Tebekeme Okoko

In vitro antioxidant and free radical scavenging activities of Garcinia kola seeds

Garcinia kola Heckel--a tropical plant which grows in moist forest, has found wide applications in traditional medicine especially in the West and Central African sub-region. The seeds have been demonstrated to possess numerous bioactivities but research is highly limited on the link between its fractions and the bioactivities. In this work, the methanolic extract of Garcinia kola seeds was subjected to silica gel column chromatography into five fractions ME1-ME5 and the free radical scavenging activities and antioxidant potentials were determined for each fraction using various in vitro models. The ME4 fraction possessed the greatest activities. It was also demonstrated that the ME4 fraction strongly inhibited nitric oxide production in lipopolysaccharide activated macrophage U937 cells. Chromatographic fractionation and spectroscopic analysis of the ME4 fraction revealed the presence of four compounds namely garcinia biflavonoids GB1 and GB2, garcinal and garcinoic acid. These findings show that these four compounds are partly responsible for the great antioxidant potential of Garcinia kola seeds. This gives further evidence to the nutraceutical and pharmaceutical potential of Garcinia kola.

Inhibitory activity of quercetin and its metabolite on lipopolysaccharide-induced activation of macrophage U937 cells.

The potential of quercetin and its metabolite 3-O-methyl quercetin in inhibiting lipopolysaccharide (LPS)-mediated activation of macrophage U937 cells was investigated. Cells were pre-incubated for different periods with 100 ng/mL phorbol myristate acetate (PMA), and later with LPS and quercetin or 3-O-methyl quercetin (30 microM). Later, the supernatant of each cell culture was assessed for catalase activity, nitric oxide, and the production of tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin 1 (IL-1). The results showed that when the cells were incubated with LPS, there were elevations in the levels of all the markers over the cells not incubated with LPS (P < 0.05). For the cells that were incubated with LPS, there were significant differences between the various cells when they were pre-incubated with PMA for various periods (P < 0.05). However, greatest production of the markers was attained when the cells were pre-treated with PMA for 48 h. Both quercetin and 3-O-methyl quercetin (at 30 mM) reduced the levels of all the markers with 3-O-methyl quercetin possessing more inhibitory potential (P < 0.05). This suggests that the flavonoids possessed significant immunomodulatory activities which depend on methylation especially at position 3.

Reduction of hydrogen peroxide-induced erythrocyte damage by Carica papaya leaf extract

OBJECTIVE To investigate the in vitro antioxidant potential of Carica papaya (C. papaya) leaf extract and its effect on hydrogen peroxide-induced erythrocyte damage assessed by haemolysis and lipid peroxidation. METHODS Hydroxyl radical scavenging activities, hydrogen ion scavenging activity, metal chelating activity, and the ferrous ion reducing ability were assessed as antioxidant indices. In the other experiment, human erythrocytes were treated with hydrogen peroxide to induce erythrocyte damage. The extract (at various concentrations) was subsequently incubated with the erythrocytes and later analysed for haemolysis and lipid peroxidation as indices for erythrocyte damage. RESULTS Preliminary investigation of the extract showed that the leaf possessed significant antioxidant and free radical scavenging abilities using in vitro models in a concentration dependent manner (P<0.05). The extract also reduced hydrogen peroxide induced erythrocyte haemolysis and lipid peroxidation significantly when compared with ascorbic acid (P<0.05). The IC50 values were 7.33 mg/mL and 1.58 mg/mL for inhibition of haemolysis and lipid peroxidation, respectively. In all cases, ascorbic acid (the reference antioxidant) possessed higher activity than the extract. CONCLUSIONS The findings show that C. papaya leaves possess significant bioactive potential which is attributed to the phytochemicals which act in synergy. Thus, the leaves can be exploited for pharmaceutical and nutritional purposes.

Glycine reduces cadmium-induced alterations in the viability and activation of macrophage U937 cells.

This study investigates the effect of glycine on cadmium-induced alterations on the viability and activation of the cell line U-937. In this experiment, U-937 cells were pre-treated with 16 microM cadmium (as cadmium chloride). These cadmium-treated cells were later incubated with or without glycine (1-16 microM). After 72 h, it was revealed that glycine significantly (P<0.05) reduced the tendency of cadmium to reduce the viability of the cells. U-937 cells were also treated with phorbol, 12-myristate, 13-acetate to enhance their transition to the macrophage form. Thereafter, the cells were treated with cadmium with or without glycine (1-16 microM). Twenty-four hours later, the supernatants of each cell culture were assessed for the production of tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 1 (IL-1), nitric oxide (NO), and catalase activity as indices of the activation of macrophages. The results show that glycine significantly (P<0.05) reduced the cadmium-induced production of all the markers of the activation of macrophages in a concentration-dependent manner. The findings support the immense antioxidant role of glycine.

Hibiscus sabdariffa extractivities on cadmium—mediated alterations of human U937 cell viability and activation

OBJECTIVE To investigate the effect of the anthocyanin-rich extract of Hibiscus sabdariffa (H. sabdariffa) calyx on the viability of cadmium-treated U937 cells and cadmium-mediated activation of U937-derived macrophages. METHODS The macrophage cell line U937 was treated with cadmium (0.1 μ mol/L) and later incubated with the anthocyanin-rich extract and cell viability was assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form by treatment with phorbol 12, myristate 13, and acetate and incubated with cadmium (10 μ mol/L). The anthocyanin-rich extract was added to the cells later and subsequently, the supernatant of each cell culture was analysed for the production of tumour necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), nitric oxide, and catalase activity as indices for the activation of macrophages. RESULTS It revealed that the anthocynanin-rich extract significantly (P < 0.05) increased the viability of the cells which was suppressed by cadmium when compared to quercetin dihydrate. The extract also reduced the cadmium-mediated production of the markers of macrophage-activation when compared to quercetin dihydrate. In both experiments, the activity of the extract was concentration-dependent (P < 0.05). CONCLUSIONS The findings show that H. sabdariffa possesses significant immunoprotective effect. These corroborate the immense reported antioxidant and medicinal potential of the calyces of the plant which could be exploited for pharmacological and neutraceutical advantages.

Protective capacity of Artemisia annua as a potent antioxidant remedy against free radical damage.

OBJECTIVE To evaluate the antioxidant capacity of four leaf-derived solvent extracts of Artemisia annua (A. annua), a medicinal plant widely touted for its vast phyto-therapeutic potential. METHODS A. annua leaves were extracted with four solvents (absolute ethanol, absolute methanol, 70% ethanol and 70% methanol), and extracts obtained studied by five complementary in vitro antioxidant test systems using ascorbic acid (vitamin C) and rutin as standard references. RESULTS The extracts remarkably inhibited lipid peroxidation (79.81%-86.70%), and erythrocyte haemolysis (40.02%-49.91%). Their IC50 values for hydroxyl, nitric oxide and hydrogen peroxide radical scavenging activities ranged from 2.39-3.81 mg/mL (superior to the standards), 107.24-144.49 µg/mL and 28.53-53.20 µg/mL, respectively. 70% alcohol extracts generally showed better antioxidant activity than absolute alcohol extracts. CONCLUSIONS The results indicate that A. annua leaf extracts have potent antioxidant activities that would have beneficial effect on human health, and aqueous organic solvents are superior to the absolute counterparts in yielding extracts with better antioxidant potential.

Kolaviron and selenium reduce hydrogen peroxide-induced alterations of the inflammatory response

The abilities of kolaviron and selenium (either separately or in combination) to prevent hydrogen peroxide-induced alterations in cell viability and activation were investigated. The cell line U937 was incubated with the antioxidants (i.e. kolaviron or selenium) for 24 h before exposure to hydrogen peroxide and cell viability was assessed via trypan blue dye exclusion assay. The U937 cells were also transformed to the macrophage form, incubated with the antioxidants before exposure to hydrogen peroxide. Subsequently, production of nitric oxide and pro-inflammatory cytokines were assessed as indices of macrophage activation. The myoblast cell line H9c2 was also incubated with Se and kolaviron for 24 h before exposure to hydrogen peroxide. Cell viability and generation of reactive oxygen species (ROS) were assessed via MTT and DCHF assays. The results revealed that hydrogen peroxide significantly reduced (p < 0.05) the viability of U937 cells which was ameliorated by kolaviron and selenium. Kolaviron and selenium also reduced hydrogen peroxide-induced secretion of nitric oxide, TNF-α, IL-1 and IL-6 by transformed U937 cells. Hydrogen peroxide also significantly reduced (p < 0.05) the viability of H9c2 cells which was significantly restored by kolaviron. Though selenium had no effect on the proliferation of H9c2 cells, co-treatment with kolaviron significantly reduced hydrogen peroxide-induced alterations. Both kolaviron and selenium also reduced hydrogen peroxide-mediated ROS production by H9c2 cells. In all cases, the combined action of kolaviron and selenium offered greater amelioration of the hydrogen peroxide-induced alterations than their separate effects (p < 0.05) but may not be synergistic or additive.

Comparative effect of selenium and glycine on hydrogen peroxide-induced cell death and activation of macrophage U937 cells

The effects of selenium and glycine (either separately or in combination) on hydrogen peroxide-induced cell death on U937 cells and activation of U937-derived macrophages were investigated. In the first instance, U937 cells were incubated with or without selenium (Se) or glycine (GLY) or both (Se + GLY) for 24 h before exposure to hydrogen peroxide. Control cells were not incubated with Se, GLY or exposed to hydrogen peroxide. Cell viability was later assessed via trypan blue and MTT assays. For the other experiment, U937 cells were transformed to the macrophage form using phorbol 12-myristate 13-acetate before incubating with or without Se, GLY, Se + GLY. Contents were subsequently exposed to hydrogen peroxide and 24 h later assessed for the production of TNF-α, IL-1, IL-6 and the expression of iNOS and NF-κB. The results revealed that hydrogen peroxide caused significant cell death which was ameliorated by both Se and GLY. Pre-incubation of the cells with both Se and GLY did not significantly enhance cell numbers compared to GLY (p > 0.05). On the other hand, Se and GLY reduced hydrogen peroxide-mediated production of TNF-α, IL-1, IL-6 and expression of iNOS and NF-κB. Incubating the U937-derived macrophages with Se + GLY significantly ameliorated hydrogen peroxide-mediated activation of macrophages when compared to pre-treatments with Se or GLY (p < 0.05). The findings demonstrate that both Se and GLY reduced hydrogen peroxide-induced alterations in U937 cells and U937-derived macrophages. Implications of the findings are discussed.