Zhong-Ji Qian

Purification and Antioxidant Properties of Bigeye Tuna (Thunnus obesus) Dark Muscle Peptide on Free Radical-Mediated Oxidative Systems

To produce bioactive peptides from by-products of fish processing, bigeye tuna dark muscle was hydrolyzed using various enzymes (alcalase, alpha-chymotrypsin, neutrase, papain, pepsin, and trypsin), and the hydrolysates were evaluated for antioxidant activity. Considering the results of degree of hydrolysis and antioxidant activities, peptic hydrolysate was used for further studies to identify a potent antioxidant peptide. Antioxidant peptide was purified using consecutive chromatographic methods and was identified as being H-Leu-Asn-Leu-Pro-Thr-Ala-Val-Tyr-Met-Val-Thr-OH (MW 1,222 Da) by quantitative time-of-flight electrospray ionization mass spectrometry. Purified antioxidant peptide from bigeye tuna dark muscle (APTDM) was investigated for its antioxidant activities using both free radical scavenging effects and polyunsaturated fatty acid (PUFA) peroxidation inhibitory activity. The results showed that APTDM effectively quenched with low 50% inhibitory concentration values compared to vitamin C as a positive control against four different free radicals: 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, superoxide, and alkyl radical. APTDM also inhibited PUFA peroxidation in a linoleic acid emulsion system, and the activity was similar to that of alpha-tocopherol. We further investigated its antioxidant activities on cellular systems, and the results showed that APTDM significantly scavenged cellular radicals and enhanced the viability of tert-butyl hydroperoxide-induced cytotoxicity. These results indicate that APTDM or a peptide fraction containing APTDM would be a beneficial ingredient for functional food and/or pharmaceuticals.

Paeonol from Hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways.

Inflammation has recently been implicated as a critical mechanism responsible for neurodegenerative diseases. In this study, paeonol (1-(2-hydroxy-4-methoxyphenyl)ethanone) isolated from the sea horse Hippocampus kuda Bleeler was studied as an agent to suppress LPS induced activation of BV-2 microglial and RAW264.7 macrophage cells. The results obtained showed that paeonol significantly suppressed LPS induced release of pro-inflammatory products such as nitric oxide (NO), prostaglandin E2 (PGE(2)), and cytokines; tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Furthermore, the compound down regulated the protein and gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β and IL-6 in both cell lines. Molecular signaling pathway studies showed that paeonol inhibited the translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits to the nucleus by blocking IKKα/β (IκB kinase α/β) mediated degradation of IκBα. Moreover, it suppressed the phosphorylation of mitogen activated protein kinase (MAPK) pathway molecules; c-Jun N-terminal kinases (JNK) and p38 in both cell lines. Collectively these results indicate that paeonol blocked the LPS stimulated inflammatory responses in BV-2 and RAW264.7 cells via modulating MAPK and NF-κB signaling pathways. Therefore, paeonol could be a promising candidate to be used in neuro-inflammatory therapy.

Protective effect of isorhamnetin 3-О-β-d-glucopyranoside from Salicornia herbacea against oxidation-induced cell damage

Isorhamnetin 3-O-beta-D-glucopyranoside (1) was isolated from Salicornia herbacea. The inhibitory effects of compound 1 on oxidative stress were evaluated in free-cellular and cellular systems. An increased concentration of compound 1 not only exhibited dose-dependent scavenging activities on the generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radicals, but also significantly decreased levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner. Further, antioxidative mechanisms by compound 1 were examined by measuring the intracellular glutathione (GSH) level and expression levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione reductase and heme oxygenase-1 (HO-1). Compound 1 significantly elevated GSH level as well as expression levels of antioxidant enzymes which were closely related with amount of cellular ROS. In addition, it significantly inhibited oxidative damage of purified genomic DNA and suppressed activity of myeloperoxidase (MPO), a generator of potent oxidant (hypochlorous acid), in tumor necrosis factor-alpha (TNF-alpha) stimulated human myeloid cells. Therefore, these results suggested that compound 1 has a therapeutic effectiveness in prevention of ROS-induced cellular damage and is a candidate worthy of being developed as a potential natural antioxidant related to oxidative stress.

Inhibitors of oxidation and matrix metalloproteinases, floridoside, and D-isofloridoside from marine red alga Laurencia undulata.

In the exploration of abundant marine biological resources, edible red alga Laurencia undulata led to two bioactive isolates: floridoside (1) and D-isofloridoside (2). For the first time, the antioxidant properties of both derivatives (1 and 2) were characterized via free radical scavenging using the ESR technique, reactive oxygen species (ROS) inhibition, membrane protein oxidation, myeloperoxidase (MPO) inhibition, gene expression levels of glutathione (GSH) and superoxide dismutase (SOD), and protein expression of MMP-2 and MMP-9. The results demonstrate that floridoside and D-isofloridoside possess significant antioxidant capacity and are potential inhibitors of MMP-2 and MMP-9. These results clarified that these components may be responsible for the relative activities of crude extract from this genus, which is used as folk medicine. Furthermore, the structure-activity relationships were also suggested. Both isomers could be effective candidates for applications in food and pharmaceutical fields as natural marine antioxidants.

Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-κB activation, and induces human osteoblastic and chondrocytic differentiation

Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptides inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-kappaB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-kappaB in MG-63 cells and MAPKs/NF-kappaB in SW-1353 cells.

Cathepsin B inhibitory activities of three new phthalate derivatives isolated from seahorse, Hippocampus Kuda Bleeler

Three new phthalate acid derivatives, 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecyl phthalate (1), 2-ethyldecyl 2-ethylundecyl phthalate (2), and bis(2-ethyldodecyl) phthalate (3), were isolated from seahorse, Hippocampus Kuda Bleeler, together with a known natural analog bis(2-ethylheptyl) phthalate (4). The structures of these compounds were elucidated mainly by means of the comprehensive analysis of their NMR spectroscopic data. The four phthalate derivatives showed dose-dependent cathepsin B inhibitions activities with IC(50) values of 0.13 mM (1), 0.21 mM (2), 0.18 mM (3), and 0.29 mM (4), respectively.

Sea cucumber, Stichopus japonicus ethyl acetate fraction modulates the lipopolysaccharide induced iNOS and COX-2 via MAPK signaling pathway in murine macrophages

The sea cucumber Stichopus japonicus is an important food and traditional medicine in Asian countries. However, ethyl acetate solvent fraction of S. japonicus (SCEA-F) is largely unknown for its anti-inflammatory activity and related molecular mechanisms. In this study, effect of SCEA-F on inflammation was investigated in LPS stimulated RAW264.7 cells. SCEA-F significantly inhibited the productions of NO and PGE(2) by inhibiting iNOS and COX-2 at their protein and gene levels. The production and the gene transcription of pro-inflammatory cytokines are also inhibited. The responsible molecular signaling for these inhibitory actions was found to be through suppression of the phosphorylation of MAPK molecules; ERK and p38 MAPK. These results indicate that SCEA-F inhibits LPS-induced inflammatory response via blocking of MAPK signaling pathway in murine macrophages, thus demonstrated its in vitro anti-inflammatory potential. Therefore it could be suggested that SCEA-F could be effectively used in functional food preparations.

Gliotoxin Isolated from Marine Fungus Aspergillus sp. Induces Apoptosis of Human Cervical Cancer and Chondrosarcoma Cells

Gliotoxin, a secondary metabolite produced by marine fungus Aspergillus sp., possesses various biological activities including anticancer activity. However, the mechanism underlying gliotoxin-induced cytotoxicity on human cervical cancer (Hela) and human chondrosarcoma (SW1353) cells remains unclear. In this study, we focused on the effect of gliotoxin induction on apoptosis, the activating expressions of caspase family enzymes in the cells. Apoptotic cell levels were measured through DAPI and Annexin V/Propidium Iodide (PI) double staining analysis. The apoptotic protein expression of Bcl-2 and caspase family was detected by Western blot in Hela and SW1353 cells. Our results showed that gliotoxin treatment inhibited cell proliferation and induced significant morphological changes. Gliotoxin induced apoptosis was further confirmed by DNA fragmentation, chromatin condensation and disrupted mitochondrial membrane potential. Gliotoxin-induced activation of caspase-3, caspase-8 and caspase-9, down-regulation of Bcl-2, up-regulation of Bax and cytochromec (cyt c) release showed evidence for the gliotoxin activity on apoptosis. These findings suggest that gliotoxin isolated from marine fungus Aspergillus sp. induced apoptosis in Hela and SW1353 cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death.

SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-κB/p38 kinase

Considerable efforts have been taken to identify natural peptides as potential bioactive substances. In this study, novel peptide (SHP-1) derived from seahorse (Hippocampus, Syngnathidae) hydrolysate was explored for its inhibitory effects on collagen release in arthritis with the investigation of its underlying mechanism of action. The efficacy of SHP-1 was determined on cartilage protective effects such as inhibition of collagen and GAG release. SHP-1 was able to suppress not only the expression of collagenases 1 and 3, but also the production of NO via down-regulation of iNOS. However, it presented an irrelevant effect on the level of GAG release in chondrocytic and osteoblastic cells. Inhibition of collagen release by SHP-1 is associated with restraining the phosphorylation of NF-kappaB and p38 kinase cascade. Therefore, it could be suggested that SHP-1 has a potential to be used in arthritis treatment.

Statistical optimization of microalgae Pavlova lutheri cultivation conditions and its fermentation conditions by yeast, Candida rugopelliculosa

In this study, sequential strategy based design was applied to optimize the microalgae, Pavlova lutheri mass culture conditions and fermentation conditions of the cultured algae by proteolytic yeast Candidia rugopelliculosa to obtain small peptide chains. This optimization of culture and fermentation conditions by response surface methodology (RSM) finally leads to effective purification of a bioactive peptide MPGPLSPL (793.01 Da) with hydroxyl radical scavenging activity. Collectively, these results indicated that microalgae P. lutheri can enhance the hydroxyl radical inhibiting effect through protein hydrolysis process under RSM optimal condition.