Protein tyrosine phosphatase 1B (PTP1B) inhibitors from the stem bark of Erythrina abyssinica

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응구엔 피 흥
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인체에서 적절한 포도당 농도를 유지하는 기능에서의 손실과 관련되는 인슐린 내성은 제2형 당뇨병의 가장 중요한 인자로, 인슐린 신호전달과정에서의 문제와 관련된다. 그러므로 인슐린 신호전달과정에서의 많은 약물 목표점들이 인슐린 감수성을 개선하기 위한 과정을 이해하기 위하여 연구되어 왔다. 인슐린 감수성 개선 여러 약물 목표점 가운데에서, protein tyrosine phosphatase 1B (PTP1B)는 제 2형 당뇨병 치료에 효과적인 약물 목표점으로 최근 부상하고 있다. PTP1B 효소는 인슐린 수용체 인산화의 negative regulator로 PTP1B 효소를 저해하는 화합물이거나 PTP1B 유전자 발현을 저해하는 화합물은 제 2형 당뇨병의 치료제로서 혹은 비만 치료제로서의 유용할 것으로 제시되었다.
따라서 본 연구는 천연 식물로부터 protein tyrosine phosphatase 1B (PTP1B) 저해 활성 물질의 탐색을 통하여 PTP1B에 저해 활성을 나타내는 Erythrina abyssinica의 줄기 껍질로부터 silica gel (입자크기, 63200 m) 와 RP-18 (입자크기, 4063 m) 및 semi-preparative HPLC [C18 column (10 × 250 mm, 입자크기, 10 m)] 등의 각종 크로마트그래피 방법을 사용하여 화합물을 분리하였고, 이러한 분리과정을 통하여 15개의 신규 화합물 (erythribyssins 1 - 15)를 얻었다. 분리한 화합물들의 구조는 각종 스펙트럼 방법 (1D, 2D-NMR, UV, IR, [α]D, CD 와 MS)과 보고된 문헌을 참고하여 동정 하였다.
분리된 화합물 중 화합물 1 – 4, 12 – 13 과 15은 새로운 prenylated pterocarpan 계열의 화합물로서 erythribyssin A – D (1 – 4), erythribyssin L – M (12 – 13)과 erythribyssin O (15)로 명명하였으며, 화합물 7, 9과 11는 flavanone 골격을 갖는 신규화합물로서 erythribyssin G (7), erythribyssin I (9)와 erythribyssin K (11)로 명명하였다. 화합물 6 과 8은 새로운 benzofuranoid 계열의 화합물로서 erythribyssin F (6)와 erythribyssin H (8)로 한 개의 특이한 화합물은 새로운 courmestan계열의 물질로서 erythribyssin N (14)로 명명 하였다.
분리한 신규 화합들 (1 - 15)를 in vitro PTP1B 효소 저해 활성을 측정하였을 때, 화합물 2 - 4, 6, 8, 9 와 11을 제외한 화합물들이 4.2  0.4 M 부터 26.7  1.2 M 범위에서 PTP1B 효소를 50% 저해함을 확인하였다. 플라보노이드 B링 부분에 3-hydroxy-2,2-dimethylpyran 구조를 갖는 화합물 3, 4 와 9는 prenyl 기가 존재하는 화합물 7이 22.6  1.5 M에서 저해작용을 갖는 것에 비하여 최종농도 50 M 이상의 농도에서도 저해활성을 나타내지 않았다. 그러나, methoxy 기나 prenyl기가 존재하는 다른 화합물 1, 5, 10, 12 - 15는 prenyl 기가 없거나 플라보노이드의 수산기가 알데하이드나 카복실기로 치환딘 화합물 6, 8, 9 와 10이 저해활성을 나타내지 않는 것에 비하여 4.2  0.4 M 부터 26.7  1.2 M 범위에서 강한 저해활성을 보여 주었다. 이러한 결과는 화합물의 작용기에 methoxy나 prenyl기의 추가가 PTP1B 효소의 활성증가와 관련되며, 알데하이드나 카복실기의 치환은 PTP1B 효소의 활성저하와 관련됨을 보여주는 것이다.
비록 prenyl기가 치환된 플라보노이드의 구조와 활성간의 관계들이 완전히 규명 되지는 않았지만, flavonoids, isoflavonoids, pterocarpans, benzofurans, courmestran을 포함한 플라보노이드 화합물에서 prenyl 기의 존재가 PTB1B 저해작용에 있어서 중요함을 이 논문에서 제안한다. 따라서, Erythrina abyssinica로 분리된 화합물들은 비만과 2형 당뇨의 개선을 위한 치료제 개발에 유익하게 이용 될 수 있다고 제안한다.|Insulin resistance resulting in loss of proper glucose homeostasis is consider as the most important factor of type 2 diabetes and it is generally due to defects in insulin-mediated signal transduction. Therefore, a number of molecular targets in insulin signaling pathway are being investigated for improving insulin sensitivity. Among the various potential drug targets, protein tyrosine phosphatase 1B (PTP1B) has recently emerged as a promising one in the effective treatment of type 2 diabetes. As a PTP1B has been considered as a major negative regulator in the insulin signaling pathway, it has been suggested that compounds reducing PTP1B activity or the genetic expression levels of PTP1B may be useful in the treatment of type-2 diabetes and possibly obesity as well.
During this screening effort we found that EtOAcsoluble extract of the stem bark of Erythrina abyssinica Harms inhibited protein tyrosine phosphatase 1B (PTP1B) activity, fifteen new compounds (115) from the stem bark of Erythrina abyssinica were isolated by bioassayguided isolation using silica gel (63200 m particle size), RP-18 (4063 m particle size) column chromatography, and semi-preparative HPLC [C18 column (10 × 250 mm, 10 m particle size)]. Their structures were elucidated on the basis of spectral (including 1D, 2D-NMR, IR, [α]D, UV, CD and MS data) and physicochemical analyses.
New compounds 1 – 4, 12 – 13 and 15 were new prenylated pterocarpans named as erythribyssins A – D (1 – 4), erythribyssins L – M (12 – 13) and erythribyssin O (15). Compounds 7, 9 and 11 were new compounds with flavanone skeleton named erythribyssin G (7), erythribyssin I (9) and erythribyssin K (11). As compounds 5 and 10 were new isoflavanones, these compounds were called as erythribyssin E (5) and erythribyssin J (10), respectively. Finally, compounds 6 and 8 were new benzofuranoids compounds, which are named erythribyssin F (6) and erythribyssin H (8), and erythribyssin N (14) is a new courmestan-type compound.
When compounds 1 – 15 were tested on in vitro PTP1B inhibitory assay, all the isolates with the exception of compounds 2 – 4, 6, 8, 9 and 11 inhibited strongly PTP1B activity with IC50 values ranging from 4.2  0.4 to 26.7  1.2 M. Compound 3, 4 and 9, which were fused as the 3-hydroxy-2,2-dimethylpyran moiety on the B ring, did not showed any inhibition at PTP1B enzyme activity over the final concentration of 50 μM with compared to that of compound 7 (22.6 ± 1.5 μM) which the prenyl group was present. However, other compounds 1, 5, 10, 12 - 15 which were attached by either mehoxyl groups or prenyl groups showed a stronger activity with IC50 value ranging from 4.2  0.4 to 26.7 ± 1.2 μM than compounds 6, 8, 9 and 11 (IC50 > 50 μM), which the prenyl groups are absent or OH group was substituted to CHO or COOH functional groups. These results indicated that the addition of prenyl group or methoxyl substituent to functional group of compounds may be responsible for increasing PTP1B enzyme activity, and substitution of hydroxyl group to the CHO or COOH groups may decrease the inhibitory activity on this enzyme.
Although structure-activity relationships of flavonoids bearing prenyl groups were not thoroughly investigated, these results indicated that substitution of prenyl groups on flavonoids may be important for in vitro PTP1B inhibitory activity. Flavonoids, isoflavonoids, pterocarpans and benzofurans, as well as courmestans with prenyl groups could be consider as promising classes of PTP1B inhibitors. Thus, its constituents from Erythrina abyssinica might be used beneficially in the treatment of diabetes as well as obesity.
Alternative Title
Erythrina abyssinica의 줄기 껍질로부터 protein tyrosine phosphatase 1B (PTP1B) 저해제의 분리
Alternative Author(s)
Nguyen Phi Hung
일반대학원 약학과
Awarded Date
Table Of Contents

1. Diabetes 7
2. Protein tyrosine phosphatase 8
3. PTP1B in human diabetes and obesity 9
4. PTP1B inhibitors 11
5. Erythrina abyssinica 16

1. Materials 18
1.1. Plant materials 18
1.2. Chemical, reagents and chromatography 18
1.3. General experimental procedures 18
2. Methods 19
2.1. Isolation of Compounds from E. abyssinica 19
2.2. in vitro PTP1B assay 25

1. Structure determination of new compounds isolated from E. abyssinica 26
Structure determination of compound 1 26
Structure determination of compound 2 31
Structure determination of compound 3 35
Structure determination of compound 4 39
Structure determination of compound 5 43
Structure determination of compound 6 47
Structure determination of compound 7 51
Structure determination of compound 8 55
Structure determination of compound 9 59
Structure determination of compound 10 63
Structure determination of compound 11 67
Structure determination of compound 12 71
Structure determination of compound 13 75
Structure determination of compound 14 79
Structure determination of compound 15 83
2. Results of PTP1B inhibitory activity 87

조선대학교 대학원
응구엔 피 흥. (2009). Protein tyrosine phosphatase 1B (PTP1B) inhibitors from the stem bark of Erythrina abyssinica.
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