Fractionation of barbados cherry (acerola fruit, a fruit of Malpighia emarginata DC.) extracts were performed by organic solvent extractions and column chromatographies, using two extraction methods. Higher cytotoxic activity was concentrated in fractions A4 and A6 (acetone extract), and H3 and HE3 (hexane extract). These four fractions showed higher cytotoxic activity against tumor cell lines such as human oral squamous cell carcinoma (HSC-2) and human submandibular gland carcinoma (HSG), when compared with that against normal cells such as human periodontal ligament fibroblasts (HPLF) and human gingival fibroblasts (HGF). HE2 (hexane extract), AE2 (ethyl acetate extract), AE3, AE4, AE5, A8, A9 and A10 showed some relatively higher anti-bacterial activity on the Gram-positive Staphylococcus epidermidis ATCC 1228 but were ineffective on the representative Gram-negative species E. coli and Ps. aeruginosa. The fractions were inactive against Helicobacter pylori, two representative Candida species, and human immunodeficiency virus (HIV). H3, H4 and HE3, which displayed higher tumor-specific cytotoxicity also showed higher multidrug resistance (MDR) reversal activity, than (+/-)-verapamil as positive control. ESR spectroscopy shows that the radical-mediated oxidation is not involved in the induction of tumor-specific cytotoxic activity. The tumor specific cytotoxic activity and MDR reversal activity of barbados cherry may suggest its possible application for cancer therapy.

We have developed a novel method for the preparation of 'recombinant proteoliposomes'. Membrane proteins were expressed on budded virus (BV) envelopes using baculovirus gene expression systems, and proteoliposomes were prepared by fusion of these viruses with liposomes. First, plasmid DNA containing the gene for the thyroid-stimulating hormone receptor (TSHR) or the acetylcholine receptor alpha-subunit (AChRalpha) was co-transfected with wild type virus [Autographa californica nuclear polyhedrosis virus (AcNPV)] genomes into insect cells [Spodoptera frugiperda (Sf9)] to obtain recombinant viruses via homologous recombination. The recombinant viruses were again infected into Sf9 cells, and the resulting BVs were shown to express TSHR and AChRalpha. Next, the fusion behaviour of AcNPV-derived BVs and liposomes was examined via a fluorescence assay, and BVs were shown to fuse with phosphatidylserine-containing liposomes below pH 5.0, the pH at which fusion glycoprotein gp64 on the virus envelope becomes active. TSHR- or AChRalpha-expressed BVs were also shown to fuse with liposomes. Finally, TSHR- and AChRalpha-recombinant proteoliposomes were immobilized on enzyme-linked immunosorbent assay plates, and their reactivities were examined via a general immunoassay, which showed that the recombinant proteoliposomes were fully active. These results successfully demonstrate the development of a method based on a baculovirus gene expression system for the preparation of recombinant and functional proteoliposomes.

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