/ml for 24 hours. Then, all RNA was isolated using Trizol (Invitrogen, USA) reagent. Total RNA was quantified, and each cDNA was synthesized from 1 μg of each and all RNA using RT-PreMix (Promega, Madison, WI, USA). mRNA expression was confirmed using a template of each cDNA and primers of iNOS, COX-2, IL-6, IL-1β, TNF-α and β-actin. The primer sequences of each gene are shown in Table 1.
웨스턴 블랏 분석Western blot analysis
RAW 264.7 세포를 뿌리 추출물 및 리포폴리사카라이드로 처리한 후 HO-1 억제제(SnPP)는 1시간 전에 전처리하였다. 얼음으로 차가운 인산완충생리식염수(ice-cold phosphate buffered saline, PBS)로 2회 세척 한 후, 방사면역침강분석 버퍼(Radioimmunoprecipitation Assay, RIPA buffer)(25 mM 트리스·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% 디옥시콜레이트 나트륨, 0.1% SDS, 프로테아제 억제제)를 넣어 얼음에서 30분간 반응시키고 전세포 용해물(Whole cell lysate)을 제조하여 20분간 원심분리하여 상층액을 모았다. 각 시료의 단백질 정량은 브래드포드 단백질 분석(Bradford protein assay) (Bio-Rad, Hercules, CA, USA)을 사용하여 595 nm에서 흡광도를 측정하여 실시하였다. 동일한 양의 단백질(30 μg)을 10% 소듐 도데실 설페이트(SDS)-폴리아크릴아마이드 겔로 분리한 후 iBlot 2 블로팅 시스템(Thermo Fisher, Waltham, MA USA)을 이용하여 변환하였다. 이 PVDF 멤브레인은 5% 탈지분유 블락 용액(non-fat milk block solution)에서 1시간 동안 반응시킨 후, 일차항체(1:1000 dilution)를 4℃에서 밤새 유지시켰다. TBST 용액으로 3번 세척한 후, 이차항체는 호스래디쉬 퍼옥시다제가 결합된 항 토끼 또는 항마우스 IgG(horseradish peroxidase (HRP)-conjugated anti-rabbit, or anti-mouse IgG) (1:10000 희석)으로 실온에서 1시간 반응시켰다. 이어서 3회 세척 후 단백질은 ChemiDoc™ 이미징 시스템(Bio-Rad, Hercules, CA, USA)을 이용하여 발현을 확인하였다. RAW 264.7 cells were treated with root extract and lipopolysaccharide and then pretreated with HO-1 inhibitor (SnPP) 1 hour before. After washing twice with ice-cold phosphate buffered saline (PBS), Radioimmunoprecipitation Assay (RIPA buffer) (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor) was added, reacted on ice for 30 minutes, whole cell lysate was prepared, and the supernatant was collected by centrifugation for 20 minutes. Protein quantification of each sample was performed by measuring absorbance at 595 nm using Bradford protein assay (Bio-Rad, Hercules, CA, USA). The same amount of protein (30 μg) was separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transformed using the
부위별 헛개나무 추출물 HPLC 분석HPLC analysis of H. serrata extract by part
HPLC 기기는 펌프, 오토 샘플, 컬럼 오븐, DAD(Agilent 1260 infinity HPLC system, Agilent Technologies, USA)를 사용하였으며, 분석에 사용된 모든 용매는 J.T. Baker(Phillipsburg, NJ, USA)로부터 구입한 HPLC급 용매를 사용하였다. 이동상으로 0.1% 포름산을 포함한 물(용매A)과 아세토니트릴(용매B)을 사용하였고 1 mL/분의 유속으로 시료 10 μl를 주입하여 254 nm 파장에서 그래디언트 조건으로 분석하였다. 헛개나무 추출물의 상이한 부위의 추출물의 분리를 위한 HPLC 조건과 주요화합물 분리를 위한 HPLC 분석 조건을 표 2에 나타내었다.As HPLC instrument, pump, auto sample, column oven, DAD (Agilent 1260 infinity HPLC system, Agilent Technologies, USA) were used, and all solvents used for analysis were J.T. An HPLC-grade solvent purchased from Baker (Phillipsburg, NJ, USA) was used. Water (solvent A) and acetonitrile (solvent B) containing 0.1% formic acid were used as mobile phases, and 10 μl of the sample was injected at a flow rate of 1 mL/min and analyzed under gradient conditions at a wavelength of 254 nm. Table 2 shows the HPLC conditions for the separation of extracts from different parts of the H. serrata extract and the HPLC analysis conditions for the separation of major compounds.
z0.1% 물 내 포름산. z 0.1% formic acid in water.
y0.1% 아세토니트릴 내 포름산 y 0.1% formic acid in acetonitrile
헛개나무 뿌리 추출물 활성성분 분리 및 구조동정Isolation and structural identification of the active ingredient of the rhododendron root extract
헛개나무 부위별 추출물 중 가장 우수한 항염증 효과를 가진 뿌리 추출물을 재순환 분취 HPLC 시스템을 사용하여 분리하였다. JAIGEL-ODS AP 컬럼(20 x 500 mm)에 추출물 1g을 주입하고 혼합용매(물:아세토니트릴=1:2, v/v)를 분당 10mL씩 용출시킨 다음 UV 검출기를 사용하여 흡광도 254 nm에서 측정하였으며, 6개의 소분획(HR1-HR6)으로 나누었다. 이중 HR4를 EtOAc:MeOH(30:1)의 혼합용매로 분취 TLC를 실시하고 세파덱스(Sephadex) LH-20(95% MeOH)로 정제하여 화합물 1(30 mg)을 얻었다. 최종 정제된 뿌리 추출물의 주요 성분은 Q Exactive plus Orbitrap LC-MS/MS(Thermo Fisher Scientific, MA, USA) 및 500MHz Bruker AM NMR(Bruker, MA, USA)을 통하여 얻은 화학 구조상의 수소 및 탄소골격에 대한 정보를 얻어 정제된 주요 성분의 구조를 동정하였다. The root extract with the best anti-inflammatory effect among the extracts for each part of H. serrata was separated using a recirculating preparative HPLC system. 1 g of the extract was injected into a JAIGEL-ODS AP column (20 x 500 mm) and a mixed solvent (water: acetonitrile=1:2, v/v) was eluted at 10 mL per minute, and then the absorbance was measured at 254 nm using a UV detector. and divided into 6 small fractions (HR1-HR6). Of these, HR4 was subjected to preparative TLC with a mixed solvent of EtOAc:MeOH (30:1), and purified by Sephadex LH-20 (95% MeOH) to obtain Compound 1 (30 mg). The main components of the final purified root extract are chemical structural hydrogen and carbon skeletons obtained through Q Exactive plus Orbitrap LC-MS/MS (Thermo Fisher Scientific, MA, USA) and 500 MHz Bruker AM NMR (Bruker, MA, USA). The structure of the purified main component was identified by obtaining information about the
통계처리Statistical processing
모든 실험 결과는 3회 이상 실시하여 그 평균값을 기초로 평균 ± 표준편차(Mean ± S.D.)로 나타내었으며, 실험결과에 대한 통계처리는 GraphPad 프리즘 5.0 소프트웨어(GraphPad Software, Inc., San Diego, CA, USA)를 이용하여 Two-way ANOVA에 준하였고 p-값이 0.05 미만일 경우 유의한 것으로 판정하였다.All experimental results were performed three or more times and expressed as mean ± standard deviation (Mean ± SD) based on the average value, and statistical processing of the experimental results was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA USA) using two-way ANOVA, and when the p-value was less than 0.05, it was judged to be significant.
비교실험예 1. NO 및 PGE2 생성 및 iNOX, COX-2 발현에 미치는 헛개나무 뿌리 추출물의 효과 확인Comparative Experimental Example 1. Confirmation of the effect of H. root extract on NO and PGE2 production and iNOX, COX-2 expression
헛개나무 부위별 추출물의 항염증 효과를 비교하기 위하여 리포폴리사카라이드(LPS)로 염증반응을 유도한 RAW 264.7 대식세포 내 부위별 추출물을 동시에 처리하여 염증의 지표인 산화질소(NO) 생성 억제효과를 측정한 결과 표 3과 같이 헛개나무 뿌리 추출물(RE)에서 탁월한 억제효과를 나타내었다. 세포독성이 나타내지 않는 농도인 10, 20, 40 μg/ml에서 뿌리 추출물을 이용하여 항염증 효과 및 조절기전을 관찰하였다. In order to compare the anti-inflammatory effects of the extracts for each part of H. serrata, the extracts of each part in RAW 264.7 macrophages induced an inflammatory response with lipopolysaccharide (LPS) were simultaneously treated to inhibit the production of nitric oxide (NO), an indicator of inflammation. As a result of measuring , as shown in Table 3, it showed an excellent inhibitory effect in the root extract (RE). Anti-inflammatory effects and regulatory mechanisms were observed using root extracts at concentrations of 10, 20, and 40 μg/ml, which do not show cytotoxicity.
z수치는 3회 반복의 평균 ± 표준편차로 표시되었다. z values were expressed as mean ± standard deviation of 3 replicates.
이 때 GAPDH가 실시간 PCR 분석에 대해 내부 대조군으로 사용되었으며(n=4), 데이터는 3번의 실험에 대하여 평균 ± 표준편차로 나타내었고 (LPS 단독으로 사용되었을 때와 비교하여 *p<0.05, **p<0.001이었다) 그 결과를 도 1a 내지 도 1d에 나타내었다.At this time, GAPDH was used as an internal control for real-time PCR analysis (n=4), and the data were expressed as mean ± standard deviation for three experiments (*p<0.05, * compared to when LPS alone was used) *p<0.001) The results are shown in FIGS. 1A to 1D.
초기 염증 반응은 유도형 산화질소 생성 효소(inducible nitric oxide synthase, iNOS)와 시클로옥시게나제(cyclooxygenase-2, COX-2)와 이들 단백질에 의한 염증 매개체들(NO, PGE2)이 유도된다. 본 발명에서는 뿌리 추출물의 항염증 효과를 알아보기 위하여 RAW 264.7 세포에 뿌리 추출물 10, 20, 40 μg/ml을 1시간 동안 전처리한 후 1 μg/ml 리포폴리사카라이드를 24시간 동안 처리하여 NO의 생성량에 미치는 영향을 같은 방법으로 조사한 결과, 10 μg/ml에서는 53.1%, 40 μg/ml에서는 94.7% 감소시켰다(도 1a). 또한, 리포폴리사카라이드에 의해 유도되는 PGE2의 생성에 미치는 영향을 조사한 결과, 10 μg/ml에서는 47.2%, 40 μg/ml에서는 90.1% 감소시켰다(도 1b).The initial inflammatory response is induced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and inflammatory mediators (NO, PGE2) by these proteins. In the present invention, in order to examine the anti-inflammatory effect of the root extract, RAW 264.7 cells were pre-treated with 10, 20, and 40 μg/ml of the root extract for 1 hour and then treated with 1 μg/ml lipopolysaccharide for 24 hours to reduce NO As a result of investigating the effect on the production amount in the same way, it was reduced by 53.1% at 10 μg/ml and by 94.7% at 40 μg/ml (FIG. 1a). In addition, as a result of examining the effect on the production of PGE2 induced by lipopolysaccharide, it was reduced by 47.2% at 10 μg/ml and by 90.1% at 40 μg/ml ( FIG. 1b ).
뿌리 추출물의 항염증 효과에 대한 작용기전을 알아보기 위하여 체내 염증발현효소(iNOS, COX-2)의 mRNA 발현을 조사하였다. iNOS에 의해서 생성된 NO는 면역반응에서 유용한 역할을 하지만, 지속적인 NO의 생성은 만성 염증 질환을 일으키는 중요한 요인이 된다. 따라서 체내에 과도한 NO를 생성하는 iNOS의 발현에 대한 뿌리 추출물의 영향을 조사하기 위해 RT-PCR을 시행하였다. 리포폴리사카라이드를 처리한 군에서 iNOS, COX-2 mRNA는 유의적으로 증가하였고, 뿌리 추출물을 처리한 군에서는 리포폴리사카라이드 처리군과 비교하여 iNOS, COX-2 mRNA의 발현량이 유의적으로 감소함으로 iNOS, COX-2 mRNA 저해효과는 NO, PGE2 생성 억제효과가 유사한 경향을 나타내었다(도 1c 및 도 1d). 그러므로 뿌리 추출물은 iNOS와 COX-2 발현을 억제함으로써 염증 억제 효과를 가짐을 확인할 수 있었다.In order to investigate the mechanism of action for the anti-inflammatory effect of the root extract, mRNA expression of the in vivo inflammatory enzymes (iNOS, COX-2) was investigated. Although NO produced by iNOS plays a useful role in the immune response, continuous NO production is an important factor in causing chronic inflammatory diseases. Therefore, RT-PCR was performed to investigate the effect of the root extract on the expression of iNOS, which produces excessive NO in the body. In the group treated with lipopolysaccharide, iNOS and COX-2 mRNA were significantly increased, and in the group treated with the root extract, the expression levels of iNOS and COX-2 mRNA were significantly increased compared to the group treated with lipopolysaccharide. As a result, the inhibitory effect of iNOS and COX-2 mRNA showed a similar tendency to the inhibitory effect of NO and PGE2 production ( FIGS. 1c and 1d ). Therefore, it was confirmed that the root extract had an anti-inflammatory effect by inhibiting iNOS and COX-2 expression.
실험예 1. 전염증성 사이토카인 생성과 mRNA 발현에 미치는 헛개나무 뿌리 추출물의 효과 확인Experimental Example 1. Confirmation of the effect of H. serrata root extract on pro-inflammatory cytokine production and mRNA expression
염증반응이 유도되는 과정에서 NO 및 PGE2와 같은 염증 매개물의 생성과 면역반응을 통한 염증성 사이토카인의 생성이 동반되며, 염증을 나타내는 중요한 지표이다. 특히 대식세포에서는 리포폴리사카라이드 등의 여러 자극 인자에 의하여 활성화가 되면 전염증성 사이토카인(TNF-α, IL-6 및 IL-1β)를 증가시키고, 이들은 iNOS의 발현을 유도한다. 따라서 뿌리 추출물이 TNF-α, IL-6, IL-1β 생성에 미치는 영향을 조사하기 위하여 효소 결합 면역 흡착검사 방법을 이용해 단백질 수준을 측정하였다. 이 때 데이터는 3번의 실험의 평균 ± 표준편차로 나타내었다(LPS를 단독으로 사용하였을 때와 비교했을 때 *p<0.05, **p<0.001이었다). In the process of inducing an inflammatory response, the production of inflammatory mediators such as NO and PGE2 and the production of inflammatory cytokines through an immune response are accompanied, and are important indicators of inflammation. In particular, in macrophages, when activated by several stimulatory factors such as lipopolysaccharide, pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) increase, and these induce the expression of iNOS. Therefore, to investigate the effect of the root extract on the production of TNF-α, IL-6, and IL-1β, the protein level was measured using the enzyme-linked immunosorbent assay method. At this time, the data were expressed as the mean ± standard deviation of three experiments (*p<0.05, **p<0.001 when compared with the LPS alone).
RAW 264.7 세포에 뿌리 추출물을 다양한 농도로 1시간 전처리하고 리포폴리사카라이드로 자극하였다. 24 시간 후 세포 상층액에서 TNF-α, IL-6 및 IL-1β를 측정한 결과를 각각 도 2a, 도 2b 또는 도 2c에 나타내었다. 리포폴리사카라이드를 처리한 군에서는 TNF-α, IL-6, IL-1β 발현이 유의성 있게 증가하였고, 뿌리 추출물을 처리한 군에서는 리포폴리사카라이드 처리군과 비교하여 염증인자들이 농도 의존적으로 강력하게 감소하는 것을 확인하였다. 위와 같은 결과로부터 착안하여 mRNA 수준에서도 전염성 인자들을 억제하는지 알아보기로 하였다. TNF-α, IL-6, IL-1β의 mRNA 발현을 정량적 중합 효소 반응 방법으로 측정한 결과 리포폴리사카라이드 자극은 mRNA의 발현을 유의성 있게 증가시켰고, 뿌리 추출물을 전처리한 실험군에서 TNF-α, IL-6 및 IL-1β mRNA 발현이 모두 농도 의존적으로 억제되었다(각각, 도 2d, 도 2e 또는 도 2f). 이 결과들을 통해 뿌리 추출물은 TNF-α, IL-1β 및 IL-6의 생성을 감소시킴으로써 iNOS의 발현을 조절하여 NO 생성을 억제하는 항염증 효과를 나타낸다는 것으로 확인하였다. 모든 실험에 사용된 뿌리 추출물의 농도별 세포독성 여부를 MTT 분석을 통하여 알아보았으며 사용된 모든 농도에서 90% 이상의 생존율을 보여 뿌리 추출물은 RAW 264.7 세포에서 독성을 나타내지 않는 것을 확인하였다(도 2g).RAW 264.7 cells were pretreated with root extracts at various concentrations for 1 hour and stimulated with lipopolysaccharide. The results of measuring TNF-α, IL-6 and IL-1β in the cell supernatant after 24 hours are shown in FIGS. 2a, 2b or 2c, respectively. In the group treated with lipopolysaccharide, the expression of TNF-α, IL-6, and IL-1β was significantly increased, and in the group treated with the root extract, the inflammatory factors were stronger in a concentration-dependent manner compared to the group treated with lipopolysaccharide. was confirmed to decrease. Based on the above results, it was decided to investigate whether infectious factors were suppressed even at the mRNA level. As a result of measuring the mRNA expression of TNF-α, IL-6, and IL-1β by a quantitative polymerase reaction method, lipopolysaccharide stimulation significantly increased mRNA expression, and in the experimental group pretreated with the root extract, TNF-α, Both IL-6 and IL-1β mRNA expression were inhibited in a concentration-dependent manner (Fig. 2d, Fig. 2e or Fig. 2f, respectively). Based on these results, it was confirmed that the root extract had an anti-inflammatory effect that suppressed NO production by regulating the expression of iNOS by reducing the production of TNF-α, IL-1β and IL-6. The cytotoxicity of each concentration of the root extract used in all experiments was checked through MTT analysis, and it was confirmed that the root extract showed no toxicity in RAW 264.7 cells by showing a survival rate of 90% or more at all concentrations used (Fig. 2g). .
실험예 2. Ik-B 발현량에 미치는 헛개나무 뿌리 추출물의 효과 확인Experimental Example 2. Confirmation of the effect of the root extract of Heotgae tree on the expression level of Ik-B
RAW 264.7 세포나 리포폴리사카라이드에 의하여 염증이 일어나게 되면 다양한 신호 전달 기전에 의하여 염증성 매개물질들을 분비하게 되는데, 대표적인 경로인 핵 인자 카파 B NF-kB, Nuclear Factor-kappa B)가 있다. NF-kB는 모든 세포에서 발현되는 유도성 전사인자로 세포외부 자극에 대한 방어 작용과 면역세포의 활성화를 일으키며 세포의 염증반응에 관련된 다양한 유전자의 발현을 조절하는 것으로 알려져 있다(Ghosh et al., 2012). 리포폴리사카라이드에 의하여 세포막에 존재하는 톨 유사 수용체(toll like receptor 4, TLR4)와 결합하게 되고, 이에 의해 세포질에 있는 전사인자 NF-kB가 활성화되면 결합해 있던 Ik-Ba(inhibitory kappa Ba)가 분해되고, 그에 따라 NF-kB가 세포질에서 핵 내로 이동하여 COX-2, iNOS 등의 전사를 유도한다(Kim et al., 2013). 본 발명에서는 뿌리 추출물 처리에 의해서 감소되는 염증 매개 인자들의 생성억제가 NF-kB 활성과의 관계를 조사하기 위해 분석한 결과는 도 3a 및 도 3b에 나타내었다. β-액틴은 각각 시토졸 및 핵 분획에 대해 내부 단백질로서 사용되었으며, 데이터는 3번의 실험의 평균 ± 표준편차로 나타내었다(LPS를 단독으로 사용하였을 때와 비교했을 때 *p<0.05, **p<0.001이었다). When inflammation occurs by RAW 264.7 cells or lipopolysaccharide, inflammatory mediators are secreted by various signal transduction mechanisms. Representative pathways include nuclear factor kappa B, NF-kB, and nuclear factor-kappa B). NF-kB is an inducible transcription factor expressed in all cells, and it is known to cause a protective action against extracellular stimuli, activate immune cells, and regulate the expression of various genes involved in cellular inflammatory responses (Ghosh et al., 2012). By lipopolysaccharide, it binds to the toll-like receptor 4 (TLR4) present in the cell membrane, and by this, when the transcription factor NF-kB in the cytoplasm is activated, the bound Ik-Ba (inhibitory kappa Ba) is degraded, and thus NF-kB moves from the cytoplasm to the nucleus to induce transcription of COX-2 and iNOS (Kim et al., 2013). In the present invention, the results of the analysis to investigate the relationship between the NF-kB activity and the inhibition of the production of inflammatory mediators reduced by the treatment of the root extract are shown in FIGS. 3A and 3B. β-actin was used as an internal protein for the cytosolic and nuclear fractions, respectively, and the data are presented as the mean ± standard deviation of three experiments (*p<0.05, ** when compared with LPS alone. p<0.001).
리포폴리사카라이드를 처리한 군에서 NF-kB의 서브유닛(subunit)인 p65의 핵 안으로의 이동이 무처리군과 비교하여 크게 감소하였고, 뿌리 추출물을 처리한 군에서는 리포폴리사카라이드 처리군과 비교하여 농도 의존적으로 발현이 감소하였다. 또한, 리포폴리사카라이드를 처리한 군에서 Ik-Ba의 분해가 무처리군과 비교하여 뚜렷이 나타났으며, 뿌리 추출물에서는 리포폴리사카라이드 처리군과 비교하여 Ik-Ba의 분해를 억제하였다. 결과적으로 뿌리 추출물은 전사인자인 NF-kB의 활성화를 감소시키고, 하위 신호전달 분자(downstream signaling molecule)인 iNOS와 COX-2의 전사를 억제하며, NO의 생성을 감소시켜 항염증 효과를 나타냄을 유추할 수 있었다. In the group treated with lipopolysaccharide, the movement of p65, a subunit of NF-kB, into the nucleus was significantly reduced compared to the untreated group. In comparison, expression was decreased in a concentration-dependent manner. In addition, in the group treated with lipopolysaccharide, the degradation of Ik-Ba was evident compared to the untreated group, and the root extract inhibited the degradation of Ik-Ba compared to the group treated with lipopolysaccharide. As a result, the root extract reduces the activation of the transcription factor NF-kB, inhibits the transcription of iNOS and COX-2, which are downstream signaling molecules, and reduces the production of NO, thereby exhibiting anti-inflammatory effects. could infer.
실험예 3. MAPK 활성에 미치는 헛개나무 뿌리 추출물의 효과 확인Experimental Example 3. Confirmation of Effect of Heotgae Root Extract on MAPK Activity
유사분열 활성화 단백질 인산화효소(Mitogen Activated Protein Kinase, MAPK)의 신호 전달 경로는 염증반응의 활성화에 중요한 역할을 한다. 특히, 리포폴리사카라이드에 의해 유도된 사이토카인의 분비에 p38 MAPK가 중요한 역할을 하고, 폐와 관련된 염증 질환에서는 p38, JNK, ERK 신호 경로가 중요한 역할을 한다고 알려져 있다(Yang et al., 2015). 특히 대식세포에서 리포폴리사카라이드는 표면에 TLR4를 자극하여 하부 세포 신호 전달 경로인 MAPK의 활성화를 유도하며 활성화된 신호전달경로는 전염증성 사이토카인, NO, PG와 같은 여러 가지 염증성 매개인자들의 발현을 유도한다(Kim et al., 2010). 본 발명에서 뿌리 추출물의 항염증 효과가 MAPK 분자조절에 의한 것인지를 조사한 결과는 도 4a 및 도 4b에 나타내었다. 데이터는 3번의 실험의 평균 ± 표준편차로 나타내었다(LPS를 단독으로 사용하였을 때와 비교했을 때 *p<0.05, **p<0.001이었다). The signaling pathway of mitogen activated protein kinase (MAPK) plays an important role in the activation of the inflammatory response. In particular, it is known that p38 MAPK plays an important role in lipopolysaccharide-induced cytokine secretion, and p38, JNK, and ERK signaling pathways play an important role in lung-related inflammatory diseases (Yang et al., 2015). ). In particular, in macrophages, lipopolysaccharide stimulates TLR4 on the surface to induce activation of MAPK, a lower cell signaling pathway, and the activated signaling pathway expresses the expression of various inflammatory mediators such as pro-inflammatory cytokines, NO and PG. induced (Kim et al., 2010). In the present invention, the results of investigating whether the anti-inflammatory effect of the root extract is due to the regulation of MAPK molecules are shown in Figs. 4a and 4b. Data are expressed as the mean ± standard deviation of three experiments (*p<0.05, **p<0.001 compared to when LPS was used alone).
뿌리 추출물 처리에 따른 대식세포 내 인산화된 MAPKs 발현량에 미치는 영향을 웨스턴 블랏으로 분석한 결과, 리포폴리사카라이드로 유도된 RAW 264.7 세포에는 MAPKs에 속한 단백질이 모두 활성화 되었음을 알 수 있었다. 뿌리 추출물의 첨가량이 높아질수록 인산화된 ERK, JNK, p-38의 발현이 리포폴리사카라이드 처리군에 비해 감소하는 것을 확인할 수 있었다. 따라서 뿌리 추출물은 MAPK의 활성화를 억제함으로써 항염증성 사이토카인, NO, 프로스타글란딘(PG)과 같은 여러 염증성 매개인자들의 발현을 억제하는 효과를 나타내었다. As a result of Western blot analysis of the effect of the root extract treatment on the expression level of phosphorylated MAPKs in macrophages, it was found that all MAPKs proteins were activated in lipopolysaccharide-induced RAW 264.7 cells. As the amount of root extract increased, it was confirmed that the expression of phosphorylated ERK, JNK, and p-38 decreased compared to the lipopolysaccharide-treated group. Therefore, the root extract showed the effect of suppressing the expression of several inflammatory mediators such as anti-inflammatory cytokines, NO, and prostaglandins (PG) by inhibiting the activation of MAPK.
실험예 4. HO-1 발현 유도에 미치는 헛개나무 뿌리 추출물의 효과 및 Nrf2 활성에 미치는 헛개나무 뿌리 추출물의 효과 확인Experimental Example 4. Effect of H. serrata root extract on induction of HO-1 expression and Effect of H. rhododendron root extract on Nrf2 activity
힘 옥시게나아제(Heme oxygenase-1, HO-1)는 힘(heme)을 산화시켜 빌베르딘, 일산화탄소(CO), 자유 철 등을 생성에 관여하는 효소로서 산화적 스트레스에 대한 보호작용을 하여 염증 반응을 약화시킨다. 활성화된 대식세포에 HO-1을 발현시키거나 CO를 처리하면 염증 촉진 사이토카인의 생산이 저해되며, 또한 염증촉진 매개체의 생산을 억제한다. HO-1 단백질 발현의 전사 조절 인자인 핵 인자-E2-관련 인자 2(nuclear factor-E2-related factor 2, Nrf2)의 핵 내로의 발현이 매우 중요한 인자로 작용한다. 뿌리 추출물이 Nrf2의 전사 여부를 조사하기 위하여 RAW 264.7 세포에 뿌리 추출물을 처리한 후 HO-1 발현과 Nrf2 활성화를 웨스턴 블롯을 이용하여 분석하였다. 뿌리 추출물의 농도에 따라 Nfr2는 점점 감소하는 반면, 핵 내부의 Nrf2는 증가하는 양상을 보이는 것을 보아 Nrf2의 핵 내로의 전사가 이루어졌음을 확인할 수 있었다(도 5a, 도 5b 및 도 5c). 데이터는 3번의 실험의 평균 ± 표준편차로 나타내었다(LPS를 단독으로 사용하였을 때와 비교했을 때 *p<0.05, **p<0.001이었다). Heme oxygenase-1 (HO-1) is an enzyme involved in oxidizing heme to produce bilberdin, carbon monoxide (CO), free iron, etc., and has a protective action against oxidative stress. weakens the inflammatory response; When HO-1 is expressed in activated macrophages or treated with CO, the production of pro-inflammatory cytokines is inhibited, and the production of pro-inflammatory mediators is also suppressed. Nuclear factor-E2-related factor 2 (Nrf2), a transcriptional regulator of HO-1 protein expression, acts as a very important factor in the expression of nuclear factor-E2-related factor 2 (Nrf2). To investigate whether the root extracts transcribed Nrf2, HO-1 expression and Nrf2 activation were analyzed using western blot after the root extract was treated on RAW 264.7 cells. While Nfr2 gradually decreased according to the concentration of the root extract, it was confirmed that Nrf2 was transcribed into the nucleus by seeing that Nrf2 inside the nucleus increased ( FIGS. 5A , 5B and 5C ). Data are expressed as the mean ± standard deviation of three experiments (*p<0.05, **p<0.001 compared to when LPS was used alone).
리포폴리사카라이드를 단독 처리하였을 때보다 뿌리 추출물을 전처리하여 리포폴리사카라이드를 처리하였더니 HO-1의 발현이 증가하는 것으로 나타났다. 도 5a 및 도 5b에서 전사조절인자 Nrf2의 세포질과 핵 내 발현양을 확인하였더니 세포질에서는 감소하였으나, 핵 내에서 증가하는 결과를 보였다. 그러므로 뿌리 추출물은 Nrf2 활성을 증가시킴으로 HO-1를 유도한다고 결론지을 수 있다. 뿌리 추출물의 항염증 효과와 HO-1의 발현 간의 직접적인 관계를 알아보기 위하여, HO-1 억제제인 Snpp를 이용하여 실험하였다. SnPP를 뿌리 추출물(40 μg/ml)과 함께 12시간 동안 처리한 실험에서 뿌리 추출물에 의해 억제되었던 iNOS와 COX-2가 SnPP를 병용투여함으로써 부분적으로 증가되었다(도 6a 및 도 6b). 데이터는 3번의 실험의 평균 ± .표준편차로 나타내었다(LPS를 단독으로 사용하였을 때와 비교했을 때 *p<0.05, **p<0.001이었다).It was found that the expression of HO-1 was increased when the root extract was pretreated and treated with lipopolysaccharide than when lipopolysaccharide was treated alone. When the expression levels of the transcriptional regulator Nrf2 in the cytoplasm and nucleus were confirmed in FIGS. 5A and 5B , it decreased in the cytoplasm, but increased in the nucleus. Therefore, it can be concluded that root extract induces HO-1 by increasing Nrf2 activity. In order to investigate the direct relationship between the anti-inflammatory effect of the root extract and the expression of HO-1, an experiment was conducted using the HO-1 inhibitor Snpp. In an experiment in which SnPP was treated with a root extract (40 μg/ml) for 12 hours, iNOS and COX-2, which were inhibited by the root extract, were partially increased by co-administration of SnPP ( FIGS. 6A and 6B ). Data are expressed as mean ± .standard deviation of three experiments (*p<0.05, **p<0.001 when compared with LPS alone).
이와 같은 결과로부터 뿌리 추출물에 의한 HO-1 발현이 NO와 PGE2의 생성을 억제하는 것을 확인하였다.From these results, it was confirmed that the expression of HO-1 by the root extract inhibited the production of NO and PGE2.
실험예 5. 헛개나무 부위별 성분분석 및 주요성분의 구조 동정Experimental Example 5. Component analysis for each part of hutgae tree and structural identification of main components
부위별 헛개나무 추출물의 주요 성분 차이를 비교하기 위하여 HPLC를 이용하여 분석하였다. 그 결과 잎, 줄기 추출물에 비해 뿌리 추출물에서 일정한 주요 피크(29.5분)를 확인할 수 있었다(도 7a 및 도 7b). 본 발명에서는 뿌리 추출물에서 추출되는 주요성분을 재순환 분취 HPLC 시스템(Recycling preparative HPLC system)을 이용하여 분리 및 정제하였다. 화합물 1(하얀 분말)은 obitrap/질량 분석기 분석을 수행하였으며 m/z 607.3649 [M]+의 분자이온 피크가 관측되어 분자량을 608.8046으로 결정하였다. In order to compare the differences in the major components of the extracts of Heotgae tree by region, analysis was performed using HPLC. As a result, a constant main peak (29.5 minutes) was confirmed in the root extract compared to the leaf and stem extract ( FIGS. 7a and 7b ). In the present invention, the main components extracted from the root extract were separated and purified using a recycling preparative HPLC system. Compound 1 (white powder) was subjected to obitrap/mass spectrometry analysis, and a molecular ion peak at m/z 607.3649 [M]+ was observed, and the molecular weight was determined to be 608.8046.
1H NMR에서 δ1.05, 0.91, 0.90 및 0.77에서 4개의 핵간 메틸(angular methyl)기와 δ3.16에서 다중항(multiplet)으로 나타나는 트리테르펜(triterpene)의 통상적인 3번 옥시메친(oxymethine) 양성자를 확인할 수 있었다. δ1.74에서 sp 2 탄소에 결합하고 있는 또 하나의 메틸기를 확인하였고, δ4.77 및 4.63에서 각각 단일항(singlet)으로 나타나는 두 개의 올레핀 양성자를 확인하여 이 화합물이 이소프로페닐기를 가지고, δ4.72, 4.55에서 이중항(doublet)으로 나타나는 하나의 함산소(oxygenate) 메틸렌기와 아로마틱 영역의 δ7.53, 6.92, 7.46에서 ABX 시스템의 양성자 커플링으로 프로토카테쿠오일(protocatechuoyl)기를 함유하고 있는 루판(lupane)계열의 트리테르페노이드(triterpenoid)임을 추정할 수 있었다. 13C-NMR 스펙트럼 (in Acetone-d6, 125 MHz)에서는 총 37개의 신호를 확인하여 트리테르펜임을 확인하였다. δ27.7, 15.3, 16.3, 16.1 및 18.8에서 5개의 메틸기를 확인하였고, δ77.7에서 3번의 옥시메친 탄소를, δ149.9 및 109.4에서 이소프로페닐 기의 올레핀 탄소를 확인하였고, δ62.8에서 함산소 메틸렌 탄소의 전형적인 신호임을 다시 한번 확인할 수 있고, δ165.9는 프로토카테쿠오일 기의 카복시 탄소를 확인할 수 있었다. 따라서 NMR 분광분석 및 MS 분석에 의하여 헛개나무 뿌리 주요성분의 화학구조가 lupane계의 트리테르페노이드인 27-O-프로토카테쿠오일베툴린산(27-O-protocatechuoylbetulinic acid)임을 규명할 수 있었다(Kang et al., 2017). In 1 H NMR, four internuclear methyl groups at δ1.05, 0.91, 0.90 and 0.77, and a multiplet of triterpene at δ3.16, the typical 3 oxymethine proton was able to check Another methyl group bonded to the sp 2 carbon at δ1.74 was identified, and two olefin protons appearing as singlets at δ4.77 and 4.63, respectively, were identified, and this compound has an isopropenyl group, δ4 A single oxygenate methylene group, which appears as a doublet at .72 and 4.55, contains a protocatechuoyl group by proton coupling of the ABX system at δ7.53, 6.92, and 7.46 of the aromatic region. It could be estimated that it was a triterpenoid of the lupane family. In the 13 C-NMR spectrum (in Acetone-d6, 125 MHz), a total of 37 signals were identified, confirming that they were triterpenes. Five methyl groups were identified at δ27.7, 15.3, 16.3, 16.1 and 18.8, three oxymethyl carbons were identified at δ77.7, and olefinic carbons of the isopropenyl group were identified at δ149.9 and 109.4, δ62.8 It can be confirmed once again that it is a typical signal of oxygenated methylene carbon, and δ165.9 can confirm the carboxy carbon of the protocatechuyl group. Therefore, by NMR spectroscopy and MS analysis, the chemical structure of the main component of the root of the H. serrata was identified as 27-O- protocatechuoylbetulinic acid, which is a triterpenoid of the lupane family (27-O-protocatechuoylbetulinic acid). Kang et al ., 2017).
본 발명에서는 헛개나무 부위별(잎, 줄기, 뿌리) 메탄올 추출물의 항염증 효과에 대해 조사하였다. 리포폴리사카라이드로 염증을 유도한 RAW 264.7 대식세포 내 부위별 추출물을 동시에 처리하여 염증 매개성 물질인 산화질소(NO) 생성 억제 효과를 확인한 결과 뿌리 추출물에서 탁월한 억제 효과가 있었다. 이러한 헛개나무 뿌리 추출물의 항염증 효과 및 관련 분자적 기전을 확인하였다. 그 결과, 염증 반응의 주요 경로인 NF-kB 및 MAPK 신호전달경로에서 뿌리 추출물이 리포폴리사카라이드로 유도된 NF-kB의 핵 이동을 억제하고, ERK, JNK, p38의 인산화를 억제함으로써 iNOS, COX-2의 발현이 감소되고, NO와 전염증성 사이토카인(IL-6, IL-1β, TNF-α)의 생성이 억제됨을 확인하였다. 또한 뿌리 추출물은 대식세포에서 Nrf2를 활성화시켜 항염증성 단백질인 HO-1 발현을 유도하여 항염증 효과를 나타내었다. 또한, 헛개나무 뿌리 추출물로부터 주요성분을 분리한 후 NMR과 MS기기를 이용하여 구조를 동정한 27-O-프로토카테쿠오일베툴린산 화합물에서도 높은 항염증 효과를 확인하였다. 따라서 헛개나무 뿌리와 그 주요성분은 염증성 질환의 조절에 중요하게 작용할 가능성이 있으며 관절염, 염증성 장질환 같은 다양한 염증 질환의 발병을 예방하거나 치료하는데 있어 효과적인 천연 항염증제로서 이용할 수 있음을 나타낸다. In the present invention, the anti-inflammatory effect of the methanol extract for each part (leaf, stem, root) of Heotgae tree was investigated. As a result of confirming the inhibitory effect on the production of nitric oxide (NO), an inflammatory mediator, by simultaneously processing the extracts of RAW 264.7 macrophages induced by inflammation with lipopolysaccharide, the root extract had an excellent inhibitory effect. The anti-inflammatory effects and related molecular mechanisms of the extracts of the H. As a result, the root extract inhibits lipopolysaccharide-induced nuclear migration of NF-kB in the NF-kB and MAPK signaling pathways, which are major pathways of the inflammatory response, and inhibits the phosphorylation of ERK, JNK, and p38. It was confirmed that the expression of COX-2 is reduced, and the production of NO and pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) is suppressed. In addition, the root extract activated Nrf2 in macrophages to induce the expression of HO-1, an anti-inflammatory protein, thereby showing an anti-inflammatory effect. In addition, a high anti-inflammatory effect was also confirmed in the 27-O-protocatequoyl betulinic acid compound, the structure of which was identified using NMR and MS after separating the main components from the root extract of Heotgae tree. Therefore, it is possible that the root and its main components may play an important role in the regulation of inflammatory diseases, indicating that they can be used as effective natural anti-inflammatory agents in preventing or treating the onset of various inflammatory diseases such as arthritis and inflammatory bowel disease.
이상과 같이 재료, 실시예, 비교실험예 및 실험예를 통하여 본 발명을 설명하였다. 본 발명이 속하는 기술 분야의 통상의 기술자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 그러므로 상술한 실험예들은 모든 면에 예시적인 것이며 한정적인 것이 아닌 것으로서 이해되어야 한다. 본 발명의 범위는 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.As described above, the present invention has been described through materials, examples, comparative experimental examples and experimental examples. Those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above-described experimental examples are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be construed as being included in the scope of the present invention.
Claims (4)
[화학식 Ⅰ]
A pharmaceutical composition for preventing or treating an inflammatory disease comprising a compound of Formula I as an active ingredient.
[Formula Ⅰ]
[화학식 Ⅰ]
A food composition for preventing or improving inflammatory diseases comprising a compound of Formula I as an active ingredient.
[Formula Ⅰ]
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