Anti-HIV and immunomodulation activities of cacao mass lignin-carbohydrate complex.

BACKGROUND: Recently, a prominent antiviral and macrophage stimulatory activity of cacao lignin-carbohydrate complex (LCC) has been reported. However, the solubility and sterility of LCC have not been considered yet. In the present study, complete solubilisation and sterilisation was achieved by autoclaving under mild alkaline conditions and the previously reported biological activities were re-examined. MATERIALS AND METHODS: LCCs were obtained by 1% NaOH extraction and acid precipitation, and a repeated extraction-precipitation cycle. Nitric oxide (NO) and cytokine productions were assayed by the Griess method and ELISA, respectively. Inducible NO synthase (iNOS) expression was determined by Western blot analysis. Superoxide anion, hydroxyl radical and nitric oxide radical-scavenging activity was determined by ESR spectroscopy. RESULTS: Cacao mass LCC showed reproducibly higher anti-HIV activity than cacao husk LCC. Cacao mass LCC, up to 62.5 µg/ml, did not stimulate mouse macrophage-like cells (RAW264.7 and J774.1) to produce NO, nor did it induce iNOS protein, in contrast to lipopolysaccharide (LPS). Cacao mass LCC and LPS synergistically stimulated iNOS protein expression, suggesting a different point of action. Cacao mass LCC induced tumour necrosis factor-α production markedly less than LPS, and did not induce interleukin-1ß, interferon-α or interferon-γ. ESR spectroscopy showed that cacao mass LCC, but not LPS, scavenged NO produced from NOC-7. CONCLUSION: This study demonstrated several new biological activities of LCCs distinct from LPS and further confirmed the promising antiviral and immunomodulating activities of LCCs.

Multiple biological complex of alkaline extract of the leaves of Sasa senanensis Rehder.

Previous studies have shown anti-inflammatory potential of alkaline extract of the leaves of Sasa senanensis Rehder (SE). The aim of the present study was to clarity the molecular entity of SE, using various fractionation methods. SE inhibited the production of nitric oxide (NO), but not tumour necrosis factor-α by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. Lignin carbohydrate complex prepared from SE inhibited the NO production to a comparable extent with SE, whereas chlorophyllin was more active. On successive extraction with organic solvents, nearly 90% of SE components, including chlorophyllin, were recovered from the aqueous layer. Anti-HIV activity of SE was comparable with that of lignin-carbohydrate complex, and much higher than that of chlorophyllin and n-butanol extract fractions. The CYP3A inhibitory activity of SE was significantly lower than that of grapefruit juice and chlorophyllin. Oral administration of SE slightly reduced the number of oral bacteria. When SE was applied to HPLC, nearly 70% of SE components were eluted as a single peak. These data suggest that multiple components of SE may be associated with each other in the native state or after extraction with alkaline solution.

DNA microarray analysis of signaling pathway in macrophages stimulated by lignin-carbohydrate complex from lentinus edodes mycelia (LEM) extract.

In order to investigate the action point of lignin-carbohydrate complex (Fr4) from Lentinus edodes mycelia extract, DNA microarray analysis was performed, using mouse macrophage-like J774.1 cells. Among seven lignin-carbohydrate complex fractions, Fr4 showed the highest stimulatory activity of tumor necrosis factor production by mouse macrophage-like J774.1 cells, as well as its previously reported anti-HIV activity. Fr4 is composed of lignin precursors such as vanillic acid, syringic acid, p-coumaric acid and ferulic acid, with trace amounts of flavonoids and tannins, and negligible amount of lipopolysaccharides (LPS), confirming the authenticity of Fr4 as a lignin. DNA microarray analysis suggested that Fr4 may affect immune response-related gene expression; however, it may not affect the expression of as many genes as LPS does.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.

Larval and juvenile development of the Echinometrid sea urchin Colobocentrotus mertensii: emergence of the peculiar form of spines.

The development of Colobocentrotus mertensii from embryos to larvae and early juveniles was observed to give the first detailed description of larval and juvenile formation and skeletal structures in echinometrid sea urchins. The first larval spicules appeared at the mesenchyme blastula stage, whereas, in many echinoids, spicules were formed after gastrulation. From late eight-armed larva to juvenile, body color of C. mertensii was deep red, which has never been described for any echinoid before. The adult form of C. mertensii is characteristic in that the spines at the aboral side are short, truncated and pavement-like. The first sign of peculiar adult features could be seen in the juvenile spines and adult spines, which are broader than those of closely related Anthocidaris crassispina. The primary podia emerged on the left side of larval body were more stout and thicker in C. mertensii than in A. crassispina. The present study shows that developmental process of larval structure of C. mertensii is in general similar to the A. crassispina and the differences is first seen in juvenile structure including the distribution of pigment spots and morphology of adult spine.