KR101718357B1 - Composition comprising compounds isolated from Salicornia herbacea for preventing or treating vascular inflammation or sepsis - Google Patents
Composition comprising compounds isolated from Salicornia herbacea for preventing or treating vascular inflammation or sepsis Download PDFInfo
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- KR101718357B1 KR101718357B1 KR1020160027905A KR20160027905A KR101718357B1 KR 101718357 B1 KR101718357 B1 KR 101718357B1 KR 1020160027905 A KR1020160027905 A KR 1020160027905A KR 20160027905 A KR20160027905 A KR 20160027905A KR 101718357 B1 KR101718357 B1 KR 101718357B1
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- Prior art keywords
- compound
- methoxy
- sepsis
- flavanone
- hydroxy
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Abstract
Description
본 발명은 함초로부터 분리된 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating vascular inflammation or sepsis which contains a compound isolated from green tea.
혈관내피는 혈관운동긴장(vasomotor tone), 혈액의 수송, 지혈 안정, 증식, 생존, 선천성 또는 적응성 면역 등을 포함하는 다양한 생리학적 기능에 대해 중요한 역할을 하고 있다. 따라서, 혈관내피의 기능장애는 생리학적인 반응이상을 초래하는데, 주로, 급성적인 폐손상, 아나필락시스(anaphylaxis), 동맥경화증, 패혈증과 같은 다양한 혈관 염증 증상으로부터 유도된다(Pharmacological Reports. 2008, 60, 139-143; Crirculation. 2007, 115, 1285-1295; Journal of Cellular Physiology. 2014, 229, 620-630).The vascular endothelium plays an important role in a variety of physiological functions including vasomotor tone, blood transport, hemostasis, proliferation, survival, congenital or adaptive immunity, and the like. Thus, vascular endothelial dysfunction results in physiological response abnormalities, which are mainly induced from a variety of vascular inflammatory conditions such as acute lung injury, anaphylaxis, atherosclerosis, sepsis (Pharmacological Reports. 2008, 60 Journal of Cellular Physiology, 2014, 229, 620-630).
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 the systemic symptoms such as fever, joint fever, 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.
함초(Salicornia herbacea)의 공식적인 명칭은 퉁퉁마디이지만 주로 함초라고 불린다. 바닷물이 잘 드나들고 비교적 땅이 잘 굳는 갯벌지에서 자란다. 줄기는 육질이고 원기둥 모양이며 가지가 마주달리고 퇴화한 비늘잎이 마주달리며 마디가 불룩하게 튀어나오므로 퉁퉁마디라는 이름이 생겼다. 우리나라 서해안과 남해안 전역에 고루 분포한다. 함초는 오래전부터 식용으로도 많이 먹었는데 줄기를 잘라다가 국을 끓이거나, 갈아서 밀가루에 함께 반죽하여 전을 부쳐서 먹기도 한다. 미네랄이 풍부하기 때문에 건강식으로 알려져 있다. 함초는 몸 안에 쌓인 독소와 숙변을 없애고, 암, 자궁근종, 축농증, 고혈압, 저혈압, 요통, 당뇨병, 기관지천식, 갑상선 기능저하, 갑상선 기능항진, 피부병, 관절염, 신장염이나 갖가지 난치병에 탁월한 치료 효과를 지니고 있는 놀라운 약초라 알려져 있다. 또한 함초는 숙변을 제거하고 비만증을 치료하는 효과가 탁월하며, 혈액순환을 좋게 하고 피를 맑게 하며 혈관을 튼튼하게 하여 염증과 관절염으로 인한 수종 등의 치료에 이용되며, 먹는 화장품이라고 할 만큼 피부미용에 효과가 탁월하다. The official name of Salicornia herbacea is Tungtung Madi, but it is mainly called Hamcho. It grows in tidal flats where the waters are well and relatively firm. The stem is fleshy and has a cylindrical shape. The branches are opposite to each other, and the degenerated scaly leaves are running against each other, and the nodes bulge out, resulting in the name Tungtungmadi. It is distributed all over the west coast and south coast of Korea. Green tea has long been eaten for many years, cutting the stem, boiling the soup, or grind it together with flour and knead it together to eat. Because it is rich in minerals, it is known as a health food. It removes toxins and succumbs that are accumulated in the body and has excellent therapeutic effect on cancer, uterine myoma, sinusitis, hypertension, hypotension, back pain, diabetes, bronchial asthma, hypothyroidism, hyperthyroidism, skin disease, arthritis, It is known as the amazing herb that it has. It is also used for treatment of diseases caused by inflammation and arthritis, such as eliminating sukbyeo and treating obesity, improving blood circulation, clearing blood, strengthening blood vessels, and so on. The effect is excellent.
함초에는 피토스테롤(phytosterol), 페놀산(phenolic acid), 플라보노이드(flavonoid), 다당류(polysaccharide), 디카페오일 퀴닉산(dicaffeoyl quinic acid), 프로카테츄익산(procatechuic acid), 페룰산(ferulic acid), 트리테르페노이드 사포닌(triterpenoid saponin) 등의 생리활성성분이 다양하게 함유되어 있다. 특히, 함초의 유효성분 중, 함초로부터 분리된 3-카페오일-4-다이하이드로카페오일 퀴닉산은 항산화 효과, 통풍치료, 항염증 효과, 비만, 지방간 등의 억제 효과가 있음이 있다고 보고되었다. The green tea contains phytosterol, phenolic acid, flavonoid, polysaccharide, dicaffeoyl quinic acid, procatechuic acid, ferulic acid, , Triterpenoid saponin, and the like. Particularly, among the active ingredients of green tea, 3-caffeoyl-4-dihydrocafeoylquinic acid isolated from green tea has been reported to have an antioxidant effect, gout treatment, anti-inflammatory effect, obesity and fatty liver.
HMGB1(high mobility group box 1)은 패혈증과 같은 혈관 손상이 있을 때 혈관 내피에서 발현되는 단백질로서(Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 228, 975-982) 혈관내피를 염증반응 이전의 모습으로 재구성하는 역할을 하는데(Science. 1999, 285, 248-251), 본 발명자들은 함초 유래의 플라바논 또는 크로몬 유도체 화합물들이 HMGB1의 발현을 억제함으로써 혈관 염증 또는 패혈증의 치료 효과가 있음을 확인하여 본 발명을 완성하였다. HMGB1 (high mobility group box 1) is a protein expressed in the endothelium in the presence of vascular injury such as sepsis (Toxicol. Appl. Pharm. 2012, 262, 91-98; J. Cell. Physio. 2013, 228, 975 (Science, 1999, 285, 248-251), the inventors of the present invention found that flavanone or chromone derivative compounds derived from Hamcho exhibited inhibition of the expression of HMGB1, Inflammatory or sepsis, and thus the present invention has been completed.
본 발명의 목적은 함초로부터 분리된 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물을 제공하는 데에 있다. It is an object of the present invention to provide a composition for preventing or treating vascular inflammation or sepsis which contains a compound isolated from green tea.
본 발명은 하기 화학식 1의 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2), 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3), 7-히드록시-6,8-디메톡시크로몬(화합물 4), 6-메톡시크로마논(화합물 5) 및 6,7-디메톡시크로몬(화합물 6)으로 이루어진 군에서 선택되는 1종 이상의 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also relates to a composition for preventing or treating vascular inflammation or sepsis.
[화학식 1][Chemical Formula 1]
상기 화합물은 함초 유래의 플라바논 또는 크로몬 유도체(flavanones or chromones derivatives)로서, HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있다. These compounds are flavanones or chromone derivatives derived from Hamcho and have an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
본 발명은 상기 화학식 1의 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2), 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3), 7-히드록시-6,8-디메톡시크로몬(화합물 4), 6-메톡시크로마논(화합물 5) 및 6,7-디메톡시크로몬(화합물 6)으로 이루어진 군에서 선택되는 1종 이상의 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 개선용 건강기능식품을 제공한다. 상기 건강기능식품은 각종 식품류, 음료, 껌, 차 및 비타민 복합제로 이루어진 군에서 선택될 수 있다. The invention of 7,2'- dihydroxy-6-methoxy -2 S Formula 1-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also provides a health functional food for preventing or ameliorating vascular inflammation or sepsis. The health functional food may be selected from the group consisting of various foods, beverages, gums, tea and vitamin complex.
본 발명은 하기 화학식 2 내지 4의 신규 화합물 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2) 또는 7-히드록시-6,8-디메톡시크로몬(화합물 4)을 제공한다.The present invention relates to novel compounds 7,2'- dihydroxy-6-methoxy -2 S of the general formula (2) to 4-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S -Flavanone (Compound 2) or 7-hydroxy-6,8-dimethoxychromone (Compound 4).
[화학식 2] (2)
[화학식 3](3)
[화학식 4][Chemical Formula 4]
따라서, 본 발명은 함초 추출물로부터 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2) 및 7-히드록시-6,8-디메톡시크로몬(화합물 4)으로 이루어진 군으로부터 선택되는 신규 화합물 1종 이상을 분리하는 방법을 제공할 수 있다. Accordingly, the present invention is 7,2'- dihydroxy-6-methoxy -2 S from Salicornia extract - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone (Compound 2) and 7-hydroxy-6,8-dimethoxychromone (Compound 4).
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 상기 화학식 1의 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2), 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3), 7-히드록시-6,8-디메톡시크로몬(화합물 4), 6-메톡시크로마논(화합물 5) 및 6,7-디메톡시크로몬(화합물 6)으로 이루어진 군에서 선택되는 1종 이상의 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물에 관한 것이다. The invention of 7,2'- dihydroxy-6-methoxy -2 S Formula 1-flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S-flavanone ( Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4), 6-methoxychroma (Compound 5) and 6,7-dimethoxychromone (Compound 6). The present invention also relates to a composition for preventing or treating vascular inflammation or sepsis.
상기 화합물은 플라바논 또는 크로몬 유도체(flavanones or chromones derivatives)로서, 함초 추출물로부터 유래된 것일 수 있다. 상기 함초 추출물은 함초를 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 함초 중량의 1~20배를 가하는 것이 바람직하며, 추출온도는 20~100℃인 것이 바람직하고, 추출시간은 1시간 내지 7일 이내가 바람직하다. 상기 함초 추출물은 상법에 따라, 물 또는 각종 유기용매에 의한 추출, 유기용매와 물의 분배, 컬럼 크로마토그래피에 의한 방법 등, 식물체 성분의 분리 추출에 이용되는 공지의 방법을 단독 또는 적합하게 조합한 방법을 이용하여 분획 또는 정제하여 사용할 수 있다. The compound may be flavanones or chromone derivatives, which are derived from a green tea extract. The green tea extract may be obtained by extracting green tea with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The extraction conditions are not limited, but it is preferable that the water, the lower alcohol having 1 to 4 carbon atoms, or the mixed solvent thereof is added 1 to 20 times as much as the weight of ammonia, and the extraction temperature is preferably 20 to 100 ° C , And the extraction time is preferably from 1 hour to 7 days. The above-mentioned green tea extract may be prepared by a known method which is used for extractive separation of plant components, such as extraction with water or various organic solvents, distribution of organic solvent and water, and column chromatography, May be fractionated or purified and used.
바람직하게는 상기 함초 추출물은 함초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 함초 추출물을 함초 추출물의 중량의 1~50배의 물을 가하여 현탁한 후, 상기 현탁물에 헥산, 클로로포름, 에틸아세테이트, 및, 부탄올로 이루어진 군에서 선택되는 용매를 가하여 얻은 함초 분획물로 제조할 수 있다. 상기 함초 분획물은 바람직하게는, 함초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 함초 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 함초 추출물 또는 함초 분획물로부터 분리된 플라바논 또는 크로몬 유도체(Flavanones or Chromones derivatives) 화합물이다. 한편, 이 외의 분획조건은 제한되지는 않으나, 상기 함초 추출물에 함초 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 green tea extract is obtained by concentrating green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, adding 1 to 50 times of water to the weight of the green tea extract, suspending the green tea extract, And a green tea fraction obtained by adding a solvent selected from the group consisting of hexane, chloroform, ethyl acetate, and butanol to water. The green tea fraction is preferably a concentrate of a hexane layer obtained by suspending a green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and then mixing with hexane, 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) of the present invention are flavanones or chromone derivatives which are separated from green tea extract or green tea leaf fraction. In addition, other conditions for the fractionation are not limited, but it is preferable to add water to the green tea extract to 1 to 50 times the weight of the green tea extract to prepare a suspension. Then, 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.
상기 함초 추출물을 분획하거나 이로부터 본 발명의 플라바논 또는 크로몬 유도체를 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 separate the fenugreek extract or to isolate the flavanone or chromone derivative of the present invention. The chromatography can be carried out by silica gel column chromatography, Lewis-20 column chromatography (LH-20 column chromatography), ion exchange resin chromatography, medium pressure liquid chromatography (MPLC), thin layer chromatography (TLC), silica gel vacuum liquid chromatography silica gel vacuum liquid chromatography, reversed phase chromatography, and high performance liquid chromatography.
상기 함초 추출물은 함초의 전초, 뿌리, 잎, 줄기 등의 어떤 부위로부터 선택되는 모든 부위로부터 추출가능하다. The green tea extract can be extracted from any part selected from a certain part such as plantago, root, leaf, stems of green tea plant.
한편, 본 발명은 신규 화합물 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2) 또는 7-히드록시-6,8-디메톡시크로몬(화합물 4)을 제공하며, 상기 신규 화합물들을 함초로부터 분리하는 방법을 제공한다. 바람직하게는 상기 신규 화합물 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2) 또는 7-히드록시-6,8-디메톡시크로몬(화합물 4)은 함초를 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매로 추출 농축하여 얻은 함초 추출물을 물에 현탁한 후 헥산과 혼합하여 얻은 헥산층, 상기 헥산층을 제거하고 남은 잔사(물층)에 에틸아세테이트를 혼합하여 얻은 에틸아세테이트층, 상기 에틸아세테이트층을 제거하고 남은 잔사(물층)의 농축물로부터 분리될 수 있다. On the other hand, the present invention relates to novel compounds 7,2'- dihydroxy-6-methoxy -2 S - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - flavanone ( Compound 2) or 7-hydroxy-6,8-dimethoxychromone (compound 4), and provides a method for separating the novel compounds from green tea. Preferably, the novel compounds 7,2'- dihydroxy-6-methoxy -2 S - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - flavanone (Compound 2) or 7-hydroxy-6,8-dimethoxychromone (Compound 4) is prepared by suspending the green tea extract obtained by extracting and concentrating green tea with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, A hexane layer obtained by mixing with hexane, an ethyl acetate layer obtained by removing the hexane layer and mixing ethyl acetate in the residue (water layer) remaining, and a concentrate of the residue (water layer) remaining after removing the ethyl acetate layer .
또한, 본 발명은 상기 화학식 1의 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2), 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3), 7-히드록시-6,8-디메톡시크로몬(화합물 4), 6-메톡시크로마논(화합물 5) 및 6,7-디메톡시크로몬(화합물 6)으로 이루어진 군에서 선택되는 1종 이상의 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 약학 조성물을 제공한다. The present invention is 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - Plastic (Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4) (5) and 6,7-dimethoxychromone (Compound (6)). The present invention also provides a pharmaceutical composition for preventing or treating vascular inflammation or sepsis.
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. 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 플라바논 또는 크로몬 유도체 화합물을 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. 상기 화합물들은 본 발명의 약학 조성물에 바람직하게는 0.001~90 중량%, 더 바람직하게는 0.001~50 중량%, 가장 바람직하게는 0.001~30 중량%로 하여 첨가될 수 있다.The dosage of the pharmaceutical composition containing the flavanone or chromone derivative compound of the present invention will depend on the age, sex, and weight of the subject to be treated, the specific disease or condition to be treated, the severity of the disease or condition, . 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. These compounds may be added to the pharmaceutical composition of the present invention in an amount of preferably 0.001 to 90% by weight, more preferably 0.001 to 50% by weight, and most preferably 0.001 to 30% by weight.
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 7,2'-디히드록시-6-메톡시-2S-플라바논(화합물 1), 2'-히드록시-6,7-메톡시-2S-플라바논(화합물 2), 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3), 7-히드록시-6,8-디메톡시크로몬(화합물 4), 6-메톡시크로마논(화합물 5) 및 6,7-디메톡시크로몬(화합물 6)으로 이루어진 군에서 선택되는 1종 이상의 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 혈관 염증 또는 패혈증의 예방 또는 개선용 건강기능식품을 제공한다. 상기 화합물들은 본 발명의 건강기능식품에 바람직하게는 0.001~90 중량%, 더 바람직하게는 0.001~50 중량%, 가장 바람직하게는 0.001~30 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. The present invention is 7,2'- dihydroxy-6-methoxy -2 S of formula (I) - flavanone (Compound 1), 2'-hydroxy-6,7-methoxy -2 S - Plastic (Compound 2), 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3), 7-hydroxy-6,8-dimethoxychromone (Compound 4) Prevention or amelioration of vascular inflammation or sepsis, including at least one compound selected from the group consisting of ethoxychromanone (Compound 5) and 6,7-dimethoxychromone (Compound 6), and a pharmaceutically acceptable food additive additive And a health functional food. The above compounds may be added to the health functional food of the present invention in an amount of preferably 0.001 to 90% by weight, more preferably 0.001 to 50% by weight, and most preferably 0.001 to 30% by weight. 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 함초 유래의 화합물을 함유하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물에 관한 것으로서, 상기 화합물은 플라바논 또는 크로몬 유도체(flavanones or chromones derivatives)로서, 상기 화합물은 HMGB1(high mobility group box 1)의 발현을 억제하고, CLP 수술법(cecal ligation and puncture operation)에 따른 마우스의 사망률을 억제하는 효과가 우수하여 혈관 염증 또는 패혈증의 예방 또는 치료용 의약 조성물이나 혈관 염증 또는 패혈증의 예방 또는 개선용 건강기능식품으로 용이하게 이용가능하다. The present invention relates to a composition for preventing or treating vascular inflammation or sepsis comprising a compound derived from Hamcho, wherein the compound is flavanones or chromone derivatives, wherein the compound is HMGB1 (high mobility group box 1) and inhibits the mortality of mice according to the CLP procedure (cecal ligation and puncture operation), thereby preventing or treating vascular inflammation or sepsis or preventing or improving vascular inflammation or sepsis It is easily available as a health functional food.
한편, 한국공개특허 제2005-0087418호에는 함초 유래의 3-카페오일-4-다이하이드로카페오일퀴닉산이 항염 효과가 있음이 개시된 바 있고, [강스미, 함초와 함초 종자 추출물의 항산화, 세포독성 및 항염 활성, 서울여자대학교, 2011년, 식품공학]에도 역시 함초 추출물이 갖는 항염 활성이 개시되어 있지만, 상기와 같은 항염증 효과가 있는 물질이 반드시 혈관 염증 또는 패혈증의 억제 효과가 있다고는 할 수 없다. 게다가, 본 발명에서는 함초 추출물이 아닌 함초 유래의 플라바논 또는 크로몬 유도체의 활성을 확인하는데다가, 상기 화합물들 또한 3-카페오일-4-다이하이드로카페오일퀴닉산과는 화학구조가 다른 화합물이기에, 본 발명의 화합물들이 갖는 혈관 염증 억제효과 또는 패혈증 치료 효과와 함초 추출물 또는 3-카페오일-4-다이하이드로카페오일퀴닉산이 갖는 효과는 전혀 다르다고 할 수 있다. On the other hand, Korean Patent Laid-Open No. 2005-0087418 discloses that 3-caffeoyl-4-dihydrocappa oil quinic acid derived from Hamcho has an anti-inflammatory effect. [Antioxidant, cytotoxic And anti-inflammatory activity, Seoul Women's University, 2011, Food Science and Technology] In addition, the anti-inflammatory activity of the green tea extract is also disclosed. However, the anti-inflammatory substance described above may be said to have an inhibitory effect on vascular inflammation or sepsis none. In addition, in the present invention, since the activity of flavanone or chromone derivative derived from green tea, which is not a green tea extract, is confirmed, and since these compounds are also compounds having different chemical structures from 3-caffeoyl-4-dihydrocappaheinquinic acid, The effects of the compounds of the present invention on the vascular inflammation inhibitory effect or the sepsis treatment and on the effects of the green tea extract or 3-caffeoyl-4-dihydrocafeoylquinic acid are completely different.
도 1은 본 발명의 화합물 1~6의 세포독성을 MTT 어세이를 통해 확인한 결과를 나타낸다.
도 2는 본 발명의 동물실험에 사용된 [μM/mouse]으로 표기한 화합물 1~6의 농도를 [㎍/mouse]로 환산하여 나타낸 것이다.
도 3은 패혈증 세포 모델(도 3A 및 도 3B) 또는 패혈증 동물 모델(도 3C 및 도 3D)에서 각각 본 발명의 화합물 1~6이 HMGB1의 발현을 억제하는 효과를 ELISA로 확인한 결과를 나타낸다.
도 4는 패혈증 세포 모델(도 4A) 또는 패혈증 동물 모델(도 4B)에서 각각 본 발명의 화합물 1~6이 혈관내피세포의 손상으로 인한 혈관 투과성을 억제하는 것을 확인한 결과를 나타낸다.
도 5는 패혈증 세포 모델에서 본 발명의 화합물 1~6이 세포유착분자인 VCAM-1, ICAM-1 및 E-selection의 발현(도 5A)과 세포표면 마커 수용체 TLR2(Toll-like receptor 2), TLR4(Toll-like receptor 4) 및 RAGE의 발현(도 5B)을 억제하는 것을 ELISA로 확인한 결과를 나타낸다.
도 6은 본 발명의 화합물 1~6이 혈관 염증을 억제하는 것을 나타내는 실험결과들로서, 도 6A은 본 발명의 화합물 1~6이 혈관 염증에 따른 혈관내피로의 단핵구의 이동을 억제하는 것을 확인한 결과를 나타내며, 도 6B는 본 발명의 화합물 1~6이 혈관 염증으로 인한 단핵구의 혈관내피로의 유착을 억제하는 것을 확인한 결과를 나타내고, 도 6C는 본 발명의 화합물 1~6이 HMGB1 또는 CLP로 유도된 마우스에서 혈관내피로의 백혈구 이동을 억제하는 것을 확인한 결과를 나타낸다.
도 7은 본 발명의 화합물 1~6이 TNF-α(도 7A), IL-1β(도 7B) 및 인산화된 NF-κB(phospho-NF-κB, 도 7C)의 발현을 억제하는 것을 ELISA로 확인한 결과를 나타낸다.
도 8은 본 발명의 화합물 1~6을 투여한 패혈증 동물들의 생존율을 나타낸다. Fig. 1 shows the cytotoxicity of the
2 is a graph showing the concentration of the
FIG. 3 shows the results of ELISA confirming the effect of the
FIG. 4 shows the results of confirming that the
FIG. 5 shows the expression of VCAM-1, ICAM-1 and E-selection (FIG. 5A) and the cell surface marker receptor TLR2 (Toll-like receptor 2) Inhibition of TLR4 (Toll-like receptor 4) and RAGE expression (Fig. 5B) by ELISA.
FIG. 6 shows experimental results showing that the
7 shows that compounds 1-6 of the present invention inhibit the expression of TNF-? (FIG. 7A), IL-1? (FIG. 7B) and phosphorylated NF-? B (phospho-NF-KB, FIG. 7C) It shows the result of confirmation.
Figure 8 shows the survival rate of sepsis animals treated with the compounds 1-6 of the present invention.
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. One. 함초로부터From Hamcho 화합물의 분리> Separation of compounds >
건조된 함초(Salicornia herbacea)의 지상부(aerial aerial) 8.3㎏을 메탄올 72ℓ로 실온에서 4일간 추출하고 이를 1회 더 반복한 뒤 얻은 액상들을 수집하여 여과한 후 농축하여 갈색의 슬러리 상태의 메탄올 추출물(1.9㎏)을 얻었다. 8.3 kg of aerial aerials of dried Salicornia herbacea were extracted with 72 L of methanol for 4 days at room temperature. After repeating this one more time, the resulting liquid phases were collected, filtered and concentrated to give a brown slurry of methanol extract 1.9 kg).
상기 메탄올 추출물을 모두 물(4.0ℓ)에 현탁한 후, n-헥산(3ℓ씩 가하여 4번 반복), 에틸아세테이트(3ℓ씩 가하여 4번 반복) 및 물로 순차적으로 분획하였다. 이렇게 해서 n-헥산 분획물(106.6g) 및 에틸아세테이트 분획물(45.7g)을 얻었다. All of the methanol extracts were suspended in water (4.0 L), and then sequentially fractionated with n -hexane (3 L portions were repeated 4 times), ethyl acetate (3 L portions were added 4 times) and water. Thus, n- hexane fraction (106.6 g) and ethyl acetate fraction (45.7 g) were obtained.
에틸아세테이트 분획물(45.7g)을 실리카겔 컬럼 VLC(25×18㎝)로 분리한 후 n-헥산:아세톤(100:0 → 0:100[100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 10:90, 0:100], 10%[v/v] increment, each step 2ℓ, final washing with 6ℓ of 100% MeOH)의 농도구배 조건으로 용출하여, 10개의 소분획물[E1(1.4㎎), E2(9.3㎎), E2(1.40g), E4(1.30g), E5(1.22g), E6(1.55g), E7(2.80g), E8(5.40g), E9(9.53g), 및 E10(18.03g)]을 얻었다. The ethyl acetate fraction (45.7 g) was separated by silica gel column VLC (25 x 18 cm) and eluted with n- hexane: acetone (100: 0 to 0: 100 [100: 0, 90:10, 80:20, , 60:40, 50:50, 30:70, 10:90, 0: 100], 10% [v / v] increment, each step 2ℓ, final washing with 6ℓ of 100% MeOH) E2 (1.40 g), E4 (1.30 g), E5 (1.22 g), E6 (1.55 g), E7 (2.80 g), E8 (5.40 g), E9 (9.53 g), and E10 (18.03 g)].
소분획물 E5(2.77g)를 MPLC를 이용한 RP-C18(column Biotage SNAP Cartridge, KP-C18-HS, 400g, 75 Psi Max)로 분리한 후, 메탄올:물(60% MeOH)의 조건으로 용출하여 7개의 소분획물[E5-1(458㎎), E5-2(275㎎), E5-3(280㎎), E5-4(220㎎), E5-5(241㎎), E5-6(620㎎) 및 E5-7(600㎎)]을 얻었다. The small fraction E5 (2.77 g) was separated by RP-C18 (column Biotage SNAP Cartridge, KP-C18-HS, 400 g, 75 Psi Max) using MPLC and eluted with methanol: water (60% MeOH) Seven fractions [E5-1 (458 mg), E5-2 (275 mg), E5-3 (280 mg), E5-4 (220 mg), E5-5 (241 mg), E5-6 Mg) and E5-7 (600 mg)].
다시 소분획물 E5-3을 semi-prep HPLC로 분리하여 메탄올:물(A-B, 0-10min, 50-70% A; 10-60min, 70-80% A; 60-70min, 80-100% A)의 농도구배 조건으로 용출하여 화합물 1(t R : 26min, 7.6㎎)과 7개의 소분획물[E5-3-1(32.3㎎), E5-3-2(9.5㎎), E5-3-3(39㎎), E5-3-4(14.4㎎), E5-3-5(33.6㎎), E5-3-6(15.5㎎) 및 E5-3-7(4.2㎎)]을 얻었다. The small fraction E5-3 was further separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-70% A; 10-60 min, 70-80% A; 60-70 min, 80-100
소분획물 E5-3-2 및 E5-3-4의 혼합물을 semi-prep HPLC로 분리하여 메탄올:물(A-B, 0-10min, 50-60% A, 10-60min, 60-68% A, 60-65min, 68-100% A) 조건으로 용출하여 화합물 2(t R : 52min, 5.2㎎)를 얻었다.The mixture of the small fractions E5-3-2 and E5-3-4 was separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-60% A, 10-60 min, 60-68% -65min, 68-100% a) elution conditions to give the compound 2 (R t to: obtain a 52min, 5.2㎎).
소분획물 E5-4를 semi-prep HPLC하여 메탄올:물(A-B, 0-10min, 50-70% A, 10-50min, 70-80% A, 50-70min, 80-100% A) 조건으로 용출하여 8개의 소분획물[E5-4-1(17.7㎎), E5-4-2(3.2㎎), E5-4-3(3.6㎎), E5-4-4(8.0㎎), E5-4-5(10.3㎎), E5-4-6(58.4㎎), E5-4-7(9.0㎎) 및 E5-4-8(21.9㎎)]을 얻었다. The small fraction E5-4 was subjected to semi-prep HPLC and eluted with methanol: water (AB, 0-10 min, 50-70% A, 10-50 min, 70-80% A, 50-70 min, 80-100% E5-4-1 (17.7 mg), E5-4-2 (3.2 mg), E5-4-3 (3.6 mg), E5-4-4 (8.0 mg), E5-4- 5 (10.3 mg), E5-4-6 (58.4 mg), E5-4-7 (9.0 mg) and E5-4-8 (21.9 mg).
소분획물 E5-4-4를 semi-prep HPLC하여 메탄올:물(A-B, 0-10min, 50-75% A, 10-50min, 75-80% A, 50-60min, 80-100% A) 조건으로 용출하여 화합물 3(t R : 32min, 5.7㎎)을 얻었다. The small fraction E5-4-4 was subjected to semi-prep HPLC under the conditions of methanol: water (AB, 0-10 min, 50-75% A, 10-50 min, 75-80% A, 50-60 min, 80-100% eluted
소분획물 E6을 MPLC를 이용한 RP-C18(column Biotage SNAP Cartridge, KP153 C18-HS, 400g, 75 Psi Max)으로 분리하고 메탄올:물(30-50% MeOH, 6ℓ)의 농도구배 조건으로 용출하여 9개의 소분획물[E6-1(121.4㎎), E6-2(739㎎), E6-3(175㎎), E6-4(312.8㎎), E6-5(258.7㎎), E6-6(48.7㎎), E6-7(90㎎), E6-8(71.8㎎) 및 E6-9(803㎎)]을 얻었다. The small fraction E6 was separated by RP-C18 (column Biotage SNAP Cartridge, KP153 C18-HS, 400 g, 75 Psi Max) using MPLC and eluted with gradient conditions of methanol: water (30-50% MeOH, 6 L) E6-1 (121.4 mg), E6-2 (739 mg), E6-3 (175 mg), E6-4 (312.8 mg), E6-5 (258.7 mg), E6-6 ), E6-7 (90 mg), E6-8 (71.8 mg) and E6-9 (803 mg)].
소분획물 E6-2를 semi-prep HPLC로 분리하고 메탄올:물(A-B, 0-10min, 25% A; 10-15min, 25-30% A; 15-20min, 30% A; 20-25min, 30-40% A, 25-70min, 40% A; 70-85min, 40-80% A; 85-90min, 80% A; 90-100min, 80-100% A; 100-110min, 100% A)의 농도구배 조건으로 용출하여 14개의 소분획물[E6-2-1(3.1㎎), E6-2-2(0.5㎎), E6-2-3(0.4㎎), E6-2-4(40.5㎎), E6-2-5(8.7㎎), E6-2-6(16.1㎎), E6-2-7(16.1㎎), E6-2-8(31.1㎎), E6-2-9(13.3㎎), E6-2-10(16.7㎎), E6-2-11(26.1㎎), E6-2-12(11.4㎎), E6-2-13(0.6㎎) 및 E6-2-14(165.5㎎)]을 얻었다.The small fraction E6-2 was separated by semi-prep HPLC and eluted with methanol: water (AB, 0-10min, 25% A; 10-15min, 25-30% A; 15-20min, 30% 40% A, 25-70 min, 40% A, 70-85 min, 40-80% A, 85-90 min, 80% A, 90-100 min, 80-100% A, 100-110 min, 100% A) (E6-2-1 (3.1 mg), E6-2-2 (0.5 mg), E6-2-3 (0.4 mg) and E6-2-4 (40.5 mg) , E6-2-5 (8.7 mg), E6-2-6 (16.1 mg), E6-2-7 (16.1 mg), E6-2-8 (31.1 mg), E6-2-9 (13.3 mg) , E6-2-10 (16.7 mg), E6-2-11 (26.1 mg), E6-2-12 (11.4 mg), E6-2-13 (0.6 mg) and E6-2-14 (165.5 mg) ].
소분획물 E6-2-8을 semi-prep HPLC로 분리하고 메탄올:물(A-B, 0-10min: 15% A, 10-15min: 30% A, 15-60min: 30% A, 60-65min: 100% A, 65-75min: 100% A)의 농도구배 조건으로 용출하여 화합물 5(t R : 18min, 3.5㎎)를 얻었다. The small fraction, E6-2-8, was separated by semi-prep HPLC and eluted with a gradient of methanol: water (AB, 0-10 min: 15% A, 10-15 min: 30% A, 15-60 min: 30% A, 60-65 min: 100 % a, 65-75min: elution with gradient conditions of 100% a) to 5 (t R compound: was obtained 18min, 3.5㎎).
화합물 4(3.0㎎)는 소분획물 E6-2-10을 결정화한 후, 다시 메탄올 상에서 재결정화하여 순수한 형태로 얻었다. Compound 4 (3.0 mg) was crystallized in a small fraction, E6-2-10, and recrystallized on methanol to give pure product.
소분획물 E6-4 (312.8㎎)를 semi-prep HPLC로 분리하고 6㎖/min의 유속을 갖는 메탄올:물(A-B, 0-10min, 25% A; 10-15min, 25-30% A; 15-20min, 30% A; 20-25min, 30-40% A, 25-70min, 40% A; 70-85min, 40-80% A; 85-90min, 80% A; 90-100min, 80-100% A; 100-110min, 100% A)의 농도구배 조건으로 용출하여 화합물 6(t R : 65min, 3.5㎎)을 얻었다. The small fraction E6-4 (312.8 mg) was separated by semi-prep HPLC and eluted with methanol / water (AB, 0-10 min, 25% A; 10-15 min, 25-30% A; 20-40 min, 30-40% A, 25-70 min, 40% A, 70-85 min, 40-80% A, 85-90 min, 80% A, 90-100 min, 80-100 % a; 100-110min, by elution with a concentration gradient conditions of 100% a) 6 (t R compound: to obtain a 65min, 3.5㎎).
* 실시예 1의 크로마토그래피 용출액의 % 비율은 모두 [v:v]로 혼합된 혼합비율(%)을 나타냄. * The percentages of the chromatographic eluant of Example 1 all represent the mixing ratio (%) mixed in [v: v].
<실시예 2. 화합물의 물리화학적 성질 확인>≪ Example 2: Identification of physicochemical properties of a compound >
실시예 2-1. 7,2'-디히드록시-6-메톡시-2Example 2-1. 7,2'-dihydroxy-6-methoxy-2 SS -플라바논(화합물 1)- flavanone (Compound 1)
7,2'-Dihydroxy-6-methoxy-2S-flavanone; 7,2'-Dihydroxy-6-methoxy- 2 S -flavanone;
1H-NMR (600 MHz, CD3OD) 및 13C-NMR (150 MHz, CD3OD) : 표 1 참조 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD): See Table 1
화합물 1(- 114.42°; c 0.05, MeOH)은 황색의 분말(yellow powder)로서 분리되었다. HR-ESI-MS 분석결과 나타난 나트륨 분자이온 피크로부터 화합물 1의 분자식은 C16H14O5로 결정된다([M+Na]+, m/z 309.1623, [calcd for C16H14O5Na]). IR 스펙트럼(KBr, νmax, ㎝-1)은 화합물 1이 히드록실 그룹(3306㎝-1), 카보닐 그룹(1656㎝-1) 및 벤젠 고리(1504, 1471, 1458㎝-1)를 갖고 있음을 나타낸다. UV-Vis 스펙트럼(MeOH, λmax(log ε))은 330 (sh), 279 (3.94) 및 237 (4.05)nm에서 최대값을 갖는 것으로 나타낸다. 화합물 1의 1H NMR 및 13C NMR 스펙트럼(표 1 참조)은 플라보노이드의 특징을 나타낸다. δ H 2.82 (dd, J = 3.3, 17.0, H-3a) 및 2.90 (dd, J = 13.0, 17.0, H-3b) (δC, 43.9, C-3)에서의 메틸렌 그룹, δH 5.73 (dd, J = 3.2, 12.9, δC, 76.7, C-2)에서의 메틴, δ C 193.8 (C-4)에서의 카보닐 그룹은 화합물 1이 플라바논임을 가리킨다. 화합물 1의 δ H 5.73 (H-2) 메틴과 δ C 155.2 (C-2')의 HMBC 상관관계 및 δ H 6.83 (H-3'), 7.17 (H-4'), 6.91 (H-5'), 7.49 (H-6')의 COSY 상관관계로부터 B-ring의 2' 위치에 히드록시 그룹을 갖는 것으로 확인된다. HMBC 분석에서 δ H 7.31 양성자와 δ C 193.8의 상관관계 신호는 5번 위치에 하이드록실 그룹의 부재를 나타내고, δ H 3.87의 메톡시 양자는 δ C 156.9의 4차 탄소에 위치하고 있음이 확인된다. 2번 위치의 절대구조는 CD 스펙트럼에서의 235.2nm의 코튼 효과(Cotton effect)에 근거하여 S 형의 절대구조인 것을 알 수 있다. 따라서, 화합물 1은 7,2'-디히드록시-6-메톡시-2S-플라바논(7,2'-dihydroxy-6-methoxy-2S-flavanone)의 구조를 갖는 신규 화합물로서 확인된다. Compound 1 ( - 114.42 DEG; c 0.05, MeOH) was isolated as a yellow powder. From the sodium molecular ion peak shown in the HR-ESI-MS analysis, the molecular formula of
실시예 2-2. 2'-히드록시-6,7-메톡시-2Example 2-2. 2'-hydroxy-6,7-methoxy-2 SS -플라바논(화합물 2)- flavanone (Compound 2)
2'-Hydroxy-6,7-methoxy-2S-flavanone;2'-Hydroxy-6,7-methoxy- 2 S -flavanone;
1H-NMR (600 MHz, CD3OD) 및 13C-NMR (150 MHz, CD3OD) : 표 1 참조 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD): See Table 1
화합물 2( - 146.75°; c 0.05, MeOH)는 노란색의 분말로서 수득되었다. 화합물 2의 HR-ESI-MS 데이터에 따른 분자식은 C17H16O5([M+Na]+, m/z 323.0895 [calcd for C17H16O5Na])로 확인된다. IR 스펙트럼(KBr, νmax, ㎝-1)은 화합물 2에 히드록실 그룹(3414㎝-1), 카보닐 그룹(1651㎝-1), 벤젠 고리(1502, 1455, 1438㎝-1) 등의 특징이 있음을 보여준다. 화합물 2의 UV-Vis 스펙트럼(MeOH, λmax(log ε))은 337 (3.85), 275 (4.10) 및 237 (4.24)에서 최대값을 갖는 것으로 확인된다. 화합물 2의 1H NMR 및 13C NMR 데이터(표 1 참조)는 δ H 3.81에서의 메톡시 양자를 제외하고 화합물 1과 매우 비슷하다. 화합물 2의 HMBC 상관관계는 δ H 3.81, δ H 3.88에서의 메톡시 양자가 δ C 158.4 및 146.1의 4차 탄소와 관련이 있음을 나타낸다. 화합물 2의 C-2의 절대구조는 CD 스펙트럼에서의 299.6nm의 양성 코튼 효과(Cotton effect)에 기반하여 S형 구조인 것으로 확인된다. 따라서, 화합물 2는 2'-히드록시-6,7-메톡시-2S-플라바논(2'-hydroxy-6,7-methoxy-2S-flavanone)의 화학구조를 갖는 신규 화합물인 것으로 확인된다. Compound 2 ( - 146.75 [deg.]; c 0.05, MeOH) was obtained as a yellow powder. The molecular formula of the HR-ESI-MS data for
실시예 2-3. 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(화합물 3)Examples 2-3. 5,2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3)
5,2'-Dihydroxy-6,7-methylenedioxyflavanone;5,2'-Dihydroxy-6,7-methylenedioxyflavanone;
1H-NMR (600 MHz, CD3OD) 및 13C-NMR (150 MHz, CD3OD) : 표 2 참조 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD): See Table 2
NMR 및 MS 분석결과 화합물 3은 5,2'-디히드록시-6,7-메틸렌디옥시플라바논(5,2'-dihydroxy-6,7-methylenedioxyflavanone)으로 확인된다.NMR and MS analysis showed that
실시예 2-4. 7-히드록시-6,8-디메톡시크로몬(화합물 4)Examples 2-4. 7-hydroxy-6,8-dimethoxychromone (Compound 4)
7-Hydroxy-6,8-dimethoxychromone;7-Hydroxy-6,8-dimethoxychromone;
1H-NMR (600 MHz, CDCl3) 및 13C-NMR (150 MHz, CDCl3) : 표 1 참조 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ): See Table 1
화합물 4는 흰색의 결정형 화합물로서 수득되었다. 화합물 4의 HR-ESI-MS 데이터에 따른 분자식은 C11H10O5([M+Na]+, m/z 245.0426 [calcd for C11H10O5Na])로 확인된다. 화합물 4에 대한 1H NMR 및 13C NMR 데이터는 δ C 135.0에서의 산화된 올레피닉 4차 탄소의 존재를 제외하고, 크로몬(chromone) 화합물과 매우 비슷하다. 화합물 4의 HMBC 상관관계는 δH 7.30에서의 1개의 메틸 양자가 δ C 176.8 (C-4)에서의 카보닐 탄소와 관련이 있음을 보여주며, 이것은 H-5임이 확인된다. 이는 화합물 4의 8번 위치(position 8)가 치환되었음을 의미한다. 또한, δH 3.92 (s)와 δH 3.98 (s)에서의 두 개의 메톡시 그룹이 δ C 145.9 (C-6) 및 135.0 (C-8)의 4차 탄소와 관련이 있음을 의미한다. 따라서 화합물 4는 7-히드록시-6,8-디메톡시크로몬(7-hydroxy-6,8-dimethoxychromone)의 구조를 갖는 신규 화합물인 것으로 확인된다.
실시예 2-5. 6-메톡시크로마논(화합물 5)Examples 2-5. 6-methoxychromanone (Compound 5)
6-Methoxychromanone;6-Methoxychromanone;
1H-NMR (600 MHz, CD3OD) 및 13C-NMR (150 MHz, CD3OD) : 표 2 참조 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD): See Table 2
NMR 및 MS 분석결과 화합물 5는 6-메톡시크로마논(6-methoxychromanone)으로 확인된다.NMR and MS analysis showed that
실시예Example 2-6. 6,7- 2-6. 6,7- 디메톡시크로몬Dimethoxychromone (화합물 6)(Compound 6)
6,7-Dimethoxychromone;6,7-Dimethoxychromone;
1H-NMR (600 MHz, CD3OD) 및 13C-NMR (150 MHz, CD3OD) : 표 2 참조 1 H-NMR (600 MHz, CD 3 OD) and 13 C-NMR (150 MHz, CD 3 OD): See Table 2
NMR 및 MS 분석결과 화합물 6은 6,7-디메톡시크로몬(6,7-dimethoxychromanone)으로 확인된다.NMR and MS analysis confirmed that
(dd, J = 12.9, 3.2) 5.73
(dd, J = 12.9, 3.2)
(d, J = 2.5)5.74
(d, J = 2.5)
(d, J = 5.9)7.79
(d, J = 5.9)
(dd, J = 17.0, 3.3)
2.90
(dd, J = 17.0, 13.0)2.82
(dd, J = 17.0,3.3)
2.90
(dd, J = 17.0, 13.0)
(d, J = 2.3)2.80
(d, J = 2.3)
(d, J = 5.9)6.23
(d, J = 5.9)
(d, J = 8.1)6.83
(d, J = 8.1)
(dd, J = 8.1, 0.7)6.83
(dd, J = 8.1, 0.7)
(dt, J = 7.8, 1.5)7.17
(dt, J = 7.8, 1.5)
(td, J = 7. 9, 1.6)7.17
(td, J = 7.9, 1.6)
(dt, J = 7.6, 0.7)6.89
(dt, J = 7.6, 0.7)
(d, J = 7.7)7.49
(d, J = 7.7)
(dd, J = 7.7, 1.4)7.51
(dd, J = 7.7,1.4)
- The coupling constant J values (in hertz) are given in parentheses.
- All assignments are based on the COSY, HMQC, and HMBC experiment.- The chemical shifts (δ) are in ppm from TSM.
- The coupling constant J values (in hertz) are given in parentheses.
- All assignments are based on the COZY, HMQC, and HMBC experiment.
2.98 dd (13.3, 17.2)2.79 dd (3.0, 17.2)
2.98 dd (13.3, 17.2)
<< 실시예Example 3. 세포독성 확인> 3. Cytotoxicity>
본 발명의 화합물들이 세포독성이 있는지를 확인하기 위해, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 어세이를 수행하였다. HUVEC(Human umbilical vein endothelial cells)을 96웰 플레이트에 웰당 5×103개로 분주한 후, 24시간 후에, ESM(effective screening medium) 배양액으로 교체하였다(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 배양 유지한다). 이 때 본 발명의 화합물 1~6을 최종농도가 1~20μM이 되도록 처리하고, 48시간 후, 100㎕의 1㎎/㎖ MTT 용액을 세포에 더하여 4시간 동안 반응시켰다. 이 후 DMSO(dimethyl sulfoxide) 150㎕를 더하여, 세포 내에 생성된 포마잔염(formazan salt)을 용해시킨 후, 이 포마잔의 양을 Microplate reader(Tecan Austria GmbH, Austria)를 이용하여 540nm에서 측정하였고, 이에 대한 결과는 도 1에 나타내었다. (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to confirm that the compounds of the present invention were cytotoxic. HUVEC (Human umbilical vein endothelial cells), and then a frequency divider in a 96-well plate per well 5 × 10 3 dogs, 24 hours later, ESM (effective screening medium) was replaced with culture medium (HUVEC cells Cambrex Bio Science (Charles City, IA, USA) and incubated with EBM-2 basal media and growth supplement (Cambrex Bio Science) supplied thereto. At this time, the
도 1을 참고하면, 본 발명의 화합물 1~6은 모두 세포독성이 없는 것으로 확인된다. Referring to FIG. 1, it is confirmed that all of the
<< 실시예Example 4. 패혈증 동물모델 유도> 4. Induction of sepsis animal models>
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㎝를 절개하여 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 배설물(장내 소화물)이 누출되게 하였고, 이 배설물을 복강에 노출시켰으며, 개복부위를 4.0-실크 봉합선으로 봉합하였다. 대조군(sham, sham-operated mice)은 맹장을 묶고 터트리는 단계를 수행하지 않고 단순히 배만 절개하고 다시 봉합하였다. 각 마우스들은 CLP 수술 24시간 후부터 패혈증 증상에 노출되었는데, 전율(shivering)이 있거나 털이 곤두서거나(bristled hair) 또는 무기력증(weakness) 등의 현상이 나타났다. 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
<< 실시예Example 5. 패혈증 모델에서의 5. In the sepsis model HMGB1HMGB1 발현 확인> Expression confirmation>
실시예Example 5-1. 패혈증 세포모델의 5-1. Sepsis cell model HMGB1HMGB1 발현 확인 Confirmation of expression
HUVEC을 6웰 플레이트(6-well plates)에서 배양한 후 LPS(lipopolysaccharide, 100ng/㎖)를 4시간 동안 처리하였으며(대조군은 saline 처리), 이 후, 본 발명의 화합물 1 내지 6을 1~10μM로 6시간 동안 처리하였다. HUVECs were cultured in 6-well plates and treated with LPS (lipopolysaccharide, 100 ng / ml) for 4 hours (control group treated with saline) For 6 hours.
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. In a 96-well plastic flat microtiter plates (Corning, NY, USA), 0.02% sodium azide was added to perform competitive enzyme-linked immunosorbent assays (HUVEC) 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 용해 버퍼(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 minutes) after decomposition by vortexing at 4 ° C for 1 hour using a protein separation reagent containing RIPA dissolution buffer (Upstate Biotechnology, USA) .
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-rabbit 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-rabbit 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 8N의 H2SO4 50㎕를 가하여 모든 반응을 중단시킨 후, 490nm에서 흡광도를 확인하였으며, 분석결과는 도 3A 및 도 3B에 나타내었다. 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, 50 μl of 8N H 2 SO 4 was added to stop all the reactions, and the absorbance was confirmed at 490 nm. The analysis results are shown in FIGS. 3A and 3B.
도 3A 및 도 3B를 참고하면, 패혈증 세포 모델에서 본 발명의 화합물들이 LPS가 처리된 HUVEC에서 본 발명의 화합물 1~6이 HMGB1의 발현을 현저하게 억제하는 효과가 있는 것으로 확인된다. 3A and 3B, it is confirmed that the compounds of the present invention in the sepsis cell model have the effect of inhibiting the expression of HMGB1 remarkably in the HUVEC treated with LPS.
실시예Example 5-2. 패혈증 동물모델의 혈중 5-2. Sepsis HMGB1HMGB1 발현 확인 Confirmation of expression
실시예 4의 방법으로 CLP 수술을 수행한 마우스에 본 발명의 화합물 1 내지 6(1~10μM/mouse)을 정맥투여하였다(CLP 수술을 수행 후 12시간 후에 투여). Mice treated with CLP according to the method of Example 4 were intravenously administered with
* 동물실험에 사용된 각 화합물의 [μM/mouse] 값과 [㎍/mouse] 값의 환산값은 도 2에 개시되어 있음.The values of [μM / mouse] and [μg / mouse] of each compound used in the animal experiment are shown in FIG.
CLP 수행 24시간 후 마우스를 희생하여 혈액을 채취한 후 2000×g에서 5분간 원심분리하여 혈청 샘플을 얻었다. ELISA 과정은 실시예 5-1과 동일하게 수행하였고, 분석 결과는 도 3C 및 도 3D에 나타내었다. After 24 hours of CLP, the mice were sacrificed and blood was collected and centrifuged at 2000 xg for 5 minutes to obtain serum samples. The ELISA procedure was performed in the same manner as in Example 5-1, and the analysis results are shown in FIGS. 3C and 3D.
도 3C 및 도 3D를 참고하면, CLP 수술법으로 유도된 패혈증 동물에서 본 발명의 화합물 1~6이 HMGB1의 발현을 현저하게 억제하는 효과가 있는 것으로 확인된다. 3C and 3D, it was confirmed that the
<< 실시예Example 6. 6. 혈관세포의Vascular cell 투과성 억제 효과 확인> Confirmation of permeation inhibiting effect>
실시예Example 6-1. 패혈증 세포모델에서의 6-1. In the sepsis cell model 혈관세포의Vascular cell 투과성 억제 효과 확인 Confirm permeation inhibiting effect
본 발명의 화합물 1~6(10μM)을 HUVEC에 처리한 후 각 화합물이 HMGB1을 통해 유도된 혈관벽 손상(혈관의 미세한 천공으로 인해 투과성이 생김)을 복구하는지를 확인하였다. 이를 위해 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.
HUVEC은 5×104/웰(well)로 3㎛의 기공(pore) 크기를 갖는 12㎜ 직경의 트랜스웰(transwell)에서 3일 동안 배양되었다. 세포가 각 트랜스웰에 가득 차면 LPS(100ng/㎖, 4시간) 또는 HMGB1(1㎍/㎖, 16시간)을 처리하여 반응시켰고, 각각의 화합물을 6시간 동안 처리하였다. 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 650nm에서 측정하였고 이에 대한 결과는 도 4A에 나타내었다. HUVECs were cultured for 3 days in a 12 mm diameter transwell with a pore size of 3 [mu] m at 5 x 10 < 4 > / well. When cells were filled in each transwell, LPS (100 ng / ml, 4 hours) or HMGB1 (1 μg / ml, 16 hours) was treated and reacted, and each compound was treated for 6 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 FIG. 4A.
도 4A를 확인하면, 본 발명의 화합물 1~6이 모두 세포 손상으로 인한 투과성(에반스 블루 용액의 흐름)을 억제하는 것을 확인할 수 있어, HMGB1로 유도된 패혈증 세포 모델에서의 혈관 염증 억제 효과가 우수함을 알 수 있다. 4A, it can be confirmed that the
실시예Example 6-2. 패혈증 동물모델에서의 6-2. In an animal model of sepsis 혈관세포의Vascular cell 투과성 억제 효과 확인 Confirm 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 각 시료(10μM/mouse)를 정맥주사를 통해 처리하고 6시간 후 살린(saline) 용액에 녹인 1%(w/v) 에반스 블루 용액과 HMGB1(2㎍/mouse)를 연이어 정맥주사하였다. Male C57BL / 6 mice were prepared (10 mice per group, 6-7 weeks old and 18-20g mice were purchased and adapted for 12 days in a laboratory, using Orient Bio Co., Sungnam, Kyungki-Do, Republic (2 μg / mouse) of Evans Blue solution and 1% (w / v) Evans Blue solution dissolved in saline solution at 6 hours after intravenous injection of each sample (10 μM / mouse) Followed by intravenous injection.
에반스 블루 용액을 정맥주사한 지 2시간 후 마우스를 희생시키고, 복강을 5㎖의 살린 용액으로 세척하면서 복막 삼출물(peritoneal exudate)을 모아 200×g에서 10분간 원심분리하였다. 상기 원심분리물의 상층액(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 4B에 나타내었다. Two hours after the intravenous injection of Evans Blue solution, the mice were sacrificed and the peritoneal exudate was collected by centrifugation at 200 xg for 10 minutes while the abdominal cavity was washed with 5 ml of saline solution. The absorbance of the supernatant of the centrifuge was confirmed at 650 nm. This is because the dye of the Evans blue solution flows into the abdominal cavity and can confirm the permeability due to the microperforations in the vascular cells (Int. Immunopharmacol. 2009, 9, 268-276.). The results are shown in FIG. 4B.
상기 도 4B를 참고하면, HMGB1이 처리된 마우스에서는 다량의 염색약 누출이 있는 것을 알 수 있는데, 상기 마우스에 화합물 1 내지 6이 처리된 군에서는 염색약의 누출이 현저하게 억제되었음을 알 수 있다. 이에 상기 화합물들이 혈관벽 손상을 보호함으로써 패혈증을 억제함을 입증할 수 있다. Referring to FIG. 4B, it can be seen that there is a large amount of dye leakage in the HMGB1-treated mouse. In the group treated with the
<< 실시예Example 7. 7. 세포유착분자Cell adhesion molecule 및 세포표면 And cell surface 마커Marker 수용체의 발현 확인> Confirmation of receptor expression>
VCAM-1(vascular cell adhesion molecule-1), ICAM-1(intercellular adhesion molecule-1), E-selection과 같은 세포유착과 관련된 단백질의 발현을 whole-cell ELISA 방법으로 확인하였다. The expression of proteins related to cell adhesion such as VCAM-1 (vascular cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1) and E-selection was confirmed by whole-cell ELISA.
세포가 90%의 confluence를 이루면 96웰 플레이트에 배양한 HUVEC에 HMGB1(1㎍/㎖)을 16시간 동안 반응시키고, 본 발명의 화합물 1~6(10μM)을 6시간 동안 처리하였다. 이 후, 세포배양액을 제거하고, 세포를 PBS(phosphate buffered saline)로 세척한 후, 각 세포를 50㎕의 1%(w/v) 파라포름알데히드(paraformaldehyde)로 15분간 25℃ 실온에서 고정하였다. 1%(w/v) 파라포름알데히드를 제거하고 세포를 세척한 후, VCAM-1, ICAM-1 또는 E-selection에 대한 마우스 항 인간 모노클로널 항체(mouse anti human monoclonal antibody, Temecula, CA, USA, 1:50 희석) 100㎕를 가하였다. 1시간 후에는 각 세포를 3번씩 세척하고, 100㎕의 1:2000 퍼옥시다아제-결합 항-마우스 IgG 항체(1:2000 peroxidase-conjugated anti-mouse IgG antibody, Sigma, Saint Louis, MO)와 1시간 동안 반응시켰고, 3번 세척한 후, o-PPD 기질(o-phenylenediamene substrate, Sigma, St. Louis, MO)과 반응시켰다. 이로 인해 나타나는 발색반응에 대한 흡광도를 490nm에서 측정하였고, 도 5A에 나타내었다. 또한, 혈관내피세포에서 혈관 염증이 일어날 때 발현하는 세포표면(cell surface) 마커인 TLR2(Toll-like receptor 2), TLR4(Toll-like receptor 4) 및 RAGE(Receptor for advanced glycation end products) 수용체의 발현도 각 마커에 대해 A-9, H-80 및 A-9 항체(Santa Cruz, CA)를 이용하여 이와 동일한 방법으로 확인하였으며, 이에 대한 결과는 도 5B에 나타내었다. When cells were confluent at 90%, HMGB1 (1 / / ml) was incubated in HUVEC cultured on a 96-well plate for 16 hours, and the
도 5A 및 도 5B를 참고하면, 본 발명의 화합물 1~6이 패혈증 세포 모델에서 세포유착분자인 VCAM-1, ICAM-1 및 E-selection(도 5A)과 세포표면 마커 수용체 TLR2(Toll-like receptor 2), TLR4(Toll-like receptor 4) 및 RAGE(도 5B)의 발현을 현저하게 억제하는 것으로 확인되어, 상기 화합물들의 혈관 염증 억제효과가 아주 우수함을 알 수 있다. 5A and 5B, Compounds 1 to 6 of the present invention inhibited cell adhesion molecules VCAM-1, ICAM-1 and E-selection (FIG. 5A) and cell surface marker receptor TLR2 (Toll-like receptor 2), TLR4 (Toll-like receptor 4) and RAGE (FIG. 5B), and thus the compounds are shown to exhibit excellent vascular inflammation inhibitory effects.
<< 실시예Example 8. 혈관 염증에 따른 혈관내피로의 단핵구 및 백혈구의 이동 확인> 8. Identification of monocyte and leukocyte migration into vascular endothelium following vascular inflammation>
실시예Example 8-1. 혈관 염증에 따른 혈관내피로의 단핵구의 이동 확인 8-1. Identification of monocyte migration into vascular endothelium following vascular inflammation
혈관 염증에 따른 혈관내피로의 단핵구(monocyte)의 이동 현상을 확인하기 위해, 8㎛의 기공(pore) 크기를 갖는 필터가 내장된 직경 6.5㎜의 트랜스웰 플레이트(transwell plate)를 이용하였다. 상기 트렌스웰 플레이트의 필터 아래의 하단 챔버에 HUVEC(6×104/cells)을 3일간 배양하여, 세포배양 플레이트에 세포가 가득차게 하였다. 단핵구인 THP-1 세포도 역시 HUVEC이 배양되는 플레이트 층의 상단 챔버에서 배양하였는데, 이 THP-1 세포를 상단 챔버에 넣기 전에 HUVEC에 HMGB1(1㎍/㎖)을 처리하였다(THP-1 세포는 2×105~1×106세포/㎖의 밀도로 L-글루타민이 포함된 RPMI1640 배지(FBS 10%[v/v], 2-머캅토에탄올 55μM 포함)에서 배양 유지됨). HMGB1을 HUVEC와 16시간 동안 반응시키고, 본 발명의 화합물 1~6(10μM)을 6시간 동안 처리하였다. 이후, 필터 아래로 이동하지 않은 위쪽 챔버에 있던 세포들을 제거하고, 하단 챔버 쪽의 필터 위로 이동한 단핵구 세포들을 8%(w/v) 글루타르알데히드(glutaraldehyde)로 고정한 후, 20%(v/v) 메탄올 수용액에 녹인 0.25%(w/v) 크리스탈 바이올렛(crystal violet) 용액으로 염색하였다. 염색결과는 광학현미경을 이용하여 육안으로 확인하였고, 이동지수(migration index)를 참고하여 단핵구의 이동정도를 도 6A에 나타내었다. To confirm the migration of monocytes to vascular endothelium following vascular inflammation, a 6.5 mm diameter transwell plate with a filter having a pore size of 8 mu m was used. HUVEC (6 x 10 < 4 > / cells) was cultured in the lower chamber below the filter of the transfer well plate for 3 days, and the cell culture plate was filled with cells. Monocyte THP-1 cells were also cultured in the upper chamber of the plate layer where the HUVECs were cultured. HUVECs were treated with HMGB1 (1 μg / ml) before the THP-1 cells were placed in the upper chamber (THP- Maintained in RPMI 1640 medium (containing 10% FBS [v / v], 55 μM 2-mercaptoethanol) containing L-glutamine at a density of 2 × 10 5 to 1 × 10 6 cells / HMGB1 was reacted with HUVEC for 16 hours, and compounds 1 to 6 (10 μM) of the present invention were treated for 6 hours. Then, the cells in the upper chamber that did not move under the filter were removed, and the mononuclear cells migrated on the filter in the lower chamber side were fixed with 8% (w / v) glutaraldehyde, v) 0.25% (w / v) crystal violet solution dissolved in an aqueous methanol solution. The results of the staining were visually confirmed using an optical microscope and the degree of migration of mononuclear cells with reference to the migration index was shown in FIG. 6A.
도 6A를 참고하면, 본 발명의 화합물 1~6이 혈관 염증에 따른 혈관내피로의 단핵구의 이동을 현저하게 억제하는 것으로 확인된다. Referring to FIG. 6A, it was confirmed that the
실시예Example 8-2. 혈관 염증으로 인한 단핵구의 혈관내피로의 유착 확인 8-2. Confirmation of monocyte adhesion to vascular endothelium due to vascular inflammation
혈관내피세포에 대한 단핵구의 유착 확인을 위해, THP-1 세포(1.5×106/㎖, 200㎕/well)를 Vybrant DiD 염료로 표지한 후, HUVEC 세포에 더하여 배양하였다. To confirm adhesion of mononuclear cells to vascular endothelial cells, THP-1 cells (1.5 × 10 6 / ml, 200 μl / well) were labeled with Vybrant DiD dye and then added to HUVEC cells.
이를 위해, 먼저 HUVEC에 HMGB1(1㎍/㎖)을 16시간 동안 반응시키고, 본 발명의 화합물 1~6(10μM)을 6시간 동안 처리하였다. 이 후, Vybrant DiD 염료로 표지된 THP-1 세포를 더하여 37℃에서 1시간 동안 배양하였으며, 유착되지 않은 세포들은 세포배지를 이용하여 세척하였고, HUVEC과 THP-1 세포의 유착 정도를 형광현미경으로 확인하였고, 이에 대한 결과는 도 6B에 나타내었다. For this, HUVEC was first reacted with HMGB1 (1 / / ml) for 16 hours, and the
도 6B를 참고하면, 본 발명의 화합물 1~6이 혈관 염증으로 인한 단핵구의 혈관내피로의 유착을 현저하게 억제하는 것으로 확인된다. Referring to FIG. 6B, it was confirmed that
실시예Example 8-3. 혈관내피로의 백혈구 이동 확인 8-3. Identification of leukocyte migration into vascular endothelium
백혈구의 이동을 측정하기 위해서는 마우스에 본 발명의 화합물 1~6(10μM/mouse)을 정맥주사한 후, HMGB1(2㎍/mouse)을 6시간 후에 정맥주사하였다. In order to measure the migration of leukocytes,
또한, 실시예 4의 방법으로 CLP 수술을 수행한 마우스에 본 발명의 화합물 1 내지 6(10μM/mouse)을 정맥투여하였다(CLP 수술을 수행 후 12시간 후에 투여). In addition, the
이 후, 각 마우스를 희생하여 복막강을 살린 용액으로 세척하여 복막 삼출액을 수집한 후, 복막 삼출액 20㎕를 0.38㎖의 Turk’ solution (0.01 % crystal violet in 3 % acetic acid)과 혼합하여 형광 현미경을 통해 확인되는 백혈구의 수를 육안으로 관찰하였다. 이에 대한 결과는 도 6C에 나타내었다. After the peritoneal effusion was collected, 20 μl of the peritoneal effusion was mixed with 0.38 ml of Turk 'solution (0.01% crystal violet in 3% acetic acid) And the number of white blood cells identified through the eye was visually observed. The results are shown in Fig. 6C.
도 6C를 참고하면, 본 발명의 화합물 1~6이 HMGB1 또는 CLP로 유도된 마우스에서 혈관내피로의 백혈구 이동이 억제되어 혈관내피에서의 백혈구 수가 현저하게 줄어들었음을 확인할 수 있다. Referring to FIG. 6C, it can be confirmed that the
<< 실시예Example 9. 염증 관련 인자의 발현 확인> 9. Expression of inflammatory factors>
HUVEC에 본 발명의 화합물 1~6(10μM)을 6시간 동안 처리하였고, 이 후 다시 HMGB1(1㎍/㎖, 16시간)을 처리하여 반응시켰다. 각 염증인자의 발현은 ELISA 키트를 이용하여 확인하였고, 이에 대한 결과는 도 7에 나타내었다.
* TNF-α 및 IL-1β(R&D Systems, Minneapolis, MN, USA), Total-NF-κB 및 phospho-NF-κB(#7174, #7173, Cell Signaling Technology, Danvers, MA, USA).TNF-? And IL-1? (R & D Systems, Minneapolis, MN, USA), Total-NF-κB and phospho-NF-κB (# 7174, # 7173, Cell Signaling Technology, Danvers, MA, USA).
도 7을 참고하면, 본 발명의 화합물 1~6이 TNF-α, IL-1β 및 인산화된 NF-κB(phospho-NF-κB)의 발현을 억제하여 혈관 염증의 치료효과가 우수함을 확인할 수 있다. 7, the
<< 실시예Example 10. 패혈증 동물모델에서의 생존율 확인> 10. Determine survival rate in sepsis animal models>
실시예 4의 방법으로 CLP 수술을 수행한 마우스에 본 발명의 화합물 1~6(10μM/mouse)을 2회 정맥투여하였다(CLP 수술을 수행 후 12시간 및 50시간, 총 2회 투여)(각 군당 20마리). 마우스의 생존은 CLP 수술을 수행한 지 6시간 후부터 126시간까지 확인하였고(카플란-마이어의 생존 분석법[Kaplan-Meier survival analysis]에 따라 관찰), 시간에 따른 마우스의 생존결과는 도 8에 나타내었다.
도 8을 참고하면, 본 발명의 화합물 1~6을 투여한 마우스 그룹은 모든 화합물에서 CLP 수술 이후 126시간째에 각각 40~60%의 생존율을 갖는 것으로 확인되어 패혈증의 치료 효과가 우수함을 알 수 있다.Referring to FIG. 8, it was found that the mouse group administered with the
<실시예 11. 독성실험> ≪ Example 11: Toxicity test &
실시예 11-1. 급성독성Example 11-1. Acute toxicity
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 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 1을 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 1을 투여한 실험군에서 마우스가 모두 생존하는 것으로 확인되었다.The toxicity of the compound of the present invention (7,2'-dihydroxy-6-methoxy-2 S -flavanone) to the animal body in an acute (within 24 hours) , And this experiment was performed to determine mortality. An ICR mouse line, a common mouse, was assigned to each group of 10 mice. In the experimental group,
실시예 11-2. 실험군 및 대조군의 장기 및 조직 독성 실험Example 11-2. Organ organs toxicity test in experimental group and control group
장기 독성 실험은 본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 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 1을 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 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention was administered to C57BL / 6J mice (10 mice per group) Respectively. (8: 1: 1, v: v: v) was administered to the test group to which
<제제예 1. 약학적 제제>≪ Formulation Example 1 >
제제예 1-1. 정제의 제조Formulation Example 1-1. Manufacture of tablets
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논) 200g을 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 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 Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) 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.
제제예 1-2. 주사액제의 제조Formulation Example 1-2. Injection preparation
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논) 1g, 염화나트륨 0.6g 및 아스코르브산 0.1g을 증류수에 용해시켜서 100㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.1 g of the
<제제예 2. 식품 제조><Formulation Example 2: Food Preparation>
제제예 2-1. 조리용 양념의 제조Formulation Example 2-1. Manufacture of cooking seasonings
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 조리용 양념에 1 중량%로 첨가하여 건강 증진용 조리용 양념을 제조하였다.Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention was added to the cooking seasoning at 1% by weight to prepare a cooking sauce for health promotion.
제제예 2-2. 밀가루 식품의 제조Formulation Example 2-2. Manufacture of flour food products
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 밀가루에 0.1 중량%로 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.The compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention was added to the wheat flour at 0.1% by weight and the mixture was used to prepare breads, cakes, cookies, crackers and noodles To produce health promotion foods.
제제예 2-3. 스프 및 육즙(gravies)의 제조Preparation Example 2-3. Manufacture of soups and gravies
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 스프 및 육즙에 0.1 중량%로 첨가하여 건강 증진용 수프 및 육즙을 제조하였다.Health enhancing soup and juice were prepared by adding Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention to soup and juice at 0.1 wt%.
제제예 2-4. 유제품(dairy products)의 제조Formulation Example 2-4. Manufacture of dairy products
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논)을 우유에 0.1 중량%로 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.The compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention was added to milk in an amount of 0.1% by weight and the milk was used to prepare various dairy products such as butter and ice cream Respectively.
제제예 2-5. 야채주스 제조Formulation Example 2-5. Vegetable juice manufacturing
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논) 0.5g을 토마토주스 또는 당근주스 1,000㎖에 가하여 건강 증진용 야채주스를 제조하였다.0.5 g of Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.
제제예 2-6. 과일주스 제조Formulation Example 2-6. Manufacture of fruit juice
본 발명의 화합물 1(7,2'-디히드록시-6-메톡시-2S-플라바논) 0.1g을 사과주스 또는 포도주스 1,000㎖에 가하여 건강 증진용 과일주스를 제조하였다.0.1 g of Compound 1 (7,2'-dihydroxy-6-methoxy-2 S -flavanone) 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]
(Compound 1), 2'-hydroxy-6,7-methoxy-2S-flavanone (Compound 2) and 5'-hydroxy-6-methoxy- , 2'-dihydroxy-6,7-methylenedioxyflavuanone (Compound 3) as an active ingredient for the prevention or treatment of vascular inflammation or sepsis, characterized in that it comprises at least one compound selected from the group consisting of Composition.
[Chemical Formula 1]
상기 화합물은 HMGB1(high mobility group box 1)의 발현을 억제하는 효과가 있는 것을 특징으로 하는 혈관 염증 또는 패혈증의 예방 또는 치료용 조성물. The method according to claim 1,
Wherein said compound has an effect of inhibiting the expression of HMGB1 (high mobility group box 1).
[화학식 1]
(Compound 1), 2'-hydroxy-6,7-methoxy-2S-flavanone (Compound 2) and 5'-hydroxy-6-methoxy- , 2'-dihydroxy-6,7-methylenedioxyflavanone (Compound 3) as an active ingredient for the prevention or amelioration of vascular inflammation or septicemia, which is characterized by comprising at least one compound selected from the group consisting of Health functional foods.
[Chemical Formula 1]
상기 건강기능식품은 각종 식품류, 음료, 껌, 차 및 비타민 복합제로 이루어진 군에서 선택되는 것을 특징으로 하는 혈관 염증 또는 패혈증의 예방 또는 개선용 건강기능식품.The method of claim 3,
Wherein said health functional food is selected from the group consisting of various foods, beverages, gums, tea and vitamin complex.
[화학식 2]
A novel compound 7,2'-dihydroxy-6-methoxy-2 S -flavanone of Formula 2 (Compound 1).
(2)
[화학식 3]
The novel compound 2'-hydroxy-6,7-methoxy-2 S -flavanone (Compound 2)
(3)
Extracting Salicornia herbacea with water, C1 to C4 lower alcohol or a mixed solvent thereof to prepare a green tea extract; Sequentially fractionating the green tea extract with hexane and ethyl acetate; And the fractions were chromatographed to yield 7,2'-dihydroxy-6-methoxy-2S-flavanone (Compound 1) and 2'-hydroxy-6,7- 2S-flavanone (Compound 2).
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