Identification of novel saponins from edible seeds of Japanese horse chestnut (Aesculus turbinata Blume) after treatment with wooden ashes and their nutraceutical activity

Natural seeds of Japanese horse chestnut (Aesculus turbinata Blume) contain large amounts of mixed triterpenoidal saponins called escins. Recent studies have shown that escins have several biological activities including anti-inflammatory action and inhibitory effects on the absorption of ethanol and glucose. For the edible utilization of the seeds, natural seeds are usually treated with wooden ashes to remove harshness. Here, we found the novel compounds derived from escins in the edible seeds after the food processing with wooden ashes. The instrumental analyses revealed the chemical structures of escins and the derivatives. These compounds are identified as four types of deacetylescins Ia, IIa, Ib, and IIb as well as two types of desacylescins I and II. To determine their biological activity, the purified compounds were tested for their potential nutraceutical activity. The oral glucose tolerance test in mice revealed that a single oral administration of the isolated components of deacetylescins at a dose of 100 mg/kg was clearly effective in attenuating the elevation of blood glucose levels. The inhibitory effects of escins and their derivatives were in the order of escins>deacetylescins>desacylescins. Moreover, we found the inhibitory activity of those compounds on pancreatic lipase. Escins were the most potent in inhibiting the enzyme activity, and followed by desacylescins and then deacetylescins. Taken together, our results suggest the potential usefulness of novel saponins including deacetylescins and desacylescins from edible seeds as novel sources for nutraceutical foods with anti-obese effects.     

Purification, characterization and potent lung lesion activity of an l-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom

L-amino acid oxidases (LAOs) are one of the major components of snake venoms, which possess numerous biological functions. However, little is known of the influence of LAOs on organ lesions. In the present study, a unique LAO from Agkistrodon blomhoffii ussurensis snake venom named ABU-LAO was purified by Heparin-Sepharose FF chromatography followed by an ion-exchange chromatography procedure. The purified ABU-LAO appears a dimer with a molecular mass of approximately 108.8kDa. Kinetics studies showed that ABU-LAO is very active towards its substrates L-Asn, L-Phe, L-Tyr, L-Leu, L-Ile and L-Trp. The most striking observation in the present study is that ABU-LAO causes severe pneumorrhagia, pulmonary interstitial edema, fusion of pulmonary alveoli, cardiac interstitial edema and bleeding when being intravenously injected into BALB/c mice. ABU-LAO also induces liver cell necrosis and release of cytokines including IL-6, IL-12 and IL-2 from highly purified human peripheral blood monocytes and T cells, respectively. In conclusion, ABU-LAO potently induces lesions in lungs and livers. The ability of ABU-LAO will contribute to the understanding of the pathogenesis of snakebite wound.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Evaluation of the antineoplastic activity of mitoxantrone–l-carnitine combination therapy on an experimental solid form of ehrlich tumour in mice

We have commenced a series of experiments to evaluate the effect of carnitine derivatives on the antineoplastic activity of mitoxantrone (MX) on various animal cancers. This report describes the therapeutic effect of MX in combination with l-carnitine (LCAR) on the growth of a solid form of Ehrlich tumour inoculated into mice. LCAR was administered subcutaneously at doses of either 200 or 100mgkg(-1) on day 6 and 13 after tumour inoculation, 1h prior to the treatment with MX. Mitoxantrone was administered intravenously at doses of 3 or 6mgkg(-1). We found that LCAR had no potentiating effect on the efficacy of MX, in terms of either slowing tumour growth or increasing the survival of mice. Nevertheless, therapeutic effects can be assumed at higher doses of both drugs based on values calculated from an index of relative hazards.     

Acute and repeated dose oral toxicity of N-acetyl-l-aspartic acid in Sprague–Dawley rats

N-acetyl-l-aspartic acid (NAA) is a constituent of the mammalian central nervous system (CNS) that has been identified in a number of commonly consumed foods. The current study reports the outcome of acute and repeated dose oral toxicology studies conducted with NAA in Sprague-Dawley (SD) rats. No mortalities or evidence of adverse effects were observed in SD rats following acute oral administration of 2000mg/kg NAA. In a separate study, NAA was added to the diets of SD rats (n=10/sex group) at concentrations corresponding to daily doses of 10, 100, or 1000mg/kg/day for 14 consecutive days and 100, 500, and 1000mg/kg/day for another 14 days. All rats survived until scheduled sacrifice and no differences in body weights, feed consumption values, or clinical signs were observed in any of the treatment groups. No biologically significant differences were observed in functional observational battery (FOB), motor activity evaluations, ophthalmologic examinations, hematology, coagulation, clinical chemistry, or organ weights of any of the NAA treatment groups. Further, no test substance-related gross or microscopic changes were observed in NAA exposure groups. Based on these results, NAA was not considered acutely toxic following oral exposure to 2000mg/kg and the no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAA is 1000mg/kg/day.     

A highly branched 1,3-β-d-glucan extracted from Aureobasidium pullulans induces cytokine production in DBA/2 mouse-derived splenocytes

We recently elucidated the structure of a highly branched 1,3-β-d-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of β-d-glucans are known to depend on their structures, the effects of a highly branched 1,3-β-d-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-γ and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-α in DBA/2 mouse-derived splenocytes in vitro.     

d-Isomer of gly-tyr-pro-cys-pro-his-pro peptide: A novel and sensitive in vitro trapping agent to detect reactive metabolites by electrospray mass spectrometry

This paper describes a d-peptide isomer-based trapping assay using an LC/MS ion-trap spectrometer with an electrospray ionization (ESI) source as the analytical tool to study bioactivation of xenobiotics. Reactive metabolites were generated from parent compounds in in vitro incubations with different sources of CYP enzymes. A short d-isomer of gly-tyr-pro-cys-pro-his-pro proved to be a sensitive trapping agent and resistant to proteases. This method was tested with 16 probe substances. Acetaminophen, 1-chloro 2,4-dinitrobenzene, clozapine, diclofenac, imipramine, menthofuran, propranolol, pulegone and ticlopidine all formed d-peptide adducts, which were analogous to the GSH adducts previously described in the literature. New adducts were identified with clopidogrel (-Cl + peptide), nicotine (-CH₃₊H + peptide), nimesulide (+peptide) and tolcapone (+peptide), i.e., no GSH adducts of those drugs have been described in the literature. No adducts were identified with ciprofloxacin, ketoconazole and verapamil. In the literature no GSH adducts have been described with ciprofloxacin and verapamil. d-Peptide-based trapping proved to be a reliable and reproducible method to identify bioactivated intermediates. d-Peptide is a new and convenient protein trapping agent for use in early phase screening of bioactivation of new chemical entities and evaluation of toxic properties of chemicals.