Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

Cyclotides are a plant-derived family of peptides that comprise approximately 30 amino acid residues, a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action mechanisms. We focus on their toxicities to insect pests and their medical and agricultural applications.

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Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world. Present therapies for COPD have limited effect on reducing the progression of COPD and suppressing the inflammatory response in the lung. Bulbs of Fritillaria cirrhosa D. Don (BFC) have been used in many Asian countries for a long time to treat pulmonary diseases, such as cough, expectoration, and asthma. Steroidal alkaloids are the major biological active constituents in BFC, whereby imperialine is one of the important steroidal alkaloids. So far, there are no studies reporting the effect of imperialine on COPD. In this study, we investigated the effect of imperialine on pulmonary function and structure and inflammation in a COPD-like rat model which was induced by the combination of exposure to CS and intratracheal administration of LPS. Our data show that imperialine mitigates pulmonary functional and structural impairment and suppressed inflammatory response in a COPD-like rat model by mediating expression of related cytokines in lung tissues of the COPD-like rats, such as IL-1β, IL-6, IL-8, TNF-α, NF-κB, TGF-β1, MMP-9, and TIMP-1.

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.