Anatomy and histochemistry of leaves and stems of Sapium glandulosum

Abstract Sapium belongs to Euphorbiaceae family and comprises 23 species. Sapium glandulosum (L.) Morong is popularly known in Brazil as "pau-leiteiro" and "leitosinha" and it is used in traditional medicine to cicatrisation. Its leaf extracts have shown analgesic, anti-inflammatory and antibacterial activities. The preliminary set of pharmacognostic tools used for quality assessment of medicinal plant parts is macro- and micro-anatomy and S. glandulosum has not anatomical and histochemical description. Thus the aim of this study was to investigate the anatomical and histochemical characteristics of the leaf and stem of S. glandulosum as a means of providing information for quality assessment of herbal industry. The leaves and stems were investigated by employing field emission scanning electron microscopy, light microscopy, and histochemistry techniques. The analysis showed that S. glandulosum had the following anatomical features: dorsiventral and amphistomatic leaves; paracytic stomata; tabular crystal druses; non-articulated and branched laticifers; midrib's biconvex shape with vascular systems in open arc with invaginated ends; petiole with a round shape and slight concavity on the adaxial side; six collateral vascular bundles in U-shaped organisation; a circular stem shape and a sclerenchymatous ring. In the histochemical tests lipophilic components were found in cuticle and in the latex; phenolic compounds were met in the mesophyll and in the latex; starch grains were found in the parenchymatous sheath; lignified elements were met in the sclerenchymatous ring in the cortex and in the perivascular sclerenchymatous caps, beyond in the vessel elements. These features are helpful when conducting a quality control process.

Cytotoxic biomonitored study of Euphorbia umbellata (Pax) Bruyns.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia umbellata latex (sap) has normally been used in folk medicine in southern Brazil to treat different types of cancers. AIM OF STUDY: To carry out a biomonitored investigation of partitioned latex using in vitro assay, to identify the main mechanisms related with the action of the most active fraction as well as to develop a phytochemical study with this material. MATERIALS AND METHODS: Biological screening was performed with hexane, chloroform, ethyl acetate and methanol fractions from the latex of E. umbellata using MTT, trypan blue, and neutral red assays to determine the cytotoxicity against HRT-18, HeLa and Jurkat cells and flow cytometry, DNA quantification, acridine orange and Hoechst 33342 staining to investigate mechanisms of action for the hexane extract. The phytochemical study of the hexane fraction was performed by chromatographic procedures and the substances were identified by NMR analysis. The isolated terpenes were evaluated using MTT to determine the cytotoxicity against Jurkat cells. RESULTS: All the fractions presented concentration and time dependent cytotoxicity. The hexane fraction showed the highest cytotoxicity; whereas the Jurkat cell was the lineage with the highest sensitivity (IC50 1.87µg/mL). Fragmentation of DNA and apoptosis are two mechanisms related with the toxicity of hexane fraction. The hexane fraction arrested the cell cycle in the G0/G1 phase, and the selectivity index was 4.30. Phytochemical study of the hexane fraction led to isolation of euphol (main compound) and germanicol acetate. Both substances demonstrated some slight cytotoxic activity against Jurkat cells after 72h; however the activity was minimal compared to vincristine (anticancer standard drug). CONCLUSION: The current research proves that the fractions of the latex from E. umbellata have a cytotoxic effect against three different cancer cells lines. The hexane fraction showed high in vitro cytotoxic effects against Jurkat cells demonstrating that the effect may be due to non-polar constituents. The two isolated terpenes (euphol and germanicol acetate) showed poor cytotoxic activity indicating that the anticancer properties of the extract may be caused by other substances present in the hexane fraction.

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom.

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.