In Vitro Selective Antibacterial and Antiproliferative Effects of Ethanolic Extracts from Cambodian and Philippine Plants Used in Folk Medicine for Diarrhea Treatment.

Bacterial diarrhea remains a global health problem, especially in developing tropical countries. Moreover, dysbiosis caused by diarrheagenic bacteria and inappropriate antimicrobial treatment has been associated with intestinal carcinogenesis. Despite the rich tradition of the use of herbs for the treatment of gastrointestinal disorders in Cambodian and Philippine folk medicine, many of them have not yet been systematically studied for their in vitro selective inhibitory effects on intestinal bacteria and cells. In the present study, in vitro inhibitory activities of 35 ethanolic extracts derived from 32 Cambodian and Philippine medicinal plants were determined by broth microdilution method against 12 pathogenic bacteria. Furthermore, cytotoxicity against intestinal cancer cells (Caco-2 and HT-29) using thiazolyl blue tetrazolium bromide cytotoxicity assay and safety to six beneficial intestinal bacteria (bifidobacteria and lactobacilli) and intestinal normal cells (FHs 74 Int) were determined for the antimicrobially active extracts. Selectivity indices (SIs) were calculated among the averages of minimum inhibitory concentrations (MICs), half-maximal inhibitory concentrations (IC50), and 80% inhibitory concentrations of proliferation (IC80) for each type of the tested agents. The extracts of Artocarpus blancoi (Elmer) Merr. (Moraceae), Ancistrocladus tectorius (Lour.) Merr. (Ancistrocladaceae), and Pentacme siamensis (Miq.) Kurz (Dipterocarpaceae) produced significant growth-inhibitory effects (MICs = 32-512 µg/ml) against intestinal pathogenic bacteria at the concentrations nontoxic to normal intestinal cells (IC80 values >512 µg/ml; SIs = 0.11-0.2). Moreover, the extract of P. siamensis (Miq.) Kurz was relatively safe to beneficial bacteria (MICs ≥512 µg/ml; SI = 0.1), and together with A. blancoi (Elmer) Merr., they selectively inhibited intestinal cancer cells (IC50 values ≥51.98 ± 19.79 µg/ml; SIs = 0.3 and 0.6). Finally, a strong selective antiproliferative effect on cancer cells (IC50 values 37.89 ± 2.68 to 130.89 ± 13.99 µg/ml; SIs = 0.5) was exerted by Ehretia microphylla Lam. (Boraginaceae), Lagerstroemia cochinchinensis Pierre ex Gagnep. (Lythraceae), and Melastoma saigonense (Kuntze) Merr. (Melastomataceae) (leaves with flower buds). The results suggest that the above-mentioned species are promising materials for the development of new selective antibacterial and antiproliferative agents for the treatment of infectious diarrhea and associated intestinal cancer diseases. However, further research is needed regarding the isolation and identification of their active constituents.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Cytokine-mimetic properties of some Philippine food and medicinal plants.

This study evaluated Philippine indigenous plants for cytokine-mimetic properties and partially characterized candidate mimetics for their stability as well as their sensitivity to variations in temperature and pH. Forty-five plant extract preparations were tested for their ability to stimulate proliferation of mouse spleen cells and bone marrow cells. Temperature and pH effects were determined by subjecting the extracts to different temperature and pH levels and measuring their residual proliferative activities. Results revealed that 24 and 14 extracts were able to stimulate proliferation of bone marrow cells and spleen cells, respectively. Extracts from yam (Dioscorea alata L.) (Dioscoreaceae) roots and leaves, taro (Colocasia esculenta L.) (Araceae) roots, and buyo (Piper betle L.) (Piperaceae) leaf were among the extracts that strongly stimulated proliferation of both bone marrow cells and splenocytes, significantly increasing cell concentrations. Heating the extracts beyond 40 degrees C markedly reduced their proliferative ability, while a pH level below 4.0 and above 8.0 also significantly decreased the extracts' activity. Three protein-mimetics with sizes of 26 kDa, 35 kDa, and 50 kDa were isolated from buyo leaf, yam root, and taro root extracts, respectively. All three molecules are biologically active and stimulate a dose-dependent proliferative response.

Inhibition of human endothelial cell proliferation by ShIF, a vacuolar H(+)-ATPase-like protein.

ShIF is a bone marrow stroma cell-derived factor originally identified to support proliferation of bone marrow cells in vitro. This protein shares high sequence homology to the yeast vacuolar H(+)-ATPase subunit, Vph1p, and the 116 kDa proton pump of the rat and bovine synaptic vesicle, Vpp1. We examined the function of ShIF in the proliferation of human umbilical vein endothelial cells (HUVEC). ShIF inhibited HUVEC proliferation in a dose-dependent manner. Recombinant ShIF added at 10 and 20 ng/ml inhibited HUVEC proliferation by 21.6 and 44.3%, respectively and increasing the concentration of ShIF to 100 ng/ml inhibited proliferation by as much as 55.5%. When HUVEC cells were cultured at various concentrations of ShIF in the presence of anti-ShIF antibody, the inhibitory effects of ShIF to HUVEC proliferation were abrogated by 89-91% indicating that the activity of ShIF to HUVEC was specific. HUVEC cultured in the presence of ShIF and bafilomycin, a specific inhibitor of ATPase, resulted to a 90% growth inhibition. Thus, ShIF may act as an antagonist to the ATPase complex by disrupting the production of cellular ATP thereby decreasing the ability of HUVEC to proliferate.

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.