Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massa.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back ~100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 µM against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

KR-12 Derivatives Endow Nanocellulose with Antibacterial and Anti-Inflammatory Properties: Role of Conjugation Chemistry.

This work combines the wound-healing-related properties of the host defense peptide KR-12 with wood-derived cellulose nanofibrils (CNFs) to obtain bioactive materials, foreseen as a promising solution to treat chronic wounds. Amine coupling through carbodiimide chemistry, thiol-ene click chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition were investigated as methods to covalently immobilize KR-12 derivatives onto CNFs. The effects of different coupling chemistries on the bioactivity of the KR12-CNF conjugates were evaluated by assessing their antibacterial activities against Escherichia coli and Staphylococcus aureus. Potential cytotoxic effects and the capacity of the materials to modulate the inflammatory response of lipopolysaccharide (LPS)-stimulated RAW 245.6 macrophages were also investigated. The results show that KR-12 endowed CNFs with antibacterial activity against E. coli and exhibited anti-inflammatory properties and those conjugated by thiol-ene chemistry were the most bioactive. This finding is attributed to a favorable peptide conformation and accessibility (as shown by molecular dynamics simulations), driven by the selective chemistry and length of the linker in the conjugate. The results represent an advancement in the development of CNF-based materials for chronic wound care. This study provides new insights into the effect of the conjugation chemistry on the bioactivity of immobilized host defense peptides, which we believe to be of great value for the use of host defense peptides as therapeutic agents.

Differential Blood-Brain Barrier Transport and Cell Uptake of Cyclic Peptides In Vivo and In Vitro.

The blood-brain barrier (BBB) poses major challenges to drug delivery to the CNS. SFTI-1 and kalata B1 are cyclic cell-penetrating peptides (cCPPs) with high potential to be used as scaffolds for drug delivery. We here studied their transport across the BBB and distribution within the brain to gauge the potential of these two cCPPs as scaffolds for CNS drugs. In a rat model, SFTI-1 exhibited, for a peptide, high extent of BBB transport with a partitioning of unbound SFTI-1 across the BBB, Kp,uu,brain, of 13%, while only 0.5% of kalata B1 equilibrated across the BBB. By contrast, kalata B1, but not SFTI-1, readily entered neural cells. SFTI-1, but not kalata B1, could be a potential CNS delivery scaffold for drugs directed to extracellular targets. These findings indicate that differences between the BBB transport and cellular uptake abilities of CPPs are crucial in the development of peptide scaffolds.

A Synthetic Cyclized Antimicrobial Peptide with Potent Effects against Drug-Resistant Skin Pathogens.

Dermal infections requiring treatment are usually treated with conventional antibiotics, but the rise of bacterial resistance to first-line antibiotics warrants alternative therapeutics. Here, we report that a backbone-cyclized antimicrobial peptide, CD4-PP, designed from the human host defense peptide LL-37, has strong direct antibacterial effects on antibiotic sensitive as well as resistant-type strains and clinical isolates of common skin pathogens in the low (<2) µM range. In addition, it influences innate immunity in keratinocytes, and treatment with CD4-PP is able to clear bacterial infections in infected keratinocytes. Additionally, CD4-PP treatment significantly reduces the wound area in a lawn of keratinocytes infected with MRSA. In conclusion, CD4-PP has the potential to serve as a future drug treating wounds infected with antibiotic-resistant bacteria.

On the structures of the penduflorines from Tabernaemontana penduliflora.

The structures of the recently published monoterpene indole alkaloids penduflorines A and B (1a and 1b), isolated from Tabernaemontana penduliflora (Apocynaceae), have been revised. Rather than an inseparable mixture of two compounds, they appear to be the known alkaloid vobasine (2). Although we could not comprehensively revise the structures of penduflorines C-E due to lacking spectral data, since their structural elucidations were based on that of 1a and 1b, their structures should also be treated with caution.

Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

Antibacterial eremophilane sesquiterpenoids from Xylaria feejeensis, an endophytic fungi of the medicinal plant Geophila repens.

Geophila repens (L.) I.M. Johnst (Rubiaceae) is a traditional medicinal plant used in Sri Lanka for the treatment of bacterial infections. Due to its rich endophytic fungi content, it was postulated that endophytically-produced specialized metabolites may be responsible for its purported antibacterial effects. To test this hypothesis, eight pure endophytic fungal cultures were isolated from G. repens then extracted and screened for antibacterial activity in a disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Large scale culturing, extraction, and purification of the most active fungal extract, obtained from Xylaria feejeensis, led to the isolation of 6',7'-didehydrointegric acid (1), 13-carboxyintegric acid (2), and four known compounds including integric acid (3). Compound 3 was isolated as the key antibacterial component (MIC = 16 µg/mL against Bacillus subtilis, 64 µg/mL against Methicillin-Resistant S. aureus). Compound 3 and its analogues were devoid of hemolytic activity up to the highest tested concentration of 45 µg/mL. This study demonstrates that specialized metabolites produced by endophytic fungi may contribute to the biological activity of some medicinal plants. Endophytic fungi should be evaluated as a potential source of antibiotics, especially from unexplored medicinal plants traditionally used for the treatment of bacterial infections.

Exploring the Limits of Cyanobactin Macrocyclase PatGmac: Cyclization of PawS-Derived Peptide Sunflower Trypsin Inhibitor-1 and Cyclotide Kalata B1.

The subtilisin-like macrocyclase PatGmac is produced by the marine cyanobacterium Prochloron didemni. This enzyme is involved in the last step of the biosynthesis of patellamides, a cyanobactin type of ribosomally expressed and post-translationally modified cyclic peptides. PatGmac recognizes, cleaves, and cyclizes precursor peptides after a specific recognition motif comprised of a C-terminal tail with the sequence motif -AYDG. The result is the native macrocyclic patellamide, which has eight amino acid residues. Macrocyclase activity can be exploited by incorporating that motif in other short linear peptide precursors, which then are formed into head-to-tail cyclized peptides. Here, we explore the possibility of using PatGmac in the cyclization of peptides larger than the patellamides, namely, the PawS-derived peptide sunflower trypsin inhibitor-1 (SFTI-1) and the cyclotide kalata B1. These peptides fall under two distinct families of disulfide constrained macrocyclic plant peptides. They are both implicated as scaffolds for drug design due to their structures and unusual stability. We show that PatGmac can be used to efficiently cyclize the 14 amino acid residue long SFTI-1, but less so the 29 amino acid residue long kalata B1.

Transforming Cross-Linked Cyclic Dimers of KR-12 into Stable and Potent Antimicrobial Drug Leads.

Is it possible to enhance structural stability and biological activity of KR-12, a truncated antimicrobial peptide derived from the human host defense peptide LL-37? Based on the mapping of essential residues in KR-12, we have designed backbone-cyclized dimers, cross-linked via a disulfide bond to improve peptide stability, while at the same time improving on-target activity. Circular dichroism showed that each of the dimers adopts a primarily alpha-helical conformation (55% helical content) when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new cross-linked cyclic form. Compared to KR-12, one of the cross-linked dimers showed 16-fold more potent antimicrobial activity against human pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans and 8-fold increased activity against Escherichia coli. Furthermore, these peptides retained antimicrobial activity at physiologically relevant conditions, including in the presence of salts and in human serum, and with selective Gram-negative antibacterial activity in rich growth media. In addition to giving further insight into the structure-activity relationship of KR-12, the current work demonstrates that by combining peptide stabilization strategies (dimerization, backbone cyclization, and cross-linking via a disulfide bond), KR-12 can be engineered into a potent antimicrobial peptide drug lead with potential utility in a therapeutic context.

Screening for Cyclotides in Sri Lankan Medicinal Plants: Discovery, Characterization, and Bioactivity Screening of Cyclotides from Geophila repens.

Cyclotides are an intriguing class of structurally stable circular miniproteins of plant origin with numerous potential pharmaceutical and agricultural applications. To investigate the occurrence of cyclotides in Sri Lankan flora, 50 medicinal plants were screened, leading to the identification of a suite of new cyclotides from Geophila repens of the family Rubiaceae. Cycloviolacin O2-like (cyO2-like) gere 1 and the known cyclotide kalata B7 (kB7) were among the cyclotides characterized at the peptide and/or transcript level together with several putative enzymes, likely involved in cyclotide biosynthesis. Five of the most abundant cyclotides were isolated, sequenced, structurally characterized, and screened in antimicrobial and cytotoxicity assays. All gere cyclotides showed cytotoxicity (IC50 of 2.0-10.2 µM), but only gere 1 inhibited standard microbial strains at a minimum inhibitory concentration of 4-16 µM. As shown by immunohistochemistry, large quantities of the cyclotides were localized in the epidermis of the leaves and petioles of G. repens. Taken together with the cytotoxicity and membrane permeabilizing activities, this implicates gere cyclotides as potential plant defense molecules. The presence of cyO2-like gere 1 in a plant in the Rubiaceae supports the notion that phylogenetically distant plants may have coevolved to express similar cytotoxic cyclotides for a specific functional role, most likely involving host defense.

Where traditional Chinese medicine meets Western medicine in the prevention of rheumatoid arthritis.

Chinese medicine has a long tradition of use against rheumatoid arthritis (RA). The formulations are based on combinations of typically 5-10 plants, which are usually boiled and administered as a decoction or tea. There are few clinical trials performed so the clinical evidence is sparse. One fundamental of traditional medicine is to prevent disease. RA is an autoimmune, inflammatory and chronic disease that primarily affects the joints of 0.5%-1% of the population. In two out of three of the cases, the patients are characterised by the presence of autoantibodies such as the rheumatoid factor and the more disease-specific autoantibody against citrullinated proteins, so-called 'ACPA' (anticitrullinated protein/peptide antibodies). ACPA positivity is also strongly associated with specific variations in the HLA-DRB1 gene, the shared epitope alleles. Together with smoking, these factors account for the major risks of developing RA. In this review, we will summarise the background using certain plant-based formulations based on Chinese traditional medicine for the treatment and prevention of RA and the strategy we have taken to explore the mechanisms of action. We also summarise the major pathophysiological pathways related to RA and how these could be analysed. Finally, we summarise our ideas on how a clinical trial using Chinese herbal medicine to prevent RA could be conducted.

A stable cyclized antimicrobial peptide derived from LL-37 with host immunomodulatory effects and activity against uropathogens.

The increasing antibiotic resistance among uropathogenic bacteria warrants alternative therapeutic strategies. We demonstrate the potential of the synthetic peptide CD4-PP, designed by dimerization and backbone cyclization of the shortest antimicrobial region of human cathelicidin, LL-37. CD4-PP is active against clinical and type strains of common uropathogens Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa at concentrations substantially below cellular cytotoxic levels and induced membrane deformation and leakage in E. coli and P. aeruginosa. Furthermore, CD4-PP treatment prevented the formation of new biofilm and dissolved mature biofilm created by E. coli and P. aeruginosa and targeted curli amyloid in E. coli biofilms. In addition, CD4-PP also induced production of LL-37 by uroepithelial cells and increased the expression of tight junction proteins claudin-14 and occludin. During uroepithelial cell infection, CD4-PP significantly reduced uropathogen survival when treatment was given at the start of infection. Low micromolar of CD4-PP treatment initiated after 2 h was successful with all tested species, except P. aeruginosa where CD4-PP was unable to reduce survival, which could be attributed by early biofilm formation. Finally, we demonstrated that urinary catheter pieces coated with saline fluid supplemented with CD4-PP reduced the attachment of E. coli, giving it a potential clinical application.

The Influence of Plant Stress Hormones and Biotic Elicitors on Cyclotide Production in Viola uliginosa Cell Suspension Cultures.

Cyclotides are macrocycle peptides produced by plants from several families, including Violaceae. These compounds have the potential for applications in medicine, bioengineering and crop protection thanks to their multiple biological activities. In most cases, cyclotides are extracted from plant material. Plant cell culture provides a viable and sustainable form of plant biomass production Cyclotides are host defense peptides. The aim of the current study was to test whether different plant stress hormones and biological elicitors have effects on cyclotide production in Viola uliginosa suspension cultures. Different concentrations of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and neutralized pathogens were tested. The cyclotide production was assessed using MALDI-MS. Five major peptides produced by V. uliginosa cultures were chosen for analysis, of which one was sequenced de novo. The treatments had little influence on the suspension's growth, with the exception of 100 µM SA, which enhanced the biomass increase, and 100 µM ABA, which was toxic. Significant increases in the production of three cyclotides (viul M, cyO13 and cyO3) were observed in suspensions primed with JA (50 µM, 100 µM, 200 µM) after 14 days of culturing. Biotic elicitors had no observable effect on cyclotide production. The current study indicates that some cyclotides in V. uliginosa are triggered in response to JA. The stress plant hormones can be used to enhance plant cell culture-based production systems.

The involvement of cyclotides in mutual interactions of violets and the two-spotted spider mite.

Plants employ different chemicals to protect themselves from herbivory. These defenses may be constitutive or triggered by stress. The chemicals can be toxic, act as repellents, phagosuppressants and/or phago-deterrents. The two-spotted spider mite (Tetranychus urticae) is a generalist arthropod herbivorous pest and its feeding causes extensive damage both to crops and wild plants. Cyclotides are cyclic peptides involved in host-plant defenses. A single Viola sp. can produce more than a hundred cyclotides with different biological activities and roles. The organ and tissue specific cyclotide patterns change over the seasons and/or with environment, but the role of biotic/abiotic stress in shaping them remains unclear. Here, we demonstrate the involvement of cyclotides in mutual interactions between violets and mites. We used immunohistochemistry and mass spectrometry imaging to show the ingested cyclotides in T. urticae and assess the Viola odorata response to mite feeding. Moreover, to assess how mites are affected by feeding on violets, acceptance and reproductive performance was compared between Viola uliginosa, V. odorata and Phaseolus vulgaris. We demonstrate that cyclotides had been taken in by mites feeding on the violets. The ingested peptides were found in contact with epithelial cells of the mite digestive system, in the fecal matter, feces, ovary and eggs. Mites preferred common bean plants (P. vulgaris) to any of the violet species; the latter affected their reproductive performance. The production of particular cyclotides in V. odorata (denoted by molecular weights: 2979, 3001, 3017, 3068, 3084, 3123) was activated by mite feeding and their levels were significantly elevated compared to the control after 5 and 21 days of infestation. Specific cyclotides may affect mites by being indigestible or through direct interaction with cells in the mite digestive tract and reproductive organs. A group of particular peptides in V. odorata appears to be involved in defense response against herbivores.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Barrettides: A Peptide Family Specifically Produced by the Deep-Sea Sponge Geodia barretti.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC50 0.64 µM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Kaempferia galanga L.: Progresses in Phytochemistry, Pharmacology, Toxicology and Ethnomedicinal Uses.

K. galanga is an aromatic medicinal herb. It is locally to India and distributed in China, Myanmar, Indonesia, Malaysia, and Thailand. K. galanga is a Traditional Chinese Herb Medicine (TCHM), which has been applied to treat cold, dry cough, toothaches, rheumatism, hypertension and so on. In addition, it has been used widely as spices since its highly aromas. The aim of this review is to compile and update the current progresses of ethnomedicinal uses, phytochemistry, pharmacology and toxicology of K. galanga. All the data on K. galanga were based on different classical literary works, multiple electronic databases including SciFinder, Web of Science, PubMed, etc. The results showed that ninety-seven compounds have been identified from rhizome of K. galanga, including terpenoids, phenolics, cyclic dipeptides, flavonoids, diarylheptanoids, fatty acids and esters. Modern pharmacology studies revealed that extracts or secondary metabolites of the herb possessed anti-inflammatory, anti-oxidant, anti-tumorous, anti-bacterial, and anti-angiogenesis effects, which were closely related to its abundant ethnomedicinal uses. In conclusion, although previous research works have provided various information of K. galanga, more in-depth studies are still necessary to systemically evaluate phytochemistry, pharmacological activities, toxicity and quality control of this herb.

Functional Characterization of the Nemertide α Family of Peptide Toxins.

Peptide toxins find use in medicine, biotechnology, and agriculture. They are exploited as pharmaceutical tools, particularly for the investigation of ion channels. Here, we report the synthesis and activity of a novel family of peptide toxins: the cystine-knotted α nemertides. Following the prototypic α-1 and -2 (1 and 2), six more nemertides were discovered by mining of available nemertean transcriptomes. Here, we describe their synthesis using solid phase peptide chemistry and their oxidative folding by using an improved protocol. Nemertides α-2 to α-7 (2-7) were produced to characterize their effect on voltage-gated sodium channels (Blatella germanica BgNaV1 and mammalian NaVs1.1-1.8). In addition, ion channel activities were matched to in vivo tests using an Artemia microwell assay. Although nemertides demonstrate high sequence similarity, they display variability in activity on the tested NaVs. The nemertides are all highly toxic to Artemia, with EC50 values in the sub-low micromolar range, and all manifest preference for the insect BgNaV1 channel. Structure-activity relationship analysis revealed key residues for NaV-subtype selectivity. Combined with low EC50 values (e.g., NaV1.1: 7.9 nM (α-6); NaV1.3: 9.4 nM (α-5); NaV1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

Cyclotide host-defense tailored for species and environments in violets from the Canary Islands.

Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC-MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC-MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 µg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis.

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.