RESUMO
OBJECTIVE: Calotropis procera latex protein (CpLP) is a popular anti-inflammatory and therefore we aimed to study its effects on inflammatory bone loss. DESIGN: Male Wistar rats were subjected to a ligature of molars. Groups of rats received intraperitoneally CpLP (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) or saline (0.9% NaCl) one hour before ligature and then daily up to 11 days, compared to naïve. Gingiva was evaluated by myeloperoxidase activity and interleukin-1 beta (IL-1ß) expression by ELISA. Bone resorption was evaluated in the region between the cement-enamel junction and the alveolar bone crest. The histology considered alveolar bone resorption and cementum integrity, leukocyte infiltration, and attachment level, followed by immunohistochemistry bone markers between 1st and 2nd molars. Systemically, the weight of the body and organs, and a leukogram were performed. RESULTS: The periodontitis significantly increased myeloperoxidase activity and the IL-1ß level. The increased bone resorption was histologically corroborated by periodontal destruction, leukocyte influx, and attachment loss, as well as the increasing receptor activator of the nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio, and Tartrate-resistant acid phosphatase (TRAP)+ cells when compared to naïve. CpLP significantly reduced myeloperoxidase activity, level of IL-1ß, alveolar bone resorption, periodontal destruction, leukocyte influx, and attachment loss. The CpLp also reduced the RANKL/OPG ratio and TRAP+ cells, when compared with the saline group, and did not affect the systemic parameters. CONCLUSIONS: CpLP exhibited a periodontal protective effect by reducing inflammation and restricting osteoclastic alveolar bone resorption in this rat model.
Assuntos
Perda do Osso Alveolar , Calotropis , Ratos , Masculino , Animais , Ratos Wistar , Látex/farmacologia , Peroxidase , Calotropis/metabolismo , Inflamação/prevenção & controle , Perda do Osso Alveolar/patologia , Osteoprotegerina/farmacologia , Processo Alveolar/metabolismo , Antioxidantes , Ligante RANK/metabolismoRESUMO
An in-depth understanding of the development and distribution of laticifer (latex secretory structure) will be important for the production of both rubber and medicines and will support studies on plant adaptations to their environments. We characterize here and describe the ontogenesis of the laticifer sytem in Calotropis procera (Apocynaceae), an invasive subshrub species in arid landscapes. Anatomical and histochemical evaluations of the primary and secondary structures of the stem were carried out on a monthly basis during a full year, with ultrastructural evaluations of laticifer on the stem apex during the rainy season. In the primary structure, laticifer differentiate early from procambium and ground meristem cells of the cortex and medulla and become concentrated adjacent to the external and internal phloem of the bicollateral bundles. In the secondary structure, laticifer differentiates from fusiform derivative cells of the phloem close to the sieve-tube elements. The laticifer is of the articulated, anastomosing, branched type, and it originates from precursor cells that loose the transversal and longitudinal walls by dissolution. Latex is a mixture of terpenes, alkaloids, flavonoids, mucilage, and proteins. The apical meristem and vascular cambium where the laticifer system begins its development are active throughout the year, including during the dry season. The vascular cambium produces phloem with laticifer precursor cells during the rainy season, with high temperatures and long days. The ability of C. procera to grow under water deficit conditions and produce laticifer throughout the year contribute to its wide distribution in arid environments.
Assuntos
Apocynaceae , Calotropis , Calotropis/metabolismo , Látex/metabolismo , Polissacarídeos/metabolismo , MeristemaRESUMO
Nature is gifted with a wide range of ornamental plants, which beautify and clean the nature. Due to its great aesthetic value, there is a need to protect these plants from a variety of biotic and abiotic stresses. Hibiscus rosa-sinensis (L.) is an ornamental plant and it is commonly known as China rose or shoeblack plant. It is affected by several fungal and bacterial pathogens. Current study was designed to isolate leaf spot pathogen of H. rosa-sinensis and its control using silver nanoparticles (AgNPs). Based on molecular and morphological features, the isolated leaf spot pathogen was identified as Aspergillus niger. AgNPs were synthesized in the leaf extract of Calotropis procera and characterized. UV-vis spectral analysis displayed discrete plasmon resonance bands on the surface of synthesized AgNPs, depicting the presence of aromatic amino acids. Fourier transform infrared spectroscopy (FTIR) described the presence of C-O, NH, C-H, and O-H functional groups, which act as stabilizing and reducing molecules. X-ray diffraction (XRD) revealed the average size (~32.43 nm) of AgNPs and scanning electron microscopy (SEM) depicted their spherical nature. In this study, in vitro and in vivo antifungal activity of AgNPs was investigated. In vitro antifungal activity analysis revealed the highest growth inhibition of mycelia (87%) at 1.0 mg/ml concentration of AgNPs. The same concentration of AgNPs tremendously inhibited the spread of disease on infected leaves of H. rosa-sinensis. These results demonstrated significant disease control ability of AgNPs and suggested their use on different ornamental plants.
Assuntos
Calotropis , Hibiscus , Nanopartículas Metálicas , Rosa , Antifúngicos , Calotropis/metabolismo , Hibiscus/metabolismo , Nanopartículas Metálicas/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/metabolismo , Rosa/metabolismo , Prata/química , Prata/metabolismo , Prata/farmacologiaRESUMO
INTRODUCTION: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. AIMS: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. METHODS: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. RESULTS: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CONCLUSION: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.
Assuntos
Calotropis , Neoplasias , Animais , Apoptose , Calotropis/genética , Calotropis/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/metabolismo , Projetos Piloto , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/farmacologia , RNA Mensageiro , Espécies Reativas de Oxigênio , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera. OBJECTIVE: We aimed to evaluate the interaction between cardenolides and Na+/K+ ATPases from herbivores. METHODS: The Na+/K+ ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera. RESULTS: The replacement of serine in sensitive Na+/K+ ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides. CONCLUSION: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera.
Assuntos
Apocynaceae , Calotropis , Calotropis/metabolismo , Cardenolídeos/química , Cardenolídeos/metabolismo , Cardenolídeos/farmacologia , Herbivoria , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.
Assuntos
Calotropis , Regulação Neoplásica da Expressão Gênica , Genes de Plantas , Pressão Osmótica , Reação em Cadeia da Polimerase em Tempo Real/normas , Calotropis/genética , Calotropis/metabolismo , Padrões de ReferênciaRESUMO
The latex of Calotropis procera has previously been reported to contain osmotin. This protein (CpOsm) inhibited phytopathogens and this was mechanistically characterized. Here, the time-course profile of CpOsm transcripts was examined in the salt-stressed cultivated callus of C. procera in order to better understand its role in the physiology of the plant. Stressed callus (80 mM NaCl) showed an unbalanced content of organic compounds (proline and total soluble sugar) and inorganic ions (Na+, Cl-, and K+). Under salt treatment, the transcripts of CpOsm were detected after 12 h and slightly increased to a maximum at day seven, followed by reduction. Interestingly, CpOsm was not detected in the soluble protein fraction recovered from the salt-stressed callus as probed by electrophoresis, dot/Western blotting and mass spectrometry. The results suggested that (1) CpOsm is not constitutive in cultivated cells (laticifer-free tissues); (2) CpOsm transcripts appear under salt-stressed conditions; (3) the absence of CpOsm in the protein fractions of stressed cultivated cells indicated that salt-induced transcripts were not used for protein synthesis and this accounts to the belief that CpOsm may be a true laticifer protein in C. procera. More effort will be needed to unveil this process. In this study we show evidences that CpOsm gene is responsive to salt stress. However the corresponding protein is not produced in cultivated cells. Therefore, presently the hypothesis that CpOsm is involved in abiotic stress is not fully supported.
Assuntos
Calotropis/metabolismo , Látex/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico/fisiologia , Calotropis/genéticaRESUMO
Calotropis procera is a C3 plant native from arid environmental zones. It is an evergreen, shrubby, non-woody plant with intense photosynthetic metabolism during the dry season. We measured photosynthetic parameters and leaf biochemical traits, such as gas exchange, photochemical parameters, A/Ci analysis, organic solutes, and antioxidant enzymes under controlled conditions in potted plants during drought stress, and following recovery conditions to obtain a better insight in the drought stress responses of C. procera. Indeed, different processes contribute to the drought stress resilience of C. procera and to the fast recovery after rehydration. The parameters analyzed showed that C. procera has a high efficiency for energy dissipation. The photosynthetic machinery is protected by a robust antioxidant system and photoprotective mechanisms such as alternative pathways for electrons (photorespiration and day respiration). Under severe drought stress, increased stomatal limitation and decreased biochemical limitation permitted C. procera to maintain maximum rate of Rubisco carboxylation (Vc,max) and photosynthetic rate (Amax). On the other hand, limitation of stomatal or mesophyll CO2 diffusion did not impair fast recovery, maintaining Vc,max, chloroplast CO2 concentration (Cc) and mesophyll conductance (gm) unchanged while electron flow used for RuBP carboxylation (Jc) and Amax increased. The ability to tolerate drought stress and the fast recovery of this evergreen C3 species was also due to leaf anti-oxidative stress enzyme activity, and photosynthetic pigments. Thus, these different drought tolerance mechanisms allowed high performance of photosynthetic metabolism by drought stressed plants during the re-watering period.
Assuntos
Calotropis/metabolismo , Fotossíntese , Estações do Ano , Estresse Fisiológico , DesidrataçãoRESUMO
O carrapato Rhipicephalus microplus é um dos principais parasitos dos rebanhos bovinos. Diferentes grupos de carrapaticidas sintéticos têm sido empregados para o controle desse ácaro, contudo, o uso constante desses produtos tem selecionado populações resistentes. Na tentativa de minimizar esses impactos tem-se buscado avaliar fitoterápicos para o controle desse parasito. Assim, objetivou-se analisar a eficácia in vitro do extrato aquoso do algodão de seda em diferentes concentrações no controle da postura e eclodibilidade dos ovos do R. microplus. Foram avaliados os efeitos das concentrações 1, 5, 10, 25, 50, 75 e 100% do extrato aquoso obtido juntamente com controles com água destilada ou com cipermetrina, 12,5 mg L-1, associada a organofosforado, 150 mg L-1, sobre a eficiência reprodutiva do carrapato. Foi realizado o biocarrapaticidograma sendo que a eficácia acaricida das concentrações 5, 25 e 100% foram superiores àquela observada para o produto comercial utilizado, que correspondeu a 93,98%, indicando a importância de futuras pesquisas para avaliar o potencial toxigênico e a eficácia in vivo dessa planta para o controle do R. microplus .
The tick Rhipicephalus microplus is one of the main parasites of bovine herds. Different groups of synthetic acaricides have been used to control this mite; however, the constant use of these products has selected resistant populations. In an attempt to minimize these impacts, researchers have tried to evaluate herbal remedies to control this parasite. Therefore, the aim of this study was to analyze the in vitro efficacy of aqueous extract of silk cotton at different concentrations to control the hatchability of R. microplus eggs. The effects of the concentrations 1, 5, 10, 25, 50, 75 and 100% aqueous extract together with controls with distilled water or with cypermethrin, 12.5 mg L-1, associated with organophosphate, 150 mg L-1, were assessed on the reproductive efficiency of the tick. Immersion test was performed and the acaricide efficacy of the 5, 25 and 100% concentrations were higher than that obtained for the commercial product, which corresponded to 93.98%, indicating the importance of further research to assess the toxigenic potential and the in vivo efficacy of this plant to control R. microplus .
Assuntos
Extratos Vegetais/análise , Calotropis/metabolismo , Ácaros e Carrapatos/classificação , Controle de Ácaros e Carrapatos/instrumentação , Zona Semiárida , Gado/anormalidadesRESUMO
A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50=67.0 µg mL⻹), Neurospora sp. (IC50=57.5 µg mL⻹) and Colletotrichum gloeosporioides (IC50=32.1 µg mL⻹). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids.
Assuntos
Antifúngicos/metabolismo , Calotropis/metabolismo , Látex/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Calotropis/química , Colletotrichum/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Fusarium/efeitos dos fármacos , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Látex/química , Espectrometria de Massas , Peso Molecular , Neurospora/efeitos dos fármacos , Imunidade Vegetal , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Isoformas de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND AND AIMS: Calotropis procera and Calotropis gigantea, originally from warm parts of Africa and Asia, are now pan-tropical and in ecological terms considered an indicator of overgrazed, disturbed lands; they grow successfully in dry areas. Variations in water relations, morphology and photosynthesis of the two species growing in the same habitat were studied to assess possible mechanisms of tolerance to drought and how these relate to their ecophysiological success. Also the hypothesis that their photosynthetic rate (A) under drought would be affected by stomatal and non-stomatal limitations was tested. METHODS: Water relations, gas exchange, water use efficiency (WUE), fluorescence parameters, pubescence and specific leaf area (SLA) of Calotropis procera and C. gigantea plants growing in the field were evaluated during the wet (WS) and dry (DS) seasons. RESULTS: The xylem water potential (ψ) was similar in both species during the WS and DS; drought caused a 28 % decrease of ψ. In C. procera, A, stomatal conductance (g(s)) and carboxylation efficiency (CE) were higher in the WS with half the values of those during the DS, this species being more affected by drought than C. gigantea. A high δ(13)C of C. gigantea (-26·2 ) in the WS indicated a higher integrated WUE, in agreement with its lower g(s). Leaves of C. gigantea were more pubescent than C. procera. Relative stomatal and non-stomatal limitation of A increased with drought in both species; no changes in maximum quantum yield of photosystem II (PSII; F(v)/F(m)) were observed. The decrease in the relative quantum yield of PSII (Ï(PSII)) and in the photochemical quenching coefficient (q(P)) was more pronounced in C. procera than in C. gigantea. CONCLUSIONS: The photosynthetic capacity of C. procera was higher than that of C. gigantea. During the DS, A was regulated by stomatal and non-stomatal factors in a coordinated manner and drought did not cause chronic photoinhibition. A higher density of trichomes and leaf angle in C. gigantea may contribute to the maintenance of A and confer more efficient protection of photochemical activity in the DS. Ecophysiological traits such as high photosynthetic rate throughout the year even during the DS, and high WUE, highly pubescent leaves and low SLA observed in both species contribute to the establishment and growth of Calotropis in dry conditions.
Assuntos
Calotropis/fisiologia , Fotossíntese , Estômatos de Plantas/fisiologia , Transpiração Vegetal , Calotropis/metabolismo , Calotropis/efeitos da radiação , Secas , Ecossistema , Processos Fotoquímicos , Estômatos de Plantas/metabolismo , Estômatos de Plantas/efeitos da radiação , Especificidade da Espécie , Clima TropicalRESUMO
The laticifer fluid of Calotropis procera is rich in proteins and there is evidence that they are involved in the pharmacological properties of the latex. However, not much is known about how the latex-containing proteins are produced or their functions. In this study, laticifer proteins of C. procera were pooled and examined by 1D and 2D electrophoresis, masses spectrometry (MALDI-TOF) and characterized in respect of proteolytic activity and oxidative enzymes. Soluble laticifer proteins were predominantly composed of basic proteins (PI>6.0) with molecular masses varying between 5 and 95 kDa. Proteins with a molecular mass of approximately 26,000 Da were more evident. Strong anti-oxidative activity of superoxide dismutase (EC 1.15.1.1) (1007.74+/-91.89 Ug(-1)DM) and, to a lesser extent ascorbate peroxidase (EC 1.11.1.1) (0.117(d)+/-0.013 microMol H(2)O(2)g(-1)min(-1)), were detected. However, catalase (EC 1.11.1.6) was absent. The strong proteolytic activities of laticifer proteins from C. procera were shown to be shared by at least four distinct cysteine proteinases (EC 3.4.22.16) that were isolated by gel filtration chromatography. Serine and metaloproteinases were not detected and aspartic proteinase activities were barely visible. Chitinases (EC 3.2.1.14) were also isolated in a chitin column and their activities quantified. The presence of these enzymatic activities in latex from C. procera may confirm their involvement in resistance to phytopathogens and insects, mainly in its leaves where the latex circulates abundantly.