Combination effect of lignin F and natural products.

We investigated the effect of lignin F, isolated from the alkaline extract of the cone of Pinus parviflora Sieb. et Zucc, on the cytotoxic activity and radical intensity (measured by ESR spectroscopy) of various natural products. Lignin F slightly inhibited the proliferation of human oral tumor cell lines (human squamous cell carcinoma HSC-2, human salivary gland tumor HSG), but not that of human gingival fibroblast HGF, suggesting its tumor specific cytotoxic action. Lignin F enhanced the cytotoxic activity of vitamin K2, vitamin K3, sodium ascorbate (vitamin C), epigallocatechin gallate (EGCG) (a major component of green tea), gallic acid (structural unit of tannin), chlorogenic acid, and 6 tea extracts (Japanese green tea, Japanese barley tea, black tea, Chinese green tea, Chinese Jasmin tea, Chinese Oolong tea), to various extents. On the other hand, lignin inhibited the cytotoxic activity of curcumin and dopamine. ESR spectroscopy demonstrated that combination of lignin and vitamin K3, EGCG or gallic acid synergistically augmented the radical intensity. Lignin F enhanced the bactericidal activity of EGCG against E. coli. These data suggest the beneficial effect of the combination of lignin F and natural products.

Diverse biological activities of healthy foods.

Diverse biological activities of 7 healthy foods [powdered pine needle, citrate-fermented sesame, powdered coffee, royal jelly, propolis, pollen and white sesame oil (extracted by super critical state (40 degrees C, 350 atmospheric pressure))] were investigated. The pine needle, sesame and powdered coffee was also extracted successively by ethanol and hot water, and lyophilized. The pine needle and coffee extracts, and propolis showed higher in vitro cytotoxic, bactericidal and oxidation activity, as compared with other 4 lipophilic healthy foods. However, propolis showed slightly lower, but significant cytotoxic and bactericidal activity with much reduced oxidation potential. ESR spectroscopy demonstrated that the cytotoxic activity of these extracts was closely related to their radical generation and O2- scavenging activities. Healthy food components may have both pro-oxidant and anti-oxidant properties. Pre-treatment of mice with pine needle, sesame or powdered coffee extract significantly reduced the lethality of bacterial infection, possibly due to their host-mediated action. These extracts failed to reduce the cytophatic effect of HIV-1 (human immunodeficiency virus) infection in MT-4 cells. No apparent acute toxicity was detected in mice by oral administration of 10 g/kg of these extracts. This data suggest the medicinal efficacy of healthy foods.

Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening.

New trend in methods for assessing pharmacological action to bacteria and cell is to measure their metabolic activities induced, while the conventional methods used population growth. We focused on respiration volume as an indicator of cell metabolism, and developed inexpensive disposable oxygen electrode sensor and multi-channel dissolved oxygen meters (DOX-10 and DOX-96KB). Using these instruments, cytotoxicity was measured for 48 hrs and the method showed superior features to conventional methods in its handiness of one step assay, and excellent adaptability to automated systems. Total usability of this oxygen electrode method is being evaluated in bacterial drug susceptibility test, anticancer drug susceptibility test, and alternatives to animal experiment.

[Anticancer susceptibility test method using general-purpose dissolved oxygen meter].

Drug susceptibility of cell can be rapidly measured by continuously monitoring its metabolic changes. We focused on respiration volume as a signal of metabolic change, and developed a new dissolved oxygen measuring system which can detect respiration volume of cells. The main feature of the system is the use of a new type bare oxygen electrode which can easily detect the changing rate of dissolved oxygen concentration. In this study, single type electrode was used to evaluate this rapid anticancer drug susceptibility test. The result obtained was almost equivalent to that with MTT method, which is a conventional method for susceptibility test of HL-60 to various kinds of anticancer drugs. We have also developed multi-type electrode plate with oxygen electrodes embedded in the bottom of 96-well plate, with which clinical evaluation of this method can be easily made.

Effect of serum components on syncytium formation and virus production by cells infected with human immunodeficiency virus in vitro.

Previously it has been reported that cocultivation of human immunodeficiency virus type 1 (HIV-1)-infected cells with uninfected cells results in formation of multinuclear giant cells, generated via an interaction of gp120 on the surface of infected cells with CD4 on the uninfected cells. Formation of multinuclear giant cells as occurring in the presence of normal fetal calf serum was not observed when HIV-infected MOLT-4 or MOLT-3 cells (chronically infected with HTLV-IIIB) and uninfected cells were cocultured in both serum-free medium and fibrinogen-depleted serum. Addition of sera (human and rabbit) as well as of fibrinogen (human and bovine), fibronectin (human), and alpha-globulin (human), but not of albumin, transferrin or gamma-globulin to serum-free medium caused formation of multinuclear giant cells. In contrast, HIV production from MOLT-3 cells proceeds also in the absence of serum. In control experiments it was established that the cells maintained at reduced serum concentration, or in serum-free medium without or with fibrinogen are viable even though displaying a lower metabolic rate (ATP formation and DNA synthesis). From these findings we conclude that serum components (e.g., fibrinogen, fibronectin, and alpha-globulin) are absolutely required for syncytium formation but are not essential for virus release.

Effect of cacao husk extract on human immunodeficiency virus infection.

A sodium hydroxide extract from cacao husk inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1) against HTLV-1-transformed T-cell lines MT-2 and MT-4. It also inhibited syncytium formation between HIV-infected and uninfected lymphoblastoid T-cell line, MOLT-4. The anti-HIV activity was concentrated by membrane filter fractionation to a fraction with molecular weight of 100-300 KDa. Anti-HIV activity of the extract was attributable to interference with the virus adsorption, rather than to inhibition of the virus replication after adsorption.

Chemical modification potentiates paramylon induction of antimicrobial activity.

Induction of antimicrobial activity by two water-insoluble polysaccharides, paramylon and TAK, was significantly potentiated by introduction of positively charged groups (such as N,N-dimethylaminoethyl; N,N-diethylaminoethyl; and 2-hydroxy-3-trimethylammoniopropyl), but not by introduction of negatively charged groups (such as carboxymethyl or sulfate). Cross-linking of these derivatives with epichlorohydrin did not increase their potentiating activity. The effects of these derivatives did not always correlate with their ability to accumulate polymorphonuclear cells and to stimulate the generation of luminol-dependent chemiluminescence by peritoneal exudate cells.

Stimulation of granulocytic cell iodination by pine cone antitumor substances.

Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). Iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed.

Antitumor activity of polysaccharide fractions from pine cone extract of Pinus parviflora Sieb. et Zucc.

Hot water extract of pine cone (PCE) of Pinus parviflora Sieb. et Zucc. dose-dependently suppressed both solid and ascites tumor cells transplanted into various mice. Acidic polysaccharides of PCE significantly increased the survival time of mice bearing ascites tumor cells, and activity increased with acidity. One of the four polysaccharide fractions obtained by NaOH extraction showed the most potent antitumor activity. This fraction significantly suppressed the growth of solid tumor cells, with occasional tumor regression and necrosis, and with little or no cytocidal effect on cultured tumor cells. All acidic polysaccharides were able to activate mouse macrophage-like cell line J774.1. There did not appear to be any correlation between the antitumor activity of these polysaccharides and their content of arabinose (or fucose), mannose, galactose, glucose, or uronic acid.

DNA synthesizing activity during induced differentiation in mouse myeloid leukemia (M1) cells.

Mouse myeloid leukemia (M1) cells were induced to differentiate in vitro by treatment with dexamethasone. After 8 h of treatment, induction of phagocytic and lysozymic activities and depression of DNA synthesis started at the same time and proceeded irreversibly. DNA synthesis in the nuclear system reflected primarily DNA replication rather than repair and this activity declined during M1 cell differentiation.