Standardized production of a homogeneous latex enzyme source overcoming seasonality and microenvironmental variables.

Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the enzymes is reported. The latex was mixed with distilled water and the mixture was cleaned through centrifugation. It was dialyzed with distilled water and centrifuged again to recover the soluble fraction [EP]. The dialyze is a key feature of the process. EP was characterized in terms of protein profile, chemical stability, among other criteria. Wild plants belonging to ten geographic regions and grown in different ecological conditions were used as latex source. Collections were carried out, spaced at three-month, according to the seasons at the site of the study. Proteolytic activity was measured as an internal marker and for determining stability of the samples. EP was also analyzed for metal content and microbiology. EP showed similar magnitude of proteolysis, chromatographic and electrophoretic profiles of proteins. Samples stored at 25 °C exhibited reduced solubility (11%) and proteolytic capacity (11%) after six months. Enzyme autolysis was negligible. Microbiological and metal analyses revealed standard quality of all the samples tested. EP induced milk clotting and hide dehairing after storage for up to six months.

Proteins from Plumeria pudica latex exhibit protective effect in acetic acid induced colitis in mice by inhibition of pro-inflammatory mechanisms and oxidative stress.

Latex proteins from P. pudica (LPPp) have anti-inflammatory activity. In the present study, LPPp was evaluated to protect animals against inflammatory ulcerative colitis (UC). UC was induced by intracolonic instillation of a 6% acetic acid solution and the animals received LPPp (10, 20 or 40 mg/kg) by intraperitoneal route 1 h before and 17 h after acetic acid injection. Eighteen hours after instillation of acetic acid, the mice were euthanized and the colons were excised to determine the wet weight, macroscopic and microscopic lesion scores, myeloperoxidase (MPO) activity, IL1-ß levels, glutathione (GSH) and malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity. The results revealed that LPPp treatment (40 mg/kg) had a protective effect on acetic acid-induced colitis by reducing the wet weight, macroscopic and microscopic scores of intestinal lesions and colonic MPO activity. Additionally, LPPp inhibited tissue oxidative stress, since decreases in GSH consumption, MDA concentration and SOD activity were observed. The treatment with LPPp reduced the levels of cytokine IL-1ß, contributing to the reduction of colon inflammation. Biochemical investigation showed that LPPp comprises a mixture of proteins containing proteinases, chitinases and proteinase inhibitors. These data suggest that LPPp has a protective effect against intestinal damage through mechanisms that involve the inhibition of inflammatory cell infiltration, cytokine release and oxidative stress.

Antidiarrheal effects of water-soluble proteins from Plumeria pudica latex in mice.

The water-soluble protein fraction obtained from Plumeria pudica (LPPp) latex has previously been demonstrated to have anti-inflammatory and antinociceptive effects. In the present study, LPPp was tested for activity against diarrhea induced by castor oil, prostaglandin E2 (PGE2) or cholera toxin. Different doses of LPPp (10, 20 or 40mg/kg) significantly inhibited the percentage of diarrheal stools (31.18%, 42.97% and 59.70%, respectively) induced by castor oil. This event was followed by significant reduction of both intestinal fluid accumulation (31.42%; LPPp 40mg/kg) and intestinal transit (68.4%; LPPp 40mg/kg). The pretreatment of animals with LPPp (40mg/kg) prevented glutathione and malondialdehyde alterations induced by castor oil. The effects of LPPp against diarrhea induced by castor oil were lost when the fraction was submitted to protein denaturing treatment with heat. LPPp (40mg/kg) also inhibited the average volume of intestinal fluid induced by PGE2 (inhibition of 46.0%). Furthermore, LPPp (40mg/kg) prevented intestinal fluid secretion accumulation (37.7%) and chloride ion concentration (50.2%) induced by cholera toxin. In parallel, colorimetric assays demonstrated that proteinases, chitinases and proteinase inhibitors were found in LPPp. Our data suggest that the antidiarrheal effect of LPPp is due to its protein content and is probably associated with its anti-inflammatory properties.

Proteins from the Rhinella schneideri parotoid gland secretion exhibit anti-nociceptive effect against nociception induced by inflammation.

As proteins isolated from the Rhinella schneideri parotoid gland secretion (RsPP) exhibit anti-inflammatory activity, the goal of this work was to investigate their anti-nociceptive effects using acetic acid-induced writhing, formalin, and hot-plate tests. The intraperitoneal administration of RsPP (2.5 or 5mg/kg) one hour prior to stimuli significantly reduced the abdominal constrictions induced by acetic acid (73.06 and 72.69% inhibition, respectively) and the inflammatory phase of paw licking time induced by formalin (69.3% inhibition, at 2.5mg/kg). However, RsPP (1, 2.5 or 5mg/kg) did not change the latency in response at the hot-plate test. The involvement of inflammatory mediators on the anti-nociceptive effect of RsPP was further demonstrated. RsPP (2.5mg/kg) significantly inhibited the inflammatory peak of paw edema induced by histamine (44.0%), bradykinin (51.3%), or prostaglandin E2 (53.7%). Our data indicate that RsPP may act on the pain process by inhibiting the effect of inflammatory mediators.

Frog Foam Nest Protein Diversity and Synthesis.

Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100 percent mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100 percent mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDF. However the toxicity seems also to involve non protein molecules present in DF.