Lipase inhibitory activity of ethyl acetate fraction from Ecklonia cava extracts

Obesity is a key contributing risk factor to cardiovascular disease, certain cancers, and diabetes. Much effort has being made to investigate potential inhibitors against lipase from natural products. The ethyl acetate (EA) extract of Ecklonia cava (EC) were tested for their ability to inhibit pancreatic lipase activity in vitro. The 22 sub-fractions from EA extract were separated using silica gel column chromatography. Among the sub-fractions, the EA6 sub-fraction exhibited the highest inhibitory activity. Dieckol compound was isolated from the EA6 sub-fraction, which inhibited the lipase activity in a concentrationdependent manner with IC₅₀ value at 0.26 mg/mL. These results suggest that EC has potential as a natural antiobesity agent.     

Molecular docking studies of a phlorotannin, dieckol isolated from Ecklonia cava with tyrosinase inhibitory activity

In this study, the phlorotannin dieckol, which was isolated from the brown alga Ecklonia cava, was examined for its inhibitory effects on melanin synthesis. Tyrosinase inhibitors are important agents for cosmetic products. We therefore examined the inhibitory effects of dieckol on mushroom tyrosinase and melanin synthesis, and analyzed its binding modes using the crystal structure of Bacillus megaterium tyrosinase (PDB ID: 3NM8). Dieckol inhibited mushroom tyrosinase with an IC(50) of 20μM and was more effective as a cellular tyrosinase having melanin reducing activities than the commercial inhibitor, arbutin, in B16F10 melanoma cells, and without apparent cytotoxicity. It was found that dieckol behaved as a non-competitive inhibitor with l-tyrosine substrates. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and dieckol. These molecular modeling studies were successful (calculated binding energy value: -126.12kcal/mol), and indicated that dieckol interacts with His208, Met215, and Gly46. These results suggest that dieckol has great potential to be further developed as a pharmaceutical or cosmetic agent for use in dermatological disorders associated with melanin.     

Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity

Matrix metalloproteinase (MMP) inhibitors have been identified as potential therapeutic candidates for metastasis, arthritis, chronic inflammation and wrinkle formation. For the first time here we report a detailed study on the inhibitory effects of phlorotannins in brown algae, Ecklonia cava (EC) on MMP activities in cultured human cell lines. A novel gelatin digestion assay could visualize complete inhibition of bacterial collagenase-1 activity at 20 microg/ml of EC extract during preliminary screening studies. Sensitive fluorometric assay revealed that EC extract can specifically inhibit both MMP-2 and MMP-9 activities significantly (P < 0.001) at 10 microg/ml. In addition, artificially induced activities of MMP-2 and MMP-9 in human dermal fibroblasts and HT1080 cells were inhibited by EC extract in a more or less similar manner to the positive control doxycycline. Even though the expression levels of MMPs differ from one cell type to the other, gelatin zymography clearly revealed that both MMP expression and activity in cells can be inhibited by EC extract. More interestingly, EC extract did not exert any cytotoxic effect even at 100 microg/ml anticipating its potential use as a safe MMP inhibitor.     

Anti-HIV-1 activity of a new scorpion venom peptide derivative Kn2-7

For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC(50) value of 2.76 μg/ml (1.65 μM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1.     

Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata

Searching for anti-HIV-1 protease (PR) inhibitors of Thai medicinal plants led to the isolation of a new cyclohexenyl chalcone named panduratin C (1) and chalcone derivatives (2-6) from the methanol extract of Boesenbergia pandurata rhizomes. The known compounds were identified to be panduratin A (2), hydroxypanduratin A (3), helichrysetin (4), 2',4',6'-trihydroxyhydrochalcone (5), and uvangoletin (6). The structures of all compounds were elucidated on the basis of chemical and spectroscopic methods. It was found that 3 possessed the most potent anti-HIV-1 PR activity with an IC50 value of 5.6 microM, followed by 2 (IC50 = 18.7 microM), whereas other compounds exhibited only mild activity. Structure-activity relationships of these compounds on anti-HIV-1 PR activity are summarized as follows: (1) hydroxyl moiety at position 4 conferred higher activity than methoxyl group; (2) prenylation of dihydrochalcone was essential for activity; (3) hydroxylation at position 4' reduced activity; and (4) introduction of double bond at C1' and C6' of chalcone gave higher activity. As regards active constituents contained in B. pandurata rhizomes, hydroxypanduratin A (3) and panduratin A (2) are active principles against HIV-1 PR.     

A phloroglucinol derivative from the brown alga Zonaria tournefortii

The major lipid component of the brown seaweed Zonaria tournefortii was identified as the acylphlorogluconol (all Z)-2′-icosa-5,8,11,14,17-pentae     

A phloroglucinol derivative of Dryopteris abbreviata

A new phloroglucinol derivative, abbreviatin BB, has been isolated from Dryopteris abbreviata. Its structure was elucidated to be methylene-bis-met     

Immunomodulatory Effects of an Enzymatic Extract from Ecklonia cava on Murine Splenocytes

We investigated whether the brown seaweed Alariaceae Ecklonia cava (E. cava) has immunological effects on splenocytes in vitro. For that purpose, we prepared an enzymatic extract from E. cava (ECK) by using the protease, Kojizyme. Here, ECK administered to ICR mice dramatically enhanced the proliferation of their splenocytes and increased the number of their lymphocytes, monocytes and granulocytes. In flow cytometry assays performed to identify in detail the specific phenotypes of these proliferating cells after ECK treatment, the numbers of CD4⁺ T cells, CD8⁺ T cells and CD45R/B220⁺ B cells increased significantly compared to those in untreated controls. In addition, the mRNA expression and production level of Th1-type cytokines, i.e., TNF-α and IFN-γ, were down-regulated, whereas those of Th2-type cytokines, i.e., IL-4 and IL-10, were up-regulated by ECK. Overall, this dramatic increase in numbers of splenocytes indicated that ECK could induce these cells to proliferate and could regulate the production of Th1- as well as Th2-type cytokines in immune cells. These results suggest that ECK has the immunomodulatory ability to activate the anti-inflammatory response and/or suppress the proinflammatory response, thereby endorsing its usefulness as therapy for diseases of the immune system. Y-J. Jeon and Y. Jee contributed equally to this study.     

Dieckol,a SARS-CoV 3CLpro inhibitor,isolated from the edible brown algae Ecklonia cava

SARS-CoV 3CL^pro plays an important role in viral replication. In this study, we performed a biological evaluation on nine phlorotannins isolated from the edible brown algae Ecklonia cava. The nine isolated phlorotannins (1–9), except phloroglucinol (1), possessed SARS-CoV 3CL^pro inhibitory activities in a dose-dependently and competitive manner. Of these phlorotannins (1–9), two eckol groups with a diphenyl ether linked dieckol (8) showed the most potent SARS-CoV 3CL^pro trans/cis-cleavage inhibitory effects (IC₅₀s = 2.7 and 68.1 μM, respectively). This is the first report of a (8) phlorotannin chemotype significantly blocking the cleavage of SARS-CoV 3CL^pro in a cell-based assay with no toxicity. Furthermore, dieckol (8) exhibited a high association rate in the SPR sensorgram and formed extremely strong hydrogen bonds to the catalytic dyad (Cys145 and His41) of the SARS-CoV 3CL^pro.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Callus formation in Ecklonia cava Kjellman (Laminariales,Phaeophyta)

Explants from stipes and meristems of Ecklonia cava were incubated on six media under several culture conditions. Both stipe and meristem explants developed calluses three to six weeks after inoculation onto all media except AS PC-1. Calluses developed on stipe explants but did not develop on meristem explants at a temperature of 23 °C. Temperatures from 8 to 13 °C were favorable for callus development. Callus development on meristem explants required light but callus development on stipe explants did not.     

Antiproliferative activity of sulfated polysaccharide isolated from an enzymatic digest of Ecklonia cava on the U-937 cell line

A sulfated polysaccharide purified from a brown alga Ecklonia cava, having high anticoagulant activity was investigated for its antiproliferative effect on murine colon carcinoma (CT-26), human leukemic monocyte lymphoma (U-937), human promyelocytic leukemia (HL-60), and mouse melanoma (B-16) cell lines. The sulfated polysaccharide isolated and purified from an enzymatic extract of E. cava had a good selective tumor cell growth inhibition effect; its effect on HL-60 and U-937 was especially promising. The IC₅₀ value for the sulfated polysaccharide from E. cava (ECSP) on U-937 was 43.9 μg mL⁻¹. The presence of the sample in the cell culture media stimulated the induction of apoptosis, revealed by nuclear staining with Hoechst 33342. The apoptosis induction was confirmed by the cell cycle analysis, while pronounced sub-G1 phase arrests of 9.5% and 13.8% were also clearly observed when the cells were treated at 15 and 30 μg mL⁻¹ of ECSP in the U-937 cell line, respectively. After a 24-h incubation period, ECSP dose-dependently enhanced the DNA fragmentation on the U-937 cell line as observed in the agarose gel electrophoresis assay. To rule out the action mechanism of ECSP for its anticancer activity, some western blot analyses were conducted with several antibodies (caspase-7, caspase-8, Bax, Bcl-xL, and PARP) and ECSP had a clear effect on the caspase -7 and 8 which cleave protein substrates, including PARP, an inducer of apoptosis responsible for DNA cleavage. Moreover, ECSP controlled the cellular transmembrane molecules like Bax and Bcl-xL. Taken together, the above results demonstrate that the apoptosis for antiproliferative effect of ECSP was clearly induced on U-937 cells.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

Anti-inflammatory activity of phlorotannin-rich fermented Ecklonia cava processing by-product extract in lipopolysaccharide-stimulated RAW 264.7 macrophages

In this study, fermentation was employed as a tool to further increase the bioactive potential of processing by-product from a brown seaweed, Ecklonia cava, which can be obtained from food and cosmetic industries after its polyphenol extraction. The fermentation process was done for 24 h using an industrially important microorganism Candida utilis prior to being extracted with 80 % ethanol. The anti-inflammatory potential of the fermented E. cava processing by-product extract (FEPBE) was evaluated in vitro. The phlorotannin-rich FEPBE dose-dependently inhibited the nitric oxide production, prostaglandin-E₂ production and suppressed the inducible nitric oxide synthase and cyclooxygenase-2 expressions in lipopolysaccharide-stimulated RAW 264.7 cells. The release of pro-inflammatory cytokines, interleukin-1β and interleukin-6, was significantly suppressed by the extract in a dose-dependent manner. Due to the profound anti-inflammatory activity, FEPBE appears as a value-added biomass fraction that can be exploited in numerous industrial applications as a source of functional ingredients.     

A new polyisoprenylated phloroglucinol derivative from Hypericum perfoliatum (Clusiaceae)

Hyperfoliatin, a new polyisoprenylated phloroglucinol derivative was isolated from the aerial parts of Hypericum perfoliatum (Clusiaceae) collected in Algeria. The structure of hyperfoliatin was elucidated on the basis of its spectral data, mainly MS and multiple-pulse NMR.     

Inhibitory effects of polyphenols isolated from marine alga Ecklonia cava on histamine release

In order to clarify the modern anti-allergy activity of crude extract of Ecklonia cava, which is an edible marine brown alga used as food ingredient and folk drug against allergy disease in Korea due to the abundant content of phlorotannin derivatives, the continuous chemical investigation led to the isolation of the two main bioactive phlorotannin derivatives 6,6′-bieckol (3) and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (4) for the first time from this genus, together with phloroglucinol (1) and dieckol (2). Their structures were confirmedly elucidated by the comprehensive analyses of MS and NMR spectroscopic data. Meanwhile, these derivatives were assessed by histamine release assay on human basophilic leukemia (KU812) and rat basophilic leukemia (RBL-2H3) cultured cell lines, respectively. Furthermore, flow cytometric analysis indicated that the potential anti-allergic mechanism is the suppression of binding activity between IgE and Fc?RI. Finally, the relationships of structure–activity were also suggested. This research indicated that these main derivatives can be used as potential candidates for foodstuff, cosmetic and pharmaceutical industries.     

Anti-HIV-1 integrase diterpenoids from Dichrocephala benthamii

Five new clerodane diterpenoid dichrocephnoids A–E (1,2, 6–8) together with three known analogues were isolated from the whole herb of Dichrocephala benthamii C.B. Clarke. Their structures were elucidated on the basis of spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). The relative configurations of the new compounds were established by NOESY correlations, and the absolute configuration of 1 was determined on the basis of CD methods. Dichrocephnoid A (1) possesses a very rare pentacyclic ring system with highly oxygenated functionalities. All of the isolated compounds exhibited inhibitory activity against HIV-1 integrase, however, dichrocephnoid C (6) showed the most promising inhibition of the enzyme with IC₅₀ value of 12.35 ± 1.27 μM.     

Anti-HIV-1 activity of poly(mandelic acid) derivatives

Homo- and heterochiral poly(mandelic acid)s (PMDAs) were synthesized under strongly acidic, mildly acidic, and nonacidic conditions. The water-soluble fractions of these polymers were evaluated with respect to their inhibitory activity against the human immunodeficiency virus (HIV-1). Polymers were prepared via a step-growth mechanism, yielding linear polyesters. The polymers were characterized by CHS elemental microanalysis, X-ray fluorescence (XRF), and FT-IR spectroscopy. Polymers prepared by the three methods have different structures. Both elemental microanalysis and XRF indicated the presence of S in those polymers prepared by treatment with concentrated H2SO4, which were the only ones exhibiting inhibitory and virucidal activity against HIV-1, mediated by their binding to cellular co-receptor binding sites on the virus envelope glycoprotein gp120. Additionally, FT-IR spectroscopy indicated the complete absence of C=O functionality in the H2SO4-prepared PMDA.     

Enzymatic synthesis of an acylated phloroglucinol derivative with phloroglucinol and vinyl octanoate in acetonitrile

Monooctanoyl phloroglucinol with anti-microbial activities was synthesized from phloroglucinol (50 mM) and vinyl octanoate (150 mM) with agitation at 40 °C using 10 mg lipase AK (Pseudomonas fluorescens) in acetonitrile (1 ml, aw=0.07). The yield was approximately 70% as conversion rate of phloroglucinol over 3 days. The product, at 205 g ml^–1, was bacteriocidal against Escherichia coli and Staphylococcus epidermidis.