갈조류 꽈배기모자반(Sargassum siliquastrum)과 쇠비름(Portulaca oleracea)으로부터 생리활성물질의 분리와 구조결정

Abstract

The life of a human being has been extended even more than before due to improvement of medical technology. Nevertheless, the incidence rate of other new forms of diseases such as cancer, hyperlipidemia, diabetes, mental disorders, etc. has also increased greatly. Following extensive research, two common, widely available, natural plants have been found to have antioxidative and antiproliferative properties which may contribute to the prevention or treatment of some diseases or may inhibit the growth of some cancers.

As a part of our search for new bioactive compounds from natural resources, the brown alga Sargassum siliquastrum and the terrestrial plant Portulaca oleracea were collected. Brown algae of the genus Sargassum (Sargassaceae) are widely distributed in the temperate and tropical oceans of the world, and often dominate benthic algal communities and occur in huge floating masses. Most species have cycles of vegetative growth and attrition. Portulaca oleracea L. (Portulacaceae) is an annual herb growing in warm areas of the world and has been used as traditional and folk remedies for thousands of years in many countries throughout the world.

Each of the collected samples was briefly dried under shade and extracted with a mixture solvent of acetone-CH2Cl2 (1:1) at room temperature for 2 days and filtered. Then, the residue was re-extracted with MeOH in the same way. The combined crude extracts of each sample were partitioned between CH2Cl2 and water. The organic layer was further partitioned between n-hexane and 85% aq. MeOH, and the aqueous layer was partitioned with n-BuOH and H2O, successively.

Previous study has shown 85% aq. MeOH solvent fraction of S. siliquastrum crude extracts has antioxidizing and antiproliferative effects. Therefore, six meroterpenoids, including three new ones were obtained from the 85% aq. MeOH fraction by bioactivity-guided separation.

In our measurement for antioxidant activities, compounds 1-3 exhibited the strong scavenging effect on DPPH radical and peroxynitrite in a non-cellular system. In addition, ROS was generated in a cellular system. The antiproliferative effect of these compounds was also measured against AGS, HT-29, HT-1080, and MCF-7 human cancer cells. In comparative analysis, all compounds showed high growth-inhibitory effects on all cancer cell lines in a dose-dependent manner.

85% aq. MeOH solvent fraction of P. oleracea crude extracts showed a significant antiproliferative inhibitory effect against HT-29 colon cancer cells. On the basis of the above result, further purification of 85% aq. MeOH led to the isolation of homoisoflavonoids 7-13. The chemical structure of these compounds was established by extensive 2D NMR experiments such as 1H gDQCOSY, TOCSY, NOESY, gHMQC, and gHMBC and by synthesis of authentic compounds.

The antiproliferative effect of compounds 7-10, 12 and 13 was also evaluated in HT1080, HT-29, AGS and MCF-7 human cancer cells using the MTT assay method. Among them, compound 10 exhibited the strongest antiproliferative inhibitory effect on the growth of human cancer cells in a dose-dependent manner.

The antioxidant activity of these compounds was also evaluated by measuring intracellular ROS level and membrane lipid peroxidation. As a result, compounds 7-10 moderately decreased intracellular ROS level and compound 10 suppressed membrane lipid peroxidation.

The latest report on extracts of P. oleracea suggested that it has a lypolitic effect through release of fatty acid and glycerol to the culture medium. Based on the above, the antiobesity effect of compounds 7-10 from P. oleracea was also examined by observing Oil-Red O stained lipid droplets in adipocyte and by measuring glycerol release and glucose contents in a culture medium. In addition, expression levels of several genes related to adipogenesis including transcription factors were examined using reverse-transcription polymerase chain reaction (RT-PCR) As a result, compounds 7 and 10 not only decreased production of lipid droplets and glucose contents but also increased glycerol release.

Further, the effect of homoisoflavonoids (7-10) on the adipogenic differentiation was investigated at the gene expression levels, compounds 9 and 10 induced down-regulationion of adipogenic transcription factors (PPAR, C/EBP, PPAR, and SREBP1c) and adipogenic target adipocyte-specific genes (FABP4, FATP1, FAS, LPL, ACS1, perilipin and HSL). Moreover, compounds 9 and 10 down-regulated adipocytokine such as leptin and perilipin, and TNF-α. In short, compounds 9 and 10 have high inhibition effect on intracellular lipid accumulation and may be a valuable potential lead compound for the treatment of obesity.

Therefore, these results suggest that S. siliquastrum and P. portulaca could be used as the valuable materials for developing biofunctional substances that may inhibit the growth of cancers, that may protect cells from the damaging effects of oxidation, or that may assist the body in maintaining a constant level of insulin, thereby preventing obesity.