Topic utilization of sesquiterpene lactone from Milleria quinqueflora on treatment of bothropic envenomation in rabbits

Estudou-se o efeito terapêutico da lactona sesquiterpênica (SL), 4,15-Epoxy-miller-9-Z-enolide, na lesão local do envenenamento botrópico experimental. Utilizaram-se três grupos de coelhos inoculados com 1.0æg de veneno de Bothrops alternatus e tratados com solução NaCl (0,85 por cento) (grupo I), SL diluída em glicerol (0,5 por cento) (grupo II) e SL diluída em vaselina (0,5 por cento) (grupo III). Todos os animais foram avaliados nos tempos 30min e 1, 2, 24, 30, 48, 54, 72, 96, 120 e 148h quanto ao grau de edema, diâmetro do halo hemorrágico e presença de necrose local. Os animais do grupo II apresentaram os menores valores de grau de edema e halo hemorrágico com desaparecimento em 54h. Apesar de a necrose ter ocorrido em todos os animais, o diâmetro também foi menor no grupo II, quando comparado com os outros grupos. A SL, extraída da Milleria quinqueflora, possui efeito antiinflamatório, que é importante no tratamento local do envenenamento botrópico.

Treatment of Bothrops alternatus envenomation by Curcuma longa and Calendula officinalis extracts and ar-turmerone

Investigou-se a eficácia do extrato de plantas no tratamento local do envenenamento botrópico. Veneno de serpentes Bothrops alternatus (1,25µg) diluído em 100µl de solução salina estéril foi inoculado (via intradérmica) entre as escápulas de 30 coelhos. Os animais foram divididos em seis grupos (tratamentos): grupo I: tratamento subcutâneo com extrato de Curcuma longa; grupo II: tratamento tópico com extrato hidroalcoólico de Curcuma longa; grupo III: tratamento tópico com ar-turmerone em vaselina; grupo IV: tratamento tópico com extrato metanólico de Curcuma longa; grupo V: tratamento tópico com pomada de Calendula officinalis e grupo VI: aplicação tópica de solução salina a 0,9 por cento (Controle). Os tratamentos foram realizados 30 minutos, 2h, 4h, 24h e 72h após a inoculação do veneno. Foram avaliados intensidade de edema local (com paquímetro), halo hemorrágico (régua com gabaritos circulares) e presença de necrose. Sete dias após a inoculação do veneno botrópico (168h) foi coletado sangue do coração, com e sem EDTA, para realização de hemograma, dosagem plasmática de fibrinogênio e dosagens séricas de proteína total, uréia e creatinina. Após as coletas de sangue, todos os animais foram anestesiados, sacrificados com inalação pelo éter etílico e necropsiados. Fragmentos de pele foram retirados para avaliação histopatológica. Os resultados obtidos mostraram que o tratamento mais eficaz para inibição da evolução do edema, necrose e hemorragia local após envenenamento com Bothrops alternatus foi a aplicação tópica de ar-turmerone.

Avaliação das lesões locais de cães envenenados experimentalmente com Bothrops alternatus após diferentes tratamentos / Evaluation of local lesions in dogs experimentally envenomed by Bothrops alternatus after different treatments

The aim of this study was to evaluate the therapeutic action of flunixin meglumine and aqueous extract of Curcuma longa (10 percent) on local lesions caused by bothropic venom, comparing with the specific antivenom. Twelve adult dogs were inoculated in the middle third of the lateral face of the thigh with 0.3mg/kg of venom. The dogs were divided into three groups of four animals and the treatment was done two hours after venom inoculation as follows: Group I- specific antivenom diluted in saline (one dosage sufficient to neutralize the venom), Group II- flunixin meglumine (1.1mg/kg, IM, once a day for five days), Group III- topical application of aqueous extract of C. longa (10 percent) (three times a day for five days). Necrosis was observed in all dogs of groups I and II, and in two dogs of group III. Edema and hemorrhagic halo were observed in all dogs. Better recovery of lesions was observed in dogs of group I and III. The results denote a satisfactory effect of C. longa on treatment of local lesions caused by bothropic venom in dogs.

[A modification of the in ovo-method for the determination of the antihemorrhagic activity of plant extracts]. / Eine Modifizierung der in ovo-Methode zur Ermittlung der antihämorrhagischen Aktivität von Pflanzenextrakten.

Up to now there has been only a method available for testing the antihaemorrhagic activity of plant extracts, in which chicken embryos had to be transferred to a special container on the fifth day of incubation. Furthermore, the test could not be conducted on the extraembryonal circulatory system until two days later. In the test method described here the embryos remain inside the egg. After removal of the eggshell through the air cell and removal of the inner shell membrane, direct access to the extraembryonal circulatory system is possible, minimising external influences on the test system. This represents a dramatic simplification in technique and permits testing already on the fourth day of incubation. The application of this method to determine the antihaemorrhagic properties of samples has the advantage of permitting the simple and quick production of haemorrhages by snake venom on the yolk sac circulatory system.

Eleganoside-A, B and C from Pseudocalymma elegans, a native of Brazil.

The toxic methanol-soluble part of Pseudocalymma elegans (leaves), a native of Brazil, yielded three new iridoidglucosides (1a-3a) as their acetate-derivatives (1-3) named eleganoside-A (1a), B (2a) and C (3a) which have been characterized with the aid of spectroscopic techniques, including 2D NMR.

Identification of a new metabolite of macrolide immunosuppressant, like rapamycin and SDZ RAD, using high performance liquid chromatography and electrospray tandem mass spectrometry.

Previously unknown metabolites from the two macrolide immunosuppressants rapamycin (sirolimus) and SDZ RAD [40-O-(2-hydroxyethyl)rapamycin] obtained after in vitro incubation with human liver microsomes have been purified. Structure elucidation was performed by nanoelectrospray ionization tandem mass spectrometry applying low energy collision activated dissociation. This ionization method is, as shown here, a powerful tool to determine metabolic pathways by analysis of even low abundance products. Product ion spectra of the isolated metabolites indicate a new kind of biotransformation reaction for rapamycin and SDZ RAD. The proposed metabolic pathway starts with an ester hydrolysis which leads to a ring-opened structure. A dehydration on C33-C34 and a supplementary hydrogenation at C33-C34 result in a structure similar to the ring-opened isomer with an single bond at C33-C34.

Influência da administraçäo de extratos de duas plantas calcinogênicas sobre a fertilidade de ratos / Effect of calcinogenic plant extracts on rat fertility

Investigou-se o efeito de extratos aquosos de Solanum malacoxylon (Sendter) Solanaceae (SM) (sin.: S. glaucophyllum) e Nierembergia veitchii (Hook) Solanaceae (NV), plantas calcinogênicas encontradas no Brasil, sobre a reproduçäo de ratos e sobre suas progênies. No experimento 1, ratos e ratas Wistar, em número variável conforme o grupo, receberam por via oral, durante os 40 dias anteriores ao acasalamento, extrato aquoso de ambas as plantas em dosagem equivalente à administraçäo diária de 10,9gxkg elevado a menos um de planta seca. Os resultados foram comparados com os de um grupo tratado diariamente com vitamina D3 (3,0mgxkg elevado a menos um) e com os de um grupo-controle (salina). No experimento 2, ratas prenhes receberam, por via oral, três diferentes doses dos extratos de ambas as plantas (equivalentes à administraçäo diária de 5,4; 10,9 e 21,7gxkg elevado a menos um de planta seca), durante toda a gestaçäo. Os resultados foram comparados com três grupos tratados com vitamina D3 (0,75; 2,25 e 3,75mgxkg elevado a menos um) e um grupo-controle (salina). Quando administradas antes da gestaçäo, o ganho de peso das fêmeas e a fertilidade foram reduzidos nos grupos tratados com SM (11 por cento) e vitamina D3 (11 por cento). No grupo tratado com NV, a fertilidade também foi reduzida (25 por cento), mas o desenvolvimento ponderal durante o tratamento näo foi afetado. Nos grupos tratados com SM e vitamina D3 anterior à gestaçäo, o número de filhotes por ninhada foi reduzido, assim como o seu peso ao nascer. Características de desenvolvimento e desempenho físico dos filhotes näo foram afetados em nenhum dos grupos. O tratamento das fêmeas com extrato aquoso de SM durante a gestaçäo causou reduçäo significativa e dose-dependente nas características reprodutivas estudadas. O mesmo foi observado com as fêmeas tratadas com vitamina D3. Com o extrato aquoso de NV näo foram observadas reduçöes nas taxas reprodutivas, quando as fêmeas foram tratadas durante a gestaçäo. Com as doses 10,9 e 21,7xkg elevado a menos um de NV foram observadas anomalias morfológicas fetais (2,3 e 0,1 por cento, respectivamente), também presentes nos filhotes de fêmeas tratadas com vitamina D3 (2,25mgxkg elevado a menos um). A presença de vitamina D3 e seus metabólitos nas plantas calcinogênicas provavelmente é responsável pela infertilidade observada, näo somente devido à toxicidade sistêmica, mas também devido aos seus efeitos específicos sobre o aparelho reprodutor

Spatial structures of the bradykinin potentiating peptide F from Agkistrodon piscivorus piscivoris venom.

Bradykinin potentiating peptides usually show two different activities, potentiation of bradykinin and inhibition of angiotensin converting enzyme (ACE). Exceptions of this rule have been found suggesting that both effects occur independently. This study of peptide F by means of NMR spectroscopy shows clearly two different main conformations of the molecule. These different conformations may be the reason for the different activities.

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom.

In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a Ki of the order of 9.7 x 10(-3) microM to BPP-II, 7 x 10(-3) microM to BPP-III, and 3.3 x 10(-3) microM to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-1Ile and 2Glu-2Gly-1Ser-3Pro-2Val-1Ile, with molecular weight of 1372, 1046, and 1078, respectively.

Isolation, characterization and biological properties of two kinin-like peptides (peptide-S and peptide-r) from Scaptocosa raptoria venom.

Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.

[Effects of calcinogenic plants--qualitative and quantitative evaluation]. / Untersuchungen der Auswirkungen von kalzinogenen Pflanzen--Qualitative und quantitative Bewertung.

Different research methods demonstrated the presence of variable quantities of Vitamin D as well as its metabolites in calcinogenic plants. Most of the experiments indicated that the active component most probably should be the metabolite 1,25 (OH)2D3 linked as a glycoside. By this research it was achieved to evaluate the presence of elements with Vitamin D-like activity in the calcinogenic plants Solanum malacoxylon, Cestrum diurnum, Trisetum flavescens and Nierembergia veitchii by testing different extracts of the above plants by oral application to rachitic chicks within the research model "Strontium added Alimentation". After the oral administration of the extracts, the serum was analysed to determine the level of the elements calcium, phosphorus and alkaline phosphatase. The results gained with chicks demonstrated the presence of substances with Vitamin D-like activity in the 4 plants. Solanum malacoxylon and Cestrum diurnum as well contained substances of hydrosoluble character with elevated activity which was indicated by the significant high levels of calcium and phosphorus combined with a reduced activity of the alkaline phosphatase. This indicated the presence of 1,25 (OH)2D3 in both plants. The hydrosoluble character of the active substance in both plants is most probably explained as a compound of the metabolite 1,25 (OH)2D3, combined as a glycoside in the position O-25 of the molecule. Nierembergia veitchii and Trisetum flavescens contained only minor concentration of elements with hydrosoluble characteristics. The results of the 4 analysed plants were evaluated quantitatively as follows: Solanum malycoxylon--82,800 IU of Vitamin D/kg, Cestrum diurnum--63,200 IU of Vitamin D/kg, Nierembergia veitchii--16,400 IU/kg and Trisetum flavescens 12,000 Vitamin D IU/kg. All concentrations are calcinogenic.

Isolation and properties of a new kallikrein inhibitor from Tityus serrulatus venom.

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar affinity to both enzymes, showing an IC50 of 14.3 microM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC50 value of 12.6 microM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide Ki = 2.5 microM. The inhibitor was heat resistant and stable at pH values less than 5.

Isolation and properties of a new kallikrein inhibitor from Tityus serrulatus venom

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar affinity to both enzymes, showing an IC(50) of 14.3 ìM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC(50) value of 12.6 ìM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide K(i) = 2.5 ìM. The inhibitor was heat resistant and stable at pH values less than 5

Isolation and characterization of a bradykinin potentiating peptide (BPP-S) isolated from Scaptocosa raptoria venom.

Venom of the spider Scaptocosa raptoria was fractionated by chromatography on Sephadex G-10 followed by HPLC, and a bradykinin potentiating peptide, BPP-S, was obtained in pure form. The amino acid sequence of this undecapeptide is presented. Peptide BPP-S significantly potentiates the effects of bradykinin on smooth muscle, and inhibits the angiotensin-converting enzyme (ACE) in vitro.

Structure and effects of a kinin potentiating fraction F (AppF) isolated from Agkistrodon piscivorus piscivorus venom.

The contractile action of bradykinin on isolated smooth muscles is known to be potentiated by special peptides isolated from snake venoms and other animal sources. A fraction F (AppF) has been derived from Agkistrodon piscivorus piscivorus venom. This fraction does not increase the depressor effect of bradykinin n blood pressure, potentiates the action of bradykinin on isolated guinea-pig ileum and prolongs the duration of relaxation in isolated rat duodenum. This fraction was able to inhibit the conversion of angiotensin I to angiotensin II in vitro. The primary structure of this decapeptide is given.