KR20150098599A - Composition comprising extract of Rhododendron brachycarpum or megastigmane glucosides isolated therefrom for preventing or treating sepsis - Google Patents
Composition comprising extract of Rhododendron brachycarpum or megastigmane glucosides isolated therefrom for preventing or treating sepsis Download PDFInfo
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- KR20150098599A KR20150098599A KR1020150096095A KR20150096095A KR20150098599A KR 20150098599 A KR20150098599 A KR 20150098599A KR 1020150096095 A KR1020150096095 A KR 1020150096095A KR 20150096095 A KR20150096095 A KR 20150096095A KR 20150098599 A KR20150098599 A KR 20150098599A
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- rhododendron
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Abstract
Description
본 발명은 만병초 추출물 또는 이로부터 분리된 메가스티그메인 배당체를 함유하는 패혈증의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating septicemia which contains a mexican plant extract or a megastigue main glycoside isolated therefrom.
패혈증(sepsis, 敗血症)은 녹농균, 대장균, 연쇄상구균, 포도상구균, 폐렴균 같은 미생물에 감염되었을 때 미생물이나 그 미생물이 생산한 독소에 의해 오한과 함께 고열, 관절통, 두통, 권태감 등의 전신적인 증상이 나타나는 상태를 말한다. 이러한 증상이 심해지면 저혈압이 동반되고 소변량이 줄며 패혈성 쇼크가 나타나기도 한다(Nat. Med. 2003, 9, 517-524). 미생물의 감염 경로를 잘 알 수 없는 경우도 있으나 맹장염, 중이염, 피부화농증, 욕창, 폐질환, 담낭염, 신우염, 골수염 등이 패혈증의 원인 병소로 알려져 있다. 혈액검사와 소변검사, 뇌척수액 검사 등을 통해 패혈증을 진단할 수 있으며 백혈구 수와 급성염증성물질이 증가한 경우도 패혈증을 진단하는데 도움을 준다. Sepsis (sepsis) is caused by toxins produced by microorganisms or microorganisms when they are infected with microorganisms such as Pseudomonas aeruginosa, Escherichia coli, Streptococcus, Staphylococcus aureus, and Pneumococcus, and it causes chills, systemic symptoms such as high fever, joint pain, headache, State that appears. If these symptoms are severe, hypotension is accompanied by decreased urine volume and septic shock (Nat. Med. 2003, 9, 517-524). Although the infection pathways of microorganisms are not well known, appendicitis, otitis media, skin pyrexia, pressure ulcer, lung disease, cholecystitis, pyelonephritis and osteomyelitis are known as the cause of sepsis. Blood tests, urine tests, and CSF tests can be used to diagnose sepsis. Increased leukocyte counts and acute inflammatory agents also help to diagnose sepsis.
아직까지 패혈증의 치료를 위한 근본적인 치료제는 확인되지 않은 상태이다. 패혈증은 통상적인 염증 억제 치료방법으로는 잘 낫지 않으며, 유일하게 FDA 승인을 받은 드로트레코긴 알파(drotrecogin alfa, Xigris®, Engl. J. Med. 2012, 366, 2055-2064)조차도 임상단계에서 패혈증에 대한 치료 효과가 명확하지 않아 이에 대한 연구가 중단되어 있는 실정이다. 현재 패혈증은 주로 항생제나 항진균제 주사로 치료하고, 치료 약제와 기간은 미생물의 종류에 따라 결정한다. 또 환자의 상태에 따라 혈액투석 또는 수혈을 하기도 한다. 항생제와 항진균제가 잘 들으면 패혈증은 완치되기도 하지만, 약제에 내성이 있는 미생물에 감염된 경우와 면역력이 약한 환자인 경우, 또 너무 늦게 치료를 시작한 경우 등에서는 치료가 어려워 환자가 사망하기도 한다. 또한, 패혈증의 사망률은 50~70%에 달해 매우 높은 편이며, 전세계적으로도 사망의 원인 중에서 높은 비중을 차지하는 것으로 알려져 있다(Nat. Med. 2003, 9, 517-524). 패혈증에 대한 합병증이 발생할 경우에는 후유증이 생길 수 있다. 합병증으로 뇌막염이 있는 경우에는 신경학적 후유증이, 화농성 관절염이 함께 나타났을 경우에는 뼈 성장에 이상이 생기기도 한다. Until now, the fundamental treatment for sepsis has not been confirmed yet. Sepsis is not well treated with conventional anti-inflammatory therapy and even the only FDA-approved drotrecogin alfa (Xigris ® , Engl. J. Med. 2012, 366, 2055-2064) The effect of the treatment is unclear. Currently, sepsis is treated mainly by injection of antibiotics or antifungal drugs, and the duration of the treatment is determined by the type of microorganism. Hemodialysis or transfusion may also be performed depending on the patient's condition. When antibiotics and antifungal agents are well tolerated, sepsis may be cured, but patients may die because they are difficult to treat when they are infected with a drug resistant microorganism, when their immunity is weak, or when they start treatment too late. In addition, the mortality rate of sepsis is very high, reaching 50-70%, and it is known that it accounts for a high percentage of deaths worldwide (Nat. Med. 2003, 9, 517-524). Complications associated with sepsis can lead to sequelae. Complications include meningitis, neurological sequelae, and pyogenic arthritis together with bone growth abnormalities may occur.
만병초(萬病草, Rhododendron brachycarpum)는 쌍떡잎식물 진달래목 진달래과(Ericaceae)의 상록관목으로서, 한국(지리산, 울릉도, 강원과 북부지방), 일본 등의 고산지대에서 자란다. 높이는 1~4 m이며, 나무껍질은 잿빛이 섞인 흰색이다. 잎은 어긋나지만 가지 끝에서는 5~7개가 모여 달리고 타원형이거나 타원 모양 바소꼴이며 혁질(革質:가죽 같은 질감)이다. 길이 8~20 cm, 너비 2~5 cm이고 가장자리는 밋밋하며 뒤로 말린다. 겉은 짙은 녹색이고 뒷면에는 연한 갈색 털이 빽빽이 난다. 잎자루는 길이 1~3 cm이다. 꽃은 6~7월에 피고 10~20개씩 가지 끝에 총상꽃차례로 달린다. 작은꽃자루는 붉은빛을 띤 갈색으로서 털이 빽빽이 난다. 화관은 깔때기 모양으로 흰색 또는 연한 노란색이고 안쪽 윗면에 녹색 반점이 있으며 5갈래로 갈라진다. 수술은 10개이고 암술은 1개이다. 씨방에는 갈색 털이 빽빽이 난다. 열매는 삭과로서 타원 모양이며 길이 약 2 cm이고 9월에 갈색으로 익는다. 진홍색 꽃이 피는 것을 홍만병초(Rhododendron brachycarpum var. roseum)라고 한다. 관상용으로 분에 심으며 잎은 이뇨제와 강장제로 쓴다. 만병초는 전통적으로 심혈관질환, 당뇨병, 고혈압, 간염, 류마티스 관절염, 두통 등 질병의 치료에 주로 사용되어 왔지만, 상기와 같은 효능에 대해서는 아직까지 많은 연구가 진행되지 않았다. Rhododendron brachycarpum is an evergreen shrub of azalea ( Azacaceae ) of the dicotyledonous plant. It grows in high mountains of Korea (Jiri, Ulleungdo, Gangwon and northern regions) and Japan. The height is 1 to 4 m, and the bark is white with ashy. Leaves are alternate but 5 ~ 7 are gathered at the end of the branch, and they are elliptical or elliptical, and they are leather (leather-like texture). Length 8 ~ 20cm,
HMGB1(high mobility group box 1)은 패혈증이 진행되어 혈관의 손상이 있을 때 발현되는 단백질로서(Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 228, 975-982), 본 발명자들은 만병초 추출물 및 만병초 유래의 화합물들이 HMGB1의 발현을 억제함으로써 패혈증의 치료 효과가 있음을 확인하여 본 발명을 완성하였다. HMGB1 (high mobility group box 1) is a protein expressed when the blood vessel is damaged due to progression of sepsis (Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 982). The present inventors completed the present invention by confirming that the compounds of the extracts of Pseudomonas sylvestris and Pseudomonas sylvestris inhibit the expression of HMGB1 and thus have a therapeutic effect on sepsis.
한편, 한국공개특허 제2009-0131399호에는 창이자, 만병초 및 우엉의 혼합추출물이 항염 및 피부자극완화 효과가 있음이 개시되어 있으며, 한국등록특허 제508402호에는 만병초를 함유하는 생약 추출물이 간염에 치료 효과가 있다는 것이 개시되어 있지만, 상기 선행문헌에는 만병초 추출물 또는 이로부터 분리된 화합물이 패혈증을 치료하는 효과가 있다는 것에 대해서는 전혀 개시되어 있지 않다. Korean Patent Laid-Open No. 2009-0131399 discloses that a mixed extract of Phellodendron japonica, Rhododendron japonica and Burdock is effective for antiinflammation and skin irritation mitigation. In Korean Patent No. 508402, a herbal medicine extract containing Rhodiola is administered to hepatitis It has been disclosed that the pioneer extract or the compound isolated therefrom is effective in treating sepsis.
본 발명의 목적은 만병초 추출물 또는 이로부터 분리된 메가스티그메인 배당체를 함유하는 패혈증의 예방 또는 치료용 조성물을 제공하는 데에 있다. It is an object of the present invention to provide a composition for preventing or treating septicemia which contains a psittacosis extract or a meggestidine main glycoside isolated therefrom.
본 발명은 하기 화학식 1의 로도덴드로사이드 A(Rhododendroside A, 화합물 1), 피크리오노사이드 A(Picrionoside A, 화합물 2) 및 이카리사이드 B6(Icariside B6, 화합물 3)로 이루어진 군으로부터 선택되는 메가스티그메인 배당체 1종 이상을 포함하는 만병초(Rhododendron brachycarpum) 추출물을 함유하는 패혈증의 예방 또는 치료용 조성물에 관한 것이다. The present invention is concerned is selected from the group consisting of formulas to as dendeuro side A of the first (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 (Icariside B 6, Compound 3) ( Rhododendron brachycarpum ) extract containing at least one meglutine main glycoside. The present invention also relates to a composition for preventing or treating sepsis.
[화학식 1][Chemical Formula 1]
상기 만병초 추출물은 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 것일 수 있다. The Rhododendron phloem extract may be obtained by extractive condensation of Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
상기 만병초 추출물은 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있다. The Rhodiola extract has an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
본 발명은 또한 상기 화학식 1의 로도덴드로사이드 A(Rhododendroside A, 화합물 1), 피크리오노사이드 A(Picrionoside A, 화합물 2) 및 이카리사이드 B6(Icariside B6, 화합물 3)로 이루어진 군으로부터 선택되는 메가스티그메인 배당체 1종 이상을 함유하는 패혈증의 예방 또는 치료용 조성물에 관한 것으로서, 상기 메가스티그메인 배당체들도 역시 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있다. The invention further also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 selected from the group consisting of (Icariside B 6, Compound 3) The present invention also relates to a composition for preventing or treating septicemia comprising at least one meglutine main glycoside, wherein the meglutine main glycosides also have an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
또한 본 발명은 상기 화학식 1의 로도덴드로사이드 A(Rhododendroside A, 화합물 1), 피크리오노사이드 A(Picrionoside A, 화합물 2) 및 이카리사이드 B6(Icariside B6, 화합물 3)로 이루어진 군으로부터 선택되는 메가스티그메인 배당체 1종 이상을 포함하는 만병초 추출물을 함유하는 패혈증의 예방 또는 개선용 건강기능식품을 제공한다.In another aspect, the present invention is selected from the group consisting of dendeuro side A (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 (Icariside B 6, Compound 3) also of the general formula (1) Which contains at least one meggystine main glycoside, which is effective for preventing or ameliorating sepsis.
한편, 본 발명은 하기 화학구조를 갖는 신규 화합물 로도덴드로사이드 A(Rhododendroside A, 화합물 1)를 제공하며, 상기 로도덴드로사이드 A를 만병초로부터 분리하는 방법을 제공한다. The present invention also provides Rhododendroside A (Compound 1) as a novel compound having the following chemical structure, and provides a method for isolating rhodendroside A from rhizome.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
상기 만병초 추출물은 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 것일 수 있다. 이 외의 추출조건은 제한되지는 않으나, 상기 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매는 만병초 중량의 1~20배를 가하는 것이 바람직하며, 추출온도는 20~100℃인 것이 바람직하고, 추출시간은 1시간 내지 7일 이내가 바람직하다. The Rhododendron phloem extract may be obtained by extractive condensation of Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The extraction conditions are not limited, but the water, the lower alcohol having 1 to 4 carbon atoms, or the mixed solvent thereof is preferably added in an amount of 1 to 20 times the weight of the vinegar, and the extraction temperature is preferably 20 to 100 ° C , And the extraction time is preferably from 1 hour to 7 days.
상기 만병초 추출물은 상법에 따라, 물 또는 각종 유기용매에 의한 추출, 유기용매와 물의 분배, 칼럼크로마토그래피에 의한 방법 등, 식물체 성분의 분리 추출에 이용되는 공지의 방법을 단독 또는 적합하게 조합한 방법을 이용하여 분획 또는 정제하여 사용할 수 있다. The above-mentioned Rhododendron phloem extract can be obtained by a known method which is used for extractive extraction of plant components, such as extraction with water or various organic solvents, distribution of organic solvent and water, column chromatography, May be fractionated or purified and used.
바람직하게는 상기 만병초 추출물은 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 만병초 추출물을 물에 현탁한 후, 상기 현탁물에 헥산, 클로로포름, 에틸아세테이트, 및, 부탄올로 이루어진 군에서 선택되는 용매를 가하여 얻은 만병초 분획물로 제조할 수 있다. 상기 만병초 분획물은 바람직하게는, 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 만병초 추출물을 물에 현탁한 후 헥산과 혼합하여 얻은 헥산층의 농축물, 상기 헥산층을 제거하고 남은 잔사(물층)에 클로로포름을 혼합하여 얻은 클로로포름층의 농축물, 또는 상기 클로로포름층을 제거하고 남은 잔사(물층)에 에틸아세테이트를 혼합하여 얻은 에틸아세테이트층의 농축물, 상기 에틸아세테이트층을 제거하고 남은 잔사(물층)에 부탄올을 혼합하여 얻은 부탄올층의 농축물, 또는 상기 부탄올층을 제거하고 남은 잔사(물층)의 농축물일 수 있다. 본 발명의 화학식 1의 화합물들은 만병초 추출물 또는 만병초 분획물로부터 분리된 메가스티그메인 배당체(megastigmane glucosides) 화합물이다. 한편, 이 외의 분획조건은 제한되지는 않으나, 상기 만병초 추출물에 만병초 추출물의 중량의 1~50배의 물을 가하여 현탁물을 제조한 후, 상기 물과 동량의 헥산, 클로로포름, 에틸아세테이트, 및, 부탄올로 이루어진 군에서 선택되는 용매를 가하여 분획할 수 있다. 또한, 헥산층을 제거한 후 남은 잔사에 클로로포름을 가하고, 클로로포름층을 제거하고 남은 잔사에 에틸아세테이트를 가하고, 에틸아세테이트를 제거하고 남은 잔사에 부탄올을 가할 때에도, 단계적으로 이루어 질 때도 역시 잔사와 동량의 각 용매(클로로포름, 에틸아세테이트 또는 부탄올)를 순차적으로 가하여 분획할 수 있다. 이 때의 각 분획시간은 특별히 제한되지는 않으나 10분 내지 1일 이내인 것이 바람직하다. Preferably, the Rhododendron phloem extract is obtained by suspending a Rhododendron phloxus extract in water by extracting and concentrating Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and then adding to the suspension, hexane, chloroform, ethyl acetate, Butanol, and the like. Preferably, the Rhizome of the Rhizome of the Rhizome is preferably a concentrate of the hexane layer obtained by suspending the Rhizome of the Rhizoma of the Rhizome by extracting and concentrating the rhizome with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, The concentrate of the chloroform layer obtained by removing the layer (chloroform) from the remaining residue (water layer) or the concentrate of the ethyl acetate layer obtained by mixing the residue (water layer) remaining after removing the chloroform layer with ethyl acetate, A concentrate of the butanol layer obtained by removing the layer and mixing butanol with the remaining residue (water layer), or a concentrate of the residue (water layer) remaining after removing the butanol layer. The compounds of formula (I) according to the present invention are megastigmane glucosides compounds isolated from the rhizome extract or Rhizoma fractions. Meanwhile, the other fractionation conditions are not limited. However, after adding a water of 1 to 50 times the weight of the Pseudomonas sp. Extract to the Pseudomonas sp. Extract to prepare a suspension, the same amount of hexane, chloroform, ethyl acetate, Butanol can be added to the reaction mixture. Further, chloroform is added to the remaining residue after removing the hexane layer, chloroform layer is removed, and ethyl acetate is added to the remaining residue. When ethyl acetate is removed and butanol is added to the remaining residue, the same amount Each solvent (chloroform, ethyl acetate or butanol) can be added sequentially to fractionation. Each fractionation time at this time is not particularly limited, but is preferably within 10 minutes to one day.
본 발명의 만병초 추출물을 분획하거나 이로부터 메가스티크메인 배당체를 분리할 때 크로마토그래피를 이용할 수 있는데, 상기 크로마토그래피는 실리카겔 컬럼 크로마토그래피(silica gel column chromatography), 엘에이취-20 컬럼 크로마토그래피(LH-20 column chromatography), 이온교환수지 크로마토그래피(ion exchange resin chromatography), 중압 액체 크로마토그래피(MPLC; medium pressure liquid chromatography), 박층 크로마토그래피(TLC; thin layer chromatography), 실리카겔 진공 액체 크로마토그래피(silica gel vacuum liquid chromatography), 역상 크로마토그래피(reversed phase chromatography) 및 고성능 액체 크로마토그래피(high performance liquid chromatography) 중에서 선택될 수 있다. Chromatography can be used to fractionate the psyllium extract of the present invention or to separate the Megastich main glycoside from the silica gel column chromatography, LH-20 column chromatography (LH Ion exchange resin chromatography, medium pressure liquid chromatography (MPLC), thin layer chromatography (TLC), silica gel gel chromatography vacuum liquid chromatography, reversed phase chromatography, and high performance liquid chromatography.
상기 만병초 추출물은 만병초의 전초, 뿌리, 잎, 가지, 줄기, 열매, 꽃으로부터 선택되는 모든 부위로부터 추출가능하다. The above-mentioned Pseudomonas sp. Extract can be extracted from all parts selected from outbreaks, roots, leaves, branches, stems, fruits and flowers of Pseudomonas sp.
한편, 본 발명은 신규 화합물 로도덴드로사이드 A(Rhododendroside A, 화합물 1)를 제공하며, 상기 로도덴드로사이드 A를 만병초로부터 분리하는 방법을 제공한다. 바람직하게는 상기 로도덴드로사이드 A는 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 만병초 추출물을 물에 현탁한 후 헥산과 혼합하여 얻은 헥산층, 상기 헥산층을 제거하고 남은 잔사(물층)에 클로로포름을 혼합하여 얻은 클로로포름층, 상기 클로로포름층을 제거하고 남은 잔사(물층)에 에틸아세테이트를 혼합하여 얻은 에틸아세테이트층, 상기 에틸아세테이트층을 제거하고 남은 잔사(물층)에 부탄올을 혼합하여 얻은 부탄올층의 농축물, 또는 상기 부탄올층을 제거하고 남은 잔사(물층)의 농축물로부터 분리될 수 있다. The present invention also provides a novel compound, Rhododendroside A (Compound 1), and provides a method for isolating Rhodendroside A from Rhodiola. Preferably, Rhodendroside A is obtained by suspending a Rhododendron aquaticus extract in water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and extracting the Rhododendron sphaeroa extract with water, and then mixing with hexane to obtain a hexane layer, A chloroform layer obtained by mixing chloroform with the residue (water layer) remaining after removal of the chloroform layer, an ethyl acetate layer obtained by mixing ethyl acetate with the residue (water layer) remaining after removing the chloroform layer, a residue (water layer) remaining after removing the ethyl acetate layer, , Or a concentrate of the butanol layer obtained by mixing butanol, or a concentrate of the residue (water layer) remaining after removing the butanol layer.
또한, 본 발명은 상기 화학식 1의 로도덴드로사이드 A(Rhododendroside A, 화합물 1), 피크리오노사이드 A(Picrionoside A, 화합물 2) 및 이카리사이드 B6(Icariside B6, 화합물 3)로 이루어진 군으로부터 선택되는 메가스티그메인 배당체 1종 이상을 포함하는 만병초 추출물 또는 이로부터 분리된 메가스티그메인 배당체를 함유하는 패혈증의 예방 또는 치료용 약학 조성물을 제공한다. 상기 약학 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. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 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. In addition, the present invention also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 from the group consisting of (Icariside B 6, Compound 3) The present invention provides a pharmaceutical composition for preventing or treating septicemia containing a meganegget extract or a meggestidine main glycoside separated therefrom, which comprises at least one selected megestigine main glycoside. The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 만병초 추출물 또는 이로부터 분리된 메가스티그메인 배당체, 또는 이를 함유한 약학 조성물의 투여량은 치료받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01 ㎎/㎏/일 내지 대략 2000 ㎎/㎏/일의 범위이다. 더 바람직한 투여량은 1 ㎎/㎏/일 내지 500 ㎎/㎏/일이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. The dosage of the phytophthora extract of the present invention or the meggestidine main glycoside isolated therefrom, or the pharmaceutical composition containing the same, depends on the age, sex, body weight of the subject to be treated, the severity of the specific disease or pathological condition to be treated, , The route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 약학 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. 본 발명의 화합물은 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다. The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the compound of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.
또한, 본 발명은 상기 화학식 1의 로도덴드로사이드 A(Rhododendroside A, 화합물 1), 피크리오노사이드 A(Picrionoside A, 화합물 2) 및 이카리사이드 B6(Icariside B6, 화합물 3)로 이루어진 군으로부터 선택되는 메가스티그메인 배당체 1종 이상을 포함하는 만병초 추출물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 패혈증의 예방 또는 개선용 건강기능식품을 제공한다. 상기 만병초 추출물은 본 발명의 건강기능식품에 0.001~100 중량%로 하여 첨가될 수 있다. 본 발명의 건강기능식품은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 본 발명의 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성식품류 등이 있다. In addition, the present invention also dendeuro side A of Formula 1 (Rhododendroside A, compound 1), blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 from the group consisting of (Icariside B 6, Compound 3) The present invention provides a health functional food for preventing or ameliorating septicemia including a psittacosis extract and a pharmaceutically acceptable food-aid additive including at least one selected megestigine main glycoside. The Rhododendron phloem extract may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.
본 발명은 만병초 추출물 또는 이로부터 분리된 메가스티그메인 배당체를 함유하는 패혈증의 예방 또는 치료용 조성물에 관한 것으로서, 상기 조성물은 혈관내피세포에서 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 우수하여 패혈증의 예방 또는 치료용 의약 조성물이나 패혈증의 예방 또는 개선용 건강기능식품으로 용이하게 이용가능하다. The present invention relates to a composition for preventing or treating septicemia, which contains a moxa agar extract or a megastigue main glycoside isolated therefrom, wherein the composition is effective for inhibiting the expression of HMGB1 (high mobility group box 1) in vascular endothelial cells Can be easily used as a health functional food for preventing or treating sepsis or for preventing or treating sepsis.
도 1은 본 발명의 화합물 1의 COSY (━) 및 HMBC (→) 상관관계를 나타낸다.
도 2는 본 발명의 화합물 1의 산가수분해에 대한 GC 분석 스펙트럼 결과이다.
도 3은 본 발명의 화합물 1에 대해 예상가능한 부분입체이성질체(diastereomers)를 나타내는 그림이다.
도 4는 본 발명의 화합물 1~3이 HMGB1으로 유도된 HUVEC 패혈증 세포 모델의 F-액틴 손상을 억제하는 것을 나타내는 면역형광염색 결과이다.
도 5는 본 발명의 만병초 메탄올 추출물(MeOH), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtOA), 부탄올 분획물(BuOH)이 HMGB1으로 유도된 HUVEC 패혈증 세포 모델에서 세포벽 투과성을 억제하는 것을 나타내는 결과이다.
도 6은 본 발명의 화합물 1~3이 HMGB1으로 유도된 HUVEC 패혈증 세포 모델에서 세포벽 투과성을 억제하는 것을 나타내는 결과이다.
도 7은 본 발명의 화합물 1~3이 LPS로 유도된 HUVEC 패혈증 세포 모델에서 HMGB1의 생성을 억제하는 것을 나타내는 결과이다.
도 8은 본 발명의 화합물 1~3이 LPS로 유도된 HUVEC 패혈증 세포 모델에서 세포벽 투과성을 억제하는 것을 나타내는 결과이다.
도 9는 본 발명의 화합물 1~3이 HMGB1으로 유도된 패혈증 동물 모델에서 혈관 내로 주입된 염료가 복강으로 누출되는 것을 억제하는 결과이다.
도 10은 본 발명의 화합물 1~3이 CLP 수술법으로 유도된 패혈증 동물들의 생존율을 확인한 결과이다.Figure 1 shows the COSY (-) and HMBC (-) correlation of
Figure 2 shows GC analysis spectral results for acid hydrolysis of
Figure 3 is a diagram showing predictable diastereomers for
Figure 4 shows immunofluorescence staining results showing that
Figure 5 is a result indicating that inhibit cell wall permeability in Rhododendron methanol extract (MeOH), chloroform fraction (CHCl 3), ethyl acetate fraction (EtOA), HUVEC sepsis cell models of butanol fraction (BuOH) is induced by HMGB1 of the invention to be.
Figure 6 shows the results of the inhibition of cell wall permeability in HMGB1-induced HUVEC sepsis cell model of Compounds 1-3 of the present invention.
Fig. 7 shows the results of inhibiting the production of HMGB1 in the HUVEC sepsis cell model in which the compounds 1-3 of the present invention were induced by LPS.
Figure 8 shows the results of the inhibition of cell wall permeability in the HUVEC sepsis cell model in which the compounds 1-3 of the present invention were induced by LPS.
FIG. 9 shows the results of inhibiting the leakage of the dye injected into the blood vessels into the abdominal cavity in the animal model of sepsis in which the
FIG. 10 shows the results of confirming the survival rate of the sepsis animals induced by CLP surgery according to
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다. Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.
<< 실시예Example 1. 만병초로부터 화합물의 분리> 1. Isolation of Compound from Pyrrhizae>
만병초의 잎을 전남 광주의 만병초 재배 농장에서 얻고, 건조된 만병초 잎(25 ㎏)을 메탄올로 실온에서 1주일 동안 2회 추출(각각 250 ℓ씩)하였고, 갈색의 슬러리 형태의 농축 추출물(6 ㎏)을 얻었다. 상기 추출물(3 ㎏)을 물(10 ℓ)에 현탁한 후, n-헥산(10 ℓ)으로 분획(3번 반복)하였고 이를 농축하여 n-헥산 분획물을 얻었다. n-헥산층과 분리된 물층(잔사)에 클로로포름을 가해 얻은 클로로포름층을 농축하여 클로로포름 분획물을 얻었다. 클로로포름층과 분리된 물층(잔사)에 에틸아세테이트를 가해 얻은 에틸아세테이트층을 농축하여 에틸아세테이트 분획물을 얻었다. 에틸아세테이트층과 분리된 물층(잔사)에 부탄올을 가하여 얻은 부탄올층을 농축하여 부탄올 분획물을 얻었다. 각 용매들은 모두 10 ℓ씩 가하고 동일한 용매를 이용하여 3번씩 반복 분획 추출하였다. 이에, 최종적으로 n-헥산 분획물 438 g, 클로로포름 분획물 140 g, 에틸아세테이트 분획물 450 g, 부탄올 분획물 320 g을 얻었다. The leaves of Rhododendron were obtained from the cultivated ranch plant in Gwangju, Jeollanam - do, and the dried rhododendron leaves (25 ㎏) were extracted with methanol twice at room temperature for one week (250 ℓ each) ). The extract (3 kg) was suspended in water (10 L), and then fractionated with n -hexane (10 L) (repeated three times) and concentrated to obtain n -hexane fraction. The chloroform layer obtained by adding chloroform to the water layer (residue) separated from the n - hexane layer was concentrated to obtain a chloroform fraction. Ethyl acetate was added to the chloroform layer and the separated water layer (residue), and the ethyl acetate layer was concentrated to obtain ethyl acetate fraction. The butanol layer obtained by adding butanol to the ethyl acetate layer and the separated water layer (residue) was concentrated to obtain a butanol fraction. Each of the solvents was added in an amount of 10 L each, and repeatedly extracted three times with the same solvent. Finally, 438 g of n -hexane fraction, 140 g of chloroform fraction, 450 g of ethyl acetate fraction and 320 g of butanol fraction were obtained.
상기 부탄올 분획물(320 g)을 실리카겔 컬럼 VLC 및 디클로로메탄:메탄올:물(10:1→2:1:0.1)의 용출 조건으로 분획하여 6개의 소분획물을 얻었다(Fr. B1-B6). 이 중에서 Fr. B1(10 g)을 MPLC(C18 SNAP Cartridge KP-C18-HS, 340 g) 및 메탄올:물(3:7→7:3)의 용출조건을 이용하여 7개의 소분획물을 얻었다(Fr. B11-B17). 이 중에서 Fr. B14(1.46 g)를 이용하여 HPLC(Luna C18(2) column: 250×21.20 ㎜, 20%(v/v) MeCN, flow rate: 6 ㎖/min)를 수행하여 화합물 2(26 ㎎, t R 40.2 min)와 화합물 3(3.8 ㎎, t R 53.6 min)을 얻었다. Fr. B2(22 g)를 MPLC(C18 SNAP Cartridge KP-C18-HS, 340 g) 및 아세톤:메탄올:물(0:0:100→12:28:60)의 용출조건을 이용하여 9개의 소분획물을 얻었다(B21-B29). 이 중에서 Fr. B26 (1.7 g)을 실리카겔 MPLC(SNAP Cartridge KP-Sil, 120 g) 및 에틸아세테이트:메탄올(98:2→90:10)의 용출조건을 이용하여 5개의 소분획물을 얻었다(B261-B265). 이 중에서 Fr. B262(92 ㎎)를 이용하여 실리카겔 컬럼 크로마토그래피(2×80 ㎝) 및 클로로포름:아세톤:물(1:2:0.1)의 용출조건을 이용하여 화합물 1(2 ㎎)을 얻었다. The butanol fraction (320 g) was fractionated by silica gel column VLC and elution conditions of dichloromethane: methanol: water (10: 1 -> 2: 1: 0.1) to obtain six small fractions (Fr. B1-B6). Of these, Fr. Seven fractions were obtained using elution conditions of B1 (10 g) with MPLC (C18 SNAP Cartridge KP-C 18 -HS, 340 g) and methanol: water (3: 7 → 7: 3) -B17). Of these, Fr. B14 (1.46 g) using HPLC (Luna C18 (2) column : 250 × 21.20 ㎜, 20% (v / v) MeCN, flow rate: 6 ㎖ / min) to perform the compound 2 (26 ㎎, t R 40.2 min) and compound 3 (3.8 mg, t R 53.6 min) were obtained. Fr. B2 (22 g) was fractionated using MPLC (C18 SNAP Cartridge KP-C18-HS, 340 g) and elution conditions of acetone: methanol: water (0: 0: 100 → 12: 28: 60) (B21-B29). Of these, Fr. B26 (1.7 g) was subjected to silica gel MPLC (SNAP Cartridge KP-Sil, 120 g) and elution conditions of ethyl acetate: methanol (98: 2 -> 90: 10) to obtain 5 small fractions (B261-B265). Of these, Fr. Compound 2 (2 mg) was obtained by using silica gel column chromatography (2 x 80 cm) and elution conditions of chloroform: acetone: water (1: 2: 0.1) using B262 (92 mg).
<< 실시예Example 2. 화합물의 물리화학적 성질 확인> 2. Identification of physicochemical properties of compounds>
실시예Example 2-1. 2-1. 로도덴드로사이드Rhododendroside A (화합물 1) A (Compound 1)
Rhododendroside A;Rhododendrosidea;
yellow amorphous powder;yellow amorphous powder;
[α] : -15.4 (c 0.1, MeOH); [alpha] : -15.4 (c 0.1, MeOH);
HR-ESI-MS : m/z 411.1996 [M+Na]+ (calcd. for C19H32O8Na, 411.1995). HR-ESI-MS: m / z 411.1996 [M + Na] + (calcd for C 19 H 32 O 8 Na, 411.1995.).
1H-NMR (methanol-d4, 600 MHz) (표 1 참조); 1 H-NMR (methanol-
13C-NMR (methanol-d4, 150 MHz) (표 1 참조). 13 C-NMR (methanol- d 4 , 150 MHz) ( see Table 1).
상기 표 1에서 Exp.는 화합물 1을 NMR spectrometer로 실측한 분석 결과이며, Cal.의 I 및 III는 화합물 1의 예상 가능한 입체이성질체(diastereomers)로서 수퍼컴퓨터를 이용하여 계산한 I 및 III의 CS 분석값을 나타낸다. In Table 1, Exp. Is the result of an analysis of
상기 화합물 1은 노란색의 비결정질 분말 상태(yellow amorphous powder)로서, HR-ESI-MS 데이터에 따른 분자식은 C19H32O8Na이다(obsd. [M+Na]+, m/z 411.1996; calcd. [M+Na]+, m/z 411.1995). 표 1의 1H NMR 스펙트럼 결과를 통해 화합물 1이 올레핀 양성자(olefinic proton, δH 4.51), 아노머 양성자(anomeric proton, δH 4.43), 옥시메틴 양성자(oxymethine proton, δH 4.16), 알릴(allylic, δH 1.66) 및 3개의 3차 메틸 그룹(tertiary methyl groups, δH 1.30, 1.07, 1.03)에 대한 공명(resonances)이 있음을 확인할 수 있다. 표 1의 13C NMR 스펙트럼 결과는 글루코피라노실 모이어티(glucopyranosyl moiety, δC 102.8, 75.1, 78.0, 71.6, 77.8, 62.7), 2개의 올레핀 탄소(δC 149.4, 97.6), 옥시메틴 탄소(δC 73.2), 3개의 4차 탄소(quaternary carbon, δC 81.5, 72.0, 40.7), 3개의 메틸렌 탄소(methylene carbon, δC 44.7, 41.4, 29.9), 1개의 알릴(δC 20.0) 및 3개의 3차 메틸 탄소(δC 27.4, 24.6, 22.7)가 속해있는 6개의 탄소 공명을 포함하는 19개의 공명이 있음을 나타낸다. 이와 같은 분광 데이터(spectroscopic data)는 이카리사이드 B2(icariside B2)(Bioorg. Med. Chem. Lett. 2012, 22, 6116-6119)라는 메가스티크메인 배당체와 비슷하다. 이는 화합물 1도 역시 메가스티그메인 배당체의 구조를 갖고 있음을 의미한다. 이카리사이드 B2와 화합물 1의 NMR 데이터의 차이는 트랜스-올레핀 그룹(conjugated trans-olefinic group)의 양자 신호 부재와 아세틸 메틸 그룹의 변화이다. 화합물 1은 이카리사이드 B2의 C-6 메틸 비닐 케톤이 고리화 되어 옥시란 모이어티(oxirane moiety)를 형성하여 생성된 최초의 환상 메가스티그메인 구조이다.이것은 H-8 (δH 4.51) 및 C-6 (δC 72.0), C-7 (δC 29.9), C-9 (δC 149.4), C-10 (δC 20.0), H-7 (δH 2.34, 1.99) 및 C-6 (δC 72.0), C-8 (δC 97.6), C-9 (δC 149.4) 사이의 HMBC 상관관계로부터 확인된다(도 1 참조). 이러한 헥사히드로-4H-크로멘 모이어티(hexahydro-4H-chromene moiety)의 생성 및 독특한 사이클로메가스티그메인 스캐폴드(unique cyclomegastigmane scaffold)는 이전에는 확인된 바가 없다. 아글리콘(aglycone)과 단당류(monosaccharide)의 치환 위치는 H-1'(δH 4.43) 및 C-3 (δC 73.2)(도 1 참조)의 HMBC 상관관계로부터 확인된다. 아노머 양자(anomeric proton)의 짝지음 상수(coupling constant, J = 7.8 Hz)는 글루코스가 β-입체 배향(β-glucosidic linkage)으로 연결되어 있음을 의미한다.The
또한, GC(gas chromatography) 분석을 이용한 산 가수분해(acid hydrolysis) 결과에 따르면(도 2 참조) 화합물 1은 D-글루코스를 가지고 있음이 확인된다(상단 오른쪽의 D-글루코스 표준품 분석 결과와 하단 중앙의 화합물 1의 분석 결과가 거의 유사함). 화합물 1의 산 가수분해법은 하기 방법을 이용하여 확인하였다. 화합물 1을 10%(v/v) HCl(1 ㎖)에 녹인 후 85℃로 3시간 동안 가열하고, 잔여물(residue)을 에탄올과 물로 분획하였다. 물층을 증발시켜 남은 잔여물을 무수 피리딘(anhydrous pyridine, 100 ㎕)으로 녹였고, 0.1 M L-시스테인 메틸 에스테르 하이드로클로라이드(L-cysteine methyl ester hydrochloride, 100 ㎕)를 가했다. 이를 60℃에서 1.5시간 동안 가열한 후, 잔여물을 말리고 1-트리메틸실일이미다졸 용액(1-trimethylsilylimidazole solution(0.5 ㎖)을 가하여 트리메틸실일화(trimethylsilylated)하였다. 이를 60℃에서 5분간 가열한 뒤 말려서 얻은 생성물을 물과 n-헥산으로 분획하였다. 이 중에서 비극성 층(n-hexane layer)을 GC 분석에 이용하였다. 단당류 모이어티(monosaccharide moiety)는 D-글루코스 표준품(t R 16.21 min)과 비교하여 확인하였다. In addition, according to the acid hydrolysis results using GC (gas chromatography) analysis (see FIG. 2),
아글리콘의 연관 구조는 짝지음 상수와 NOE 상관관계를 통해 확인된다. H-3 (tt, 12.6, 4.1 Hz)의 짝지음 상수는 의자형 구조(diastereomer I) 또는 보트형 구조(diastereomer II)의 사이클로헥산 고리(cyclohexane ring)가 있음을 의미한다(도 3A 참조). C-5의 연관 구조는 H-3 (δH 4.16) 및 H-12 (δH 1.07) 사이의 강한 NOE 상관관계를 기반으로 배치되어 있고, H-3 (δH 4.16) 및 H-13 (δH 1.30)에는 NOE 상관관계가 없는 것을 의미한다(도 3A 참조). 이에, 화합물 1에 대한 상기 자료들과 함께 MMFF94 force field(Schrodinger LLC.)를 이용한 2면각의 제한(dihedral constraints) 및 공간 거리 정보(spatial distance information)에 대한 정보를 이용하여 2개의 가능한 부분입체이성질체(diastereomers I: 3S, 5R, 6R; II: 3S, 5R, 6S)를 제안하였다(도 3A 참조). 부분입체이성질체 I에서 H-12 (δH 1.07) 및 H-8 (δH 4.51) 사이에는 NOE 상관관계가 있고, H-11 (δH 1.03) 및 H-13 (δH 1.30) 사이에는 NOE 상관관계가 없다. 그러나 부분입체이성질체 II에서는 이것이 가능하다. NOE 실험결과, 화합물 1은 부분입체이성질체(diastereomer) I에 상응하는 NOE 상관관계를 나타내었으므로 화합물 1의 상대입체구조를 부분입체이성질체(diastereomer) I로 결정하였다(도 3A 참조).The association structure of aglycons is confirmed by the correlation of NOE with a pair constant. The coupling constants of H-3 (tt, 12.6, 4.1 Hz) means that there is a cyclohexane ring of the diastereomer I or diastereomer II (see FIG. 3A). The association structure of C-5 is arranged based on strong NOE correlation between H-3 (δ H 4.16) and H-12 (δ H 1.07), and H-3 (δ H 4.16) and H-13 < / RTI > H < RTI ID = 0.0 > 1.30) < / RTI > Using the information on the dihedral constraints and spatial distance information using the MMFF94 force field (Schrodinger LLC.) With the above data for
화합물 1의 양적 제한 때문에, DP4 분석에 따른 GIAO NMR CS(gauge-invariant atomic orbital NMR chemical shift) 계산을 이용하여 절대입체구조(absolute configuration)를 결정하였다. 한편, 화합물 1이 갖는 D-글루코피라노실 모이어티(D-glucopyranosyl moiety) 때문에, 거울상 이성질체의 아글리콘 모이어티(enantiomeric aglycone moiety)를 갖는 부분입체이성질체 I 및 III이 화합물 1의 구조로 고려되었다(도 3B 참조). 부분입체이성질체 I 및 III에 대한 확증은 매크로모델(Macromodel, Schrodinger LLC.)의 이용 및 가우시안 09 패키지(Gaussian 09 package, Gaussian Inc. B3LYP/6-31G(d,p) level)를 이용한 GIAO 쉴드 상수 계산(GIAO shielding constant calculations)을 통해 수행하였다. Because of the quantitative limitation of
CS값은 MMFF94 위치 에너지(MMFF94 potential energy)에 근거하여 볼츠만-평균화(Boltzmann-averaged)하였다. 평균화된 값은 DP4 통계분석을 통해 계산하였다.CS values were Boltzmann-averaged based on MMFF94 potential energy. The averaged values were calculated by DP4 statistical analysis.
도 3C에 개시된 DP4 통계분석 결과, 화합물 1의 절대입체구조가 I의 입체구조와 일치할 확률이 98.4%인 것으로 나타났다. Statistical analysis of DP4 disclosed in FIG. 3C showed that the absolute stereochemistry of
화합물 1의 절대입체구조는 화합물 2 및 3으로부터 생합성되는 경로를 화학적으로 예측해 봄으로써 다시 한 번 입증되었다. 즉, 하기 반응식 1을 통해, 화합물 3의 효소 에폭시화(enzymatic epoxidation)가 3β-적도상 치환기(3β-equatorial substituent)를 통해 유도되고, 옥시란(oxirane, 화합물 4)이 형성된다. 이 후, 화합물 5를 통해 고리화 및 탈수 반응을 이용하여 화합물 1이 형성되는 것을 알 수 있다. The absolute stereostructure of
[반응식 1][Reaction Scheme 1]
따라서, 상기 결과들을 종합하여, 화합물 1을 (3S,5R,6R)-1,1,5,9-테트라메틸-3-β-D-글루코피라노실옥시-2,3,4,5-테트라하이드로-7H-크로멘-6-올((3S,5R,6R)-1,1,5,9-tetramethyl-3-β-D-glucopyranosyloxy-2,3,4,5-tetrahydro-7H-chromen-6-ol)의 화학구조를 갖는 로도덴드로사이드 A(rhododendroside A)로 결정하였다. Thus, the synthesis by the above results, the compound 1 (3 S, 5 R, 6 R) -1,1,5,9- tetramethyl -3- β -D- glucopyranosyl-oxy -2,3,4, 5-tetrahydro--7 H - chromen-6-ol ((3 S, 5 R, 6 R) -1,1,5,9-tetramethyl-3- β -D-glucopyranosyloxy-2,3,4, also has a chemical structure of the 5-tetrahydro-7 H -chromen- 6-ol) was determined by dendeuro side a (rhododendroside a).
실시예Example 2-2. 2-2. 피크리오노사이드Picronoside A (화합물 2) A (Compound 2)
Picrionoside A; Picrionosidea;
Yellow amorphous powder; Yellow amorphous powder;
ESI-MS m/z 371 [M+H]+; ESI-MS m / z 371 [M + H] < + >;
[α]:+108.8(c0.1,MeOH); [[alpha]]: + 108.8 (c0.1, MeOH);
1H-NMR (250 MHz, pyridine-d 5) δH 6.63(1H, dd, J = 15.8, 10.0 Hz, H-7), 6.17 (1H, d, J = 15.8 Hz, H-8), 5.91 (1H, brs, H-4), 4.98 (1H, d, J = 7.7 Hz, H-1'), 4.50 (1H, m, H-3), 4.58-4.02 (6H, m, H-2'~H-6'), 2.42 (1H, d, J = 9.9 Hz, H-6), 2.24 (3H, s, H-10), 1.84 (1H, d, J = 13.6, 5.9 Hz, H-2a), 1.69 (1H, dd, J = 13.6, 5.8 Hz, H-2b), 1.49 (3H, s, H-13), 1.01 (3H, s, H-12), 0.78 (3H, s, H-10); 1 H-NMR (250 MHz, pyridine- d 5) δ H 6.63 (1H, dd, J = 15.8, 10.0 Hz, H-7), 6.17 (1H, d, J = 15.8 Hz, H-8), 5.91 (1H, brs, H-4), 4.98 (1H, d, J = 7.7 Hz, H-1 '), 4.50 (1H, m, H-3), 4.58-4.02 ~ H-6 '), 2.42 (1H, d, J = 9.9 Hz, H-6), 2.24 (3H, s, H-10), 1.84 (1H, d, J = 13.6, 5.9 Hz, H-2a ), 1.69 (1H, d, J = 13.6, 5.8 Hz, H-2b), 1.49 (3H, s, H- 10);
13C-NMR (63 MHz, pyridine-d 5) δC 197.8 (C-9), 147.5 (C-7), 135.5 (C-5), 134.3 (C-8), 126.1 (C-4), 103.6 (C-1'), 79.11 (C-3'), 78.9 (C-5'), 75.6 (C-2'), 72.7 (C-3), 72.2 (C-4'), 63.3 (C-6'), 54.8 (C-6), 40.4 (C-2), 34.0 (C-1), 29.5 (C-12), 27.4 (C-10), 25.3 (C-11), 22.9 (C-13). 13 C-NMR (63 MHz, pyridine- d 5 )? C 197.8 (C-9), 147.5 (C-7), 135.5 (C-3 '), 78.9 (C-5'), 75.6 (C-2 '), 72.7 (C-6), 54.8 (C-6), 40.4 (C-2), 34.0 (C-1), 29.5 -13).
실시예Example 2-3. 2-3. 이카리사이드Ikarishide B B 6 6 (화합물 3)(Compound 3)
Icariside B6;Icariside B 6 ;
Yellow amorphous powder; Yellow amorphous powder;
ESI-MS m/z 373 [M+H]; ESI-MS m / z 373 [M + H];
[α]:-59(c0.1, MeOH); [[alpha]]: 59 (c0.1, MeOH);
1H-NMR (600 MHz, MeOD) δH 4.44 (1H, d, J = 7.8 Hz, H-1'), 4.08 (1H, m, H-3), 3.88 (1H, m, H-6'a), 3.69 (1H, m, H-6'b), 3.34 (2H, m, H-3',H-5'), 3.28 (1H, m, H-4'), 3.16 (1H, m, H-2'), 2.56 (2H, ddd, J = 9.9, 6.2, 3.4 Hz, H-8), 2.36 (1H, m, H-4a), 2.30 (1H, m, H-7a), 2.22 (1H, m, H-7b), 2.15 (3H, s, H-10), 2.05 (1H, m, H-4b), 1.86 (1H, m, H-2a), 1.64 (3H, s, H-13), 1.52 (1H, t, J = 12.1 Hz, H-2b), 1.06 (6H, s, H-11, H-12); 1 H-NMR (600 MHz, MeOD)? H 4.44 (1H, d, J = 7.8 Hz, H-1 '), 4.08 m, H-4 '), 3.16 (1H, m, H-5'), 3.69 M, H-2 '), 2.56 (2H, ddd, J = 9.9, 6.2, 3.4 Hz, H-8), 2.36 (1H, m, H-7b), 2.15 (3H, s, H-10), 2.05 -13), 1.52 (1H, t, J = 12.1 Hz, H-2b), 1.06 (6H, s, H-11, H-12);
13C-NMR (150 MHz, MeOD) δC 211.4 (C-9), 137.5 (C-6), 126.1 (C-5), 102.3 (C-1'), 78.1 (C-3'), 77.8 (C-5'), 75.1 (C-2'), 73.1 (C-3), 71.6 (C-4'), 62.7 (C-6'), 47.3 (C-2), 44.9 (C-8), 39.7 (C-4), 38.7 (C-1), 30.0 (C-12), 29.7 (C-10), 28.6 (C-11), 22.8 (C-7), 19.8 (C-13). 13 C-NMR (150 MHz, MeOD)? C 211.4 (C-9), 137.5 (C-6), 126.1 (C-5), 102.3 (C-5 '), 75.1 (C-2'), 73.1 (C-3), 71.6 ), 39.7 (C-4), 38.7 (C-1), 30.0 (C-12), 29.7 .
<< 실시예Example 3. 패혈증 세포모델에서의 F- In the sepsis cell model, F- 액틴Actin 손상 억제 효과 확인> Confirmation of damage inhibition effect>
화합물 1~3이 HMGB1을 매개로 한 혈관의 손상을 억제함으로써 패혈증의 치료 효과가 있는지 확인하였다. 화합물 1~3을 HUVEC(human umbilical vein endothelial cells)에 처리하고 F-액틴의 손상(패혈증에 따른 혈관벽 손상에 대한 모델)을 위해서는 HMGB1을 처리한 뒤, 팔로이딘(phalloidin)을 이용하여 세포내의 F-액틴(F-actin)에 대한 면역형광염색(immunofluorescence staining)을 하였다.
HUVEC 세포는 Cambrex Bio Science(Charles City, IA, USA)에서 얻었으며, EBM-2 기본 배지(basal media) 및 이에 공급되는 성장 보충물(Cambrex Bio Science)을 첨가하여 배양하였다. 면역형광염색을 위해서는 HUVEC을 0.05%(w/v) 폴리-L-라이신(poly-L-lysine)으로 코팅된 유리커버슬립(glass cover slip)에서 10%(v/v) FBS가 포함된 세포배양배지(EGM-2)를 이용하여 48시간 동안 배양하였다. 세포벽 손상을 유발하기 위해, HUVEC에 각 화합물(10μM)을 6시간 동안 전처리한 뒤, 1 ㎍/㎖의 HMGB1(Abnova)을 처리하여 16시간 동안 배양하였다. 화합물 처리를 마친 후에는 4% 포름알데하이드(4% formaldehyde in PBS[phosphate buffered saline], v/v)를 처리하고 실온에서 15분 동안 반응시켜 세포를 고정하였다. 세포 고정액을 제거한 후에는 0.05% 트리톤 X-100(0.05% Triton X-100 in PBS, v/v)을 15분간 처리하여 세포에 항체가 투과되도록 반응시키고(permeabilization), 이를 제거한 뒤, 5% BSA(bovine serum albumin in PBS, w/v)를 넣고 4℃에서 18시간 이상 밤새 반응시켰다. 항체(mouse monoclonal antibody, 1:400, diluted in 5%[w/v] BSA in PBS) 및 형광물질 팔로이딘이 표지된 F-액틴항체(fluorescein phalloidin, F432, Molecular Probes, Invitrogen)는 4℃에서 18시간 이상 밤새 반응시켰고, 형광 현미경(fluorescence microscopy, Carl Zeiss, AG, Germany)으로 400×의 배율로 확인하였다. HUVEC cells were obtained from Cambrex Bio Science (Charles City, IA, USA) and cultured with EBM-2 basal media and growth supplement (Cambrex Bio Science) supplied thereto. For immunofluorescence staining, HUVECs were incubated with 10% (v / v) FBS in glass cover slips coated with 0.05% (w / v) poly-L-lysine And cultured for 48 hours using a culture medium (EGM-2). To induce cell wall damage, each compound (10 μM) was pretreated with HUVEC for 6 hours and then treated with 1 μg / ml HMGB1 (Abnova) for 16 hours. After the compound treatment, cells were fixed by treatment with 4% formaldehyde in PBS (phosphate buffered saline, v / v) at room temperature for 15 min. After removing the cell fixation solution, the cells were treated with 0.05% Triton X-100 in PBS (v / v) for 15 minutes to permeabilize the cells. Then, the cells were washed with 5% BSA (bovine serum albumin in PBS, w / v) and incubated at 4 ° C overnight for more than 18 hours. (Fluorescein phalloidin, F432, Molecular Probes, Invitrogen) labeled with fluorochrome and mouse monoclonal antibody (1: 400, diluted in 5% [w / v] The reaction was carried out overnight over 18 hours and confirmed by fluorescence microscopy (Carl Zeiss, AG, Germany) at a magnification of 400 ×.
세포막의 손상부위는 도 4에서 화살표로 표시하였다. 도 4를 참고하면, 화합물 1~3이 HMGB1이 유도한 F-액틴 손상을 복구시켰음을 확인할 수 있다. The damaged area of the cell membrane is indicated by an arrow in Fig. Referring to FIG. 4, it can be seen that the
<< 실시예Example 4. 패혈증 세포 모델에서의 4. In the sepsis cell model 혈관세포의Vascular cell 투과성 억제 효과 확인> Confirmation of permeation inhibiting effect>
본 발명의 만병초 메탄올 추출물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물(1~10 ㎍/㎖)과 화합물 1~3(1~10 μM)을 HUVEC에 농도별로 처리한 후 각 화합물이 HMGB1을 통해 유도된 혈관벽 손상(혈관의 미세한 천공으로 인해 투과성이 생김)을 복구하는지를 다시 한번 확인하였다. 이를 위해 HUVEC에 대한 투과성을 확인하였으며, 2-챔버 모델(2-compartment chamber model, Blood 2011, 118, 3952-3959)을 이용해 배양된 세포층에서의 에반스 블루와 결합된 알부민(Evans blue-bound albumin)의 흐름을 정량적으로 측정하였다. (1 ~ 10 / / ml) and
HUVEC은 5×104/웰(well)로 3 μm pore size의 12 ㎜ 직경의 트랜스웰( transwell)에서 3일 동안 배양되었다. 세포가 각 트랜스웰에 가득 차면 각각의 화합물을 6시간 동안 처리하였고, 이 후 다시 HMGB1(1 ㎍/㎖)을 처리하여 16시간 동안 반응시켰다. 이 후 각 트렌스웰을 PBS(pH 7.4)로 세척하였고, 4%(w/v) BSA가 포함된 세포배양배지에 희석된 0.5 ㎖의 에반스 블루 용액(0.67 ㎎/㎖)을 준비하였다. 신선한 세포배양배지가 하단의 챔버에 더해졌고, 상단의 챔버에는 상기 에반스 블루 용액으로 교체되었다. 10분 후, 하단 챔버의 OD(Optical density)를 650 nm에서 측정하였고 이에 대한 결과는 도 5 및 6에 나타내었다. HUVECs were cultured for 3 days in a transwell of 12 mm diameter with 3 占 pore size at 5 占04 / well. When cells were filled in each transwell, each compound was treated for 6 hours, then treated with HMGB1 (1 μg / ml) for 16 hours. Each transwell well was then washed with PBS (pH 7.4) and 0.5 ml of Evans Blue solution (0.67 mg / ml) diluted in cell culture medium containing 4% (w / v) BSA was prepared. Fresh cell culture medium was added to the lower chamber, and the upper chamber was replaced with the above Evans blue solution. After 10 minutes, the OD (optical density) of the lower chamber was measured at 650 nm and the results are shown in FIGS. 5 and 6. FIG.
도 5 및 6을 확인하면, 본 발명의 만병초 메탄올 추출물(MeOH), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtOA), 부탄올 분획물(BuOH)(1~10 ㎍/㎖)과 화합물 1~3(1~10 μM)이 모두 혈관벽 손상으로 인한 투과성을 억제하는 것을 확인할 수 있어, HMGB1 유도된 패혈증 모델에서의 혈관 염증 억제 효과가 우수함을 알 수 있다(화합물 1[10 μM = 3.88 ㎍/㎖], 화합물 2[10 μM = 3.7 ㎍/㎖], 화합물 3[10 μM = 3.72 ㎍/㎖]은 만병초 추출물 또는 분획물[10 ㎍/㎖]에 비해 약 3배 가량 활성이 높음). 5 and 6, the methanol extracts of Phenological Veterinary Methanol (MeOH), the chloroform fraction (CHCl 3 ), the ethyl acetate fraction (EtOA) and the butanol fraction (BuOH) (1 to 10 μg / (10 μM = 3.88 ㎍ / ㎖) in the HMGB1-induced septicemia model, indicating that the inhibition of the permeability due to damage to the blood vessel wall is superior to that of the HMGB1-induced sepsis model (1 to 10 μM) , Compound 2 [10 μM = 3.7 μg / ml] and compound 3 [10 μM = 3.72 μg / ml] are about three times more active than the Rhizome extract or fraction [10 μg / ml].
<< 실시예Example 5. 5. LPSLPS 유도 패혈증 반응의 억제 효과 확인> Identification of inhibitory effects of induced sepsis>
HUVEC을 6웰 플레이트(6-well plates)에서 배양한 후 LPS(lipopolysaccharide, 100ng/㎖)를 100시간 동안 처리하였다(대조군은 saline 처리). HUVEC의 세포배양액을 이용하여 ELISA(competitive enzyme-linked immunosorbent assays)를 수행하기 위해 96웰 플레이트(96-well plastic flat microtiter plates, Corning, NY, USA)에 0.02% 소듐 아자이드(sodium azide)가 포함된 20mM 카보네이트/바이카보네이트완충용액(carbonate/bicarbonate buffer, pH 9.6)에 녹인 HMGB1 단백질(Abnova)을 코팅하여 4℃에서 18시간 이상 밤을 지새워 반응하였다. 이 후, 플레이트를 PBS-T(PBS-0.05%[w/w] Tween 20)로 3번 세척한 후, PBS-T를 넣어서 4℃에서 18시간 이상 밤을 지새워 보관하였다. HUVECs were cultured in 6-well plates and treated with LPS (lipopolysaccharide, 100 ng / ml) for 100 h (control treated with saline). To perform competitive enzyme-linked immunosorbent assays (ELISA) using HUVEC cell culture media, 0.02% sodium azide was added to 96-well plastic flat microtiter plates (Corning, NY, USA) HMGB1 protein (Abnova) dissolved in 20 mM carbonate / bicarbonate buffer (pH 9.6) was coated and reacted overnight at 4 ° C for 18 hours or more. After that, the plate was washed 3 times with PBS-T (PBS-0.05% [w / w] Tween 20), then PBS-T was added and kept overnight at 4 ° C for 18 hours or more.
한편, HMGB1 단백질(Abnova, 단백질 정량용, PBS-T에 희석), HUVEC의 세포배양액(conditioned culture media)(LPS 처리된 샘플 포함)은 각각 96웰 플레이트에서 항-HMGB1 항체(Abnova, PBS-T에 1:1000 희석)와 37℃에서 90분간 전반응시켰다. 상기 HUVEC의 세포용출액은 1mM PMSF, 1mM Na3VO4, 1mM NaF, 1㎍/㎖ 아프로티닌(aprotinin), 1㎍/㎖ 펩스타틴(pepstatin), 1㎍/㎖ 류펩틴(leupeptin) 및 10% RIPA lysis buffer(Upstate Biotechnology, USA)를 함유하고 있는 단백질 분리 시약을 이용해 4℃에서 1시간 동안 볼텍싱(vortexing)하여 분해시킨 후 원심분리(15000 rpm, 4℃, 20분)하여 얻은 상층액이다.HMGB1 protein (for Abnova, dilution in PBS-T for protein determination) and HUVEC conditioned culture media (including LPS-treated sample) were incubated with anti-HMGB1 antibody (Abnova, PBS-T At 1: 1000 dilution) at 37 < 0 > C for 90 minutes. The HUVEC cell extracts contained 1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / ml leupeptin and 10% The supernatant was obtained by centrifugation (15000 rpm, 4 ° C, 20 min) using a protein separation reagent containing RIPA lysis buffer (Upstate Biotechnology, USA) at 4 ° C for 1 hour by vortexing .
상기 항체가 전반응된 HMGB1 단백질, HUVEC의 세포배양액을 HMGB1이 코팅된 플레이트로 옮겨 실온에서 30분간 반응시켰다. 이 후, 각 플레이트들은 PBS-T로 3번 세척하였고, 실온에서 90분간 2차 항체(peroxidase-conjugated anti-goat IgG antibodies, R&D Systems, PBS-T에 1:2000 희석)와 반응시켰다. The cell culture of HMGB1 protein, HUVEC, which had been reacted with the antibody, was transferred to a plate coated with HMGB1, and reacted at room temperature for 30 minutes. Each plate was then washed three times with PBS-T and reacted with secondary antibodies (peroxidase-conjugated anti-goat IgG antibodies, 1: 2000 dilution in R & D Systems, PBS-T) for 90 minutes at room temperature.
각 플레이트들을 PBS-T로 3번 세척한 후, 실온의 암실에서 60분간 200 ㎕씩의 기질용액(100 ㎍/㎖ o-phenylenediamine & 0.003% H2O2)을 처리하였다. 다음으로는 50 ㎕의 8 N H2SO4으로 모든 반응을 중단시킨 후, 490 nm에서 흡광도를 확인하였다. Each plate was washed three times with PBS-T and treated with 200 μl of substrate solution (100 μg / ml o- phenylenediamine & 0.003% H 2 O 2 ) for 60 minutes in a dark room at room temperature. Next, all reactions were stopped with 50 μl of 8 NH 2 SO 4 and the absorbance was confirmed at 490 nm.
도 7을 참고하면, 본 발명의 화합물 1~3이 LPS로 유도된 HUVEC의 패혈증 반응에서, HMGB1의 생성을 억제하는 효과가 매우 우수함을 알 수 있다. Referring to FIG. 7, it can be seen that the
또한, 상기와 동일하게 LPS 처리(100 ng/㎖, 100시간)된 HUVEC을 이용하여 실시예 4에 개시된 방법으로 세포 투과성 실험을 수행하여 이에 대한 결과를 도 8에 나타내었다. In addition, the cell permeability test was carried out by the method described in Example 4 using HUVEC treated with LPS (100 ng / ml, 100 hours) as described above, and the results are shown in FIG.
도 8을 참고하면, 본 발명의 화합물 1~3이 LPS로 유도된 패혈증 반응이 일어난 이후에도 세포 투과성을 억제하는 것을 알 수 있다.Referring to FIG. 8, it can be seen that the
<< 실시예Example 6. 패혈증 동물모델에서의 6. Sepsis In animal models 혈관세포의Vascular cell 투과성 억제 효과 확인> Confirmation of permeation inhibiting effect>
수컷 C57BL/6 마우스를 준비하고(각 군당 10마리씩 준비), 6-7주령 및 체중 18~20g의 마우스를 구입하고 12일간 실험실에서 순응화한 후 사용, Orient Bio Co. Sungnam, Kyungki-Do, Republic of Korea), 상기 마우스에 화합물 1~3(7.7 ㎍/mouse)을 정맥주사를 통해 처리하고 6시간 후 살린(saline) 용액에 녹인 1%(w/v) 에반스 블루 용액과 HMGB1(2 ㎍/mouse)을 연이어 정맥주사하였다. 16시간 후 마우스를 희생시킨 후 복강을 5㎖의 살린 용액으로 세척하면서 복막 삼출물(peritoneal exudate)을 모아 200×g에서 10분간 원심분리하였다. 상기 원심분리물의 상청액(supernatant)의 흡광도는 650nm에서 확인하였다. 이는 에반스 블루 용액의 염료가 복강 내로 흘러나온 것을 통해 혈관세포에 생긴 미세천공으로 인한 투과성을 확인할 수 있기 때문이다(Int. Immunopharmacol. 2009, 9, 268-276.). 상기 결과는 도 9에 나타내었다. Male C57BL / 6 mice were prepared (10 mice per group), 6-7 week old mice and 18-20 g body weight were purchased and adapted for laboratory use for 12 days. (W / v) Evans blue (1 mg / mouse) which had been treated with intravenous injection of
상기 도 9에서 음성대조군은 화합물의 용매인 DMSO만 처리한 것으로서, 상기 음성대조군과 화합물 1~3만 처리한 군과 음성대조군의 염색약 누출양이 거의 동일하다. 즉, HMGB1(2 ㎍/mouse)을 처리하지 않은 그룹들에서는 화합물의 처리 유무와 상관없이 염색약의 누출(dye leakage)이 없는 것을 알 수 있다. 반면, HMGB1을 처리한 양성대조군에서는 다량의 염색약 누출이 있는 것을 알 수 있는데, 화합물 1~3이 HMGB1으로 인한 염색약의 누출을 현저하게 억제하는 효과가 있는 것으로 확인된다. 이에 상기 화합물들이 혈관벽 손상을 보호함으로써 패혈증을 억제함을 입증할 수 있다. In FIG. 9, the negative control group was treated with DMSO, which is a solvent of the compound, and the leakage amount of the dye between the negative control group and the negative control group was almost the same. In other words, in the groups not treated with HMGB1 (2 μg / mouse), dye leakage did not occur regardless of treatment with the compound. On the other hand, the positive control group treated with HMGB1 showed a large amount of dye leaching, and it was confirmed that the
<< 실시예Example 7. 패혈증 동물모델에서의 생존율 확인> 7. Identification of survival rate in animal models of sepsis>
마우스의 맹장을 터트리는 CLP 수술법(cecal ligation and puncture operation)을 이용하여 마우스에 패혈증을 유도하였다. CLP 수술에 이용된 마우스는 수컷 C57BL/6 마우스를 이용하였고, 각 군당 10마리씩 이용하였다(6~7주령 및 체중 18~20g의 마우스를 구입하고 12일간 실험실에서 순응화한 후 사용, Orient Bio Co. Sungnam, Kyungki-Do, Republic of Korea). 마우스를 졸레틸 50(zoletil 50) 및 3%(w/v) 이소플루란(isoflurane, Forane®, Choongwae Pharma. Corp., Seoul, Korea)으로 마취시켰다. 패혈증 모델을 유도하기 위해 배 부위 2㎝ 절개를 하여 맹장과 근처의 창자를 노출시켰다. 맹장 끝으로부터 5 ㎜ 부위의 맹장을 3.0-실크 봉합선으로 강하게 묶고, 22-게이지 바늘로 이를 터트렸다. 맹장을 부드럽게 짜 내어 천공 부위를 통해 소량의 배설물이 누출되게 하였고, 이 배설물을 복강에 노출시켰으며, 개복부위를 4.0-실크 봉합선으로 봉합하였다. 음성대조군(sham, sham-operated mice, ○)은 맹장을 묶고 터트리는 단계를 수행하지 않고 단순히 배만 절개하고 다시 봉합하였다. 상기 CLP 수술법으로 마우스에 패혈증을 유도한 후, 각 화합물(7.7 ㎍/mouse)을 CLP 수술 12시간 및 50시간 뒤 정맥주사를 통해 각 마우스(군당 10마리씩)에 처리하였다. CLP만 수행한 양성 대조군(●)에는 화합물 대신 살린(saline) 용액을 처리하였다. 각 마우스들은 CLP 수술 24시간 후부터 패혈증 증상에 노출되었는데, 전율(shivering)이 있거나 털이 곤두서거나(bristled hair) 또는 무기력증(weakness) 등의 현상이 나타났다. 마우스의 생존은 CLP 수술 6시간 후부터 132시간까지 확인하였고(카플란-마이어의 생존 분석법[Kaplan-Meier survival analysis]에 따라 관찰), 시간에 따른 마우스의 생존결과는 도 10에 나타내었다. Sepsis was induced in mice using a cecal ligation and puncture operation that exploded the mouse's cecum. The mice used for CLP surgery were male C57BL / 6 mice, and 10 mice were used per group (6 ~ 7 weeks old and 18 ~ 20g mice were purchased and adapted for 12 days in a laboratory. After using Orient Bio Co Sungnam, Kyungki-Do, Republic of Korea). Mice were anesthetized with
도 10을 참고하면, 화합물 1~3을 투여한 마우스 그룹은 CLP 수술 이후 132시간 째에 각각 60%(화합물 1), 40%(화합물 2) 및 40%(화합물 3)의 생존율을 갖는 것으로 확인되어 패혈증의 치료 효과가 아주 우수함을 알 수 있다.10, it was confirmed that the mouse group to which the compounds 1-3 were administered had a survival rate of 60% (Compound 1), 40% (Compound 2) and 40% (Compound 3) at 132 hours after CLP surgery And the treatment effect of sepsis is excellent.
<< 실시예Example 8. 독성실험> 8. Toxicity test>
실시예Example 8-1. 8-1. 급성독성Acute toxicity
본 발명의 화합물 1(로도덴드로사이드 A) 및 만병초 메탄올 추출물을 단기간에 과량을 섭취하였을 때 급성적(24시간 이내)으로 동물체내에 미치는 독성을 조사하고, 치사율을 결정하기 위하여 본 실험을 수행하였다. 일반적인 마우스인 ICR 마우스 계통을 각 군당 10마리씩 배정하였다. 대조군에는 PEG-400:Tween-80:에탄올(8:1:1, v:v:v) 만을 투여하고, 실험군은 본 발명의 화합물 1 및 만병초 메탄올 추출물을 상기 PEG-400:Tween-80:에탄올(8:1:1, v:v:v)에 녹여 각각 경구투여하였다. 투여 24시간 후에 각각의 치사율을 조사한 결과, 대조군과 2g/㎏/day 농도의 본 발명의 화합물 1 및 만병초 메탄올 추출물을 투여한 실험군에서 마우스가 모두 생존하는 것으로 확인되었다.This experiment was conducted to investigate the toxicity of the compounds of the present invention (Rhodendroside A) and methanol extract of Rhododendron vinegar to an animal body in an acute manner (within 24 hours) in a short period of time and to determine the mortality rate . An ICR mouse line, a common mouse, was assigned to each group of 10 mice. In the experimental group,
실시예Example 8-2. 8-2. 실험군Experimental group 및 대조군의 장기 및 조직 독성 실험 And control organ organs and tissue toxicity experiments
장기 독성 실험은 본 발명의 화합물 1(로도덴드로사이드 A) 및 만병초 메탄올 추출물을 각 농도로 8주 동안 C57BL/6J 마우스(각 군당 10마리)에 투여하여 실험하였다. 동물의 각 장기(조직)에 미치는 영향을 조사하기 위하여 본 발명의 화합물 1 및 만병초 메탄올 추출물을 투여한 실험군과 PEG-400:Tween-80:에탄올(8:1:1, v:v:v)만을 투여한 대조군의 동물들로부터 8주 후 혈액을 채취하여 GPT(glutamate-pyruvate transferase) 및 BUN(blood urea nitrogen)의 혈액 내 농도를 Select E(Vital Scientific NV, Netherland) 기기를 이용하여 측정하였다. 그 결과, 간독성과 관계있는 것으로 알려진 GPT와 신장독성과 관계있는 것으로 알려진 BUN의 경우, 대조군과 비교하여 실험군은 별다른 차이를 보이지 않았다. 또한, 각 동물로부터 간과 신장을 절취하여 통상적인 조직절편 제작과정을 거쳐 광학현미경으로 조직학적 관찰을 시행하였으며 모든 조직에서 특이한 이상이 관찰되지 않았다. In the long-term toxicity test, Compound 1 (Rhodendroside A) and Rhodiola methanol extract of the present invention were administered to C57BL / 6J mice (10 mice per group) for 8 weeks at each concentration. (8: 1: 1, v: v: PEG-400: Tween-80: ethanol) was administered to the experimental group to which
<< 제제예Formulation example 1. 약학적 제제> 1. Pharmaceutical preparations>
제제예Formulation example 1-1. 정제의 제조 1-1. Manufacture of tablets
본 발명의 부탄올 분획물 200 g을 락토오스 175.9 g, 감자전분 180 g 및 콜로이드성 규산 32 g과 혼합하였다. 이 혼합물에 10%(w/v) 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다. 200 g of the butanol fraction of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. A 10% (w / v) gelatin solution was added to the mixture, followed by pulverization and passed through a 14 mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.
제제예Formulation example 1-2. 주사액제의 제조 1-2. Injection preparation
본 발명의 부탄올 분획물 1 g, 염화나트륨 0.6 g 및 아스코르브산 0.1 g을 증류수에 용해시켜서 100 ㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.1 g of the butanol fraction of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.
<< 제제예Formulation example 2. 식품 제조> 2. Food Manufacturing>
제제예Formulation example 2-1. 조리용 양념의 제조 2-1. Manufacture of cooking seasonings
본 발명의 부탄올 분획물을 조리용 양념에 1 중량%로 첨가하여 건강 증진용 조리용 양념을 제조하였다.The butanol fraction of the present invention was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.
제제예Formulation example 2-2. 밀가루 식품의 제조 2-2. Manufacture of flour food products
본 발명의 부탄올 분획물을 밀가루에 0.1 중량%로 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.The butanol fraction of the present invention was added to wheat flour at 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare a food for health promotion.
제제예Formulation example 2-3. 2-3. 스프soup 및 육즙(gravies)의 제조 And gravies
본 발명의 부탄올 분획물을 스프 및 육즙에 0.1 중량%로 첨가하여 건강 증진용 수프 및 육즙을 제조하였다.The butanol fraction of the present invention was added to the soup and juice at 0.1 wt% to prepare health promotion soup and juice.
제제예Formulation example 2-4. 유제품(dairy products)의 제조 2-4. Manufacture of dairy products
본 발명의 부탄올 분획물을 우유에 0.1 중량%로 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.The butanol fraction of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.
제제예Formulation example 2-5. 2-5. 야채주스Vegetable juice 제조 Produce
본 발명의 부탄올 분획물 0.5 g을 토마토주스 또는 당근주스 1,000 ㎖에 가하여 건강 증진용 야채주스를 제조하였다.0.5 g of the butanol fraction of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare health promotion vegetable juice.
제제예Formulation example 2-6. 2-6. 과일주스Fruit juice 제조 Produce
본 발명의 부탄올 분획물 0.1 g을 사과주스 또는 포도주스 1,000 ㎖에 가하여 건강 증진용 과일주스를 제조하였다.0.1 g of the butanol fraction of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.
Claims (8)
[화학식 1]
To Rhododendron containing the target Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 (Icariside B 6, Compound 3) Mega-styryl the main glycoside one or more selected from the group consisting of Formula 1 (Rhododendron brachycarpum ) Extract. ≪ / RTI >
[Chemical Formula 1]
상기 만병초 추출물은 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 추출물인 것을 특징으로 하는 패혈증의 예방 또는 치료용 조성물.The method according to claim 1,
The composition for preventing or treating septicemia according to any one of claims 1 to 4, wherein the extract is obtained by extracting and extracting Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
상기 만병초 추출물은 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있는 것을 특징으로 하는 패혈증의 예방 또는 치료용 조성물.The method according to claim 1,
The composition for preventing or treating septicemia according to any one of claims 1 to 3, wherein the extract of Phenotypes is effective for inhibiting the expression of HMGB1 (high mobility group box 1).
[화학식 1]
To characterized in that it contains a blood Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 (Icariside B 6, Compound 3) Mega-styryl the main glycoside one or more selected from the group consisting of formula I A composition for preventing or treating sepsis.
[Chemical Formula 1]
상기 메가스티그메인 배당체는 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있는 것을 특징으로 하는 패혈증의 예방 또는 치료용 조성물.5. The method of claim 4,
Wherein the megestigin main glycoside has an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
[화학식 1]
To Rhododendron containing the target Cri Ono side A (Picrionoside A, Compound 2) and Ikari side B 6 (Icariside B 6, Compound 3) Mega-styryl the main glycoside one or more selected from the group consisting of Formula 1 (Rhododendron brachycarpum ) Extract of the present invention for preventing or ameliorating sepsis.
[Chemical Formula 1]
상기 만병초 추출물은 만병초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 추출물인 것을 특징으로 하는 패혈증의 예방 또는 개선용 건강기능식품.The method according to claim 6,
Wherein the extract of the Rhododendron phloem is extract obtained by extracting Rhododendron with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and a health functional food for preventing or ameliorating sepsis.
상기 만병초 추출물은 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있는 것을 특징으로 하는 패혈증의 예방 또는 개선용 건강기능식품.The method according to claim 6,
Wherein the extract of Rhododendron japonica has an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
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