Phlorotannins are polyphenols formed by the polymerization of phloroglucinol and are thought to have multiple functions including herbivore deterrence, antifouling with epiphytes, UV protection, cell wall component, wound healing and adhesion to the substrate. Phlorotannins occur only in brown algae.
The edible brown alga Ecklonia cava, one of the main sources of phlorotannins, belongs to the family of Laminariaceae as a perennial plant and is widely distributed along the coast of South Korea and Japan. The collected samples of E. cava were air-dried on the shade and ground into powder. The powder was extracted repeatedly with MeOH for 3 hours under reflux condition. The crude extracts were partitioned between CH2Cl2 and H2O. The organic layer was evaporated and re-partitioned between n-hexane and 85% aq. MeOH, and the aqueous layer was also re-partitioned between n-BuOH and H2O.
Among four solvent fractionsof the crude extract, total phlorotannins contents increased in the order of n-BuOH > 85% aq. MeOH > H2O > n-hexane, showing concentration of 68.78, 22.89, 3.86, and 1.16 mg/mL, respectively. Antioxidant activities of crude extract and its solvent fractions were evaluated using 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical, authentic ONOO- and ONOO-generated from SIN-1, occurrence of intracellular reactive oxygen species (ROS) and NO. The scavenging effect of each fraction on DPPH radical increased in the order of n-BuOH > n-hexane > 85% aq. MeOH > H2O fractions. The excellent scavenging effects on both authentic ONOO- and induced ONOO-from SIN-1 were shown for all fractions except H2O fraction. The n-BuOH fraction, inter alia, showed the highest scavenging activity, comparable with L-ascorbic acid at 0.1 ㎍/ml.
The levels of intracellular reactive oxygen species (ROS) were measured for the crude extract and its solvent fractions using 2',7'-dichlorofluorescin diacetate (DCF-DA) as fluorescence probe in Raw 264.7 cells. All fractions except H2O fraction significantly decreased level of intracellular ROS.
Reactive NO has been known to generate peroxynitrite that is a latent oxidizing agent of biomolecules, by the chemical reaction between NO and superoxide. Therefore, NO production is an important step in the regulation of NO-mediated diseases. To measure the inhibitory effects of crude extract and its solvent fractions on NO production in LPS-induced Raw 264.7 cells, nitrite accumulation was measured. Among them, n-BuOH and 85% aq. MeOH fractions exhibited potent inhibitory activities compared to control.
It has been well-known that elevated nitric oxide (NO) production and oxidative stress in macrophages contribute to development of chronic disease and the inflammatory process. During inflammatory process, large amounts of proinflammatory mediator NO is generated by the iNOS. The mRNA expression level of iNOS in LPS-treated macrophage was examined in order to evaluate whether crude extract and its solvent fractions reveal antiinflammatory effects. The expression level of iNOS was significantly reduced by 85% aq.MeOH and n-BuOH fractions.
Matrix metalloproteinases (MMPs), a family of zinc-dependent endopoptidss, have been associated with tumor cell invasion and metastasis due to their ability to hydrolyze a variety of extracellular matrix (ECM). ROS can positively activate several matrix metalloproteinases (MMPs) that contribute vitally to an inflammatory network, and this activation can be blocked by an antioxidant. Of the MMPs, MMP-2 (the 72-kDa type Ⅳcollagenase) and MMP-9 (92-kDa type Ⅳ collagenase) have been explored extensively because they play a pivotal role in the degradation of ECM. The inhibitory effects of crude extract and its solvent fractions on MMP-2 and -9 were evaluated in HT1080cells using RT-PCR and gelatin zymography. n-BuOH fraction significantly down-regulated mRNA expression levels of MMP-9 and -2, especially showing ~50% inhibition ratio for MMP-9. Phlorotannins isolated from n-BuOH fraction were also estimated for their inhibitory effect on MMP-2 and -9. All compounds inhibited dose-dependently mRNA expression levels of MMP-2 and -9. Particularly, compounds 3 and 6 exerted the potent inhibitory effects on both MMP-2 and -9. On the other hand, compounds 4 and 5 strongly inhibited MMP-9 and -2, respectively. However, they seldom inhibited MMP-2 and -9, respectively.
In conclusion, present work demonstrated the neutraceutical properties of E. cava, especially the potential for its use as an antioxidant and antiinflammatory agent, and chemopreventive ingredient for cancer through MMP-2 and -9 mechanisms in addition to its original usage as food source.