Exploring the Sequence Diversity of Cyclotides from VietnameseViolaSpecies

Abstract

Viola, the largest genus in the Violaceae plant family, is known for its ubiquitous natural production of cyclotides. Many Viola species are used as medicinal herbs across Asia for the treatment of diseases, including ulcers and asthma, and are often consumed by humans in teas. Previous studies have isolated cyclotides from Viola species in many countries in the hope of discovering novel compounds with anticancer activities; however, Viola species from Vietnam have not been investigated to date. Here we report the discovery of cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam. In total, 10 cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. We show that the NMR structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), we tested the cytotoxicity of the cyclotides isolated from V. arcuata. All tested cyclotides were cytotoxic, albeit to varying degrees. The sequences discovered in this study significantly expand our understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis

“…Although ribes 21 and 22 lack a cyclic backbone, they show similar conserved 3D folds to the cyclic analogues ribe 33 and cycloviolacin O2. The sequences of ribes 21, 22, and 33 show most variation at hydrophobic loop 3, which has been reported to correlate to the cytotoxic activities of cyclotides …”

“…The cytotoxic activity of the isolated cyclotide and acyclotides from R. bengalensis was tested against human cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table ). Two cancer cell lines (PC-3, a human prostate cancer cells; MDA-MB-231, human breast cancer cells) and one noncancerous cell line (HUVEC; human umbilical vein endothelial cells) were used in the assays. Kalata B1 and cycloviolacin O2, reported in the plants Oldenlandia affinis and V. odorata , respectively, were isolated to use as positive controls in the cytotoxicity and hemolytic assays. ,,, …”

“…Next, the supernatant obtained from this extraction was collected and filtered, before being lyophilized on a freeze-dryer (CHRIST Alpha 2-4 LD). The lyophilized crude extract was reduced with 10 mM dithiothreitol, alkylated with 25 mM iodoacetamide, and digested with endo Glu-C for 16 h; it was then desalted with C 18 Ziptips (Millipore) . Peptide mass signals were analyzed using MALDI-TOF/MS (Bruker UltrafleXtreme TOF-TOF/MS).…”

From farm to pharm: taking plant pharmaceuticals from research to production

“…Whereas plants generally do not harbor human pathogens, the safety of closed and sterile bioreactors is much higher compared to field grown plants in terms of product contamination with endotoxins and mycotoxins as well as possible release of genetically modified material into the wild. Serendipitously, plant viruses typically cannot infect suspension cultures, as their means of transportation, the plasmodesmata, is not available [55]. Table 2 compares mammalian cells with various plant-based production systems and outlines the opportunities provided by plant cell suspensions.…”

“…and Viola austrosinensis (cyO2) [55]. Establishing suspension cultures of these three species would be a first step to test the prediction.…”