Bullfrog oil is a natural product extracted from the Rana catesbeiana Shaw adipose tissue and used in folk medicine for the treatment of several diseases. The aim of this study was to evaluate the extraction process of bullfrog oil, to develop a suitable topical nanoemulsion and to evaluate its efficacy against melanoma cells. The oil samples were obtained by hot and organic solvent extraction processes and were characterized by titration techniques and gas chromatography mass spectrometry (GC-MS). The required hydrophile-lipophile balance and the pseudo-ternary phase diagram (PTPD) were assessed to determine the emulsification ability of the bullfrog oil. The anti-tumoral activity of the samples was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for normal fibroblast (3T3) and melanoma (B16F10) cell lines. Both extraction methods produced yielded around 60% and the oil was mainly composed of unsaturated compounds (around 60%). The bullfrog oil nanoemulsion obtained from PTPD presented a droplet size of about 390 nm and polydispersity = 0.05 and a zeta potential of about´25 mV. Both the bullfrog oil itself and its topical nanoemulsion did not show cytotoxicity in 3T3 linage. However, these systems showed growth inhibition in B16F10 cells. Finally, the bullfrog oil presented itself as a candidate for the development of pharmaceutical products free from cytotoxicity and effective for antineoplastic therapy.

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Polysaccharides from algae are also proper candidates with therapeutic properties and immunomodulatory and antitumor effects. The brown seaweed Lobophora variegata synthesizes different groups of anionic polysaccharides with several biological properties. Sulfated polysaccharides were obtained by delipidation of seaweed, proteolysis, and fractionation with different volumes of acetone. A fraction of sulfated polysaccharides, fucans or fucoidans, was extracted with 0.8 v acetone and named L. variegata (LV). This fucan was assessed in the inflammatory process in rats, antitumor action on human colon adenocarcinoma (HT-29 cells), apoptosis, and its effect on the cell cycle. LV fraction, a galactofucan, exhibited a high ratio of total sugar/sulfate (1.5) and a very low level of proteins. This polysaccharide showed an antiinflammatory effect on two models of inflammation induced by croton oil and oxazolone in rats. LV was analyzed in cellular proliferation of HT-29. We also demonstrated the cytotoxic action against this cell line and induction in the apoptosis and decreased the cell cycle in phases S and G 2 /M and the accumulation of cells in the G 1 phase. Our studies with LV showed a marked immunomodulatory action without and with lipopolysaccharide (LPS) on RAW 264.7 cells. Comparative studies of two fucans, LV and FV (Fucus vesiculosus), with different structures were assessed on viability in macrophages, RAW 264.7 cells. Both showed cytotoxicity in these cells. We observed high levels of nitric oxide (NO) production, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) when treated at 0.25, 0.5, and 1.0 mg mL −1 of LV. Data also suggest that LV has potential antitumor effects on HT-29 and antiinflammatory and immunomodulatory effects on RAW 264.7 cells.

A beta-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 degrees C, Km 2.7 x 10(-6) and Vmax 15.3 microM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. beta-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.