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Study of the activity of extracts and compounds of Moquiniastrum polymorphun, Blutaparon portulacoides, Doliocarpus dentatus and Piper sp on leucocytes and in vitro coagulation system of patients with rheumatological diseases.
Federal University of Grande Dourados – UFGD
University Hospital of Federal University of Grande Dourados . R. Ivo Alves da Rocha, 558.Altos do Indaia, Dourados, MS, 79823501
Caroline Andre de Souza Jorge
Candida Aparecida Leite Kassuya,Elisangela Santos,Laura Priscila Toledo Bernal,Armando Jorge Junior,Marcia Midori,Marica Strassburger, Lidiane Schutz, Luis Fernando Marcorini
portuguese and english
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), are characterized by chronic inflammation of different organs. It is evidenced in the literature that the presence of inflammatory process activates mechanisms that favor the formation of thrombi, through the presence of cytokines that induce macrophages to the activation of the coagulation process (Esmon, 2008). Some plants such as Moquiniastrum polymorphum, Blutaparon portulacoides, Doliocarpus dentatus and Piper sp showed anti-inflammatory and anti-immunogenic activity in experimental models (Limeiras et al., 2016; Oliveira et al., 2016). It is relevant to test the extracts and compounds in human material, in vitro, in relation to anticoagulant activity and chemotaxis, viability of leukocytes, especially neutrophils.
What is the aim of the project?
To determine if extracts and compounds obtained from Moquiniastrum polymorphum, Blutaparon portulacoides, Doliocarpus dentatus and Piper sp affect in vitro the coagulation system and the leukocyte levels of patients with systemic lupus erythematosus and rheumatoid arthritis and its models of inflammation and pain in animals.
What techniques and methods are used?
In human samples 1. Collection of biological samples: The biological samples will be collected from the patients of the UFGD University Hospital, through the signing of the Informed Consent Term. 2. Separation of plasma and leukocytes: The separation will be done through the centrifugation process with specific equipment. 3. Cytokine dosing: The dosage will be performed by enzyme-linked immunosorbent assay (ELISA) 4. Testing of extracts and compounds on anticoagulant activity: Will be evaluated through the Prothrombin Activity Time (TAP) and Activated Partial Thromboplasmin Time (TTAP) methods through Quick-Timer analysis. 5. Test of extracts and compounds in leukocytes isolated from human blood: 1) Isolation of neutrophils from patients' blood: Through centrifugation 2) Chemotaxis and neutrophil viability assay: Evaluated from the monitoring of the evolution of the infection and presence of phlogistic signs and blood levels of plasma levels of leukocytes In animals: 1. Models of inflammation: The animals will receive an injection of 50 ulsc in the right hind paw of carrageenan (300 ug / paw) which will be suspended in sterile 0.9 per cent saline solution. The contralateral paw will receive only saline solution and will be used as control. 2. Pain models: Pain will be induced by intraplantar injection of capsaicin (CAP) and intraperitoneal acetic acid in mice. 3. Toxicity: The animals will be treated with different dosages of the plant extract for weeks to perform the biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (gamma-GT), bilirubins, albumin, total protein, nitrogen serum creatinine, sodium, potassium, globulin, calcium, cholesterol, HDL cholesterol and glucose, with COBAS integrates 400 Plus (Roche Diagnostics); the weighing of vital organs (heart, lung, kidney, adrenal, liver and spleen) and reproductive organs (testis, epididymis, seminal vesicle and vas deferens); and analysis of the organs removed, the tissues (kidney, liver, testis and epididymis) will be fixed in Bouin's solution, the sections will be stained with hematoxylin and eosin and observed by light microscopy to evaluate the general histopathological parameters. Statistical analyses: The data will be presented as the mean plus minus standard error of the mean (SEM). Student's T-test will be used for comparisons between two experimental groups (acute toxicity). Differences between 3 or more groups will be determined by analysis of variance (ANOVA) followed by the Tukey or Newman-Keuls or Kruskal-Wallis test followed by the Dunn test. Significant differences will be considered significant at p minor 0.05. The graphics will be produced using GraphPad Prism software.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student will accompany the collection of biological samples from the patients; The student will accompany in the analysis of the effects of plant extracts on the experimental models of pain and inflammation induced in rats in the laboratory; The student will accompany the analysis of toxicities of plant extracts in experimental models in vivo in rats and in vitro in human biological samples; The student will accompany the in vitro analysis of the effects of plant extracts on the blood coagulation system and the leukocyte levels of biological samples from patients with systemic lupus erythematosus and rheumatoid arthritis.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes. Tutors will offer training to exchange students. The tutor will present the laboratory to the exchange students, the procedures performed, the methods used and the team present. The tutor will show how to handle the materials and equipment and explain the project objectives and expected results, as well as the mechanisms to achieve them.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a poster - The student will prepare a presentation - The student will prepare a scientific report - The student will prepare an abstract - The student’s name will be mentioned in a future publication - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
The student should already have knowledge of laboratory biosafety, brings his/her own lab coat and logbook. Appropriate clothing (lab coat, closed shoes, long pants) and hair stuck inside the lab.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Students in biomedical fields
- da Silva Balin P; Zanatta FC; Jorge BC; Leitao M; Kassuya RM; Cardoso CAL; Kassuya CAL; Arena AC. Toxicological evaluation and anti-inflammatory potential of an ethanolic extract from Bromelia balansae (Bromeliaceae) fruit. J Ethnopharmacol. 2018 Aug 10;222:79-86. doi: 10.1016/j.jep.2018.04.049. Epub 2018 May 3. PubMed PMID: 29729384.
- Ishikawa RB; Leitao MM; Kassuya RM; Macorini LF; Moreira FMF; Cardoso CAL; Coelho RG; Pott A; Gelfuso GM; Croda J; Oliveira RJ; Kassuya CAL. Anti-inflammatory; antimycobacterial and genotoxic evaluation of Doliocarpus dentatus. J Ethnopharmacol. 2017 May 23;204:18-25. doi: 10.1016/j.jep.2017.04.004. Epub 2017 Apr 6. PubMed PMID: 28392458.
- Limeiras SM; Oliveira BC; Pessatto LR; Pesarini JR; Kassuya CA; Monreal AC; Cantero WB; Antoniolli-Silva R; Antoniolli-Silva AC; Stefanello ME; Oliveira RJ. Effects of Moquiniastrum polymorphum ssp floccosum ethnolic extract on colorectal carcinogenesis induced by 1;2-dimethylhydrazine. Genet Mol Res. 2017 Mar 16;16(1). doi: 10.4238/gmr16019518. PubMed PMID: 28362989
- Branquinho LS; Santos JA; Cardoso CAL; Mota JDS; Junior UL; Kassuya CAL; Arena AC. Anti-inflammatory and toxicological evaluation of essential oil from Piper glabratum leaves. J Ethnopharmacol. 2017 Feb 23;198:372-378. doi: 10.1016/j.jep.2017.01.008. Epub 2017 Jan 18. PubMed PMID: 28109914.
- Oliveira RJ; de David N; Pesarini JR; Nogueira TD; Kassuya CA; Strapasson RL; Stefanello ME; Monreal AC; Matuo R; Antoniolli-Silva AC. Moquiniastrum polymorphum subsp floccosum extract: screening for mutagenic and antimutagenic activity. Genet Mol Res. 2016 Oct 24;15(4). doi: 10.4238/gmr15048976. PubMed PMID: 27813611
- Hoff Brait DR; Mattos Vaz MS; da Silva Arrigo J; Borges de Carvalho LN; Souza de Araujo FH; Vani JM; da Silva Mota J; Cardoso CA; Oliveira RJ; Negrao FJ; Kassuya CA; Arena AC. Toxicological analysis and anti-inflammatory effects of essential oil from Piper vicosanum leaves. Regul Toxicol Pharmacol. 2015 Dec;73(3):699-705. doi: 10.1016/j.yrtph.2015.10.028. Epub 2015 Nov 7. PubMed PMID: 26545326.
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