Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

Small Molecules with Thiourea Skeleton Induce Ethylene Response in Arabidopsis.

Ethylene is the only gaseous plant hormone that regulates several aspects of plant growth, from seedling morphogenesis to fruit ripening and organ senescence. Ethylene also stimulates the germination of Striga hermonthica, a root parasitic weed that severely damages crops in sub-Saharan Africa. Thus, ethylene response stimulants can be used as weed and crop control agents. Ethylene and ethephon, an ethylene-releasing compound, are currently used as ethylene response inducers. However, since ethylene is a gas, which limits its practical application, we targeted the development of a solid ethylene response inducer that could overcome this disadvantage. We performed chemical screening using Arabidopsis thaliana "triple response" as an indicator of ethylene response. After screening, we selected a compound with a thiourea skeleton and named it ZKT1. We then synthesized various derivatives of ZKT1 and evaluated their ethylene-like activities in Arabidopsis. Some derivatives showed considerably higher activity than ZKT1, and their activity was comparable to that of 1-aminocyclopropane-1-carboxylate. Mode of action analysis using chemical inhibitors and ethylene signaling mutants revealed that ZKT1 derivatives activate the ethylene signaling pathway through interactions with its upstream components. These thiourea derivatives can potentially be potent crop-controlling chemicals.

D-Pinitol Improved Glucose Metabolism and Inhibited Bone Loss in Mice with Diabetic Osteoporosis.

Diabetic osteoporosis (DO) has been increasingly recognized as an important complication of diabetes. D-pinitol, a natural compound found in various legumes, is known for its anti-diabetic function, but its effect on DO has not been investigated. Two doses of pinitol (50 and 100 mg/kg Bw/d) were administered orally to experimentally induce the DO mouse model for 5 weeks. The results indicated that pinitol suppressed fasting blood glucose levels and tended to enhance impaired pancreatic function. Pinitol also suppressed serum bone turnover biomarkers, and improved dry femur weight, cancellous bone rate, and bone mineral content in the DO mice. Based on the inositol quantification using GC-MS in serum, liver, kidney, and bone marrow, the pinitol treatment significantly recovered the depleted D-chiro-inositol (DCI) content or the decreased the ratio of DCI to myo-inositol caused by DO. In short, our results suggested that pinitol improved glucose metabolism and inhibited bone loss in DO mice via elevating the DCI levels in tissues.

Preparation, Purification and Characterization of Antibacterial and ACE Inhibitory Peptides from Head Protein Hydrolysate of Kuruma Shrimp, Marsupenaeus japonicus.

Kuruma shrimp (Marsupenaeus japonicus) heads, as the main by-product of the seafood processing industry, are rich in underutilized high-quality protein. After papain hydrolysis at 50 °C for 4 h, the protein hydrolysate of shrimp heads was found to show notable antibacterial and angiotensin I-converting enzyme (ACE) inhibitory activities. After purification using two stages of revered-phase high-performance liquid chromatography (RP-HPLC), the antibacterial peptide VTVP and the ACE inhibitory peptide ARL/I were successfully identified from most active fractions by LC-MS/MS. Peptide VTVP was a desirable hydrophobic peptide, with a MIC value in the range from 1.62 to 8.03 mM against all tested pathogens. Peptide ARL/I exhibited potent ACE inhibitory activity, with an IC50 value of 125.58 µM, and was found to be a competitive inhibitor based on the Lineweaver-Burk plot. Moreover, the result of the molecular docking simulation indicated that the interaction binding between ARL/I and ACE was mainly stabilized by hydrogen bonds, as well as forming a coordinate bond with the Zn2+ site. The purified peptides did not show hemolytic activity toward rabbit erythrocytes. To sum up, the bioactive peptides isolated from shrimp heads could be applicable for food or pharmaceutical areas as promising ingredients.

Isolation and Characterization of Collagen and Collagen Peptides with Hyaluronidase Inhibition Activity Derived from the Skin of Marlin (Istiophoridae).

Type I and V collagens are the major components of fibrillogenic proteins in fish skin, and their hydrolysis products possess hyaluronidase inhibitory activity. In this study, for the first time, type I and V collagens were isolated from the skin of shortbill spearfish and striped marlin. Type I (2α1[I]α2[I]) and type V (α1[V]α3[V]α2[V]) collagens composed of distinct α-peptide chains with comparable structures were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometric chromatography. After enzymatic digestion, the collagen peptides were purified by using ultrafiltration (30 KDa) and high-performance liquid chromatography (RP-HPLC) to yield CPI-F3 and CPV-F4 fractions with strong hyaluronidase inhibition rates (42.17% and 30.09%, respectively). Based on the results of simulated gastrointestinal fluid, temperature, and pH stability assays, CPI-F3 and CPV-F4 exhibited stability in gastric fluid and showed no significant changes under the temperature range from 50 to 70 °C (p > 0.05). The results of this first research on the bioactivity of type V collagen peptides provide valuable information for the biomedical industry and show the potential for future bioactivity investigations of type V collagen and its peptides.

Cholesterol-binding ability of saponin from Japanese starfish.

Steroidal gylcosides are the predominant metabolites of starfish and are responsible for various biological activities. Some of these activities are recognized as a part of self-defense mechanism of starfish. Cholesterol-binding ability was evaluated with seven starfish crude extracts, where significantly (p < 0.05) highest ability (34%) was observed in Asterias amurensis and the lowest (16%) was attributed in Distolasterias nippon. To characterize the active compound exists in crude saponin from A. amurensis, the extract was subjected to thin layer chromatography following silica gel column chromatography. As the results, seven fractions (fr. A-G) were separated and frs. D and F demonstrated the highest cholesterol-binding ability (32% and 33%, respectively), equivalent to that of the A. amurensis extract. The isolated component (fr. F) was further separated (fr. F1-F3) for structural analysis. Based on cholesterol-binding ability result (29%), fr. F2 was analysed by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) and then nuclear magnetic resonance spectroscopy (NMR). The compound was identified as thornasteroside A, one of the major bioactive compounds already found in A. amurensis. The discovery of a saponin with cholesterol-binding ability has important implications not only for the utilization of starfish but also for food and pharmaceutical research.

Total Syntheses of Lepadiformine Marine Alkaloids with Enantiodivergency, Utilizing Hg(OTf)2 -Catalyzed Cycloisomerization Reaction and their Cytotoxic Activities.

The enantioselective total syntheses of lepadiformine marine alkaloids, azatricyclic natural products isolated from marine tunicates, were completed. These alkaloids have a unique chemical structure characterized by the trans-1-azadecalin (AB ring system) fused with the spirocyclic ring (AC ring system). Here we found that a cycloisomerization reaction from functionalized linear substrates to a 1-azaspiro[4.5]decane framework corresponding to the AC ring in lepadiformines is promoted by a catalytic amount of mercury(II) triflate (Hg(OTf)2 ). The total syntheses of (-)-lepadiformines A and B were achieved in 28 % and 21 % overall yields, respectively, through the novel cycloisomerization reaction. The syntheses of (+)- and (-)-lepadiformine C hydrochloride salts also enabled us to determine the absolute configuration of natural lepadiformine C. It has been found that a phenomenon of enantiodivergence occurs in lepadiformine alkaloids from a single species of marine tunicate, Clavelina moluccensis. The cytotoxic activities of synthesized lepadiformine hydrochloride salts and their synthetic intermediates were evaluated.

Zonarol, a sesquiterpene from the brown algae Dictyopteris undulata, provides neuroprotection by activating the Nrf2/ARE pathway.

Seaweed-origin electrophilic compounds are proposed as a class of neuroprotective compounds that provide neuroprotection through activation of the Nrf2/ARE pathway. Electrophilic hydroquinones are of particular interest due to their ability to become electrophilic quinones upon auto-oxidation. Although many marine plants produce a variety of electrophilic compounds, the detailed mechanism of action of these compounds remain unknown. Here, we focused on the neuroprotective effects of zonarol (ZO), a para-hydroquinone-type pro-electrophilic compound from the brown algae Dictyopteris undulata. We show that ZO activates the Nrf2/ARE pathway, induces phase-2 enzymes, and protects neuronal cells from oxidative stress. ZO is the first example of a neuroprotective pro-electrophilic compound obtained from brown algae.

Potential anti-osteoporotic effects of herbal extracts on osteoclasts, osteoblasts and chondrocytes in vitro.

BACKGROUND: Osteoporosis (OP) is one of the most serious diseases in the modern world, and OP patients frequently suffer from fragility fractures in the hip, spine and wrist, resulting in a limited quality of life. Although bisphosphonates (BPs) are the most effective class of anti-bone-resorptive drugs currently available and the most commonly prescribed for the clinical treatment of OP, they are known to cause serious side effects such as bisphosphonate-related osteonecrosis of the jaw. Novel therapeutic materials that can replace the use of BPs have therefore been developed. METHODS: We commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only in OP, but also in oral and skeletal diseases. In the present study, we report on 3 Chinese medical herbal extracts from the root barks of Melia azedarach, Corydalis turtschaninovii, and Cynanchum atratum. RESULTS: All of these extracts inhibited osteoclast proliferation and induced apoptosis by up-regulation of caspase activity and increase of mitochondrial pro-apoptotic proteins expression. Furthermore, the extracts enhanced differentiation, but did not affect proliferation of both osteoblasts and chondrocytes. The osteo-inducible effect was also observed in cultured primary bone marrow cells. CONCLUSIONS: Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects in OP. In this study, we elucidate the potency of these herbal extracts as novel candidates for OP therapy.

Enzyme from Padina arborescens Holmes Synthesizes Parinaric Acid, a Conjugated Tetraenoic Fatty Acid, from α-Linolenic Acid.

Conjugated fatty acids have anticancer effects. Therefore, the establishment of a synthetic method for conjugated fatty acids is important for overcoming cancer. Here, we attempted to synthesize conjugated fatty acids using enzymes extracted from seaweeds containing these fatty acids. Lipids from 12 species of seaweeds from the seas around Japan were analyzed, and Padina arborescens Holmes was found to contain conjugated fatty acids. Then, we synthesized parinaric acid, a conjugated tetraenoic acid, from α-linolenic acid using the enzyme of P. arborescens. This method is expected to have a variety of potential applications for overcoming cancer.

D-Pinitol Ameliorated Osteoporosis via Elevating D-chiro-Inositol Level in Ovariectomized Mice.

A natural sugar alcohol, D-pinitol, has been reported to be a potential compound for osteoporosis treatment via inhibiting osteoclastgenesis. However, research on the effects of pinitol on osteoporosis in vivo is still limited. The present study investigated the protective effects of pinitol on ovariectomized mice and attempted to elucidate this mechanism in vivo. Four-week-old female ovariectomized ICR mice were employed as a postmenopausal osteoporosis model and treated with pinitol or estradiol (E2) for 7 wk. Thereafter, serum calcium content, phosphorus content, tartrate-resistant acid phosphatase (TRAcP) and bone-specific alkaline phosphatase activity (BALP) were measured. Bilateral femurs were isolated, and bone marrow protein was collected through centrifuge. Dry femurs were weighed, while femur length, cellular bones, and bone mineral content were measured. D-chiro-Inositol (DCI) and myo-inositol (MI) content in serum and bone marrow was measured by GC-MS. At the end of experiment, the serum BALP and TRAcP activities of the OVX mice were suppressed significantly by treatment with either pinitol or E2. Femur weight, cellular bone rate, Ca and P content were improved by pinitol or E2. The DCI content of the serum of OVX decreased significantly, although it recovered to some extent after pinitol treatment. Pinitol significantly increased the ratio of DCI to MI in serum or bone marrow protein in the observed OVX mice. Besides, pinitol had no significant effects on osteoblast viability and differentiation. The present results showed that continuous pinitol intake exerts potent anti-osteoporosis activity via elevating DCI content in serum and bone marrow in OVX mice.

Cycloprop-2-ene-1-carboxylates: Potential chemical biology tools in the early growth stage of Arabidopsis thaliana.

Cyclopropene derivatives have been used as extremely reactive units in organic chemistry owing to their high ring-strain energy. They have become popular reagents both for bioorthogonal chemistry and for chemical biology because of their small size and ability to be genetically encoded. In this context, we conducted an exploratory study to identify the biologically active cyclopropenes that affect normal plant growth. We synthesized several cycloprop-2-ene-1-carboxylic acid derivatives and evaluated their effects on the early growth stage of Arabidopsis thaliana. Eventually, we identified the chemicals that affect apical hook development in Arabidopsis thaliana. Their mode of action is different from those of ethylene receptor inhibition and gibberellin biosynthesis inhibition. We expect that some of the chemicals reported here can be new tools in chemical biology to determine useful molecular targets for herbicides or plant growth regulators.

Pharmacokinetic Analysis of Zonarol, a Marine Algal Hydroquinone, in Mice Using HPLC with Fluorescence Detection.

Zonarol, which was discovered in the brown algae Dictyopteris undulata, has antibiotic, antioxidative, anti-inflammatory, and neuroprotective hydroquinone properties. Additionally, a daily treatment of zonarol taken orally has been proven to prevent ulcerative colitis and nonalcoholic fatty liver disease in experimentally induced mice models. In this study, to elucidate the physiological behavior of zonarol in vivo, the establishment of quantitative methods for the determination of zonarol in biological samples and basic pharmacokinetics parameters after oral or intravenous administration with purified zonarol to mice were investigated. The zonarol (20-600 ng/mL) in this study was dose-dependently detected using an HPLC-FI system as a single peak on the ODS column with 80% aqueous methanol at 332 nm with an excitation of 293 nm. The pharmacokinetic parameters were derived from a non-compartment analysis of the plasma concentration of zonarol following oral or intravenous treatment in mice. The absolute bioavailability of zonarol was calculated as 25.0%. Interestingly, the maximal distribution of zonarol in the brain (2.525 ± 1.334 µg/g tissue) at 30 min was observed to be higher and slower than that in the liver and kidney at 15 min after bolus intravenous administrations to the mice (10 mg/kg BW). Based on these results, zonarol might be a candidate for a potential drug, an effective tool for drug delivery, or enhancing the treatment of cerebral disease.

Peach leaf contains multiflorin a as a potent inhibitor of glucose absorption in the small intestine in mice.

Peach leaf extract has anti-hyperglycemic effects on the postprandial blood glucose level in glucose-loaded mice. In our previous study, the mechanism of action was considered to be the inhibition of glucose absorption in the small intestine. To elucidate the active principle in peach leaf, purification of the active compound and a structure determination were performed. With the use of bioassay-guided fractionation using glucose-loaded mice, the acetylated kaempferol glycoside multiflorin A (MFA), a potent inhibitor of glucose absorption from the intestine, was isolated from the MeOH extract of leaf of the edible peach Prunus persica. The structure was identified by HPLC using thiazolizine derivatives and by an analysis of its spectral data. The inhibitory effect of MFA against glucose absorption was demonstrated in the dose dependent manner in mice. However, as the deacetylated analog of MFA, multiflorin B did not show the activity at the in vivo, the activity of MFA was suggested to depend on the acetyl group on the sugar moiety. This is the first report of anti-hyperglycemic activity of MFA in peach leaf extract. MFA may be useful in functional foods or medicines for preventing the postprandial absorption of glucose in hyperglycemia.

Suppressive effect of peach leaf extract on glucose absorption from the small intestine of mice.

The crude extract of peach leaves dose-dependently suppressed the postprandial elevation in the blood glucose level after an oral administration of soluble starch to mice. This study examines the mechanism for this suppressive effect in vivo. An oral carbohydrate-loading test on mice showed that the peach leaf extract suppressed the glucose-induced increase in the blood level of glucose, but without affecting the insulin level. An enteral soluble starch and glucose loading test on mice also showed that the crude extract (1,000 mg/kg) significantly suppressed the postprandial elevation of the blood glucose level and increased the amount of glucose that remained in the intestine to within the same range as that with phloridzin (500 mg/kg), a natural sodium-dependent glucose transporter (SGLT)-specific inhibitor. In contrast, the extract did not suppress the postprandial elevation of the blood triglyceride and cholesterol levels in mice, and did not affect the normal blood glucose level in a feeding test for 21 d. These results reveal that the extract of peach leaves suppressed the postprandial elevation of blood glucose level by inhibiting the absorption of glucose in the small intestine of mice.

Suppressive effects by leaves of the Dypsis lutescens palm on fat accumulation in 3T3-L1 cells and fat absorption in mice.

The methanol extract of Dypsis lutescens leaves showed inhibitory effects on lipase activity in vitro and on triglyceride accumulation in 3T3-L1 pre-adipocytes. Further experiments using the extract on mice demonstrated a suppressive effect on the postprandial elevation of blood triglyceride level and an anti-obesity effect on obese mice induced by a high-fat diet. D. lutescens will accordingly be useful for preventing obesity.

Emerging technologies for the chemical control of root parasitic weeds.

Parasitic plants in the Orobanchaceae family include devastating weed species, such as Striga, Orobanche, and Phelipanche, which parasitize major crops, drastically reduces crop yields and cause economic losses of over a billion US dollars worldwide. Advances in basic research on molecular and cellular processes responsible for parasitic relationships has now achieved steady progress through advances in genome analysis, biochemical analysis and structural biology. On the basis of these advances it is now possible to develop chemicals that control parasitism and reduce agricultural damage. In this review we summarized the recent development of chemicals that can control each step of parasitism from strigolactone biosynthesis in host plants to haustorium formation.

Functional-food constituents in the fruiting bodies of Stropharia rugosoannulata.

Nine compounds (1-9) were isolated from the fruiting bodies of Stropharia rugosoannulata. Compounds 1-5, 8, and 9 suppressed the formation of osteoclast. Compounds 2 and 5 showed anti-fungal activity, and their MIC were 250 µM and 500 µM respectively. Compounds 2-6 showed inhibitory effects on thapsigargin toxicity.

Anti-hyperglycaemic effects of the Japanese red maple Acer pycnanthum and its constituents the ginnalins B and C.

The anti-hyperglycaemic effects of the leaves of Acer pycnanthum K. Koch, and the purification and identification of the active compounds were investigated. Extracts of the leaves showed a potent inhibitory effect on the α-glucosidase in both in vivo and in vitro experiments. The fractionation of the crude extract gave two active compounds, ginnalin B (6-O-galloyl-1,5-anhydro-D-glucitol) and ginnalin C (2-O-galloyl-1,5-anhydro-D-glucitol), by spectroscopic analysis. This is the first report that A. pycnanthum and its constituents may be useful for the prevention or treatment of diabetes mellitus.

Pycnalin, a new α-glucosidase inhibitor from Acer pycnanthum.

A new compound, pycnalin (1), together with four known compounds, ginnalins A (2), B (3), C (4), and 3,6-di-O-galloyl-1,5-anhydro-D-glucitol (3,6-di-GAG) (5), were isolated from Acer pycnanthum. The structure of 1 was determined on the basis of 2D-NMR spectral data and synthesis of 1. Pycnalin (1) is the first 1,5-anhydro-D-mannitol linked to a gallic acid, while compounds 2-5 were 1,5-anhydro-D-glucitol linked to gallic acids. All compounds were tested in vitro for α-glucosidase inhibitory and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. Pycnalin (1) exhibited moderate α-glucosidase inhibitory activity as well as free radical scavenging activity. Ginnalin A (2) and 3,6-di-GAG (5), which have two galloyl groups, exhibited potent α-glucosidase inhibition, compared to those of other compounds 1, 3, and 4 containing a galloyl group. These results suggest that α-glucosidase inhibition is influenced by the number of galloyl groups.