Natalia P. Mishchenko

Echinochrome A Protects Mitochondrial Function in Cardiomyocytes against Cardiotoxic Drugs

Echinochrome A (Ech A) is a naphthoquinoid pigment from sea urchins that possesses antioxidant, antimicrobial, anti-inflammatory and chelating abilities. Although Ech A is the active substance in the ophthalmic and cardiac drug Histochrome®, its underlying cardioprotective mechanisms are not well understood. In this study, we investigated the protective role of Ech A against toxic agents that induce death of rat cardiac myoblast H9c2 cells and isolated rat cardiomyocytes. We found that the cardiotoxic agents tert-Butyl hydroperoxide (tBHP, organic reactive oxygen species (ROS) inducer), sodium nitroprusside (SNP; anti-hypertension drug), and doxorubicin (anti-cancer drug) caused mitochondrial dysfunction such as increased ROS level and decreased mitochondrial membrane potential. Co-treatment with Ech A, however, prevented this decrease in membrane potential and increase in ROS level. Co-treatment of Ech A also reduced the effects of these cardiotoxic agents on mitochondrial oxidative phosphorylation and adenosine triphosphate level. These findings indicate the therapeutic potential of Ech A for reducing cardiotoxic agent-induced damage.

Echinochrome A Increases Mitochondrial Mass and Function by Modulating Mitochondrial Biogenesis Regulatory Genes

Echinochrome A (Ech A) is a natural pigment from sea urchins that has been reported to have antioxidant properties and a cardio protective effect against ischemia reperfusion injury. In this study, we ascertained whether Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation in rat cardio myoblast H9c2 cells. To study the effects of Ech A on mitochondrial biogenesis, we measured mitochondrial mass, level of oxidative phosphorylation, and mitochondrial biogenesis regulatory gene expression. Ech A treatment did not induce cytotoxicity. However, Ech A treatment enhanced oxygen consumption rate and mitochondrial ATP level. Likewise, Ech A treatment increased mitochondrial contents in H9c2 cells. Furthermore, Ech A treatment up-regulated biogenesis of regulatory transcription genes, including proliferator-activated receptor gamma co-activator (PGC)-1α, estrogen-related receptor (ERR)-α, peroxisome proliferator-activator receptor (PPAR)-γ, and nuclear respiratory factor (NRF)-1 and such mitochondrial transcription regulatory genes as mitochondrial transcriptional factor A (TFAM), mitochondrial transcription factor B2 (TFB2M), mitochondrial DNA direct polymerase (POLMRT), single strand binding protein (SSBP) and Tu translation elongation factor (TUFM). In conclusion, these data suggest that Ech A is a potentiated marine drug which enhances mitochondrial biogenesis.

Echinochrome A Improves Exercise Capacity during Short-Term Endurance Training in Rats

Echinochrome A (Echi A) improves mitochondrial function in the heart; however, its effects on skeletal muscle are still unclear. We hypothesized that Echi A administration during short-term exercise may improve exercise capacity. Twenty-four male Sprague-Dawley rats were randomly divided into the following groups: control group (CG), Echi A-treated group (EG), aerobic exercise group (AG), and aerobic exercise treated with Echi A group (AEG) (n = 6 per group). Echi A was administered intra-peritoneally (0.1 mg/kg of Echi A in 300 µL phosphate-buffered saline) daily 30 min before each exercise training. The AG and AEG groups performed treadmill running (20 m/min, 60 min/day) five days/week for two weeks. The exercise capacity was significantly higher in the AG and AEG groups compared to other groups. Interestingly, the exercise capacity increased more effectively in the AEG group. The body weight in the EG tended to be slightly lower than that in the other groups. There were no significant changes in the plasma lipids among the groups. However, the gastrocnemius muscle mitochondria content was greater in the EG and AEG groups. These findings show that Echi A administration after short-term endurance training enhances exercise capacity, which was associated with an increase in skeletal muscle mitochondrial content.

Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A from Sea Urchin Scaphechinus mirabilis

Echinochrome A (EchA) is a dark-red pigment of the polyhydroxynaphthoquinone class isolated from sea urchin Scaphechinus mirabilis. Acetylcholinesterase (AChE) inhibitors are used in the treatment of various neuromuscular disorders, and are considered as strong therapeutic agents for the treatment of Alzheimer’s disease (AD). Although EchA is clinically used to treat ophthalmic diseases and limit infarct formation during ischemia/reperfusion injury, anti-AChE effect of EchA is still unknown. In this study, we investigated the anti-AChE effect of EchA in vitro. EchA and its exhausted form which lost anti-oxidant capacity did not show any significant cytotoxicy on the H9c2 and A7r5 cells. EchA inhibited AChE with an irreversible and uncompetitive mode. In addition, EchA showed reactive oxygen species scavenging activity, particularly with nitric oxide. These findings indicate new therapeutic potential for EchA in treating reduced acetylcholine-related diseases including AD and provide an insight into developing new AChE inhibitors.

Diversity of Polyhydroxynaphthoquinone Pigments in North Pacific Sea Urchins

Using high‐performance liquid chromatography with diode‐array detection and mass spectrometry (HPLC‐DAD/MS) we investigated the composition of polyhydroxynaphthoquinone (PHNQ) pigments from sea urchins Strongylocentrotus pallidus, St. polyacanthus, St. droebachiensis, Brisaster latifrons and Echinarachnius parma, collected in the Sea of Okhotsk and the Bering Sea. Identification of PHNQ pigments from sea urchins St. polyacanthus, B. latifrons, and E. parma was performed for the first time. Among the usual PHNQ pigments, mono‐ and dimethoxy derivatives of spinochrome E, not previously found in other sea urchins, were discovered in St. polyacanthus and St. droebachiensis. In St. droebachiensis, two monomethoxy derivatives of echinochrome A were detected, isolated previously from only tropical sea urchins. It was found that the composition and total content of pigments of St. droebachiensis depends on the collection area of the sea urchins and its depth and varies from 88 to 331 μg/g of dry shells. Sea urchins St. pallidus, B. latifrons and E. parma had average values for PHNQ pigment content, approximately 30 μg/g, and St. polyacanthus had a low PHNQ content, 13 μg/g.

Naphthoquinones of the spinochrome class: occurrence, isolation, biosynthesis and biomedical applications

Quinones are widespread in nature and have been found in plants, fungi and bacteria, as well as in members of the animal kingdom. More than forty closely related naphthoquinones have been found in echinoderms, mainly in sea urchins but occasionally in brittle stars, sea stars and starfish. This review aims to examine controversial issues on the chemistry, biosynthesis, functions, stability and application aspects of the spinochrome class, a prominent group of secondary metabolites found in sea urchins. The emphasis of this review is on the isolation and structure of these compounds, together with evaluation of their relevant biological activities, source organisms, the location of origin and methods used for isolation and identification. In addition, the studies of their biosynthesis and ecological function, stability and chemical synthesis have been highlighted. This review aims to establish a focus for future spinochrome research and its potential for benefiting human health and well-being.

Extraction, structural characterization and stability of polyhydroxylated naphthoquinones from shell and spine of New Zealand sea urchin (Evechinus chloroticus)

The extraction of polyhydroxylated naphthoquinone (PHNQ) pigments from the shell and spines of the New Zealand sea urchin Evechinus chloroticus was evaluated using six different macroporous resins as an alternative to using organic solvent extraction alone. Four of the resins evaluated in this study (D4006, D4020, D101 and NKA-9) provided the best extraction of PHNQ pigments in terms of the overall adsorption and desorption of E. chloroticus PHNQ pigments from the resins. Organic solvents alone had a higher yield of PHNQs than the resins. The PHNQ composition was characterised by high-performance liquid chromatography (HPLC) with diode-array detection and mass spectrometry. Five PHNQ compounds (spinochromes E, B, C, A and echinochrome A), and three aminated PHNQ compounds (spinamine E, echinamines A and B) were identified. The pigments were found to be prone to degradation on exposure to light, with the aminated PHNQ pigments being the least stable.

A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.

Cytotoxicity of quinonoid pigments from sea urchins

T study was to confirm the effects of anti-melanogenesis and anti-inflammatory effects from Opuntia humifusa fruit and stem extracts. A potent anti-oxidant activity was showed from the leaf extract at IC50 value of 38.33±1.07 μg/mL and fruit extract at IC50 value of 40.23±2.21 μg/mL by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Also, phenolic contents were confirmed through total phenolic assay by High Performance Liquid Chromatography (HPLC). Fraction of taxifolin from leaf extract was identified using HPLC and gas chromatography/mass spectrometry. The extracts of Opuntia humifusa fruit and stem were confirmed about toxicity effect in B16 F1 by cell viability. It was also observed that the melanin contents were decreased. Opuntia humifusa fruit and stem extracts had a positive effect on melanin synthesis inhibition for skin whitening. In investigating the anti-inflammatory activities of Opuntia humifusa, the results of cell viability indicated that taxifolin did not show any cytotoxicity effect on RAW264.7 cells at 500 μM of concentration. The result show that taxifolin inhibited Lipo Polysaccharide (LPS)-induced production of Nitrite Oxide (NO). In addition, taxifolin indicated the inhibition of Lipo Polysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-6 productions by cytokine assay and Cyclooxygenase (COX)-2 expressions by Western blot analysis, meaning that taxifolin had a significant anti-inflammatory effect. Our results suggested that taxifolin from Opuntia humifusa has anti-melanogenesis and anti-inflammatory activities.T investigation of natural products isolated from marine sources is an interesting approach in the development of new antiviral agents. Sulfated Polysaccharides (SPs) and Polyphenols (PPs) extracted from five Mexican seaweeds (Macrocystis pyrifera, Eisenia arborea, Pelvetia compressa, Ulva intestinalis and Solieria filiformis) were tested in this study in order to evaluate their effect on the Measles Virus (MeV) infection in vitro. The effect of SPs and PPs on cell viability was determined by MTT assay; their antiviral activity on MeV infection was evaluated by syncytia reduction assays and confirmed by qPCR. The tested metabolites showed antiviral activity and no cytotoxicity at inhibitory concentrations, resulting in therapeutic indexes >1500 in all cases. Looking at the mode of action of these metabolites, time of addition experiments and viral penetration assays suggest that SPs tested in this study are acting at different levels within the first stages in viral infection, while Virucidal assays showed that PPs have a direct inactivation effect on the virions. SP2, SP5, PP2 and PP5 showed the highest antiviral activities and were selected to determine their combined effect. The best synergistic effect was observed for combinations of SP2 at IC50 with SP5 at IC25, and SP5 at IC25 with PP2 at IC50. The synergistic effect observed in this study would allow reducing the treatment dose and minimizing or delaying the induction of antiviral resistance. SPs and PPs of the selected seaweed species thus appear as promising candidates for the development of natural antiviral agents.

Antioxidant quinonoid pigments from ceolomic fluid of Far Eastern sea urchins

 Collection of T.bellerica fruits in Jan-Feb 2015 (Voucher LWG 98572).  Fruits were grilled at 150 deg cent, extracted with 50% ethanol and the extract was partitioned into hexane, chloroform, ethyl acetate and butanol fractions.  Phytochemical studies: 1. Estimation of phenols, flavonoids and tannins. 2. Non-targeted metabolic profile of ethyl acetate fraction (GC-MS) of dried (DF) & grilled fruits (GF). 3. HPTLC profile (gallic and ellagic acid).  In-vitro studies 1. DPPH free radical scavenging 2. Antilipid peroxidation assay 3. Antibacterial activity (E.coli).  In-vivo studies 1. Anti-diarrheal activity (castor oil induced diarrhea). 2. Antiulcer activity: aspirin induced, pylorus ligation induced and ethanol induced ulcer models. 3. Estimation of pro-inflammatory cytokines TNFα and IL-6 and anti-inflammatory cytokine IL-10. (a) Inhibition of DPPH free radical; (b) IC50 of 50% ethanolic extract and successive fractions of dried and grilled fruits of T. bellerica a b Anti-lipid peroxidation using goat liver homogenate. Estimation of phenols, flavonoids and tannins