3β-Hydroxy-urs-11-en-28,13β-olide의 항당뇨와 항염증 활성

Abstract

Ursolic acid (UA), 3β-hydroxy-urs-12-en-28-oic-acid, is a pentacyclic triterpenoid compound that is widely distributed in higher plant kingdoms. UA belongs to the cyclosqualenoid family and naturally occurs in leaves and berries as medicinal plants and in the protective wax-like coatings of apples, pears, prunes and other fruits. Medicinal plants containing ursolic acid has been used as a folk medicine since ancient times. UA has been reported to have several pharmacological effects, including antioxidant, anti-inflammatory, antitumor, anti-diabetic and neuroprotective properties. Chemical modifications of ursolic acid have been conducted to obtain new derivatives with enhanced biological activities as mentioned above. In this respect, we have initiated chemical modification of UA with peroxynitrite (ONOO−), a reactive nitrogen species, which afforded an oxidized derivative of UA. The oxidized derivative of UA was identified as 3β-hydroxy-urs-11-en-28,13β-olide (UAL) by EI-MS, 1H-NMR, 13C-NMR as well as by comparisons with spectral published data. Although UAL has been known to exert Ca2+ antagonist activity, neurotrophic factor-potentiating activity and cytotoxic effect on C6 rat glioma cell, A431 skin carcinoma cell, A2780 human ovarian cancer cell, however, its activity regarding anti-diabetic and anti-inflammatory has not been elucidated yet. Therefore, the present study was undertaken to investigate the pharmacological activities of UAL related to anti-diabetic activity via protein tyrosine phosphatase (PTP1B), α-glucosidase inhibition, anti-inflammatory activity through reduction of nitric oxide (NO) production, as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. UAL was also evaluated by ONOO− scavenging activity assay and inhibitory activity against ONOO− mediated tyrosine nitration for anti-diabetic and anti-inflammatory activities, compared to UA. UAL demonstrated high and moderate inhibitory activities for PTP1B and α-glucosidase with IC50 values of 5.11 ± 0.25 µM and 20.68 ± 0.11 µM, respectively. UAL was investigated using the enzyme kinetic assay, Dixon plot and Linweaver-Burk plot. It exhibited non-competitive inhibition against PTP1B with Ki value of 4.45 µM and mixed inhibition against α-glucosidase with Ki value of 10.87 µM. Furthermore, UAL did not exhibit cytotoxicity below 50 µM while UA was cytotoxic above 12.5µM and reduced NO production with IC50 value of 39.78 ± 0.54 µM as well as iNOS and COX-2 protein inhibition in LPS-stimulated RAW 264.7 cells. Although UAL showed less ONOO− scavenging activity than UA, it indicated inhibitory activity against 3-nitrotyrosine as well as UA via western blot analysis. It was worthy that inhibitory activities of UAL and UA on formation of 3-nitrotyrosine were found for the first time. These findings indicate that UAL, an oxidized derivative of UA, exhibited potent biological activities including anti-diabetic and anti-inflammatory activities. We estimated that carboxyl group at C-28 of UA may be responsible fot potent inhibitory activities against PTP1B and α-glucosidase of UA when compared with UAL. Likewise, UAL exhibited less cytotoxicity than UA due to epoxidation or condensation reaction taken place in between C-(12-13) and carboxyl group at C-28 regarding structure-activity relationship. In addition, a double bond at C-(12-13) and carboxyl group at C-28 of UA may be related to ONOO− scavenging activity due to its electron donating ability. Taken together these result, UAL may be considered as a useful therapeutic and preventive approach to prevent various diabetic and inflammatory diseases and a potential candidate as a substitute for UA.