KR101503600B1

KR101503600B1 - Composition for inhibiting cellular senescence comprising erthro-austrobailignan-6 isolated from Saururus chinensis

Info

Publication number: KR101503600B1
Application number: KR1020130101713A
Authority: KR
Inventors: 김재룡; 손종근; 양효현; 박동근
Original assignee: 영남대학교 산학협력단
Priority date: 2013-08-27
Filing date: 2013-08-27
Publication date: 2015-03-19
Also published as: KR20150025520A

Abstract

본 발명은 삼백초로부터 분리된 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)을 유효성분으로 함유하는 세포 노화 억제용 조성물에 관한 것으로, 아드리아마이신에 의해 유도되는 것을 특징으로 하는 세포 노화 억제용 약학 조성물을 제공한다. 이렇게 섬유아세포의 세포노화 과정을 억제함으로써 노화관련 질환, 예를 들어 피부노화, 류마티스성 관절염, 골관절염, 간염, 만성 피부손상 조직, 동맥경화, 전립샘 증식증 및 간암 등과 같은 질환 치료에 유용하게 사용될 수 있다. 또한 세포노화를 저해할 수 있는 항노화 기능성 식품, 항혈관노화 약물 개발, 화장품 개발에 활용될 수 있을 것으로 기대된다.The present invention relates to a composition for inhibiting cell senescence comprising erythro-austrobailignan-6 isolated from Saururus chinensis as an active ingredient, and relates to a composition for inhibiting cell senescence, which is characterized by being induced by adriamycin A pharmaceutical composition is provided. By inhibiting the cell aging process of the fibroblast, it can be useful for treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer . It is also expected to be useful for development of anti-aging functional food, anti-aging drug, and cosmetics which can inhibit cell senescence.

Description

The present invention relates to a composition for inhibiting cellular senescence comprising erythro-austrobilignan-6 isolated from Saururus chinensis as an active ingredient. (Composition for inhibiting cellular senescence comprising erthro-austrobailignan-6 isolated from Saururus chinensis)

본 발명은 삼백초로부터 분리된 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)을 유효성분으로 함유하는 세포 노화 억제용 조성물에 관한 것이다.The present invention relates to a composition for inhibiting cell senescence comprising erythro-austrobailignan-6 isolated from Saururus chinensis as an active ingredient.

정상 체세포는 일정 횟수 분열하면 더 이상 분열할 수 없게 되면서 세포노화 상태가 된다. 이는 염색체 말단의 텔로미어가 세포분열 과정에서 점점 짧아지면서 DNA 손상이 생기기 때문에 일어나는데, 이를 복제노화라고 한다. 텔로미어의 단축뿐만 아니라, 암유전자 및 암 억제 유전자의 기능 이상, 염증반응, 산화스트레스, 항암제, 자외선 및 방사선 등에 의해서도 세포노화가 유도된다. 노화 세포는 크기가 크고 모양이 더 편평해지며, 세포성장이 멈추고, 핵에 DNA 손상 흔적이 많으며, 다양한 염증성 단백질을 분비한다. 그리고 생화학적으로 노화 베타-갈락토시다제(senescence-associated β-galactosidase; SA-β-gal) 활성이 증가하는 것으로 알려져 있다. 다양한 인자들에 의해 노화가 유도되지만 p53과 Rb/p16 암억제 유전자 신호전달 경로를 통하여 노화가 조절되는 것으로 밝혀져 있다.When normal somatic cells are divided a certain number of times, they can no longer divide and become aged. This is because the telomere at the end of the chromosome is becoming shorter and shorter in the process of cell division, resulting in DNA damage. Cellular senescence is induced not only by shortening of telomere but also by dysfunction of cancer gene and cancer suppressor gene, inflammation reaction, oxidative stress, anticancer agent, ultraviolet ray and radiation. Aging cells are large in size, flatter in shape, stop cell growth, have many signs of DNA damage in the nucleus, and secrete a variety of inflammatory proteins. And biochemically it is known that senescence-associated β-galactosidase (SA-β-gal) activity increases. Although senescence is induced by a variety of factors, it has been shown that senescence is regulated through the signaling pathway of p53 and Rb / p16 tumor suppressor genes.

세포노화 현상은 암을 억제하거나 촉진하기도 하며, 조직 재생과 복구, 조직/개체 노화와 노화관련 질환의 중요한 기전으로 제시되고 있다. 아울러 세포노화는 암, 동맥경화, 피부노화, 퇴행성 신경질환, 근감소증, 골다골증, 전립선비대증 등과 같은 다양한 노화관련 질환의 병인에 기여한다. 최근의 연구결과들은 세포노화를 선택적으로 조절하면 조직, 장기의 노화, 건강 수명, 노화관련 질환의 발생을 조절할 수 있는 것으로 보고되고 있다. 텔로머라제 결핍 생쥐는 노화가 빨리 오는 것으로 알려져 있는데, 늙은 텔로미어 결핍 생쥐에서 텔로머라제 발현을 증가시키면 노화에 따른 조직 또는 장기의 퇴행성 변화를 역전시킴을 확인하였다. 노화가 빨리 오는 생쥐 모델에 있어서 노화세포에서 발현이 증가하는 것으로 알려진 p16을 발현하는 세포를 선택적으로 제거한 결과 노화로 인한 조직 병변이 억제되며, 노화관련 질환의 발생이 감소하는 것을 확인하였다. 생쥐에서 간 섬유화가 일어나는 과정에서 간 성상세포의 노화가 나타나는데, 간성상세포의 노화가 과다한 간 섬유화를 억제하는 기능을 하는 것으로 알려져 있다. p53 활성이 적절하게 조절되지 않은 상태에서 지나치게 높아지면 노화가 빨리 나타나지만, 적절한 p53의 활성은 오히려 노화를 억제하는 것으로 알려져 있다. Cell senescence also inhibits or promotes cancer, and is suggested as an important mechanism of tissue regeneration and restoration, tissue / individual aging and aging related diseases. In addition, cellular senescence contributes to the pathogenesis of various aging-related diseases such as cancer, arteriosclerosis, skin aging, degenerative neurological diseases, myopenia, osteodystrophy, and hyperplasia of the prostate. Recent studies have shown that selective regulation of cell senescence can regulate tissue, organ aging, health life span, and the development of aging-related diseases. Telomerase-deficient mice are known to be aging rapidly, confirming that increasing telomerase expression in old telomeres-deficient mice reverses the degenerative changes of tissues or organs due to aging. In a mouse model with rapid senescence, selective removal of cells expressing p16, which is known to increase expression in senescent cells, inhibits senescence-induced tissue lesions and reduces the incidence of aging-related diseases. In the course of hepatic fibrosis in mice, aging of hepatic stellate cells occurs. It is known that aging of hepatic stellate cells inhibits excessive liver fibrosis. If the p53 activity is not properly regulated, the aging will occur quickly, but the proper p53 activity is known to inhibit aging.

그리고 세포노화를 억제하는 효능이 있는 물질들에 대한 연구 결과도 보고되고 있다. 비타민 C, N-아세틸시스테인(N-acetylcysteine), NS398 및 에피프리에데라놀(epifriedelanol)과 같은 약물 또는 단일 성분들이 이 세포노화를 억제한다. 그리고 라파마이신(rapamycin)이 생쥐모델에서, 4,4'-디아미노디페닐설폰(4,4'-diaminodiphenylsulfone)이 꼬마선충에서 노화관련 질환의 발생을 억제하며, 건강수명을 늘리는 것으로 보고되었다.Studies have also been reported on substances that have the potential to inhibit cellular senescence. Drugs or single components such as vitamin C, N-acetylcysteine, NS398 and epifriedelanol inhibit this cell senescence. In rapamycin-induced mouse models, 4,4'-diaminodiphenylsulfone has been reported to inhibit the development of aging-related diseases in small nematodes and to increase health life span.

삼백초(Saururus chinensis BAILL.)는 삼백초과 식물로 여러해살이풀로서 중국, 일본 및 한국에 많이 분포되어있다. 한방에서 삼백초는 수종, 황달, 성인병, 해열, 이뇨, 간암 그리고 항염증 등에 효과가 있다고 한다. 삼백초에서 분리된 리그난유도체인 테트라히드로푸란류는 세포부착 억제, 항염증, 간보호 및 섭식저해 활성 등이 보고된바 있다.
Saururus chinensis BAILL. Is a perennial herbaceous plant widely distributed in China, Japan and Korea. It is said that Sambucoptera is effective for several species, jaundice, adult disease, fever, diuretic, liver cancer and anti-inflammation. Tetrahydrofuran, a lignan derivative isolated from Saururus chinensis, has been reported to inhibit cell adhesion, antiinflammation, liver protection and feeding inhibition activity.

한편, 한국등록특허 제10-0749675호에서는 삼백초 추출물 또는 이로부터 분리된 활성성분을 포함하는 면역억제용 조성물에 관한 것으로서, 상세하게는 삼백초(Saururus chinensis) 추출물, 또는 이로부터 분리된 (-)-소서네올[(-)-saucerneol], 소서네올 C(saucerneol C), 마나산틴 A(manassantin A) 또는 마나산틴 B를 유효성분으로 함유하는 면역억제용 조성물에 대해 개시하고 있으나, 본원 발명과 같이 삼백초로부터 분리된 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)의 섬유아세포 노화 억제 효과에 대한 언급은 없다.Korean Patent No. 10-0749675 discloses an immunosuppressive composition comprising Saururus chinensis extract or an active ingredient isolated therefrom. More particularly, the present invention relates to a Saururus chinensis extract or a (-) - Discloses a composition for immunosuppression comprising as an active ingredient, docus neol [(-) - saucerneol], saucerneol C, manassantin A or manasantin B. However, There is no mention of the erythro-austrobailignan-6 inhibitory effect on fibroblastogenesis,

본 발명의 목적은 삼백초로부터 분리된 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)을 유효성분으로 함유하는 세포 노화 억제용 조성물을 제공하는 데에 있다.It is an object of the present invention to provide a composition for inhibiting cell senescence comprising erythro-austrobailignan-6 isolated from Saururus chinensis as an active ingredient.

상기 목적을 달성하기 위하여, 본 발명자들은 사람 섬유아세포를 대상으로 삼백초에서 분리한 23가지 단일 성분 (SC1-13)의 세포노화 저해 효능을 조사하였다. 세포노화 저해 효능은 SA-b-gal활성염색, p53 발현 분석, 활성산소 분석으로 확인하였다. 그 결과 사람 섬유아세포에서 삼백초 단일성분 SC-9 (erthro-austrobailignan-6)이 세포노화 저해 효능이 있음을 확인하고 본 발명을 완성하였다.
In order to achieve the above object, the inventors investigated the cell aging inhibitory effect of 23 single components (SC1-13) isolated from Saururus chinensis in human fibroblasts. Cell aging inhibition efficacy was confirmed by SA-b-gal active staining, p53 expression analysis, and reactive oxygen analysis. As a result, it was confirmed that the single component SC-9 (erthro-austrobailignan-6) inhibits cell senescence in human fibroblasts and completed the present invention.

본 발명은 하기 화학식 1로 표시되는 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)을 유효성분으로 함유하는 세포 노화 억제용 약학 조성물을 제공한다. 상세하게는, 상기 세포는 섬유아세포인 것을 특징으로 한다.
The present invention provides a pharmaceutical composition for inhibiting cell senescence comprising erythro-austrobailignan-6 represented by the following formula (1) as an active ingredient. Specifically, the cell is a fibroblast.

< 화학식 1 >&Lt; Formula 1 >

상세하게는, 상기 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)은 삼백초 추출물로부터 분리된 것을 특징으로 하고, 보다 상세하게는, 삼백초 메탄올 추출액에 증류수 및 헥산(n-hexane)을 첨가하여 분획화한 증류수 층에 에틸아세테이트(EtOAc)를 첨가하고 분획화하여 추출된 에틸아세테이트(EtOAc) 분획 추출물인 것을 특징으로 한다.
Specifically, the erythro-austrobailignan-6 is characterized in that it is separated from the Saururus chinensis extract. More specifically, the distilled water and n-hexane are added to the Saururus chinensis methanol extract (EtOAc) fraction extract obtained by adding ethyl acetate (EtOAc) to the fractionated distilled water layer and fractionating it.

상기 화학식 1의 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)은 천연물질, 바람직하게는 식물로부터 분리할 수 있다. 천연, 잡종, 변종 식물의 다양한 기관, 뿌리, 줄기, 잎, 꽃뿐만 아니라 식물 조직 배양물을 추출하여 분리가능하다. 가장 바람직하게는 삼백초로부터 분리할 수 있다.
The erythro-austrobailignan-6 of the above formula (1) can be isolated from a natural substance, preferably a plant. It can be extracted by extracting various organ, root, stem, leaf, flower as well as plant tissue culture of natural, hybrid, and variant plants. Most preferably from Saururus chinensis.

상세하게는, 상기 세포 노화는 아드리아마이신에 의해 유도되는 것을 특징으로 하고, 세포 노화 억제는 노화 베타-갈락토시다제(senescence-associated β-galactosidase; SA-β-gal) 활성 억제를 측정하는 것을 특징으로 한다.
Specifically, the cell senescence is characterized by being induced by adriamycin, and inhibition of cell senescence is measured by measuring senescence-associated beta-galactosidase (SA-beta-gal) .

본 발명의 약학적 조성물인 경우, 상기 약학적 조성물은 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6) 이외에 약제학적으로 허용되는 담체를 포함할 수 있는데, 이러한 약학적으로 허용되는 담체는 약품 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘, 미네랄 오일 등을 포함할 수 있으나, 이에 한정되는 것은 아니다. 또한, 상기 약학적 조성물은 첨가제로서 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier other than erythro-austrobailignan-6, Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, and the like. , Water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

상기 약학적 조성물은 세포 노화의 증상 정도에 따라 투여 방법이 결정되는데, 통상적으로는 국소 투여 방식이 바람직하다. 또한, 상기 약학적 조성물 중 유효성분의 투여량은 투여경로, 질병의 정도, 환자의 나이, 성별, 체중 등에 따라 달라질 수 있으며, 일일 1회 내지 수회 투여할 수 있다.In the above pharmaceutical composition, the administration method is determined according to the degree of symptom of cell senescence. Usually, local administration method is preferable. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

상기 약학적 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내(intracerebroventricular)주사에 의해 투여될 수 있다.The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

상기 약학적 조성물은 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때, 제형은 용액, 현탁액 또는 유화액 형태이거나 엘렉시르제, 엑스제, 분말제, 과립제, 정제, 경고제, 로션제, 연고제 등의 형태일 수 있다.
The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

한편, 상기 약학적 조성물은 피부노화, 류마티스성 관절염, 골관절염, 간염, 만성 피부손상 조직, 동맥경화, 전립샘 증식증 및 간암으로 이루어진 군에서 선택된 어느 하나의 질환을 치료할 수 있지만, 이에 한정되는 것은 아니다.Meanwhile, the pharmaceutical composition may treat any one selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, arteriosclerosis, prostatic hyperplasia and liver cancer, but is not limited thereto.

본 발명자들은 삼백초로부터 분리된 에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)이 아드리아마이신에 의한 세포 노화를 억제하고, 아울러 세포 분열로 인한 복제노화를 저해함을 확인하였다. 이렇게 세포노화 과정을 억제함으로써 노화관련 질환, 예를 들어 피부노화, 류마티스성 관절염, 골관절염, 간염, 만성 피부손상 조직, 동맥경화, 전립샘 증식증 및 간암 등과 같은 질환 치료에 유용하게 사용될 수 있다. 또한, 섬유아세포의 세포노화를 저해할 수 있는 항노화 기능성 식품, 항혈관노화 약물 개발, 화장품 개발에 활용될 수 있을 것으로 기대된다. The present inventors have confirmed that erthro-austrobailignan-6 isolated from Saururus chinensis inhibits cell senescence by adriamycin and inhibits the replication senescence due to cell division. By inhibiting the cell senescence process, it can be useful for treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer. It is also expected to be useful for the development of anti-aging functional food, anti-vascular aging drug, and cosmetics that can inhibit cellular senescence of fibroblasts.

도 1은 삼백초 추출물의 분리 모식도를 나타낸다.
도 2는 삼백초로부터 분리된 단일성분의 화학구조를 나타낸다.
도 3은 사람 섬유아세포에서 삼백초 단일 성분의 세포독성 및 아드리아마이신에 의한 세포노화 저해 효과를 나타낸다. A, 삼백초 단일 성분의 세포독성 효과. 각 성분을 10ug/ml 처리 후, 3일 동안 배양하여 MTT법으로 세포독성 효과를 조사하였다. B, SA-β-gal 활성 염색 백분율. 아드리아마이신 처리 후, SC-7, SC-8, SC-10 및 SC-14를 10ug/ml로 처리하고, 3일 후 SA-β-gal 활성 염색을 시행하였다. 결과는 각 실험을 독립적으로 3회 이상 반복시행한 후 평균과 표준편차로 나타냈다. Cont, 대조군; DMSO, 디메틸설폭사이드(dimethylsulfoxide); NAC, N-아세틸시스테인(N-acetylcysteine). *p<0.05 vs DMSO.
도 4는 사람 섬유아세포에서 SC-9 [에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)]의 세포독성 및 아드리아마이신에 의한 세포노화 저해 효과를 나타낸다. A, SC-9의 세포독성 효과를 나타낸다. SC-9를 0.1 ug/ml 처리 후, 3일 동안 배양하여 MTT법으로 세포독성 효과를 조사하였다. B 및 C, SA-β-gal 활성 염색 사진. 아드리아마이신 처리 후, SC-9를 0.1 ug/ml로 처리하고, 3일 후 SA-β-gal 활성 염색을 시행하였다. 결과는 각 실험을 독립적으로 3회 이상 반복시행한 후 평균과 표준편차로 나타냈다. D, p53, 인산화 S6K, p21의 발현을 나타낸다. 아드리아마이신 처리 후, SC-9를 0.1 ug/ml로 처리하고, 웨스턴 블랏법으로 각 단백질의 발현 정도를 조사하였다. NT, 미처리; Cont, 대조군; DMSO, 디메틸설폭사이드(dimethylsulfoxide); NAC, N-아세틸시스테인(N-acetylcysteine); Rap, 라파마이신(rapamycin).
도 5는 사람 섬유아세포에서 활성산소 생성에 미치는 SC-9 [에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)]의 효과를 나타낸다. A, 활성산소 유세포분석. B, 평균 활성산소량 비교. 섬유아세포에 아드리아마이신을 처리하고, SC-9를 0.1 ug/ml 처리하여, 세포내 활성산소 정도를 유세포분석기로 조사하였다. 각 실험을 독립적으로 3회 이상 반복시행 한 후 평균과 표준편차로 나타냈다. ADR, 아드리아마이신(adriamycin); Y, 젊은 세포(young cells); Cont, 대조군; DMSO, 디메틸설폭사이드(dimethylsulfoxide); NAC, N-아세틸시스테인(N-acetylcysteine); Rap, 라파마이신(rapamycin).
도 6은 사람 섬유아세포에서 SC-9 [에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)]의 복제세포노화 저해 효과를 나타낸다. A 및 B, SA-β-gal 활성 염색 사진 및 SA-β-gal 활성 염색 백분율을 나타낸다. C, SC-9의 세포독성 효과를 나타낸다. 복제노화 세포에 SC-9를 0.1 ug/ml 처리하고, 3일 후 SA-β-gal 활성 염색을 시행하였다. 세포독성은 MTT법으로 조사하였다. 결과는 각 실험을 독립적으로 3회 이상 반복시행한 후 평균과 표준편차로 나타냈다. Old, 늙은 세포(Old cells); DMSO, 디메틸설폭사이드(dimethylsulfoxide); NAC, N-아세틸시스테인(N-acetylcysteine); Rap, 라파마이신(rapamycin).Fig. 1 shows a schematic diagram of separation of Saururus chinensis extract.
Figure 2 shows the chemical structure of a single component separated from Saururus chinensis.
FIG. 3 shows the cytotoxicity of a single component of Saururus chinensis in human fibroblasts and the effect of inhibiting cell senescence by adriamycin. A, Cytotoxic effect of single component of Saururus chinensis. Each component was treated with 10 ug / ml, cultured for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After adriamycin treatment, SC-7, SC-8, SC-10 and SC-14 were treated with 10 ug / ml and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p < 0.05 vs. DMSO.
Fig. 4 shows the cytotoxicity of SC-9 (erthro-austrobailignan-6) in human fibroblasts and the cytotoxic effect of adriamycin on cell senescence. A and SC-9. SC-9 was treated with 0.1 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B and C, SA-β-gal active staining photograph. After treatment with adriamycin, SC-9 was treated with 0.1 μg / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. D, p53, phosphorylated S6K, p21. After treatment with adriamycin, SC-9 was treated with 0.1 ug / ml, and the degree of expression of each protein was examined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.
Figure 5 shows the effect of SC-9 [erythro-austrobailignan-6] on the production of reactive oxygen species in human fibroblasts. A, active oxygen flow cytometry. B, the average amount of active oxygen. The fibroblasts were treated with adriamycin, treated with SC-9 at 0.1 ug / ml, and the degree of active oxygen in the cells was examined by flow cytometry. Each experiment was repeated three times or more independently and then expressed as mean and standard deviation. ADR, adriamycin; Y, young cells; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.
Fig. 6 shows the effect of SC-9 (erthro-austrobailignan-6) on human cell fibroblasts against clonal cell senescence. A and B, SA-beta-gal active staining and SA-beta-gal active staining percentage. C and SC-9. SC-9 was treated with 0.1 μg / ml of replicating senescent cells, and SA-β-gal active staining was performed after 3 days. Cytotoxicity was assessed by MTT assay. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.

이하, 하기 실시예를 통해 본 발명을 보다 상세하게 설명한다. 다만, 이러한 실시예에 의해 본 발명이 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< < 실시예Example 1 > 삼백초 추출물 분리 및 단일성분의 화학구조 1> Separation of Saururus chinensis extract and chemical composition of single component

1. 삼백초 추출물 및 단일성분 분리1. Saururus chinensis extract and single component separation

건조한 삼백초 지상부 10.0 kg을 70%, 90%, 100% 에탄올(13L×3회)로 약 60℃에서 12 시간 환류냉각하면서 추출하여 총 추출액을 감압 농축하여 에탄올 추출물 1.2 kg을 얻었다. 이 에탄올 추출물에 증류수 (1.5 L) 및 헥산(hexane) (1.5 L)을 가하여 분획 깔때기로 증류수 층 및 헥산(hexane) 층으로 분획하는 조작을 3회 실시하고 각 분획을 감압 농축하여 증류수 추출물 및 헥산 추출물을 얻었으며, 다시 수층을 상기와 같은 방법으로 EtOAc, BuOH순으로 추출하여 헥산 추출물 51.3 g, 에틸아세테이트(EtOAc) 추출물 248.4 g, 부탄올(BuOH) 추출물 189.4 g 및 H₂O 추출물 346.7 g을 얻었다. 이들 분획 중 EtOAc 추출물 100 g을 normal phase column chromatography를 실시하였다. column (100×12 cm)에 silica gel (No.9385, 230-400 mesh, Merck)을 약 30 cm 정도 채우고 헥산(hexane) 3 L로 용출(elution)시켜 고정상(stationary phase)을 균일한 상태로 만든 후 시료 100 g을 silica gel (No.7734, 70-230 mesh, Merck) 200 g에 흡착시켜 컬럼에 로딩(loading)시켰다. 이 후 hexane-EtOAc (gradient from hexane 100 % to EtOAc 100 %), EtOAc-MeOH (gradient from EtOAc 100 % to MeOH 100 %)로 용출(elution)시켜 26개의 분획 (SCFE1～26)을 얻었다. 상기 분획중 SCFE7(2.5 g)을 100% n-hexane으로 재결정하여 물질 SC-1(1933 mg)을 얻었다. SCFE21(500 mg)을 sephades LH-20 column (4×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 물질 SC-2 (150 mg)을 얻었다. SCFE14 (3 g)을 sephades LH-20 column (4×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 얻은 분획 SCFE14-1 (479 mg)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=40:60에서 MeOH 100 %로 흘려 물질 SC-3 (120 mg)을 얻었다. SCFE17 (500 mg)을 sephades LH-20 column (4×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 얻은 분획 SCFE17-1 (174 mg)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=50:50에서 MeOH 100 %로 흘려 물질 SC-4 (19 mg)와 물질 SC-5 (76 mg)을 얻었다. SCFE19 (500 mg)을 sephades LH-20 column (4×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 얻은 분획 SCFE19-1 (213 mg)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=60:40에서 MeOH 100 %로 흘려 물질 SC-6 (120 mg)을 얻었다. SCFE20 (500 mg)을 sephades LH-20 column (4×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 얻은 분획 SCFE20-5 (20 mg)을 reverse-phase column (3×50 cm, LiChroprep RP-18)을 MeOH:H₂O=50:50에서 MeOH 100 %로 흘려 물질 SC-7 (120 mg)을 얻었다.10.0 kg of the dried Saururus chinensis was extracted with 70%, 90% and 100% ethanol (13L × 3 times) at 60 ° C for 12 hours while refluxing and cooling. The total extract was concentrated under reduced pressure to obtain 1.2 kg of ethanol extract. Distilled water (1.5 L) and hexane (1.5 L) were added to the ethanol extract, and the mixture was divided into a distilled water layer and a hexane layer with a fractionation funnel. The fractions were concentrated under reduced pressure to obtain distilled water extract and hexane And the aqueous layer was further extracted with EtOAc and BuOH in the same manner as described above to obtain 51.3 g of hexane extract, 248.4 g of ethyl acetate (EtOAc) extract, 189.4 g of butanol (BuOH) extract and 346.7 g of H ₂ O extract . 100 g of EtOAc extract in these fractions were subjected to normal phase column chromatography. The silica gel (No. 9385, 230-400 mesh, Merck) was filled to about 30 cm in a column (100 × 12 cm) and eluted with 3 L of hexane to obtain a stationary phase in a uniform state After making the sample, 100 g of the sample was adsorbed on 200 g of silica gel (No. 7734, 70-230 mesh, Merck) and loaded on the column. Then 26 fractions (SCFE 1-26) were obtained by eluting with hexane-EtOAc (gradient from hexane 100% to EtOAc 100%) and EtOAc-MeOH (gradient from EtOAc 100% to MeOH 100%). SCFE7 (2.5 g) in the above fraction was recrystallized from 100% n- hexane to obtain the product SC-1 (1933 mg). SCFE21 (500 mg) was poured into 100% MeOH using a sephadex LH-20 column (4 x 90 cm, Sephadex LH-20) to obtain substance SC-2 (150 mg). Fraction SCFE14-1 (479 mg) obtained by flowing SCFE14 (3 g) to 100% MeOH using a sephadex LH-20 column (4 x 90 cm, Sephadex LH-20) LiChroprep RP-18) was run from MeOH: H ₂ O = 40: 60 to 100% MeOH to obtain material SC-3 (120 mg). A fraction SCFE17-1 (174 mg) obtained by flowing SCFE17 (500 mg) to 100% MeOH using a sephadex LH-20 column (4 × 90 cm, Sephadex LH-20) (19 mg) and SC-5 (76 mg) were obtained by pouring 100% of MeOH into a mixture of MeOH: H ₂ O = 50:50 in MeOH: LiChroprep RP-18. The fraction SCFE19-1 (213 mg) obtained by flowing SCFE19 (500 mg) to 100% MeOH using a sephadex LH-20 column (4 x 90 cm, Sephadex LH-20) LiChroprep RP-18) was run from MeOH: H ₂ O = 60: 40 to 100% MeOH to obtain material SC-6 (120 mg). The fraction SCFE20-5 (20 mg) obtained by flowing SCFE20 (500 mg) to 100% MeOH using a sephadex LH-20 column (4 x 90 cm, Sephadex LH-20) LiChroprep RP-18) was poured from MeOH: H ₂ O = 50: 50 to 100% MeOH to obtain material SC-7 (120 mg).

건조한 삼백초 뿌리 9.7 kg을 70 % MeOH (13L×3회)로 약 60℃에서 24 시간 환류냉각하면서 추출하여 총 추출액을 감압 농축하여 메탄올 추출물(MeOH extract) 1 kg을 얻었다. 이 메탄올 추출물에 증류수 (1.4 L) 및 헥산(hexane) (1.4 L)을 가하여 분획 깔때기로 증류수 층 및 헥산(hexane) 층으로 분획하는 조작을 3회 실시하고 각 분획을 감압 농축하여 증류수 추출물 및 헥산 추출물을 얻었으며, 다시 수층을 상기와 같은 방법으로 EtOAc, BuOH순으로 추출하여 헥산 추출물 136.75 g, EtOAc 추출물 182.9 g, BuOH 추출물 42.8 g 및 H₂O 추출물 623.3 g을 얻었다. 이들 분획 중 EtOAc 추출물 130 g을 normal phase column chromatography를 실시하였다. column (120×600 mm)에 silica gel (No.9385, 230-400 mesh, Merck)을 약 30 cm 정도 채우고 헥산(hexane) 3 L로 용출(elution)시켜 stationary phase를 균일한 상태로 만든 후 시료 130 g을 silica gel (No.7734, 70-230 mesh, Merck) 250 g에 흡착시켜 컬럼에 로딩(loading)시켰다. 이 후 hexane-EtOAc (gradient from hexane 100 % to EtOAc 100 %), EtOAc-MeOH (gradient from EtOAc 100 % to MeOH 100 %)로 elution시켜 39개의 분획 (SCE1～39)을 얻었다. 상기 분획 중 SCE4, 11, 22, 32 및 34에서 물질 SC-8 (300 mg), SC-9 (430 mg), SC-13 (2 g), SC-20 (18 g) 및 SC-21 (8 g)을 각각 얻었다. 분획 중 SCE15 (3.5 g)와 SCE18 (6.2 g)을 100 % 헥산(hexane)으로 재결정하여 물질 SC-10 (3 g)와 SC-11 (490 mg)를 각각 얻었다. SCE19 (970 mg)를 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=60:40에서 MeOH 100 %로 흘려 얻은 분획 SCE19-10 (160 mg)을 sephades LH-20 column (3×90 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 물질 SC-18 (90 mg)을 얻었다. SCE20 (1.3 g)과 SCE26 (1.0 g)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=40:60에서 MeOH 100 %로 흘려 물질 SC-12 (70 mg)과 SC-16 (40 mg)을 각각 얻었다. SCE25 (1.3 g)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=50:50에서 MeOH 100 %로 흘려 물질 SC-15 (230 mg)과 SC-17 (70 mg)을 각각 얻었다. SCE27 (700 mg)을 reverse-phase column (4×50 cm, LiChroprep RP-18)을 MeOH:H₂O=50:50에서 MeOH 100 %로 흘려 물질 SC-14 (200 mg)을 얻었다. 또한 SCE29 (500 mg)을 sephadex LH-20 column (4.5×80 cm, Sephadex LH-20)을 이용하여 MeOH 100 %로 흘려 물질 SC-19 (400 mg)를 얻었다 (도 1). 분리된 23종의 단일 물질들은 MS, NMR (1H, 13C, DEPT, 1H-1H COSY, HMQC, HMBC) 등 분광학적 분석방법을 이용하여 화합물의 화학구조를 결정하고 문헌과 비교하여 동정하였다. 각 분획들은 디메틸 설폭사이드(Dimethyl sulfoxide)에 녹여 세포에 처리하였다.
9.7 kg of dried Saururus chinensis root was extracted with 70% MeOH (13 L × 3 times) at about 60 ° C under refluxing for 24 hours, and the total extract was concentrated under reduced pressure to obtain 1 kg of methanol extract (MeOH extract). Distilled water (1.4 L) and hexane (1.4 L) were added to the methanol extract, and the mixture was divided into a distilled water layer and a hexane layer with a fractionation funnel. The fraction was concentrated under reduced pressure to give a distilled water extract and hexane And the aqueous layer was further extracted with EtOAc and BuOH in the same manner as described above to obtain 136.75 g of hexane extract, 182.9 g of EtOAc extract, 42.8 g of BuOH extract and 623.3 g of H ₂ O extract. 130 g of EtOAc extract from these fractions were subjected to normal phase column chromatography. The silica gel (No. 9385, 230-400 mesh, Merck) was filled in a column (120 × 600 mm) with about 30 cm length and eluted with 3 L of hexane to make the stationary phase uniform. 130 g was adsorbed onto 250 g of silica gel (No. 7734, 70-230 mesh, Merck) and loaded onto the column. After that, 39 fractions (SCE1-39) were obtained by eluting with hexane-EtOAc (gradient from hexane 100% to EtOAc 100%) and EtOAc-MeOH (gradient from EtOAc 100% to MeOH 100%). SC-8 (300 mg), SC-9 (430 mg), SC-13 (2 g), SC-20 (18 g) and SC-21 8 g). SCE15 (3.5 g) and SCE18 (6.2 g) were recrystallized from hexane to obtain SC-10 (3 g) and SC-11 (490 mg), respectively. Fraction SCE19-10 (160 mg) obtained by flowing SCE19 (970 mg) through a reverse-phase column (4 × 50 cm, LiChroprep RP-18) in MeOH: H ₂ O = 60:40 to 100% SC-18 (90 mg) was obtained by pouring 100% of MeOH using a 20 column (3 x 90 cm, Sephadex LH-20). SC-20 (1.3 g) and SCE26 (1.0 g) were poured into a reverse-phase column (4 x 50 cm, LiChroprep RP-18) in MeOH: H ₂ O = ) And SC-16 (40 mg), respectively. SC-25 (1.3 g) was poured into a reverse-phase column (4 × 50 cm, LiChroprep RP-18) in MeOH: H ₂ O = 50:50 to 100% 70 mg), respectively. SCE27 (700 mg) was poured into a reverse-phase column (4 x 50 cm, LiChroprep RP-18) in MeOH: H ₂ O = 50:50 to 100% MeOH to obtain material SC-14 (200 mg). In addition, SCE29 (500 mg) was poured into MeOH 100% using a sephadex LH-20 column (4.5 x 80 cm, Sephadex LH-20) to obtain 400 mg of substance SC-19 (Fig. 1). The chemical structures of the 23 isolated compounds were determined using MS and NMR (1H, 13C, DEPT, 1H-1H COZY, HMQC and HMBC) Each fraction was dissolved in dimethyl sulfoxide and treated with cells.

2. 분리된 물질의 물리화학적 특성2. Physico-chemical properties of separated materials

삼백초의 EtOAc 분획에서 분리한 23 종의 화합물은 기지물질로서 MS, NMR (¹H, ¹³C, DEPT, ¹H-¹H COSY, HMQC, HMBC) 등 각종 분광학적 분석방법으로 분석하고 문헌과 비교하여 SC-1 [사우치논(sauchinone)], SC-2 [퀘르시트린(quercitrin)], SC-3 [오도라티솔 C(odoratisol C)], SC-4 [사우세르네올(saucerneol)], SC-5 [마나산틴 B(manassantin B)], SC-6 [마나산틴 A(manassantin A)], SC-7 [아프젤린(afzelin)], SC-8 [아리산(arisan)], SC-9 [에리스로-오스트로바이리그난-6(erthro-austrobailignan-6)], SC-10 [사우치논(sauchinone)], SC-11 [사우치논 B(sauchinone B)], SC-12 [사우세르네올 G(saucerneol G)], SC-13 [메소-디하이드로구아이아레틱산(meso-dihydroguaiaretic acid)], SC-14 [마칠린 D(machilin D)], SC-15 [사우세르네올 F(saucerneol F)], SC- 16 [사우세르네올 H(saucerneol H)], SC-17 [넥탄드린 B(nectandrin B)], SC-18 [사우세르네올 I(saucerneol I)], SC-19 [사우세르네올 D(saucerneol D)], SC-20 [마나산틴 B(manassantin B)], SC-21 [마나산틴 A(manassantin A)]로 확인하였다 (도 2).
The 23 compounds isolated from the Saururus chinensis EtOAc fraction were analyzed by various spectroscopic methods such as MS and NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC and HMBC) SC-1 [sauchinone], SC-2 [quercitrin], SC-3 [odoratisol C], SC-4 [saucerneol] SC-5 [manassantin B], SC-6 [manassantin A], SC-7 [afzelin], SC-8 [arisan] SC-10 [sauchinone], SC-11 [sauchinone B], SC-12 [erythro-austrobailignan-6] SC-13 [meso-dihydroguaiaretic acid], SC-14 [machilin D], SC-15 [saucerneol F SC-16 [saucerneol H], SC-17 [nectandrin B], SC-18 [saucerneol I], SC-19 [saucerneol F] Saucerne D (saucerneol D)], were identified as SC-20 [mana xanthine B (manassantin B)], SC-21 [mana xanthine A (manassantin A)] (Fig. 2).

SCSC -1 (-One ( sauchinonesauchinone ))

Colorless powder; C₂₀H₂₀O₆; ¹H-NMR (CDCl₃, 250 MHz) δ 6.82 (1H, s, H-6), 6.38 (1H, s, H-3), 5.91 (1H, d, J = 1.3 Hz, Ar-OCH₂O-), 5.87 (1H, d, J = 1.3 Hz, Ar-OCH₂O-), 5.66 (1H, s, Al-OCH₂O-), 5.60 (1H, s, Al-OCH₂O-), 5.50 (1H, s, H-3´), 3.04 (1H, d, J = 4.2 Hz, H-7), 2.58 ~ 2.39 (3H, m, H-1´, 6´, 8), 1.93 (1H, m, H-7a´), 1.88 (1H, m, H-8´), 1.62 (1H, m, H-7b´), 1.21 (3H, d, J = 7.3 Hz, H-9), 0.73 (3H, d, J = 7.3 Hz, H-9´); ¹³C-NMR (CDCl₃, 62.9 MHz) δ 199.6 (C-2´), 168.5 (C-4´), 146.6 (C-5), 144.9 (C-2), 143.1 (C-4), 115.6 (C-1), 106.4 (C-6), 101.2 (C-3´), 100.3 (Ar-OCH₂O-), 100.1 (C-3´), 99.1 (C-3), 98.5 (Al-OCH₂O-), 37.5 (C-6´), 37.4 (C-1´), 34.9 (C-7), 34.7 (C-8), 33.3 (C-8´), 25.1 (C-7´), 21.2 (C-9), 20.8 (C-9´); Positive FABMS m/z 356 [M]⁺.Colorless powder; C _₂₀ H ₂₀ O _6; ^{_{1 H-NMR (CDCl 3,}} 250 MHz) δ 6.82 (1H, s, H-6), 6.38 (1H, s, H-3), 5.91 (1H, d, J = 1.3 Hz, Ar-OCH 2 O -), 5.87 (1H, d , J = 1.3 Hz, Ar-OCH 2 O-), 5.66 (1H, s, Al-OCH 2 O-), 5.60 (1H, s, Al-OCH 2 O-), 5.50 (1H, s, H- 3'), 3.04 (1H, d, J = 4.2 Hz, H-7), 2.58 ~ 2.39 (3H, m, H-1', 6', 8), 1.93 (1H , m, H-7a'), 1.88 (1H, m, H-8'), 1.62 (1H, m, H-7b'), 1.21 (3H, d, J = 7.3 Hz, H-9), 0.73 (3H, d, J = 7.3 Hz, H-9 '); ^{_{13 C-NMR (CDCl 3,}} 62.9 MHz) δ 199.6 (C-2'), 168.5 (C-4'), 146.6 (C-5), 144.9 (C-2), 143.1 (C-4), 115.6 (Al-OCH ₂ O-), 100.1 (C-3), 99.1 (C-3), 98.5 OCH ₂ O-), 37.5 (C-6 '), 37.4 (C-1'), 34.9 (C-7), 34.7 ), 21.2 (C-9), 20.8 (C-9 '); Positive FABMS m / z 356 [M] ^< ⁺ & gt ^; .

SCSC -2 (-2 ( quercitrinquercitrin ))

Green powder; C₂₁H₂₀O₁₁; ¹H-NMR (CD₃OD, 250 MHz) δ 7.32 (1H, d, J = 1.7 Hz, H-2´), 7.30 (1H, dd, J = 2, 8.5 Hz, H-6´), 6.90 (1H, d, J = 8.1 Hz, H-5´), 6.35 (1H, d, J = 2 Hz, H-8), 6.18 (1H, d, J = 1.9 Hz, H-6), 5.33 (1H, d, J = 1.4 Hz, H-1´´), 4.21 (1H, dd, J = 1.6, 3.1 Hz, H-2´´), 3.74 (1H, dd, J = 3.3, 9.0 Hz, H-3´´), 3.46 ~ 3.38 (2H, m, H-4´´, 5´´), 0.93 (3H, d, J = 5.8 Hz, H-6´´); ¹³C-NMR (CD₃OD, 62.9 MHz) δ 179.6 (C-4), 165.9 (C-7), 163.2 (C-5), 159.3 (C-9), 158.5 (C-2), 149.8 (C-3´), 146.4 (C-4´), 136.2 (C-3), 122.9 (C-1´), 122.8 (C-6´), 116.9 (C-2´), 116.3 (C-5´), 105.8 (C-10), 103.5 (C-1´´), 99.8 (C-6), 94.7 (C-8), 73.2 (C-4´´), 72.1 (C-3´´), 72.0 (C-5´´), 71.9 (C-2´´), 17.6 (C-6´´); Positive FABMS m/z 449 [M]⁺.Green powder; C ₂₁ H ₂₀ O ₁₁ ; ^{_{1 H-NMR (CD 3 OD}} , 250 MHz) δ 7.32 (1H, d, J = 1.7 Hz, H-2'), 7.30 (1H, dd, J = 2, 8.5 Hz, H-6'), 6.90 (1H, d, J = 8.1 Hz, H-5'), 6.35 (1H, d, J = 2 Hz, H-8), 6.18 (1H, d, J = 1.9 Hz, H-6), 5.33 ( 1H, d, J = 1.4 Hz , H-1''), 4.21 (1H, dd, J = 1.6, 3.1 Hz, H-2''), 3.74 (1H, dd, J = 3.3, 9.0 Hz, H 3 ''), 3.46-3.38 (2H, m, H-4 '', 5 ''), 0.93 (3H, d, J = 5.8 Hz, H-6 ''); ¹³ C-NMR (CD ₃ OD, 62.9 MHz) ? 179.6 (C-4), 165.9 (C-7), 163.2 (C-5), 159.3 C-3 '), 146.4 (C-4'), 136.2 (C-3), 122.9 ), 105.8 (C-10), 103.5 (C-1 "), 99.8 (C-6), 94.7 , 72.0 (C-5 "), 71.9 (C-2 "), 17.6 (C-6 "); Positive FABMS m / z 449 [M] ^< ⁺ & gt ^; .

SCSC -3 (-3 ( odoratisolodoratisol C) C)

Brown gum; C₂₀H₂₄O₅; ¹H-NMR (CDCl₃, 250 MHz) δ 7.02 ~ 6.75 (6H, m, aromatic protons), 5.09 (1H, d, J = 8.7 Hz, H-7´), 4.38 (1H, d, J = 9.3 Hz, H-7), 3.88 (3H, s, 3-OCH3), 3.83 (3H, s, 3´-OCH3), 2.22 (1H, dq, J = 8.7, 7 Hz, H-8´), 1.76 (1H, dq, J = 9.3, 6.5 Hz, H-8), 1.03 (3H, d, J = 6.5 Hz, H-9), 0.64 (3H, d, J = 7 Hz, H-9´); ¹³C-NMR (CDCl₃, 62.9 MHz) δ 146.5 (C-3), 146.1 (C-3´), 145.1 (C-4), 144.5 (C-4´), 133.2 (C-1), 132.7 (C-1´), 119.8 (C-6´), 119.2 (C-6), 114.2 (C-5), 113.8 (C-5´), 109.6 (C-2´), 109.4 (C-2), 87.3 (C-7), 83.1 (C-7´), 55.8 (C-MeO-3´), 55.8 (C-MeO-3), 47.6 (C-8), 45.9 (C-8´), 14.9 (C-9´, 9); Positive FABMS m/z 345 [M]⁺.Brown gum; C ₂₀ H ₂₄ O ₅ ; ^{_{1 H-NMR (CDCl 3,}} 250 MHz) δ 7.02 ~ 6.75 (6H, m, aromatic protons), 5.09 (1H, d, J = 8.7 Hz, H-7'), 4.38 (1H, d, J = 9.3 (1H, d, J = 8.7 Hz, H-7), 3.88 (3H, s, 3-OCH3), 3.83 (1H, d, J = 9.3, 6.5 Hz, H-8), 1.03 (3H, d, J = 6.5 Hz, H-9), 0.64 (3H, d, J = 7 Hz, H-9 '); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) ? 146.5 (C-3), 146.1 (C-3 '), 145.1 (C-1 '), 119.8 (C-6'), 119.2 (C-6), 114.2 ), 87.3 (C-7), 83.1 (C-7 '), 55.8 (C-MeO-3'), 55.8 , 14.9 (C-9 ',9); Positive FABMS m / z 345 [M] ^< ⁺ & gt ^; .

SCSC -4 (-4 ( saucerneolsaucerneol ))

Yellow powder; C₃₁H₃₈O₈; ¹H-NMR (CDCl₃, 250 MHz) δ 6.98 ~ 6.73 (9H, m, aromatic protons), 5.42 (2H, d, J = 4 Hz, H-7, 7´), 4.62 (1H, d, J = 8.4 Hz,H-7´´), 4.09 (1H, m, H-8´´), 3.90 (3H, s, -OCH₃), 3.87 (3H, s, -OCH₃), 3.86 (3H, s, -OCH₃), 3.85 (3H, s, -OCH₃), 2.25 (2H, m, H-8, 8´), 1.14 (3H, d, J = 6.2, H-9´´), 0.68 (6H, d, J = 6.2, H-9, 9´); ¹³C-NMR (CDCl₃, 62.9 MHz) δ 150.5 (C-3´), 148.9 (C-3´´), 148.7 (C-4´´), 146.3 (C-4´), 146.2 (C-3), 144.4 (C-4), 136.6 (C-1´), 133.2 (C-1), 132.4 (C-1´´), 120.0 (C-6´´), 119.0 (C-6), 118.7 (C-6´), 118.6 (C-5´), 113.9 (C-5), 110.7 (C-5´´), 110.0 (C-2´), 109.8 (C-2´´), 108.7 (C-2), 84.1 (C-8´´), 83.5 (C-7), 83.3 (C-7´), 78.4 (C-7´´), 55.8 (-OCH₃×4), 44.0 (C-8, 8´), 17.0 (C-9´´), 14.8 (C-9, 9´); Positive FABMS m/z 538 [M]⁺.Yellow powder; C ₃₁ H ₃₈ O ₈ ; ^{_{1 H-NMR (CDCl 3,}} 250 MHz) δ 6.98 ~ 6.73 (9H, m, aromatic protons), 5.42 (2H, d, J = 4 Hz, H-7, 7'), 4.62 (1H, d, J = 8.4 Hz, H-7'') , 4.09 (1H, m, H-8''), 3.90 (3H, s, -OCH 3), 3.87 (3H, s, -OCH 3), 3.86 (3H, _{s, -OCH 3), 3.85 (} 3H, s, -OCH 3), 2.25 (2H, m, H-8, 8'), 1.14 (3H, d, J = 6.2, H-9''), 0.68 (6H, d, J = 6.2, H-9, 9 '); ^{_{13 C-NMR (CDCl 3,}} 62.9 MHz) δ 150.5 (C-3'), 148.9 (C-3''), 148.7 (C-4''), 146.3 (C-4'), 146.2 (C- 3), 144.4 (C-4), 136.6 (C-1), 133.2 (C-1), 132.4 118.7 (C-6 '), 118.6 (C-5'), 113.9 (C-5), 110.7 (C-2), 84.1 ( C-8''), 83.5 (C-7), 83.3 (C-7'), 78.4 (C-7''), 55.8 (-OCH 3 × 4), 44.0 ( C-8, 8 '), 17.0 (C-9''), 14.8 (C-9, 9'); Positive FABMS m / z 538 [M] ^< ⁺ & gt ^; .

SCSC -5 (-5 ( manassantinmanassantin B) B)

Colorless powder; C₄₁H₄₈O₁₁; m.p 83-86 ℃; [a]²⁵ _D -99.8° (c 0.5, CHCl₃); ¹H-NMR (CDCl₃, 250 MHz) δ 6.99 ~ 6.73 (12H, m, aromatic protons), 5.92 (2H, s, -OCH₂O-), 5.45 (2H, d, J = 5.8 Hz, H-7, 7´), 4.64 (1H, d, J = 8.2 Hz, H-7´´), 4.61 (1H, d, J = 8.2 Hz, H-7´´´), 4.10 (2H, m, H-8´´, 8´´´), 3.90 (3H, s, OCH₃), 3.89 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 3.85 (3H, s, OCH₃), 2.27 (2H, m, H-8, 8´), 1.16 (3H, d, J = 6.2 Hz, H-9´´), 1.14 (3H, d, J = 6.2 Hz, H-9´´´), 0.71 (6H, d, J = 6.0 Hz, H-9, 9´); ¹³C-NMR (CDCl3, 62.9 MHz) δ 150.5 (C-3, 3´), 148.9 (C-3´´), 148.8 (C-3´´´), 147.7 (C-4´´´), 147.3 (C-4´), 146.4 (C-4), 146.3 (C-4´´), 136.5 (C-1), 136.4 (C-1´), 133.9 (C-1´´´), 132.5 (C-1´´), 121.0 (C-6´´´), 119.9 (C-6´´), 118.8 (C-5), 118.6 (C-5´, 6, 6´), 110.7 (C-5´´), 109.9 (C-2, 2´, 2´´), 108.0 (C-5´´´), 107.5 (C-2´´´), 101.0 (-OCH₂O-), 84.1 (C-8´´), 84.0 (C-8´´´), 83.3 (C-7, 7´), 78.3 (C-7´´, 7´´´), 55.8 (OCH₃×4), 44.1 (C-8, 8´), 17.0 (C-9´´), 16.9 (C-9´´´), 14.8 (C-9, 9´); Positive FABMS m/z 716 [M]⁺.Colorless powder; C ₄₁ H ₄₈ O ₁₁ ; mp 83-86 [deg.] C; ^{_{[a] 25 D -99.8 ° (}} c 0.5, CHCl 3); ^{_{1 H-NMR (CDCl 3,}} 250 MHz) δ 6.99 ~ 6.73 (12H, m, aromatic protons), 5.92 (2H, s, -OCH 2 O-), 5.45 (2H, d, J = 5.8 Hz, H- 7, 7'), 4.64 (1H , d, J = 8.2 Hz, H-7''), 4.61 (1H, d, J = 8.2 Hz, H-7'''), 4.10 (2H, m, H -8'', 8'''), 3.90 (3H , s, OCH 3), 3.89 (3H, s, OCH 3), 3.87 (3H, s, OCH 3), 3.85 (3H, s, OCH 3) , 2.27 (2H, m, H -8, 8'), 1.16 (3H, d, J = 6.2 Hz, H-9''), 1.14 (3H, d, J = 6.2 Hz, H-9''' ), 0.71 (6H, d, J = 6.0 Hz, H-9, 9 '); ¹³ C-NMR (CDCl 3, 62.9 MHz) ? 150.5 (C-3, 3 '), 148.9 (C-3 "), 148.8 136.3 (C-1), 133.9 (C-1 "), 132.5 (C-4) (C-1), 121.0 (C-6 "), 119.9 (C-6 "), 118.8 -5''), 109.9 (C-2 , 2', 2''), 108.0 (C-5'''), 107.5 (C-2'''), 101.0 (-OCH 2 O-), 84.1 (C-8''), 84.0 ( C-8'''), 83.3 (C-7, 7'), 78.3 (C-7'', 7'''), 55.8 (OCH 3 × 4), 44.1 (C-8, 8 '), 17.0 (C-9''), 16.9 (C-9''), 14.8 (C-9, 9'); Positive FABMS m / z 716 [M] ^< ⁺ & gt ^; .

SCSC -6 (-6 ( manassantinmanassantin A) A)

Colorless powder; C₄₂H₅₂O₁₁; m.p 82-85 ℃; [a]²⁵ _D -102.1° (c 0.5, CHCl₃); ¹H-NMR (CDCl₃, 250 MHz) δ 6.98 ~ 6.79 (12H, m, aromatic protons), 5.45 (2H, d, J = 5.8 Hz, H-7, 7´), 4.64 (2H, d, J = 8.3 Hz, H-7´´, H-7´´´), 4.05 (2H, m, H-8´´, 8´´´), 3.90 (6H, s, -OCH₃×2), 3.86 (6H, s, -OCH₃×2), 3.85 (6H, s, -OCH₃×2), 2.27 (2H, m, H-8, 8´), 1.15 (6H, d, J = 6.2 Hz, H-9´´, 9´´´), 0.71 (6H, d, J = 6.4 Hz, H-9, 9´); ¹³C-NMR (CDCl₃, 62.9 MHz) δ 150.5 (C-3, 3´), 148.9 (C-3´´, C-3´´´), 148.8 (C-4´´, 4´´´), 146.4 (C-4, 4´), 136.4 (C-1, 1´), 132.5 (C-1´´, 1´´´), 119.9 (C- 6´´, 6´´´), 118.6 (C- 6, 6´, 5, 5´), 110.7 (C-5´´, C-5´´´), 110.0 (C-2, 2´), 109.9 (C-2´´, 2´´´), 84.0 (C-7, 7´), 83.3 (C-8´´, 8´´´), 78.3 (C-7´´, 7´´´), 55.8 (-OCH₃×6), 44.1 (C-8, 8´), 17.0 (C-9´´, 9´´´), 14.8 (C-9, 9´); Positive FABMS m/z 732 [M]⁺.　Colorless powder; C ₄₂ H ₅₂ O ₁₁ ; mp 82-85 [deg.] C; ^{_{[a] 25 D -102.1 ° (}} c 0.5, CHCl 3); ^{_{1 H-NMR (CDCl 3,}} 250 MHz) δ 6.98 ~ 6.79 (12H, m, aromatic protons), 5.45 (2H, d, J = 5.8 Hz, H-7, 7'), 4.64 (2H, d, J = 8.3 Hz, H-7'', H-7'''), 4.05 (2H, m, H-8'', 8'''), 3.90 (6H, s, -OCH 3 × 2), 3.86 _{(6H, s, -OCH 3 ×} 2), 3.85 (6H, s, -OCH 3 × 2), 2.27 (2H, m, H-8, 8'), 1.15 (6H, d, J = 6.2 Hz, H-9 ", 9 "'), 0.71 (6H, d, J = 6.4 Hz, H-9, 9'); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) ? 150.5 (C-3, 3 '), 148.9 (C-3 " ), 146.4 (C-4, 4 '), 136.4 (C-1,1'), 132.5 (C-1 ", 1" C-2 ', 109.9 (C-2 ", 2 '),'''), 84.0 (C-7 , 7'), 83.3 (C-8'', 8'''), 78.3 (C-7'', 7'''), 55.8 (-OCH 3 × 6 ), 44.1 (C-8, 8 '), 17.0 (C-9', 9 ''), 14.8 (C-9, 9 '); Positive FABMS m / z 732 [M] ^< ⁺ & gt ^; .

SCSC -7 (-7 ( afzelinafzelin ))

Yellow powder; C₂₁H₂₀O₁₀; ¹H-NMR (CD₃OD, 250 MHz) δ 7.76 (2H, d, J = 8.7 Hz, H-2´, 6´), 6.92 (2H, d, J = 8.7 Hz, H-3´, 5´), 6.36 (1H, d, J = 1.7, H-8 Hz), 6.18 (1H, d, J = 1.8 Hz, H-6), 5.36 (1H, d, J = 1.3 Hz, H-1´´), 4.21 (1H, dd, J = 1.5, 3.4 Hz, H-3´´), 3.70 (1H, dd, J = 3.4, 8.8 Hz, H-3´´), 3.35~3.25 (2H, m, H-4´´, 5´´), 0.91 (3H, d, J=4.4 Hz, H-6´´); ¹³C-NMR (CD₃OD, 62.9 MHz) δ 179.6 (C-4), 166.2 (C-7), 163.2 (C-5), 161.6 (C-4´), 159.2 (C-2), 158.6 (C-9), 136.2 (C-3), 131.9 (C-2´, 6´), 122.6 (C-1´), 116.5 (C-3´, 5´), 105.8 (C-10), 103.5 (C-1´´), 99.9 (C-6), 94.8 (C-8), 73.1 (C-4´´), 72.0 (C-3´´, 5´´), 71.9 (C-2´´), 17.7 (C-6´´); Positive FABMS m/z 433 [M]⁺.Yellow powder; C ₂₁ H ₂₀ O ₁₀ ; ^{_{1 H-NMR (CD 3 OD}} , 250 MHz) δ 7.76 (2H, d, J = 8.7 Hz, H-2', 6'), 6.92 (2H, d, J = 8.7 Hz, H-3', 5 '), 6.36 (1H, d , J = 1.7, H-8 Hz), 6.18 (1H, d, J = 1.8 Hz, H-6), 5.36 (1H, d, J = 1.3 Hz, H-1' ), 4.21 (1H, dd, J = 1.5, 3.4 Hz, H-3 ''), 3.70 (1H, dd, J = 3.4, 8.8 Hz, H- , H-4 ", 5 "), 0.91 (3H, d, J = 4.4 Hz, H-6 "); ^{_{13 C-NMR (CD 3 OD}} , 62.9 MHz) δ 179.6 (C-4), 166.2 (C-7), 163.2 (C-5), 161.6 (C-4'), 159.2 (C-2), 158.6 (C-9), 136.2 (C-3), 131.9 (C-2 ', 6'), 122.6 (C-6), 94.8 (C-8), 73.1 (C-4 "), 72.0 (C-3 ", 5 "), 71.9 ''), 17.7 (C-6 ''); Positive FABMS m / z 433 [M] ^< ⁺ & gt ^; .

SCSC -8 (-8 ( arisanarisan ))

Colorless oil; C₁₁H₁₂O₃; 1H-NMR (CDCl₃, 250 MHz) δ 6.36 (1H, s, H-3), 6.33 (1H, s, H-6), 5.91 (2H, s, -OCH₂O-), 5.89 (1H, m, H-8), 5.07 (2H, m, H-9), 3.87 (3H, s, OCH₃), 3.26 (2H, d, J = 6.67 Hz, H-7); 13C-NMR (CDCl₃, 62.9 MHz) δ 148.8 (C-4), 143.5 (C-5), 137.3 (C-8), 134.6 (C-2), 133.4 (C-1), 115.8 (C-9), 107.6 (C-3), 102.6 (C-6), 101.2 (-OCH₂O-), 56.5 (OCH₃), 40.2 (C-7); FABMS m/z 192 [M]⁺.Colorless oil; C ₁₁ H ₁₂ O ₃ ; _{1H-NMR (CDCl 3, 250} MHz) δ 6.36 (1H, s, H-3), 6.33 (1H, s, H-6), 5.91 (2H, s, -OCH 2 O-), 5.89 (1H, m, H-8), 5.07 (2H, m, H-9), 3.87 (3H, s, OCH 3), 3.26 (2H, d, J = 6.67 Hz, H-7); 13 C-NMR (CDCl ₃ , 62.9 MHz)? 148.8 (C-4), 143.5 (C-5), 137.3 9), 107.6 (C-3 ), 102.6 (C-6), 101.2 (-OCH 2 O-), 56.5 (OCH 3), 40.2 (C-7); FABMS m / z 192 [M] ^< ⁺ & gt ^; .

SCSC -9 (-9 ( erythroerythro -- austrobailignanaustrobailignan -6)-6)

Yellow oil; C₂₀H₂₄O₄; mp 68.5-69.0 ℃; [α]²⁵ _D +4.8° (c 1, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.81 (1H, d, J = 7.8 Hz, H-5), 6.70 (1H, d, J = 7.8 Hz, H-5´), 6.60 ~ 6.51 (4H, m, H-6´, 6, 2, 2´), 5.90 (2H, s, -OCH₂O-), 5.47 (1H, s, Ar-OH), 3.82 (3H, s, OCH₃), 2.50 (2H, dd, J = 6.6, 13.5 Hz, H-7a, 7a´), 2.34 (2H, dd, J = 6.6, 13.5 Hz, H-7b, 7b´), 1.70 (2H, m, H-8, 8´), 0.81 (6H, d, J = 6.1 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 147.3 (C-3´), 146.2 (C-3), 145.3 (C-4´), 135.5 (C-1´), 133.4 (C-1), 121.7 (C-6´), 121.61 (C-6), 113.9 (C-5), 111.2 (C-2), 109.3 (C-2´), 107.8 (C-5´), 100.6 (OCH₂O), 55.8 (C-OCH₃), 41.1 (C-7), 41.0 (C-7´), 37.8 (C-8), 37.7 (C-8´), 13.8 (C-9), 13.7 (C-9´); FABMS m/z 328 [M]⁺.Yellow oil; C ₂₀ H ₂₄ O ₄ ; mp 68.5-69.0 [deg.] C; [?] ²⁵ _D + 4.8 (c 1, CHCl ₃ ); _{1H-NMR (CDCl 3, 250} MHz) δ 6.81 (1H, d, J = 7.8 Hz, H-5), 6.70 (1H, d, J = 7.8 Hz, H-5'), 6.60 ~ 6.51 (4H, m, H-6', 6, 2, 2'), 5.90 (2H, s, -OCH 2 O-), 5.47 (1H, s, Ar-OH), 3.82 (3H, s, OCH 3), 2.50 (2H, d, J = 6.6,13.5 Hz, H-7a, 7a '), 2.34 (2H, dd, J = 6.6, 13.5 Hz, , 8 '), 0.81 (6H, d, J = 6.1 Hz, H-9, 9'); 13 C-NMR (CDCl ₃ , 62.9 MHz)? 147.3 (C-3 '), 146.2 (C-3), 145.3 (C-6'), 121.61 ( C-6), 113.9 (C-5), 111.2 (C-2), 109.3 (C-2'), 107.8 (C-5'), 100.6 (OCH 2 O) _{, 55.8 (C-OCH 3)} , 41.1 (C-7), 41.0 (C-7'), 37.8 (C-8), 37.7 (C-8'), 13.8 (C-9), 13.7 (C- 9 '); FABMS m / z 328 [M] ^< ⁺ & gt ^; .

SCSC -10 (-10 ( sauchinonesauchinone ))

Colorless powder; C₂₀H₂₀O₆; mp 224-226 ℃; [α]²⁶ _D -140° (c 1, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.82 (1H, s, H-6), 6.38 (1H, s, H-3), 5.91 (1H, d, J = 1.3 Hz, Ar-OCH₂O-), 5.87 (1H, d, J = 1.3 Hz, Ar-OCH₂O-), 5.66 (1H, s, Al-OCH₂O-), 5.60 (1H, s, Al-OCH₂O-), 5.50 (1H, s, H-3´), 3.04 (1H, d, J = 4.2 Hz, H-7), 2.58 ~ 2.39 (3H, m, H-1´, 6´, 8), 1.93 (1H, m, H-7a´), 1.88 (1H, m, H-8´), 1.62 (1H, m, H-7b´), 1.21 (3H, d, J = 7.3 Hz, H-9), 0.73 (3H, d, J = 7.3 Hz, H-9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 199.6 (C-2´), 168.5 (C-4´), 146.6 (C-5), 144.9 (C-2), 143.1 (C-4), 115.6 (C-1), 106.4 (C-6), 101.2 (C-3´), 100.3 (Ar-OCH₂O-), 100.1 (C-3´), 99.1 (C-3), 98.5 (Al-OCH₂O-), 37.5 (C-6´), 37.4 (C-1´), 34.9 (C-7), 34.7 (C-8), 33.3 (C-8´), 25.1 (C-7´), 21.2 (C-9), 20.8 (C-9´); FABMS m/z 356 [M]⁺.Colorless powder; C _₂₀ H ₂₀ O _6; mp 224-226 [deg.] C; [?] ²⁶ _D -140 (c 1, CHCl ₃ ); _{1H-NMR (CDCl 3, 250} MHz) δ 6.82 (1H, s, H-6), 6.38 (1H, s, H-3), 5.91 (1H, d, J = 1.3 Hz, Ar-OCH 2 O- ), 5.87 (1H, d, J = 1.3 Hz, Ar-OCH 2 O-), 5.66 (1H, s, Al-OCH 2 O-), 5.60 (1H, s, Al-OCH 2 O-), 5.50 (1H, s, H-3 '), 3.04 (1H, d, J = 4.2 Hz, H-7), 2.58-2.39 (3H, m, H- m, H-7a'), 1.88 (1H, m, H-8'), 1.62 (1H, m, H-7b'), 1.21 (3H, d, J = 7.3 Hz, H-9), 0.73 ( 3H, d, J = 7.3Hz, H-9 '); (CDCl ₃ , 62.9 MHz)? 199.6 (C-2 '), 168.5 (C-4'), 146.6 (C-5), 144.9 C-1), 106.4 (C -6), 101.2 (C-3'), 100.3 (Ar-OCH 2 O-), 100.1 (C-3'), 99.1 (C-3), 98.5 (Al-OCH _{2 O-), 37.5 (C-} 6'), 37.4 (C-1'), 34.9 (C-7), 34.7 (C-8), 33.3 (C-8'), 25.1 (C-7') , 21.2 (C-9), 20.8 (C-9 '); FABMS m / z 356 [M] ^< ⁺ & gt ^; .

SCSC -11 (-11 ( sauchinonesauchinone B) B)

Colorless powder; C₂₀H₂₀O₆; mp 235-237 ℃; [α]²⁵ _D -100° (c 0.1, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.76 (1H, s, H-6), 6.33 (1H, s, H-3), 5.89 (1H, s, Ar-OCH₂O-), 5.87 (1H, s, Ar-OCH₂O-), 5.60 (1H, s, Al-OCH₂O-), 5.59 (1H, s, Al-OCH₂O-), 5.56 (1H, s, H-3´), 3.15 (1H, dd, J = 8.77, 1.6 Hz, H-7), 2.86 (1H, t, J = 8.88, 1.6 Hz, H-6´), 2.64 (1H, td, J = 12.7, 2.9 Hz, H-1´), 2.24 (1H, dd, J = 7.3, 14.75 Hz, H-8), 2.01 (1H, m, H-7a´), 1.56 (1H, m, H-8´), 1.21 (3H, d, J = 7.5 Hz, H-9), 1.10 (1H, m, H-7b´), 0.69 (3H, d, J = 7.04 Hz, H-9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 199.5 (C-2´), 168.2 (C-4´), 146.4 (C-5), 144.9 (C-2), 143.2 (C-4), 118.7 (C-1), 107.3 (C-6), 101.2 (C-5´), 100.7 (Ar-OCH₂O-), 100.3 (C-3), 99.4 (C-3´), 98.2 (Al-OCH₂O-), 42.1 (C-6´), 39.4 (C-1´), 36.1 (C-7), 35.2 (C-8), 32.7 (C-8´), 30.7 (C-7´), 24.5 (C-9), 23.6 (C-9); FABMS m/z 356 [M]⁺.Colorless powder; C _₂₀ H ₂₀ O _6; mp 235-237 [deg.] C; [?] ²⁵ _D -100 (c 0.1, CHCl ₃ ); _{1H-NMR (CDCl 3, 250} MHz) δ 6.76 (1H, s, H-6), 6.33 (1H, s, H-3), 5.89 (1H, s, Ar-OCH 2 O-), 5.87 (1H _{, s, Ar-OCH 2 O-} ), 5.60 (1H, s, Al-OCH 2 O-), 5.59 (1H, s, Al-OCH 2 O-), 5.56 (1H, s, H-3') , 3.15 (1H, dd, J = 8.77, 1.6 Hz, H-7), 2.86 (1H, t, J = 8.88, 1.6 Hz, H-6'), 2.64 (1H, td, J = 12.7, 2.9 Hz , H-1 '), 2.24 (1H, dd, J = 7.3,14.75Hz, H-8), 2.01 (3H, d, J = 7.5 Hz, H-9), 1.10 (1H, m, H-7b), 0.69 (3H, d, J = 7.04 Hz, H-9 '); (CDCl ₃ , 62.9 MHz)? 199.5 (C-2 '), 168.2 (C-4'), 146.4 (C-5), 144.9 C-1), 107.3 (C -6), 101.2 (C-5'), 100.7 (Ar-OCH 2 O-), 100.3 (C-3), 99.4 (C-3'), 98.2 (Al-OCH _{2 O-), 42.1 (C-} 6'), 39.4 (C-1'), 36.1 (C-7), 35.2 (C-8), 32.7 (C-8'), 30.7 (C-7') , 24.5 (C-9), 23.6 (C-9); FABMS m / z 356 [M] ^< ⁺ & gt ^; .

SCSC -12 (-12 ( saucerneolsaucerneol G) G)

Sticky solid; C₂₀H₂₀O₆; [α]²² _D +13° (c 0.588, CHCl₃); UV (MeOH) λmax (logε) 229 (4.14), 276 (3.69), 306 (3.88) nm; IR (KBr) Vmax 3424, 2954, 2903, 1658, 1504, 1442, 1251, 1173, 1038 cm^-1; 1H-NMR (CDCl₃, 250 MHz) δ 7.59 (1H, dd, J = 1.55, 8.2 Hz, H-6), 7.45 (1H, d, J = 1.52 Hz, H-2), 6.86 (1H, d, J = 8.2 Hz, H-5), 6.49 (1H, s, H-2´), 6.48 (1H, s, H-5´), 6.04 (2H, s, -OCH₂O-), 5.84 (2H, s, -OCH₂O-), 3.17 (1H, m, H-8), 2.60 (1H, d, J = 13.5 Hz, H-7a´), 2.15 (1H, m, H-8´), 2.00 (1H, dd, J = 10.1, 13.5 Hz, H-7b´), 1.21 (3H, d, J = 7.2 Hz, H-9), 0.97 (3H, d, J = 6.4 Hz, H-9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 204.7 (C-7), 152.2 (C-3), 149.9 (C-3´), 148.3 (C-4), 146.7 (C-4´), 140.3 (C-1´), 130.6 (C-1), 125.1 (C-6), 117.4 (C-6´), 110.1 (C-2´), 108.5 (C-2), 108.0 (C-5), 102.0 (-OCH₂O-), 100.8 (-OCH₂O-), 98.6 (C-5´), 46.3 (C-8), 37.7 (C-7´), 35.6 (C-8´), 16.5 (C-9), 16.2 (C-9´); HRFABMS m/z 357.1335 ([M+H]⁺, calcd. for C₂₀H₂₁O₆, 357.1338).Sticky solid; C _₂₀ H ₂₀ O _6; ^{_{[α] 22 D + 13 °}} (c 0.588, CHCl 3); UV (MeOH)? Max (log?) 229 (4.14), 276 (3.69), 306 (3.88) nm; IR (KBr)? Max 3424, 2954, 2903, 1658, 1504, 1442, 1251, 1173, 1038 cm ^-1 ; _{1H-NMR (CDCl 3, 250} MHz) δ 7.59 (1H, dd, J = 1.55, 8.2 Hz, H-6), 7.45 (1H, d, J = 1.52 Hz, H-2), 6.86 (1H, d , J = 8.2 Hz, H- 5), 6.49 (1H, s, H-2'), 6.48 (1H, s, H-5'), 6.04 (2H, s, -OCH 2 O-), 5.84 ( _{2H, s, -OCH 2 O-)} , 3.17 (1H, m, H-8), 2.60 (1H, d, J = 13.5 Hz, H-7a'), 2.15 (1H, m, H-8') , 2.00 (1H, dd, J = 10.1, 13.5 Hz, H-7b'), 1.21 (3H, d, J = 7.2 Hz, H-9), 0.97 (3H, d, J = 6.4 Hz, H-9 '); C-NMR (CDCl ₃ , 62.9 MHz)? 204.7 (C-7), 152.2 (C-3), 149.9 C-1 '), 130.6 (C-1), 125.1 (C-6), 117.4 (C-6'), 110.1 _{102.0 (-OCH 2 O-), 100.8} (-OCH 2 O-), 98.6 (C-5'), 46.3 (C-8), 37.7 (C-7'), 35.6 (C-8'), 16.5 (C-9), 16.2 (C-9 '); HRFABMS m / z 357.1335 ([M + H] +, calcd. For C 20 H 21 O 6, 357.1338).

SCSC -13 (-13 ( mesomeso -- dihydroguaiareticdihydroguaiaretic acidacid ))

Green powder; C₂₀H₂₆O₄; mp 88-89.5 ℃; [α]²⁵ _D 0° (c 1.44, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.77 (2H, d, J = 7.9 Hz, H-5, 5´), 6.58 (2H, d, J = 8.0 Hz, H-6, 6´), 6.50 (2H, d, J = 1.7 Hz, H-2, 2´), 3.78 (6H, s, -OCH₃×2), 2.55 ~ 2.32 (4H, m, H-7, 7´), 1.72 (2H, m, H-8, 8´), 0.80 (6H, d, J = 6.7 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 146.2 (C-3, 3´), 143.4 (C-4, 4´), 133.5 (C-1, 1´), 121.5 (C-6, 6´), 113.8 (C-5, 5´), 111.2 (C-2, 2´), 55.7 (-OCH₃×2), 41.0 (C-7, 7´), 37.4 (C-8, 8´), 13.8 (C-9, 9´); HRFABMS m/z 330.1842 ([M]⁺, calcd. for C₂₀H₂₆O₄, 330.1831).Green powder; C ₂₀ H ₂₆ O _4; mp 88-89.5 DEG C; ^{_{[α] 25 D 0 ° (}} c 1.44, CHCl 3); _{1H-NMR (CDCl 3, 250} MHz) δ 6.77 (2H, d, J = 7.9 Hz, H-5, 5'), 6.58 (2H, d, J = 8.0 Hz, H-6, 6'), 6.50 (2H, d, J = 1.7 Hz, H-2, 2 '), 3.78 (6H, s, -OCH ₃ x 2), 2.55 to 2.32 , m, H-8, 8 '), 0.80 (6H, d, J = 6.7 Hz, H-9, 9'); 13 C-NMR (CDCl ₃ , 62.9 MHz)? 146.2 (C-3, 3 '), 143.4 (C-4, 4'), 133.5 , 113.8 (C-5, 5 '), 111.2 (C-2, 2'), 55.7 (-OCH ₃ x 2), 41.0 13.8 (C-9, 9 '); HRFABMS m / z 330.1842 ([M ] +, calcd. For C 20 H 26 O 4, 330.1831).

SCSC -14 (-14 ( machilinmachilin D) D)

Colorless oil; C₂₀H₂₄O₅; [α]²⁵ _D -160° (c 0.7, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.93 ~ 6.82 (6H, m, aromatic protons), 6.36 (1H, dd, J = 1.1, 15.7 Hz, H-7´), 6.17 (1H, m, H-8´), 5.67 (1H, brs, -OH), 4.61 (1H, d, J = 8.4 Hz, H-7), 4.10 (1H, m, H-8), 3.89 (3H, s, OCH₃), 3.85 (3H, s, OCH₃), 1.87 (3H, dd, J = 1.2, 6.4 Hz, H-9´), 1.15 (3H, d, J = 6.2 Hz, H-9); 13C-NMR (CDCl₃, 62.9 MHz) δ 150.7 (C-3´), 146.7 (C-3), 146.5 (C-4´), 145.4 (C-4), 133.4 (C-1´), 131.8 (C-1), 130.4 (C-7´), 124.9 (C-8´), 120.7 (C-6), 118.9 (C-6´), 118.7 (C-5´), 114.1 (C-5), 109.3 (C-2´), 109.1 (C-2), 84.2 (C-8), 78.4 (C-7), 55.9 (OCH₃), 55.7 (OCH₃), 18.4 (C-9´), 17.0 (C-9); FABMS m/z 344 [M]⁺.Colorless oil; C ₂₀ H ₂₄ O ₅ ; [?] ²⁵ _D -160 (c 0.7, CHCl ₃ ); _{1H-NMR (CDCl 3, 250} MHz) δ 6.93 ~ 6.82 (6H, m, aromatic protons), 6.36 (1H, dd, J = 1.1, 15.7 Hz, H-7'), 6.17 (1H, m, H- 8'), 5.67 (1H, brs , -OH), 4.61 (1H, d, J = 8.4 Hz, H-7), 4.10 (1H, m, H-8), 3.89 (3H, s, OCH 3) , 3.85 (3H, s, OCH 3), 1.87 (3H, dd, J = 1.2, 6.4 Hz, H-9'), 1.15 (3H, d, J = 6.2 Hz, H-9); _{13C-NMR (CDCl 3, 62.9} MHz) δ 150.7 (C-3'), 146.7 (C-3), 146.5 (C-4'), 145.4 (C-4), 133.4 (C-1'), 131.8 (C-1), 130.4 (C-7 '), 124.9 (C-8'), 120.7 (C-6), 118.9 ), 109.3 (C-2') , 109.1 (C-2), 84.2 (C-8), 78.4 (C-7), 55.9 (OCH 3), 55.7 (OCH 3), 18.4 (C-9') , 17.0 (C-9); FABMS m / z 344 [M] ^< ⁺ & gt ^; .

SCSC -15 (-15 ( saucerneolsaucerneol F) F)

Amorphous powder; C₃₀H₃₂O₈; mp 63-65 ℃; [α]²⁵ _D -60.6° (c 0.2, CHCl₃); UV (MeOH) λmax (logε) 234 (4.36), 284 (4.12) nm; IR (KBr) Vmax 3468, 2963, 2891, 1505, 1443, 1249, 1038 cm^-1; 1H-NMR (CDCl₃, 250 MHz) δ 6.74 ~ 6.93 (9H, m, aromatic protons), 5.93 (2H, s, -OCH₂O-), 5.92 (2H, s, -OCH₂O-), 5.40 (2H, d, J = 6.0 Hz, H-7, H-7´), 4.61 (1H, d, J = 8.4 Hz, H-7´´), 4.09 (1H, m, H-8´´), 3.90 (3H, s, -OCH₃), 2.20 ~ 2.30 (2H, m, H-8, 8´), 1.14 (3H, d, J = 6.2 Hz, H-9´´), 0.69 (6H, d, J = 6.3 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 150.9 (C-3´), 148.1 (C-3´´), 147.9 (C-4´´), 147.8 (C-3), 146.8 (C-4), 146.7 (C-4´), 137.1 (C-1´), 135.8 (C-1), 134.4 (C-1´´), 121.5 (C- 6´´), 119.7 (C- 5´), 119.3 (C-6), 119.1 (C-6´), 110.5 (C-2´), 108.5 (C-5´´), 108.2 (C-5), 108.0 (C-2´´), 107.3 (C-2), 101.4 (-OCH₂O-), 101.3 (-OCH₂O-), 84.5 (C-8´´), 84.1 (C-7), 83.9 (C-7´), 78.8 (C-7´´), 56.2 (-OCH₃), 44.3 (C-8), 44.2 (C-8´), 17.4 (C-9´´), 15.1 (C-9, 9´); HRFABMS m/z 543.2000 ([M+Na]⁺, calcd. for C₃₀H₃₂O₈Na, 543.1995).Amorphous powder; C ₃₀ H ₃₂ O ₈ ; mp 63-65 [deg.] C; [?] ²⁵ _D -60.6 (c 0.2, CHCl ₃ ); UV (MeOH)? Max (log?) 234 (4.36), 284 (4.12) nm; IR (KBr)? Max 3468, 2963, 2891, 1505, 1443, 1249, 1038 cm ^-1 ; _{1H-NMR (CDCl 3, 250} MHz) δ 6.74 ~ 6.93 (9H, m, aromatic protons), 5.93 (2H, s, -OCH 2 O-), 5.92 (2H, s, -OCH 2 O-), 5.40 (2H, d, J = 6.0 Hz, H-7, H-7 '), 4.61 (1H, d, J = 8.4 Hz, H- , 3.90 (3H, s, -OCH 3), 2.20 ~ 2.30 (2H, m, H-8, 8'), 1.14 (3H, d, J = 6.2 Hz, H-9''), 0.69 (6H, d, J = 6.3 Hz, H-9, 9 '); (CDCl ₃ , 62.9 MHz)? 150.9 (C- _{3 '} ), 148.1 (C-3''), 147.9 , 146.7 (C-4 '), 137.1 (C-1'), 135.8 (C-1), 134.4 109.3 (C-6), 119.1 (C-6), 110.5 (C-2 '), 108.5 (C-5 "), 108.2 C-2), 101.4 (-OCH 2 O-), 101.3 (-OCH 2 O-), 84.5 (C-8''), 84.1 (C-7), 83.9 (C-7'), 78.8 (C _{-7''), 56.2 (-OCH 3)} , 44.3 (C-8), 44.2 (C-8'), 17.4 (C-9''), 15.1 (C-9, 9'); HRFABMS m / z 543.2000 ([M + Na] +, calcd. For C 30 H 32 O 8 Na, 543.1995).

SCSC - 16 (- 16 ( saucerneolsaucerneol H) H)

Sticky solid; C₂₀H₂₂O₆; [α]²⁵ _D -51.9° (c 0.346, CHCl₃); UV (MeOH) λmax (logε) 231 (4.10), 290 (3.93) nm; IR (KBr) Vmax 3424, 2954, 2903, 1658, 1504, 1442, 1251, 1173, 1038 cm^-1; 1H-NMR (CDCl₃, 250 MHz) δ 6.80 (1H, s, H-2), 6.72 (2H, s, H-5, H-6), 6.52 (1H, s, H-2'), 6.33 (1H, s, H-5'), 5.84 (2H, s, -OCH₂O-), 5.93 (2H, s, -OCH₂O-), 4.27 (1H, d, J = 9.7 Hz, H-7), 2.81 (1H, dd, J = 3.8, 13.3, H-7b'), 2.25 (1H, m, H-7a'), 2.18 (1H, m, H-8'), 1.74 (1H, m, H-8), 0.85 (3H, d, J = 6.5 Hz, H-9'), 0.56 (3H, d, J = 7.0 Hz, H-9); 13C-NMR (CDCl₃, 62.9 MHz) δ 148.7 (C-3'), 147.8 (C-3), 147.1 (C-4), 146.2 (C-4'), 140.7 (C-1'), 138.0 (C-1), 120.6 (C-6), 119.0 (C-6'), 110.3 (C-2'), 108.0 (C-5), 107.0 (C-2), 101.0 (-OCH₂O-), 100.8 (-OCH₂O-), 98.4 (C-5'), 79.5 (C-7), 43.1 (C-8), 38.0 (C-7'), 34.0 (C-8'), 14.6 (C-9'), 12.0 (C-9); HREIMS m/z 340.1316 ([M-H2O]⁺, calcd. for C₂₀H₂₀O₅, 341.1311).Sticky solid; C ₂₀ H ₂₂ O _6; ^{_{[α] 25 D -51.9 ° (}} c 0.346, CHCl 3); UV (MeOH)? Max (log?) 231 (4.10), 290 (3.93) nm; IR (KBr)? Max 3424, 2954, 2903, 1658, 1504, 1442, 1251, 1173, 1038 cm ^-1 ; _{1H-NMR (CDCl 3, 250} MHz) δ 6.80 (1H, s, H-2), 6.72 (2H, s, H-5, H-6), 6.52 (1H, s, H-2 '), 6.33 (1H, s, H-5 '), 5.84 (2H, s, -OCH 2 O-), 5.93 (2H, s, -OCH 2 O-), 4.27 (1H, d, J = 9.7 Hz, H- M, H-8 '), 1.74 (1H, m, H-7'), 2.81 (1H, dd, J = 3.8, 13.3, , H-8), 0.85 (3H, d, J = 6.5 Hz, H-9 '), 0.56 (3H, d, J = 7.0 Hz, H-9); _{13C-NMR (CDCl 3, 62.9} MHz) δ 148.7 (C-3 '), 147.8 (C-3), 147.1 (C-4), 146.2 (C-4'), 140.7 (C-1 '), 138.0 (C-1), 120.6 ( C-6), 119.0 (C-6 '), 110.3 (C-2'), 108.0 (C-5), 107.0 (C-2), 101.0 (-OCH 2 O- ), 100.8 (-OCH ₂ O-), 98.4 (C-5 '), 79.5 (C-7), 43.1 (C-9 '), 12.0 (C-9); HREIMS m / z 340.1316 ([M -H2O] +, calcd. For C 20 H 20 O 5, 341.1311).

SCSC -17 (-17 ( nectandrinnectandrin B) B)

Colorless oil; C₂₀H₂₄O₅; 1H-NMR (MeOD, 250 MHz) δ 6.71 ~ 6.85 (6H, m, aromatic protons), 5.43 (2H, d, J = 6.5 Hz, H-7, 7´), 3.85 (6H, s, -OCH₃×2), 2.18 ~ 2.31 (2H, m, H-8, 8´), 0.67 (6H, d, J = 6.4 Hz, H-9, 9´); 13C-NMR (MeOD, 62.9 MHz) δ 148.7 (C-3, 3´), 146.6 (C-4, 4´), 134.2 (C-1, 1´), 120.2 (C-2, 2´), 115.8 (C-6, 6´), 111.3 (C-5, 5´), 85.7 (C-7, 7´), 56.4 (-OCH₃×2), 44.6 (C-8, 8´), 14.8 (C-9, 9´); FABMS m/z 345 [M+H]⁺.Colorless oil; C ₂₀ H ₂₄ O ₅ ; 1H-NMR (MeOD, 250 MHz ) δ 6.71 ~ 6.85 (6H, m, aromatic protons), 5.43 (2H, d, J = 6.5 Hz, H-7, 7'), 3.85 (6H, s, -OCH 3 × 2), 2.18-2.31 (2H, m, H-8, 8 '), 0.67 (6H, d, J = 6.4 Hz, H-9, 9'); C-NMR (MeOD, 62.9 MHz)? 148.7 (C-3, 3 '), 146.6 (C-4, 4'), 134.2 115.8 (C-6, 6') , 111.3 (C-5, 5'), 85.7 (C-7, 7'), 56.4 (-OCH 3 × 2), 44.6 (C-8, 8'), 14.8 (C-9, 9 '); FABMS m / z 345 [M + H] < ⁺ >.

SCSC -18 (-18 ( saucerneolsaucerneol I) I)

white powder (MeOH-H2O); C₂₀H₂₂O₆; mp 138-142 °C; [α]²⁵ _D -70.3 (c 0.20, CHCl₃); UV (MeOH) λmax (log ε) 235 (4.07), 287 (4.00) nm; IR (KBr) νmax 3333, 2968, 2919, 1503, 1488, 1443, 1248, 1040 cm^-1; 1H NMR (CDCl₃, 250 MHz) δ 6.86 (2H, s, H-2, 2′), 6.77 (2H, d, J = 8.8 Hz, H-6, H-6′), 6.73 (2H, d, J = 8.1 Hz, H-5, H-5′), 5.92 (4H, s, OCH₂O × 2), 4.26 (2H, d, J = 9.9 Hz, H-7, H-7′), 2.44 (2H, m, H-8, H-8′), 0.56 (6H, d, J = 6.7 Hz, H-9, H-9′); 13C NMR (CDCl₃, 62.9 MHz) δ 147.8 (C, C-3, C-3′), 147.0 (C, C-4, C-4′), 138.4 (C, C-1, C-1′), 120.5 (CH, C-6, C-6′), 107.9 (CH, C-5, C-5′), 107.0 (CH, C-2, C-2′), 100.9 (CH₂, OCH₂O × 2), 77.1 (CH, C-7, C-7′), 39.1 (CH, C-8, C-8′), 10.4 (CH₃, C-9, C-9′); HREIMS m/z 358.1414 [M]⁺ (calcd for C₂₀H₂₁O₅, 358.1416).white powder (MeOH-H2O); C ₂₀ H ₂₂ O _6; mp 138-142 [deg.] C; ^{_{[α] 25 D -70.3 (c}} 0.20, CHCl 3); UV (MeOH)? Max (log?) 235 (4.07), 287 (4.00) nm; IR (KBr)? Max 3333, 2968, 2919, 1503, 1488, 1443, 1248, 1040 cm ^-1 ; _{1H NMR (CDCl 3, 250 MHz} ) δ 6.86 (2H, s, H-2, 2 '), 6.77 (2H, d, J = 8.8 Hz, H-6, H-6'), 6.73 (2H, d , J = 8.1 Hz, H- 5, H-5 '), 5.92 (4H, s, OCH 2 O × 2), 4.26 (2H, d, J = 9.9 Hz, H-7, H-7'), 2.44 (2H, m, H-8, H-8 '), 0.56 (6H, d, J = 6.7 Hz, H-9, H-9'); _{13C NMR (CDCl 3, 62.9 MHz} ) δ 147.8 (C, C-3, C-3 '), 147.0 (C, C-4, C-4'), 138.4 (C, C-1, C-1 ' ), 120.5 (CH, C- 6, C-6 '), 107.9 (CH, C-5, C-5'), 107.0 (CH, C-2, C-2 '), 100.9 (CH 2, OCH _{2 O × 2), 77.1 (} CH, C-7, C-7 '), 39.1 (CH, C-8, C-8'), 10.4 (CH 3, C-9, C-9 '); HREIMS m / z 358.1414 [M] + (calcd for C 20 H 21 O 5, 358.1416).

SCSC -19 (-19 ( saucerneolsaucerneol D) D)

Amorphous brown powder; C₃₁H₃₆O₈; mp 71-72 ℃; [α]²⁵ _D-75.7° (c 0.65, CHCl₃); 1H-NMR (CDCl₃, 900 MHz) δ 6.99 (1H, d, J = 8.1 Hz, H-5´), 6.95 (1H, d, J = 1.3 Hz, H-2´´), 6.93 (1H, dd, J = 8.2, 1.6 Hz, H-6´´), 6.91 (1H, d, J = 1.3 Hz, H-2´), 6.85 (1H, d, J = 8.2 Hz, H-5´´), 6.83 (1H, dd, J = 8.1, 1.3 Hz, H-6´), 6.82 (1H, bs, H-2), 6.80 (1H, d, J = 7.9 Hz, H-5), 6.76 (1H, d, J = 7.8 Hz, H-6), 5.96 (2H, s, -OCH₂O-), 5.44 (1H, d, J = 6.6 Hz, H-7´), 5.42 (1H, d, J = 6.7 Hz, H-7), 4.65 (1H, d, J = 8.3 Hz, H-7´´), 4.13 (1H, dq, J = 8.1, 6.3 Hz, H-8´´), 3.93 (3H, s, -OCH₃), 3.90 (3H, s, -OCH₃), 3.88 (3H, s, -OCH₃), 2.28 (1H, ddq, J = 13.6, 6.8, 6.8 Hz, H-8´), 2.27 (1H, ddq, J = 13.6, 6.8, 6.8 Hz, H-8), 1.17 (3H, d, J = 6.2 Hz, H-9´´), 0.71 (6H, d, J = 6.5 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 150.4 (C-3´), 148.9 (C-3´´), 148.8 (C-4´´), 147.4 (C-3), 146.4 (C-4, 4´), 136.5 (C-1´), 135.3 (C-1), 132.5 (C-1´´), 120.0 (C- 6´´), 119.3 (C- 6), 118.7 (C-6´), 118.6 (C-5´), 110.7 (C-5´´), 110.0 (C-2´), 109.9 (C-2´´), 107.8 (C-5), 106.9 (C-2), 100.8 (-OCH₂O-), 84.0 (C-8´´), 83.7 (C-7), 83.4 (C-7´), 78.4 (C-7´´), 55.8 (-OCH₃×3), 43.9 (C-8), 43.8 (C-8´), 17.0 (C-9´´), 14.6 (C-9, 9´); FABMS m/z 536 [M]⁺.Amorphous brown powder; C ₃₁ H ₃₆ O ₈ ; mp 71-72 [deg.] C; ^{_{[α] 25 D -75.7 ° (}} c 0.65, CHCl 3); _{1H-NMR (CDCl 3, 900} MHz) δ 6.99 (1H, d, J = 8.1 Hz, H-5'), 6.95 (1H, d, J = 1.3 Hz, H-2''), 6.93 (1H, dd, J = 8.2, 1.6 Hz , H-6''), 6.91 (1H, d, J = 1.3 Hz, H-2'), 6.85 (1H, d, J = 8.2 Hz, H-5'') , 6.83 (1H, dd, J = 8.1, 1.3 Hz, H-6'), 6.82 (1H, bs, H-2), 6.80 (1H, d, J = 7.9 Hz, H-5), 6.76 (1H , d, J = 7.8 Hz, H-6), 5.96 (2H, s, -OCH 2 O-), 5.44 (1H, d, J = 6.6 Hz, H-7'), 5.42 (1H, d, J = 6.7 Hz, H-7), 4.65 (1H, d, J = 8.3 Hz, H-7 "), 4.13 (1H, dq, J = 8.1, 6.3 Hz, H- _{, s, -OCH 3), 3.90} (3H, s, -OCH 3), 3.88 (3H, s, -OCH 3), 2.28 (1H, ddq, J = 13.6, 6.8, 6.8 Hz, H-8') , 2.27 (1H, ddq, J = 13.6, 6.8, 6.8 Hz, H-8), 1.17 (3H, d, J = 6.2 Hz, H-9''), 0.71 (6H, d, J = 6.5 Hz, H-9, 9 '); (C- ₃ ), 148.9 (C-4), 147.4 (C-3), 146.4 (C- ), 118.3 (C-6), 119.7 (C-6), 118.7 (C-1) ), 118.6 (C-5 '), 110.7 (C-5''), 110.0 (C-2'), 109.9 _{100.8 (-OCH 2 O-), 84.0} (C-8''), 83.7 (C-7), 83.4 (C-7'), 78.4 (C-7''), 55.8 (-OCH 3 × 3) , 43.9 (C-8), 43.8 (C-8 '), 17.0 (C-9''), 14.6 (C-9, 9'); FABMS m / z 536 [M] ^< ⁺ & gt ^; .

SCSC -20 (-20 ( manassantinmanassantin B) B)

Amorphous brown powder; C₄₁H₄₈O₁₁; mp 82-83 ℃; [α]²⁵ _D-95.8° (c 0.62, CHCl3); 1H-NMR (CDCl₃, 250 MHz) δ 6.99 ~ 6.73 (12H, m, aromatic protons), 5.92 (2H, s, -OCH₂O-), 5.45 (2H, d, J = 5.8 Hz, H-7, 7´), 4.64 (1H, d, J = 8.2 Hz, H-7´´), 4.61 (1H, d, J = 8.2 Hz, H-7´´´), 4.10 (2H, m, H-8´´, 8´´´), 3.90 (3H, s, -OCH₃), 3.89 (3H, s, -OCH₃), 3.87 (3H, s, -OCH₃), 3.85 (3H, s, -OCH₃), 2.27 (2H, m, H-8, 8´), 1.16 (3H, d, J = 6.2 Hz, H-9´´), 1.14 (3H, d, J = 6.2 Hz, H-9´´´), 0.71 (6H, d, J = 6.0 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 150.5 (C-4, 4´), 148.9 (C-4´´´), 148.8 (C-4´´), 147.7 (C-3´´´), 147.3 (C-3´´), 146.4 (C-3´), 146.3 (C-3), 136.5 (C-1´), 136.4 (C-1), 133.9 (C-1´´´), 132.5 (C-1´´), 121.0 (C-6´´´), 119.9 (C-6´´), 118.8 (C-5´´´), 118.6 (C-5´´, 6, 6´), 110.7 (C-2´´´), 109.9 (C-2, 2´, 2´´), 108.0 (C-5), 107.5 (C-5´), 101.0 (-OCH₂O-), 84.1 (C-8´´), 84.0 (C-8´´´), 83.3 (C-7, 7´), 78.3 (C-7´´, 7´´´), 55.8 (-OCH₃×4), 44.1 (C-8, 8´), 17.0 (C-9´´), 16.9 (C-9´´´), 14.8 (C-9, 9´); FABMS m/z 716 [M]⁺. Amorphous brown powder; C ₄₁ H ₄₈ O ₁₁ ; mp 82-83 [deg.] C; ^{_{[α] 25 D -95.8 ° (}} c 0.62, CHCl3); _{1H-NMR (CDCl 3, 250} MHz) δ 6.99 ~ 6.73 (12H, m, aromatic protons), 5.92 (2H, s, -OCH 2 O-), 5.45 (2H, d, J = 5.8 Hz, H-7 , 4 '), 4.64 (1H, d, J = 8.2 Hz, H-7''), 4.61 (1H, d, J = 8.2 Hz, H- 8'', 8'''), 3.90 (3H , s, -OCH 3), 3.89 (3H, s, -OCH 3), 3.87 (3H, s, -OCH 3), 3.85 (3H, s, - _{OCH 3), 2.27 (2H,} m, H-8, 8'), 1.16 (3H, d, J = 6.2 Hz, H-9''), 1.14 (3H, d, J = 6.2 Hz, H-9 ''), 0.71 (6H, d, J = 6.0 Hz, H-9, 9 '); 14 C-NMR (CDCl ₃ , 62.9 MHz)? 150.5 (C-4, 4 '), 148.9 (C-4' 136.3 (C-3), 146.3 (C-3), 146.3 (C-3) (C-1 ''), 121.0 (C-6 '''), 119.9 (C-6''), 118.8 , 110.7 (C-2'''), 109.9 (C-2, 2', 2''), 108.0 (C-5), 107.5 (C-5'), 101.0 (-OCH 2 O-), 84.1 (C-8''), 84.0 ( C-8'''), 83.3 (C-7, 7'), 78.3 (C-7'', 7'''), 55.8 (-OCH 3 × 4) , 44.1 (C-8, 8 '), 17.0 (C-9''), 16.9 (C-9''), 14.8 (C-9, 9'); FABMS m / z 716 [M] ^< ⁺ & gt ^; .

SCSC -21 (-21 ( manassantinmanassantin A) A)

Amorphous brown powder; C₄₂H₅₂O₁₁; mp 80-82 ℃; [α]²⁵ _D -107.6° (c 0.64, CHCl₃); 1H-NMR (CDCl₃, 250 MHz) δ 6.98 ~ 6.79 (12H, m, aromatic protons), 5.45 (2H, d, J = 5.8 Hz, H-7, 7´), 4.64 (2H, d, J = 8.3 Hz, H-7´´, H-7´´´), 4.05 (2H, m, H-8´´, 8´´´), 3.90 (6H, s, -OCH₃×2), 3.86 (6H, s, -OCH₃×2), 3.85 (6H, s, -OCH₃×2), 2.27 (2H, m, H-8, 8´), 1.15 (6H, d, J = 6.2 Hz, H-9´´, 9´´´), 0.71 (6H, d, J = 6.4 Hz, H-9, 9´); 13C-NMR (CDCl₃, 62.9 MHz) δ 150.5 (C-4´´, 4´´´), 148.9 (C-3´´, C-3´´´), 148.8 (C-1´´, 1´´´), 146.4 (C-4, 4´), 136.4 (C-3, 3´), 132.5 (C-1, 1´), 119.9 (C- 6´´, 6´´´), 118.6 (C- 5´´, 5´´´, 2´´, 2´´´), 110.7 (C-6, C-6´), 110.0 (C-2, 2´), 109.9 (C-5, 5´), 84.0 (C-7´´, 7´´´), 83.3 (C-7, 7´), 78.3 (C-8´´, 8´´´), 55.8 (-OCH₃×6), 44.1 (C-8, 8´), 17.0 (C-9´´, 9´´´), 14.8 (C-9, 9´); FABMS m/z 732 [M]⁺.
Amorphous brown powder; C ₄₂ H ₅₂ O ₁₁ ; mp 80-82 [deg.] C; ^{_{[α] 25 D -107.6 ° (}} c 0.64, CHCl 3); _{1H-NMR (CDCl 3, 250} MHz) δ 6.98 ~ 6.79 (12H, m, aromatic protons), 5.45 (2H, d, J = 5.8 Hz, H-7, 7'), 4.64 (2H, d, J = H-7 ''), 4.05 (2H, m, H-8 ", 8 "'), 3.90 (6H, s, -OCH ₃ x 2), 3.86 _{6H, s, -OCH 3 × 2} ), 3.85 (6H, s, -OCH 3 × 2), 2.27 (2H, m, H-8, 8'), 1.15 (6H, d, J = 6.2 Hz, H -9 ", 9 "'), 0.71 (6H, d, J = 6.4 Hz, H-9, 9'); 13 C-NMR (CDCl ₃ , 62.9 MHz)? 150.5 (C-4 '', 4'''), 148.9 (C-3''C-3'''), 148.8 ''), 146.4 (C-4, 4 '), 136.4 (C-3,3''132.5 (C-5 '', 5 ''',2'',2'''), 110.7 (C-6, C- (C-7 ', 7''), 78.0 (C-8'',8'''), 55.8 (-OCH ₃ x 6) , 44.1 (C-8, 8 '), 17.0 (C-9', 9 ''), 14.8 (C-9, 9 '); FABMS m / z 732 [M] ^< ⁺ & gt ^; .

< < 실시예Example 2 > 삼백초 단일 성분의 세포 노화 저해 효능 조사 2> Effect of single component of Saururus chinensis on cell senescence inhibition

1. 실험 재료1. Experimental material

사람 섬유아세포와 제대정맥혈관내피세포는 Lonza (Walkersville, MD, 미국)에서 구입하였다. 둘베코스-변형 이글스 배지(Dubeccos-Modified Eagle's medium; DMEM), 우태아혈청, 항생제 용액 페니실린-스트렙토마이신(Penicillin-Streptomycin)은 WelGene (Daegu, Korea), 내피세포성장 배양액-2(endothelial cell growth medium-2, EGM-2)는 Lonza (Walkersvill, MD, 미국)에서 구입하였다. p53에 대한 항체는 SantaCruz Biotech, Inc. (SantaCruz, CA, 미국)에서 구입하였으며, p21과 pS6에 대한 항체는 Cell Signaling Technology Inc.(Beverly, MA, 미국)에서 구입하였다. GAPDH 항체는 한국생명공학연구원 권기선 박사로부터 분양받았다. 아드리아마이신은 일동제약주식회사 제품을 사용하였다.
Human fibroblasts and umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA). (Penicillin-Streptomycin, WelGene (Daegu, Korea), Endothelial Cell Growth Medium-2 (DMEM), Dulbecco's Modified Eagle's Medium -2, EGM-2) were purchased from Lonza (Walkersville, MD, USA). Antibodies to p53 were obtained from Santa Cruz Biotech, Inc. (SantaCruz, CA, USA), and antibodies against p21 and pS6 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The GAPDH antibody was obtained from Dr. Kwon Sun - sun of Korea Biotechnology Research Institute. Adriamycin was manufactured by Ildong Pharmaceutical Co., Ltd.

2. 세포 배양2. Cell culture

사람 섬유아세포는 10% 우태아혈청과 1% 항생제 [페니실린(penicillin) 10,000unit/ml, 스트렙토마이신(stretomycin) 10,000ug/ml)가 포함된 DMEM 배양액을 이용하여 100 mm 배양접시에 세포를 1 × 10⁵개로 분주한 후, 37℃, 5% 이산화탄소 배양기에서 배양하였다. 배양접시의 바닥에 세포가 80-90% 정도 자라면, 트립신-EDTA 용액 (2.5X) 을 넣어 세포를 분리한 후, 계대 배양하였다. 제대혈관내피세포는 EGM-2를 배양액으로 사용하여 사람 섬유아세포와 같은 방법으로 세포를 배양하였다. 세포를 계대할 때마다 세포 수를 측정하여 세포가 몇 회 분열하였는지 분열 횟수를 조사하였다. 세포의 분열 횟수 (population doubling, PD)는 PD= log₂F/log₂I (F=마지막 세포수, I=처음 세포수)의 식을 이용하여 계산하였다. 실험에 사용한 세포들은 분열횟수가 사람 섬유아세포의 경우 PD<35 또는 PD>75, 제대혈관내피세포는 PD<30 또는 PD>50회의 것을 사용하였다.
Human fibroblasts were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin 10,000 цm / ml, streomycin 10,000 ug / ml) 10 ⁵ , and then cultured in a 5% carbon dioxide incubator at 37 ° C. If the cells were grown at the bottom of the culture dish by 80-90%, trypsin-EDTA solution (2.5X) was added to separate the cells, followed by subculture. Cells in umbilical cord blood were cultured in the same manner as human fibroblasts using EGM-2 as a culture medium. The number of cells was measured every time the cells were transferred, and the number of times of division of the cells was examined. The population doubling (PD) was calculated using the equation: PD = log ₂ F / log ₂ I (F = final cell number, I = initial cell number). The cells used for the experiment were PD <35 or PD> 75 in the case of human fibroblasts, and PD <30 or PD> 50 in the cord blood.

3. 아드리아마이신 처리에 의한 세포노화 유도3. Induction of cell aging by treatment with adriamycin

직경 100mm 배양접시에 사람 섬유아세포, 제대정맥혈관내피세포를 1.5x10⁵개 분주하였다. 3일간 37℃, 5% 이산화탄소배양기에서 배양한 후, 세포 배양액을 제거하였다. 세포를 항생제가 포함된 DMEM 배양액으로 2회 세척하였다. 세포에 500 nM 아드리아마이신을 4시간 처리한 후, 항생제가 포함된 DMEM 배양액으로 3회 세척하였다. 사람 섬유아세포는 10% 우태아혈청과 1% 항생제가 포함된 DMEM 배양액으로, 사람 제대정맥혈관내피세포는 EGM-2 배양액으로 배양하였다. 4일 후, 노화 베타-갈락토시다제(senescence-associated β-galactosidase; SA-β-gal) 활성 염색으로 세포노화가 유도됨을 확인하였다.
Human fibroblasts and umbilical vein endothelial cells were plated at a density of 1.5x10 ⁵ cells in a 100 mm diameter culture dish. After culturing for 3 days at 37 ° C in a 5% carbon dioxide incubator, the cell culture medium was removed. Cells were washed twice with DMEM containing antibiotics. Cells were treated with 500 nM adriamycin for 4 hours and then washed three times with DMEM medium containing antibiotics. Human fibroblasts were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic, and human umbilical vein endothelial cells were cultured in EGM-2. After 4 days, it was confirmed that senescence-associated β-galactosidase (SA-β-gal) staining resulted in cell senescence.

4. 3-(4, 5-디메틸티아졸-2일)-2, 5-디페닐테트라졸리움 브로마이드(3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide; MTT) 측정 방법4. Measurement of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT)

삼백초 단일 화합물이 세포의 성장속도에 미치는 영향은 MTT법으로 조사하였다. 0.1% MTT 용액을 96 웰(well) 배양용기의 각 웰(well) 당 50 ul씩 넣고 3시간 동안 37℃, 5% 이산화탄소배양기에서 반응시켰다. 배양액과 MTT 용액을 제거 한 후, 디메틸설폭사이드(dimethyl sulfoxide) 100 ul를 첨가하여 형성된 결정을 녹였다. 마이크로플레이트 리더(microplate reader)를 이용하여 550 nm에서 흡광도를 측정하여 세포의 성장속도를 측정하였다.
The effect of a single compound on the growth rate of cells was investigated by MTT method. 0.1% MTT solution was added to each well of a 96-well culture vessel in an amount of 50 μl per well and reacted in a 5% carbon dioxide incubator at 37 ° C for 3 hours. After removing the culture solution and MTT solution, 100 μl of dimethyl sulfoxide was added to dissolve the crystals formed. The cell growth rate was measured by measuring the absorbance at 550 nm using a microplate reader.

5. 아드리아마이신에 의한 세포노화에서 삼백초 단일성분의 효과 조사5. Investigation of the effect of single component of Saururus chinensis in cell senescence by adriamycin

아드리아마이신에 의해 노화된 세포에 삼백초로부터 분리된 단일 화합물들이 효과가 있는지 조사하였다. 아드리아마이신을 4시간 처리한 세포들을 트립신-EDTA로 배양접시에서 분리하여 96well, 12well, 24well 세포배양용기로 분주하였다. 96well 세포배양용기에 각 well당 섬유아세포는 500개, 제대혈관내피세포는 1,000개씩 분주하였다. 24well에는 섬유아세포는 3000개/well, 제대혈관내피세포는 5000개/well로 분주하였으며, 12well은 섬유아세포 5000개/well, 재대혈관내피세포 7000개/well을 분주하였다. 하루 동안 37℃, 5% 이산화탄소배양기에서 배양하였다. 96well에는 각 well에 10% 우태아혈청과 1% 항생제가 포함된 DMEM 배양액과 EGM-2배양액을 100ul씩 더 넣어 주고 12well과 24well은 배양액을 교환한 후, 추출물은 100ug/ml, 단일 화합물은 10 ug/ml로 처리하였다. 음성 대조군으로 디메틸설폭사이드(dimethyl sulfoxide)를, 양성 대조군으로 N-아세틸시스테인(N-acetylcysteine) 5mM과 라파마이신(rapamycin) 500nM을 첨가하였다. 3일 동안 37℃, 5% 이산화탄소배양기에서 배양한 후, 세포의 성장 정도는 MTT법으로, 세포노화 정도는 노화 베타-갈락토시다제(senescence-associated β-galactosidase; SA-β-gal) 활성 염색법으로 조사하였다.
We examined the effect of single compounds isolated from Saururus chinense on cells aged by adriamycin. Cells treated with adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA and dispensed into 96-well, 12-well, 24-well cell culture vessels. In a 96-well cell culture container, 500 fibroblasts and 1,000 cells were placed in each well. 24 wells were plated at 3000 cells / well, cord blood cells were plated at 5000 cells / well and 12 wells were plated at 5000 cells / well and 7000 cells / well of vascular endothelial cells. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. In 96 wells, 100 μl of DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic and 100 μl of EGM-2 culture medium were added to each well. After 12 and 24-well cultures were exchanged, < / RTI > ug / ml. Dimethylsulfoxide was used as a negative control, and 5 mM N-acetylcysteine and 500 nM rapamycin were added as a positive control. After 3 days of incubation at 37 ° C in a 5% carbon dioxide incubator, the degree of cell growth was measured by MTT method and the degree of cell senescence was measured by senescence-associated β-galactosidase (SA-β-gal) Staining method.

6. 노화 베타-갈락토시다제(senescence-associated β-galactosidase; SA-β-gal) 활성 염색6. senescence-associated β-galactosidase (SA-β-gal) active staining

세포노화에 대한 효과는 SA-β-gal 활성 염색으로 조사하였다. 24 well 또는 12 well 배양용기에 단일 화합물을 3일 동안 처리한 후, 세포를 인산완충액으로 세척하였다. 3.7% 파라포름알데히드(paraformaldehyde)로 세포를 고정한 후, 고정액을 제거하고 다시 인산완충액으로 세척하였다. SA-β-gal 염색 용액 [40 mM 시트릭산(citric acid)/포스페이트(phosphate); pH 5.8, 5 mM 포타슘 페로시아나이드(potassium ferrocyanide), 5 mM 포타슘 페리시아나이드(potassium ferricyanide), 150 mM NaCl, 2 mM MgCl₂, X-gal 1 mg/ml]을 24 well 배양용기에는 각 well 당 250 ul, 12 well 배양용기에는 각 well 당 500ul를 넣어 주었다. 은박지로 싸서 37℃에서 17시간 동안 반응시켰다. 인산완충용액(PBS)으로 2번 세척한 후, 1% 에오진 용액으로 1분간 염색하였다. 인산완충용액으로 2회 세척 한 후, 광학현미경으로 파란색으로 염색된 세포를 관찰하였다. SA-β-gal 활성 정도는 총 50~100개의 세포 중에서 세포질에 파란색으로 염색된 세포 수를 측정하여 백분율 (%)로 표시하였다.
The effect on cell senescence was examined by SA-β-gal active staining. After 3 days of treatment with a single compound in a 24 well or 12 well culture vessel, the cells were washed with phosphate buffer. The cells were fixed with 3.7% paraformaldehyde, and the fixative solution was removed and washed again with phosphate buffer solution. SA-β-gal staining solution [40 mM citric acid / phosphate; pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / ml] was added to each well 250 ul per well and 500 ul per well in a 12 well culture vessel. Wrapped in silver foil and reacted at 37 캜 for 17 hours. After washing twice with phosphate buffered saline (PBS), the cells were stained with 1% ezine solution for 1 minute. After washing twice with phosphate buffer, blue-stained cells were observed under an optical microscope. The degree of SA-β-gal activity was expressed in percentage (%) by measuring the number of cells stained blue in the cytoplasm among 50-100 cells in total.

7. 세포 단백질 추출7. Cell protein extraction

각 세포를 60 mm 배양접시에 1x10⁵개로 분주한 후 37℃, 5% 이산화탄소 배양기에서 배양하였다. 세포를 항생제가 포함된 DMEM 배양액으로 2회 세척한 후, 삼백초 단일성분을 농도 별로 1시간 전 처리하고, 아드리아마이신 500 nM을 4시간 동안 처리하였다. 배양액을 제거한 후, 인산완충액으로 2회 세척하였다. 배양접시 당 세포 용해 용액 [25mM Tris-HCl(pH 7.6), 150mM Nacl, 1% 트리톤(Tryton) X-100, 0.5% 소듐 데옥시콜레이트(sodium deoxycholate), 0.1% SDS, 1mM 소듐 바나데이트(Sodium vanadate), 5mM NaF, 프로테아제 억제제(protease inhibitor) 또는 1mM PMSF]을 50 ul를 넣었다. 세포 긁개를 이용하여 배양접시를 긁어 용액과 세포를 모은 후 미세원침관으로 옮겼다. 얼음에서 30분간 반응시키면서 매 10분마다 용액을 진탕하였다. 12,000 rpm에서 15분간 원침하여 상청액을 새 튜브로 옮겼다. 용액 속의 단백질 양은 우혈청알부민을 표준단백질로 사용하여 바이신코니닉산(bicinchoninic acid; BCA) 법 (Pierce Biotechnology Inc., Rockford IL, 미국)으로 정량하였다.
Each cell was placed in a 60 mm culture dish at 1 × 10 ⁵ cells and cultured at 37 ° C. in a 5% CO 2 incubator. Cells were washed twice with DMEM medium containing antibiotics, treated with a single component of Saururus chinensis one hour prior to concentration and treated with 500 nM adriamycin for 4 hours. After the culture medium was removed, the cells were washed twice with phosphate buffer. The cell lysate solution (25 mM Tris-HCl, pH 7.6), 150 mM Nacl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate vanadate), 5 mM NaF, protease inhibitor or 1 mM PMSF]. The cells were scraped using a cell scraper to scrape the culture dish, and then transferred to a microneedle tube. The solution was shaken every 10 minutes while reacting on ice for 30 minutes. The supernatant was transferred to a new tube by spinning at 12,000 rpm for 15 minutes. The amount of protein in the solution was quantified by bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford IL, USA) using bovine serum albumin as a standard protein.

8. 웨스턴 블랏(Western blot) 분석8. Western blot analysis

단백질 (30μg)을 10% SDS-폴리아크릴아미드(SDS-polyacrylamide) 겔에서 전기영동하여 분리하였다. 니트로셀룰로스 막으로 단백질을 이동시킨 후, 5% 전지분유가 포함된 트윈-20-트리스 완충된 식염수(Tween-20-Tris buffered saline; TTBS)에서 30분 동안 반응시켰다. 니트로셀룰로스 막을 p53 또는 p21에 대한 일차항체가 포함된 5% 전지분유-TTBS 용액에서 밤새도록 반응시켰다. TTBS 용액으로 10분씩 3회 세척 한 후, 겨자무 과산화효소(horseradish peroxidase)가 결합된 2차 항체와 1시간 30분 동안 반응시켰다. TTBS로 막을 7분씩 5회 세척 한 후, 향상된 화학발광(enhanced chemiluminescence) 용액을 이용하여 p53 또는 p21의 양을 측정하였다. 각 항체와 반응한 특정 단백질의 양은 LAS-3000 영상장치 (Fujifilm Corp., Stanford, CT, 미국)을 사용하여 측정하였다. 각 실험에 동일한 양의 단백질이 사용되었음은 글리세르알데히드-3-포스페이트 디하이드로게나아제(glyceraldehyde-3-phosphate dehydrogease; GAPDH) 항체를 이용해 비교하였다.
Protein (30 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and allowed to react for 30 minutes in Tween-20-Tris buffered saline (TTBS) containing 5% whole milk powder. Nitrocellulose membranes were reacted overnight in 5% whole milk powder-TTBS solution containing primary antibody against p53 or p21. After washing three times for 10 minutes with TTBS solution, the cells were reacted with a secondary antibody conjugated with horseradish peroxidase for 1 hour and 30 minutes. The membranes were washed five times for 7 min with TTBS and the amount of p53 or p21 was measured using an enhanced chemiluminescence solution. The amount of specific protein reacted with each antibody was measured using a LAS-3000 imaging device (Fujifilm Corp., Stanford, CT, USA). The same amount of protein used in each experiment was compared using glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

9. 세포 내 활성산소(ROS) 농도 측정9. Measurement of intracellular reactive oxygen (ROS) concentration

세포를 100mm 배양접시에 1.5x10⁵개로 분주한 후 37℃, 5% 이산화탄소 배양기에서 3일 동안 배양하였다. 세포를 항생제가 포함된 DMEM 배양액으로 2회 세척한 후, 아드리아마이신 500 nM을 4시간 동안 처리하였다. 인산완충액으로 한 번 세척하고, 트립신-EDTA 용액 (2.5%) 을 처리하여 세포를 분리한 후, 60mm 배양접시에 1 X 10⁵개로 분주하였다. 하루 동안 37℃, 5% 이산화탄소배양기에서 배양하였다. 배양액을 갈아주고 단일화합물을 각각의 농도로 처리하였다. 음성 대조군으로 디메틸설폭사이드(dimethyl sulfoxide)를 양성 대조군으로 N-아세틸시스테인(N-acetylcysteine) 5mM과 라파마이신(rapamycin) 500nM을 첨가하였다. 3일 동안 37℃, 5% 이산화탄소배양기에서 배양한 후, 항생제가 포함된 DMEM 배양액으로 2회 세척하고 H₂DCFDA 250uM을 20분 동안 처리하였다. 인산완충용액으로 1회 세척하고 트립신-EDTA 용액을 넣어 세포를 분리하여 미세원침관으로 옮겼다. 5,000×g 에서 1분간 원침하여 상청액을 버리고 2% 우태아혈청을 포함한 인산완충용액을 1ml 넣어 세포를 세척하고 다시 5,000×g에서 1분간 원침하였다. 세척과정을 2회 반복한 후, 1% 파라포름알데히드(paraformaldehyde)를 1ml 넣어 주었다. 세포 내 ROS 양은 BD FACS CantoⅡ 유세포 분류기 (BD Biosciences, San Jose, CA)를 사용하여 측정하였다.
Cells were plated at ^1.5 × 10 ⁵ in a 100 mm culture dish and cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days. Cells were washed twice with DMEM medium containing antibiotics and treated with 500 nM adriamycin for 4 hours. The cells were washed once with phosphate buffer and treated with trypsin-EDTA solution (2.5%), and then the cells were divided into 1 × 10 ⁵ cells in a 60 mm culture dish. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. The culture medium was changed and a single compound was treated at each concentration. As a negative control, dimethyl sulfoxide was added as a positive control, 5 mM N-acetylcysteine and 500 nM rapamycin. After incubation for 3 days in a 5% CO 2 incubator at 37 ° C, the cells were washed twice with DMEM containing antibiotics and treated with 250 μM H ₂ DCFDA for 20 minutes. The cells were washed once with phosphate buffered saline, and the trypsin-EDTA solution was added to the cells. The supernatant was discarded at 5,000 × g for 1 minute, and the cells were washed with 1 ml of phosphate buffer solution containing 2% fetal bovine serum, and further washed at 5,000 × g for 1 minute. The washing procedure was repeated twice, followed by the addition of 1 ml of 1% paraformaldehyde. The amount of intracellular ROS was measured using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, Calif.).

10. 결과10. Results

섬유아세포를 대상으로 삼백초에서 추출한 단일 성분 23가지 중에서 어떤 성분이 아드리아마이신에 의한 세포노화를 억제하는지 조사하기 하였다. 각 단일성분을 10 ug/ml로 처리하여, 세포독성을 조사하였다. 그 중에서 SC-4, SC-9, SC-13, SC-23을 제외한 단일성분들은 세포독성이 나타나지 않았다 (도 3A). 이 중 SC-7, SC-8, SC-10, SC-14를 아드리아마이신에 의해 나타나는 세포노화를 저해하는지 조사하였으나, 세포노화를 저해하는 효능을 관찰할 수 없었다 (도 3B).To investigate the effect of adriamycin on cell senescence, we investigated the effect of adipocyte on cell proliferation. Each single component was treated with 10 ug / ml to investigate cytotoxicity. Among them, unicellular components except for SC-4, SC-9, SC-13 and SC-23 showed no cytotoxicity (FIG. 3A). Among them, SC-7, SC-8, SC-10 and SC-14 were examined for inhibition of cell senescence induced by adriamycin, but no effect of inhibiting cell senescence could be observed (Fig. 3B).

SC-9의 농도를 1, 0.1 ug/ml로 처리했을 때, 0.1 ug/ml에서 세포독성이 나타나지 않았다 (도 4A). SC-9 0.1 ug/ml로 처리하여 아드리아마이신에 의해 유도되는 세포노화를 저해하는지 조사하였다. 그 결과 SC-9이 아드리아마이신에 의해 증가되는 SA-β-gal 활성을 저해하였다 (도 4B, 4C). SC-9이 아드리아마이신에 의한 세포노화과정에서 발현이 증가하는 p53, p21 단백질 발현을 억제하는지 웨스턴 블랏(Western blot) 분석으로 조사한 결과, p53, p21의 발현이 감소되었다 (도 4D). SC-9는 아드리아마이신에 의해 증가되는 세포 내 ROS의 양도 감소시켰다 (도 5A, 5B). SC-9가 아드리아마이신에 의한 세포노화뿐만 아니라, 복제노화가 유도된 세포에서도 세포노화를 저해하는지 조사하였다. 그 결과, SC-9이 복제노화 세포의 SA-β-gal 활성을 감소시켰다 (도 6A, 6B). When the concentration of SC-9 was treated at 1, 0.1 ug / ml, no cytotoxicity was observed at 0.1 ug / ml (Fig. 4A). SC-9 0.1 ug / ml to examine whether it inhibits adiamycin-induced cell senescence. As a result, SC-9 inhibited SA-β-gal activity which was increased by adriamycin (FIG. 4B, 4C). Western blot analysis revealed that SC-9 inhibits the expression of p53 and p21 protein, which are expressed in the cell aging process by adriamycin, and the expression of p53 and p21 is decreased (Fig. 4D). SC-9 also reduced the amount of intracellular ROS that was increased by adriamycin (Figures 5A, 5B). We investigated whether SC-9 inhibits adipocyte-induced cell senescence as well as adipocyte-induced cell senescence. As a result, SC-9 reduced the SA-beta-gal activity of the replicating senescent cells (Fig. 6A, 6B).

이상의 결과로부터 섬유아세포에서 SC-9 (erthro-austrobailiganan-6)가 아드리아마이신에 의한 세포노화 뿐만 아니라, 복제노화로 인한 세포노화도 저해하는 효능이 있음을 확인하였다.From the above results, it was confirmed that SC-9 (erthro-austrobailiganan-6) in fibroblasts has an effect of inhibiting not only cell aging by adriamycin but also cell senescence due to reproductive aging.

Claims

A pharmaceutical composition for prevention or treatment of skin aging characterized by comprising erythro-austrobailignan-6 (erthro-austrobailignan-6) represented by the following formula 1 as an active ingredient isolated from Saururus chinensis extract and inhibiting cell senescence Composition.
&Lt; Formula 1 >

delete

The method according to claim 1, wherein the Saururus chinensis extract is prepared by adding ethyl acetate (EtOAc) to a distilled water layer fractionated by adding distilled water and n-hexane to a methanol extract of Saururus chinensis and fractionating the extracted ethyl acetate (EtOAc) Wherein the composition is an extract.

The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the cell is a fibroblast.

The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the cell aging is induced by adriamycin.

The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the inhibition of cell senescence measures the inhibition of senescence-associated beta-galactosidase (SA-beta-gal) activity.

delete