Anti-diabetic potential of Urena lobata leaf extract through inhibition of dipeptidyl peptidase IV activity

Objective: To evaluate the anti-diabetic potential of leaf extract from Urena lobata(U. lobata) through dipeptidyl peptidase IV(DPP-IV) inhibitory activity.Methods: U. lobata leaf was extracted in hot water and ethanol. The activity of DPPIV inhibitor was tested by in vitro study using gly-pro-p-nitroanilide as substrat of DPPIV and vildagliptin, as standard reference. A product of the reactions between gly-pro-pnitroanilide and DPP-IV, was observed by microplate readers with λ = 405 nm. All data were expressed as mean ± SD and the IC50 value was determined by non linear regression curve fit. Active substances in leaf extract of U. lobata was analyzed by liquid chromatography-mass spectrometry. DPP-IV inhibitory activity of active compounds was evaluated in silico using docking server. Results: The ethanolic extract of U. lobata showed stronger DPP-IV inhibitor activity than water extract with the IC50 values of 1 654.64 and 6 489.88 μg/mL, respectively. Vildagliptin, based on standard reference for DPP-IV inhibitor activity, has IC50 value of 57.44 μg/mL. Based on in silico analysis, mangiferin, stigmasterol and β-sitosterol in U. lobata extract have a strong inhibitory activity on DPP-IV. Conclusions: The results showed that DPP-IV inhibitory activity of U. lobata is related to its active compounds such as mangiferin, stigmasterol and β-sitosterol.

Decreasing a-synuclein aggregation by methanolic extract of Centella asiatica in zebrafish Parkinson's model

Objective: To observe the effects of Centella asiatica(C. asiatica) methanolic extract on a-synuclein aggregation and its expression in rotenone-exposed zebra fish.Methods: Zebra fish(Danio rerio) were exposed to 5 m g/L rotenone for 28 days and coincubated with 2.5, 5.0 and 10.0 m g/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebra fish were sacrificed on the ice block and protein was isolated from zebra fish brain for ELISA of dopamine and Western blotting of a-synuclein. Immunohistochemistry was conducted to observe the a-synuclein expressions from histopathological preparation of zebra fish brain. The head were soaked in 10% formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using anti a-synuclein antibody. We also measured zebra fish motility for 5 min in each week.Results: C. asiatica has important bioactive compounds such as asiaticoside that has antiin flammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack to a-synuclein protein that caused aggregation and increase of its expression.The motility of zebra fish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebra fish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increased a-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebra fish, but C. asiatica increased dopamine level.Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson's such as a-synuclein aggregation and expression, increasing motility and dopamine as well.

Prediction of protein structure of novel protein (116 kDa) from human sperm membrane

Objective:To predict the protein similarity, biological function and structure of 116 kDa protein that was isolated from human sperm membrane of acrosome by usingin silico. Methods:Predictions for 116 kDa protein similarity was done through comparative analysis of its isoelectric point (pI) and molecular weight to the UniProtKB/TrEMBL protein database. The three-dimensional structure of the protein was built using SWISS-MODEL.Results:The 116 kDa protein with pI 4.4 was expected to have similarities with CDH23 proteins that had pI 4.38, thus they likely had similar physical and biochemical properties. The predicted protein had an extracellular N-terminal that likely acted as a receptor, and showed cell recognition characteristic, playing a role in fertilization.Conclusions:The 116 kDa protein isolated from acrosome membrane has similarity on its biophysical character to the CDH23.

Molecular interaction of zp3 to zp3r reveals a cross-species fertilization mechanism

Objective: To evaluate the role of ZP3R in the species-specific fertilization mechanism. Methods:ZP3/ZP3R protein sequences ofMus musculus,Rattus norvegicus, andCavia porcellus were downloaded from UNIPROT. Percentage of amino acids that was calculated by using the SIAS program. Protein sequences modeled was established by using the Modeller 9.14 program and glycosylation of the ZP3 using GlyProt program. Docking simulation of the ZP3R-ZP3 was performed between the same species and different species with PatchDock program.Results:Comparison of the ZP3R and ZP3 structure between species showed that ZP3 in these three species was more similar than ZP3R. Docking simulations of protein showed that changes in the pattern of the ZP3-ZP3R domain for interaction on cross-species compared to the same species. Changes in the pattern of binding ZP3R-ZP3 made sperm-egg binding was not functional and could inhibit cross-fertilization.Conclusions: ZP3R-ZP3 interaction is species-specific, and the role of ZP3R is greater than ZP3 in determining the species-specific recognition stage and sperm-egg binding.

Identification of DNA Photolyase Gene of a Drought Tolerance Branching Line of Kenaf (<i>Hibiscus cannabinus</i>L.)

DNA photolyase is a photoreactivation enzyme which has a role in the mechanism of DNA repair. The existence of DNA photolyase gene could affect the success of mutation process. SM026H is a drought tolerance branching line of kenaf identified by Research Institute for Tobacco and Fibre Crops, Karangploso, Malang, Indonesia which has a potential as a source for mutation breeding. In this research, cloning and sequencing of the DNA photolyase gene of line SM026H were conducted. The DNA of SM026H leaves was isolated using DNeasy Kit, then was amplified by Polymerase Chain Reaction (PCR) using AC1 as a forward primer, paired with AC3R or AC4R as reverse primers and checked using electrophoresis on a 0.7% agarose gel. The AC1-AC3R primer pair produced a DNA fragment with a size of 750 bp, whereas the AC1-AC4R primer pair produced a DNA fragment of 1000 bp in size. Cloning of the AC1-AC3R and AC1-AC4R fragments was done prior to sequencing. Preparation for cloning was done by running and extracting the PCR products from a 0.7% Low Melting Agarose (LMA), and then ligating them to a pCR21 plasmid using an electroporation method. Two sub-fragments of each AC1-AC3R and AC1-AC4R fragments were identified. They were 600 bp and 500 bp, resulting from the AC1-AC3R fragment, and 1300 bp and 500 bp, resulting from the AC1-AC4R fragment. The sequencing result of those sub-fragments was analyzed using a Basic Local Alignment Search Tool (BLAST) program. It was shown that the sequences have a degree of homology to DNA photolyase sequence of Arabidopsis thaliana A. thaliana, Cucumis sativus, Spinacia oleracea, Stellaria longipes and Oryza sativa. These suggested that the kenaf line SM0026H possesses the DNA photolyase gene. However, further study needs to be done to ascertain that the gene was not the cryptochrome.