Latex proteins downregulate inflammation and restores blood-coagulation homeostasis in acute Salmonella infection

BACKGROUND Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels. OBJECTIVES To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII). METHODS Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels. FINDINGS LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood. MAIN CONCLUSIONS LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.

Chemical constituents of Calotropis procera latex and ultrastructural effects on Haemonchus contortus / Constituintes químicos do látex de Calotropis procera e efeitos ultraestruturais em Haemonchus contortus

Abstract This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.

Resumo Este estudo objetivou avaliar os efeitos anti-helmínticos e ultraestruturais do látex de Calotropis procera sobre Haemonchus contortus. Látex de C. procera foi centrifugado duas vezes à a 10.000xg e dialisado para obter uma fração rica em proteínas, denominada proteínas do látex (LP). E centrifugado e centrifugado a 3.000 rpm, para obter uma fração rica em metabólitos secundários, denominada LNP (látex não proteico). Espécimes de H. contortus expostos à LNP, LP e PBS no Teste de Motilidade dos Nematoides Adultos (TMNA) foram submetidos a microscopia eletrônica de varredura (MEV) e de transmissão (MET), para verificar alterações em sua ultraestrutura. Testes fitoquímicos em LNP indicaram a presença de fenóis, esteroides, alcaloides e cardenolídeos. A presença dos compostos ácido gálico e quercetina em LNP foi caracterizada por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo de proteínas em LP foi de 43,1 ± 1,1 mg/mL e de 7,7 ± 0,3 mg/mL em LNP. No TMNA, LNP e LP inibiram a motilidade de 100% dos nematoides, sendo LNP mais eficaz que LP, e a ivermectina mais eficaz que ambos (p <0,05). Alterações na cutícula de nematoides tratados com LP e LNP foram observadas por MEV e MET. O látex de C. procera apresenta efeito anti-helmíntico sobre H. contortus, causando danos à sua cutícula e outras alterações em sua ultraestrutura.

Aislamiento y caracterizacion parcial de fracciones con actividad ribonucleasa a partir del latex de calotropis procera (Aiton) W.T. Aiton y pedilanthus tithymaloides (L.) poit / Isolation and partial characterization of fractions with ribonuclease activity from latex of calotropis procera (Aiton) W.T. Aiton and pedilanthus tithymaloides (L.) poit

Con el objetivo de aislar y caracterizar parcialmente las enzimas ribonucleasas (RNasas) contenidas en el látex de Calotropis procera y Pedilanthus tithymaloides, se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con acetato de sodio y centrifugación a 16.000 x g durante 15 min y fraccionadas por cromatografía de intercambio iónico. Se estimó la masa molecular a través de ecuaciones de regresión lineal. Se realizaron pruebas de glicosilación. En ambas especies, las proteínas con actividad RNasa presentaron una masa molecular entre 28 y 30 kDa. No existe evidencia de proteínas glicosiladas en el látex de C. procera. En P. tithymaloides la RNasa es una proteína glicosilada.

In order to isolate and characterize partially ribonucleases (RNases) enzymes contained in the latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from mature plants. Soluble proteins were extracted with sodium acetate and centrifugation at 16,000 xg for 15 min and fractionated by ion exchange chromatography. Molecular mass was estimated by linear regression equations. Glycosylation tests were conducted. In both species, proteins with RNase activity showed a molecular mass between 28 and 30 kDa. No evidence of glycosylated proteins in latex from C. procera. In P. tithymaloides, RNase may be a glycosylated protein.

Citotoxicidad in vitro de extractos laticiferos de Calotropis procera (Aiton) W.T. Aiton y Pedilanthus tithymaloides (L.) Poit / In vitro cytotoxicity of laticifers extracts from Calotropis procera (Aiton) WT and Pedilanthus tithymaloides (L) Poit

In order to assess the anticancer action of extracts obtained by latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from adult plants. Soluble proteins were extracted with 16 uL of 50 mM sodium acetate pH 5/ug integral latex and centrifugation at 16,000 x g for 15 min, the supernatant was named "latex crude extract" (LCE). The "latex methanolic extract" (LME) was obtained on dried latex. Both extracts were tested in vitro by cytotoxic and cytostatic activity in Jurkat cell cultures. Cellular viability, proliferation, necrosis and apoptosis were evaluated. LCE and LME of C. procera were found with cytotoxic and cytostatic activity after 24 incubation hours (p < 0,05) with doses from 1ug/mL. The LCE and LME of P. tithymaloides presented cytotoxic effect (p < 0,05) from 50 ug/mL and from 1ug/mL, respectively.

Con el objetivo de evaluar el potencial anticanceroso de extractos de látex de Calotropis procera y Pedilanthus tithymaloides se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con 16 uL de acetato de sodio 50 mM pH 5/ug de látex integral y centrifugación a 16.000 x g durante 15 min, denominándose al sobrenadante “extracto crudo de látex” (ECL). El “extracto metanólico de látex” (EML) se obtuvo sobre látex deshidratado. Ambos extractos fueron probados en su actividad citotóxica y citostática in vitro sobre cultivos de células Jurkat. Se realizaron estudios de viabilidad, proliferación, necrosis y apoptosis celular. El ECL y el EML de C. procera presentaron actividad citotóxica y citostática después de 24 y 48 horas de incubación (p < 0,05) con dosis desde 1 ug/mL. Los ECL y EML de P. tithymaloides presentaron efectos citotóxicos (p < 0,05) a partir de 50 ug/mL y desde 1 ug/mL respectivamente.

Protective effect of aqueous suspension of dried latex of Calotropis procera against oxidative stress and renal damage in diabetic rats

Calotropis species have been used in the traditional medicinal system for the treatment of diseases of the liver and abdomen. In view of the antioxidant and anti-hyperglycemic properties of an aqueous suspension obtained from the dried latex of Calotropis procera, the present study was carried out to evaluate its efficacy in affording protection against alloxan induced changes in rat kidney. A single intraperitoneal injection of alloxan (150 mg/kg) in rats produced hyperglycemia within 3 days and altered kidney functions over a period of 90 days. Daily oral administration of the aqueous suspension (100 and 400 mg/kg) in diabetic rats produced anti-hyperglycemic effect that was comparable to that of glibenclamide (10 mg/kg). Unlike glibenclamide, the aqueous suspension did not increase the serum insulin levels in diabetic rats. However, it produced a marked reduction in the levels of urinary glucose and protein and normalized the renal tissue levels of thiobarbituric acid-reactive substances (TBARS) and glutathione (GSH) in diabetic rats and the effect was comparable to that of glibenclamide. The protection afforded by the aqueous suspension was also evident from the histological analysis of the renal tissue. Our study shows that by exhibiting antioxidant and anti-hyperglycemic property the aqueous suspension of dried latex of C. procera affords protection against the complications associated with diabetes.

Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers

The Calotropis procera seed fibers provide an excellent model system to study the genes involved in fiber elongation, fineness and strength. Expansins constitute one of the important gene families involved in plant cell expansion and other cell wall modification processes. Four homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 were isolated from the cDNA library obtained from fast growing Calotropis procera fibers. These homologs represented typical Expansin A family. Each of them had two conserved domains including GH45 like domain and the putative polysaccharide binding domain. The deduced amino acid sequences of the homologs indicated three conserved motifs: i) eight cysteine residues at N-terminus, ii) four tryptophan residues at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the center of the sequence. The presence of N-terminal signal peptide consisting of hydrophobic amino acids and a transmembrane region in all these expansin isoforms suggests their cotranslational insertion into the endoplasmic reticulum and then transportation to the cell wall by secretory pathway. The relative quantification of the four expansins in root, stem, fiber and leave tissues indicated that the transcripts of CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these tissues. The lowest transcription of all the four Expansin A isoforms was observed in elongating roots indicating that root tissue might be having specific expansins other than those confined to air grown organs.

In vitro and in vivo antiproliferative activity of Calotropis procera stem extracts

The cytotoxic potential of stem organic extracts from Calotropis procera (Asclepiadaceae) was firstly evaluated against cancer cell lines by MTT assay. Subsequently, samples considered cytotoxic were tested for antimitotic activity on sea urchin egg development and for in vivo antiproliferative activity in mice bearing Sarcoma 180 tumor. Among the five extracts (hexane, dichloromethane, ethyl acetate, acetone and methanol), ethyl acetate and acetone extracts displayed higher cytotoxic potential against tumor cells, with IC50 ranging from 0.8 to 4.4 μg/mL, while methanolic extract was weakly cytotoxic. Cytotoxic extracts also exhibited cell division inhibition capacity by antimitotic assay, revealing IC50 values lower than 5 μg/mL. In the in vivo antitumor assessments, ethyl acetate- and acetone-treated animals showed tumor growth inhibition ratios of 64.3 and 53.1 percent, respectively, with reversible toxic effects on liver and kidneys. Further studies are in progress in order to identify C. procera cytotoxic compound(s) and to understand the mechanism of action responsible for this tumor-decreasing potential.

O potencial citotóxico de extratos orgânicos do caule de Calotropis procera (Asclepiadaceae) foi primeiramente avaliado frente a linhagens de células tumorais através do ensaio de MTT. Aquelas amostras consideradas citotóxicas foram sub-sequentemente testadas para atividade antimitótica sobre o desenvolvimento de ovos de ouriço-do-mar e para atividade antiproliferativa in vivo em camundongos transplantados com tumor Sarcoma 180. Dentre os cinco extratos estudados (hexano, diclorometano, acetato de etila, acetona e metanol), os extratos acetato de etila e acetona mostraram maior potencial citotóxico contra células tumorais, com CI50 variando de 0,8 to 4,4 μg/mL, enquanto o extrato metanólico revelou ser fracamente citotóxico. s extratos citotóxicos também exibiram capacidade de inibição da divisão celular com valores de CI50 menores que 5 μg/mL. Nas avaliações antitumorais in vivo, os animais tratados com os extratos acetato de etila e acetona mostraram taxas de inibição do crescimento tumoral de 64,3 e 53,1 por cento, respectivamente, com efeitos tóxicos reversíveis sobre o fígado e os rins.

Caracterização dos taninos condensados das espécies maniçoba (Manihot pseudoglazovii), flor-de-seda (Calotropis procera), feijão-bravo (Capparis flexuosa, L) e jureminha (Desmanthus virgatus) / Characterization of condensed tannin of the species maniçoba (Manihot pseudoglaziovii), flor-de-seda (Calotropis procera), feijão-bravo (Capparis flexuosa) and jureminha (Desmanthus virgatus)

Caracterizaram-se os taninos condensados (TC) e determinaram-se a composição bromatológica e a digestibilidade in vitro da matéria seca (DIVMS) das espécies maniçoba (Manihot pseudoglazovii Pax & Hoffman), flor-de-seda (Calotropis procera), feijão-bravo (Capparis flexuosa, L) e jureminha (Desmanthus virgatus (L.) Willd). As concentrações em tanino solúvel, tanino ligado ao resíduo e tanino total (TT) das espécies foram determinadas pelo método butanol-HCL e a adstringência pelo método de difusão radial. Para a DIVMS, foi utilizado método de dois estágios. Foram observadas diferenças entre as espécies (P<0,01) quanto à concentração e adstringência dos taninos. As maiores concentrações foram encontradas na jureminha, 2,4 por cento TT e 13,7-3 de adstringência. Não foi detectado tanino na flor-de-seda. O teor em PB foi acima de 16 por cento em todas as espécies estudadas, e o FDA e lignina abaixo de 39 e 15 por cento, respectivamente. A DIVMS foi baixa na jureminha, 43 por cento, e alta na flor-de-seda, 80 por cento. A correlação entre a DIVMS e a concentração em taninos condensados foi baixa (r=0,097)

Condensed tannins (CT) of the species maniçoba (Manihot pseudoglaziovii), flor-de-seda (Calotropis procera), feijão-bravo (Capparis flexuosa) and jureminha (Desmanthus virgatus) were characterized and the chemical composition and in vitro dry matter digestibility (IVDMD) of those species were determined. Concentration of soluble tannin (ST), bound tannin (BT) and total tannin (TT) of the plants was determined using butanol-HCL method; astringency was determined, using radial diffusion method, and the IVDMD was determined using two-stages method. Concentration and astringency of condensed tannins varied between species (P<0.01). Jureminha was the species that presented the highest value (2.4 percent TT and 13.7-3 astringency). No tannins were detected in flor-de-seda. Crude protein was higher than 16 percent in all studied species, and the ADF and lignin values were lower than 39 and 15 percent, respectively. The IVDMD was low in jureminha (43 percent) and high in flor-de-seda (80 percent). The correlation between IVDMD and TT was low (r²=0.097)

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.

Evaluation of cytotoxic potential of latex of Calotropis procera and Podophyllotoxin in Allum cepa root model

In the present study we have utilized the Allium cepa root tip meristem model to evaluate the cytotoxic and anti-mitotic activities of latex of Calotropis procera (DL) and podophyllotoxin. Standard cyto-toxic drug cyclophosphamide and non-cytotoxic drugs cyproheptadine and aspirin served as controls. Like cyclophosphamide, both DL and podophyllotoxin significantly inhibited the growth of roots and mitotic activity in a dose-dependent manner. However, podophyllotoxin was more potent in this regard and produced root decay. Cyproheptadine and aspirin, on the other hand, showed a marginal effect on the root growth and mitotic activity at much higher concentrations

Laboratory study on larvicidal properties of leaf extract of Calotropis procera (Family-Asclepiadaceae) against mosquito larvae.

Fresh leaf extract of milkweed (Calotropis procera) showed larvicidal properties against mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. Methanolic extracts of the same plant were, however, more effective as larvicide. C. procera is a weed growing in uncultivated soils as well as in dry, arid zones in Indian subcontinent throughout the year. The results of this study suggest the utility of milkweed as potential technology for control of mosquito larvae.