Identification of Cyclotides From Butterfly Pea ( Clitoria ternatea ) Petals and Its Biological Activities

Abstract

Background

Cyclotides are macrocyclic peptides characterized by a head-to-tail cyclized backbone and a cyclic cystine knot motif, and are found in the petals of butterfly pea (Clitoria ternatea).

Objective

Clitoria ternatea cv. “Theparatpailin 63” is a newly established butterfly pea cultivar with large petals rich in anthocyanins. This study aimed to identify, quantify, and evaluate the biological activities of cyclotides present in this cultivar.

Methods

Dried petals and sepals of C. ternatea cv. “Theparatpailin 63” were ground in liquid nitrogen and extracted with methanol/dichloromethane. Following filtration, the extracts were fractionated by column chromatography using Amberlite XAD7-100G and OASIS HLB, and further purified by preparative reversed-phase high-performance liquid chromatography. The molecular masses of the isolated compounds were determined by MALDI-TOF mass spectrometry. Nucleotide sequences of individual cyclotides were analyzed using cyclotide-specific primers with genomic DNA and total RNA isolated from flowers as templates. Biological activities were assessed by examining the effects of purified cyclotides on the growth of HL-60 cells and Escherichia coli.

Results

Transcripts encoding cyclotides: cliotide T1 (cT1), cT2, cT3, cT4, and cT5 were detected in flowers. Mass spectrometric analysis revealed that cT3, cT4, cT5, and a methionine-oxidized form of cT3 [cT3(O)] were predominantly present in petals, whereas cT3 and cT3(O) were mainly detected in sepals. Purified cT5 and cT3(O) slightly promoted the growth of E. coli, while no significant effect was observed for cT3 and cT4. In contrast, the growth of HL-60 cells was slightly inhibited by the purified cyclotide mixture.

Conclusion

The major cyclotides present in the flowers of C. ternatea cv. “Theparatpailin 63” are cT3, cT4, cT5, and the methionine-oxidized form of cT3. These cyclotides exhibit modest and differential biological activities toward bacterial and human leukemia cells.

Keywords

cyclotide peptide quantification MALDI-MS anticancer

Introduction

In plants, metabolites are organic compounds that participate in metabolic processes. These metabolites are broadly classified into primary and secondary metabolites according to their physiological roles. Primary metabolites are directly involved in the growth, development, and reproduction of plants and are essential for fundamental cellular functions. They are ubiquitous and present in all living plant cells. Secondary metabolites, in contrast, are not directly involved in primary metabolism but play critical roles in the interaction between plants and their environment. Although not essential for basic survival, they confer adaptive, protective, and competitive advantages to plants.¹

Several cyclic peptides have been identified as plant secondary metabolites.² Plant cyclic peptides constitute a diverse group of ribosomally synthesized peptides characterized by a covalently closed backbone that confers exceptional structural stability. Based on their biosynthetic origin, sequence features, and structural motifs, plant cyclic peptides are broadly classified into several families, including cyclotides, orbitides, and sunflower trypsin inhibitor (SFTI)-like peptides. Among these, cyclotides are the most extensively studied and are distinguished by a head-to-tail cyclized backbone combined with a cyclic cystine knot (CCK) motif formed by three interlocking disulfide bonds.^2,3 Orbitides represent a class of small, cysteine-free cyclic peptides that are widely distributed across higher plants. In contrast to cyclotides, orbitides lack disulfide bonds and exhibit greater structural flexibility, although they still maintain enhanced stability relative to linear peptides due to backbone cyclization. SFTI-like peptides are characterized by a compact cyclic structure stabilized by a single disulfide bond and are best known for their potent protease inhibitory activity.⁴

Cyclotides are macrocyclic peptides of approximately 30 amino acids and relatively compactly folded. This compact structure endows cyclotides with remarkable stability against thermal, chemical, and enzymatic degradation.^4,5 Unlike many plant secondary metabolites, cyclotides are directly encoded in the genome. They are translated as larger precursor proteins that undergo specific processing and folding events to yield mature cyclotides.^4,5 Biosynthetically, cyclotides are produced as precursor proteins that undergo extensive post-translational processing, including proteolytic cleavage and backbone cyclization mediated by asparaginyl endopeptidases.⁶ This ribosomal origin, coupled with precise enzymatic maturation, enables the generation of considerable sequence diversity while maintaining a conserved structural framework.

Functionally, cyclotides are primarily implicated in plant defense, exhibiting a wide spectrum of biological activities such as antimicrobial, antifungal, insecticidal, cytotoxic, and antiviral effects.^4,5 Many cyclotides exert their bioactivity through interactions with biological membranes, often in a lipid-dependent manner, which can lead to membrane disruption or modulation of membrane-associated processes. Their expression is frequently tissue-specific and inducible by environmental stimuli, further supporting their role in protecting plants against biotic stress.⁷

Because of their extraordinary stability, well-defined structure, and functional versatility, cyclotides have attracted significant interest as molecular scaffolds for drug design and agricultural applications. A deeper understanding of cyclotide diversity, biosynthesis, and biological function is therefore essential for elucidating their roles in plant biology and for harnessing their potential in biotechnology.⁸

The first cyclotide, kalata B1, was identified in Oldenlandia affinis (Rubiaceae).^2-5,8 To date, more than 300 cyclotides have been identified across various plant families, including Rubiaceae (coffee family), Violaceae (violet family), Cucurbitaceae (gourd family), Fabaceae (legume family), and Solanaceae (nightshade family). Moreover, genes encoding cyclotide-like molecules have also been discovered in Poaceae (grass family), which includes major cereal crops such as wheat, maize, and rice.^9,10 These findings suggest that the diversity of cyclotides may exceed that of the well-known plant defensins. Cyclotides exhibit a wide range of biological activities, including hemolytic, anti-neurotensin, anti-HIV, cytotoxic, antibacterial, antifouling, and nematocidal properties.^5,8,11-15 However, individual cyclotides do not necessarily display all of these activities, and the specific biological roles of many distinct cyclotide molecules remain poorly understood.

Clitoria ternatea, commonly known as butterfly pea, is a perennial leguminous plant distinguished by its vivid blue petals.¹⁶ In recent years, it has attracted considerable attention for its potential applications in modern medicine and agriculture, as well as for its use as a source of natural colorants and antioxidants.¹⁶ Traditionally, C. ternatea has been cultivated as an animal feed and herbal tea, and has long been utilized in traditional medicine, particularly for its reported cognitive-enhancing effects and for alleviating symptoms associated with fever, inflammation, pain, and diabetes.¹⁷ It has been found that C. ternatea also contains cyclotides.^16-19 Their immunostimulatory effects and antimicrobial activities were demonstrated, raising anticipations for their pharmacological applications.^8,12

A new butterfly pea cultivar, C. ternatea cv. “Theparatpailin 63”, was generated by selective breeding from a commonly cultivated butterfly pea variety at the Phichit Agricultural Research and Development Center in 2014 to enhance petal yield per plant. Indeed, the petals of this variety are known to contain approximately 1.37 times more ternatin,^16,17,20 a blue anthocyanin, than standard butterfly pea petals. These results indicate that this cultivar represents a promising source of natural food colorants and antioxidants. However, no information has been obtained regarding the cyclopeptide analogues contained in this cultivar.

Therefore, this study aimed to identify the molecular species of cyclopeptide analogues present in the petals and sepals of C. ternatea cv. “Theparatpailin 63” and to evaluate their biological activities.

Material and Methods

Plants Materials

Fresh petals and sepals of C. ternatea cv “Theparatpailin 63″ plants were collected from Thailand and then naturally dried. For DNA and mRNA isolation, Seeds of C. ternatea cv “Theparatpailin 63 were sown in a mixture of Akadama soil (16 L) and potting soil (40 L) and then grown for two months in a natural-light phytotron at 30/25 °C (day/night).

Isolation and Purification of Cyclotides

Cyclotides were extracted from petals and sepals using a previously described method with some modifications.²¹ Dried petals and sepals of Clitoria ternatea cv. “Theparatpailin 63” were separated and ground in liquid nitrogen using a mortar and pestle. For extraction, 10 ml each of methanol and dichloromethane were added to the petal powder, and 5 ml each of methanol and dichloromethane were added to the sepal powder. The mixtures were incubated overnight in the dark with 5% ethanol. After filtration, the extracts were subjected to column chromatography using Amberlite XAD7-100G resin (Merck, Germany). The columns were washed with 1% ethanol and eluted sequentially with 20%, 40%, 60%, and 80% ethanol. Cyclotides were recovered in the 60% ethanol eluate, which was designated as the cyclotide-enriched fraction.

Cyclotide-enriched fractions from petal and sepal extracts were further purified using OASIS HLB 1 cc extraction cartridges (Waters, Milford, MA, USA) and eluted with 1 ml of 10% acetonitrile and 1 ml of 40% acetonitrile, respectively. The eluted fractions were subjected to preparative high-performance liquid chromatography (HPLC) on a JASCO system (Tokyo, Japan) equipped with a 5C18MS-II column (4.6 mm ID × 250 mm, Nacalai Tesque). Elution was carried out at a flow rate of 1 ml/min using a linear gradient from buffer A (1% acetonitrile, 0.09% trifluoroacetic acid) to 80% buffer B (100% acetonitrile, 0.08% trifluoroacetic acid). Elution was monitored by UV detection at 215 nm. Fractions corresponding to cyclotide peaks were collected, and their purity was confirmed by re-chromatography, yielding a single peak in each case.

Mass Spectrometry

Insulin B-chain (oxidized form), angiotensin II, and adrenal corticosteroids were used as internal standards for mass calibration. Oxidized insulin B-chain MALDI-MS standard (Merck) was dissolved in 50% acetonitrile containing 0.05% trifluoroacetic acid (TFA) to a concentration of 10 pmol/μl. Angiotensin II MALDI-MS standard (Merck) was dissolved in 0.1% TFA to 10 pmol/μl, and adrenal corticosteroids (Merck) were dissolved in ultrapure water to 586 nmol/μl.

The matrix solution consisted of 10 mg/ml sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid; Nacalai Tesque) dissolved in 40% acetonitrile. For MALDI sample preparation, 1 μl of each HPLC fraction was mixed with 10 μl of the matrix solution in a 1.5 ml low-adsorption microtube. Then, 1 μl of the mixture was spotted onto the target plate and air-dried. MALDI-TOF mass spectrometry was performed using a JMS-S3000 instrument (JEOL, Japan) in spiral mode.

Cloning of Cyclotide Genes

Total RNA was extracted from fresh C. ternatea flowers using RNeasy Plant Mini Kit (QIAGEN, Inc.) as per manufacturer’s guidelines. cDNA was generated from total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). To amplify DNA fragments from cDNA, primers based on previously reported cyclotides: cliotide T1 (cT1)–cT4, cT12 and Cter A were used.¹⁸ The primer for cT1 (5′-CAAGAAGGAAGCATCGCAA-3′ and 5′-CCTAGCTTATGTAAGTCGTGAA-3′) and cT2 (5′-ATGGCATACGTTAGGCTTACT-3′ and 5′-CAGGATGCATCATACATAATTACTTT-3′), cT3(5′-ATGGCTTACGTTAGACTTACTTC-3′ and 5′-TTAGTTGGTACTTTCCAAAGGC-3′), cT4 (5′-GCAAAGCATCAACTTAATCCATT-3′ and 5′-ATGACTACTTTCAGTTGGTGATAG-3′), cT12 (5′-ATGGCTTCCCTTCGCATTGC-3′ and 5′-GATAGTACATGCATCATGGGGATCT-3′), Cter A (5′-ATGGCTTCCCTTCGCATTGC-3′ and 5′-GCATGTGTGACTATTTTCAGTTGGT-3′). Amplified fragments were cloned into the pCR-Blunt vector (Invitrogen). The resulting plasmids were separately transformed into the DH5α strains. After transformation, the bacterial cells were plated on LB agar plates containing 50 µg ml^-1 kanamycin and incubated for 16 h at 37 °C. The DNA fragments were amplified using the following PCR primer sets designed on the vector using the kanamycin-resistant colonies as templates: 5′-CGTTGTAAAACGACGGCCAG-3′ and 5′- CAGGAAACAGCTATGACCATG-3’. Amplified products were subjected to sequence analysis by using DNA sequencer (3500 Genetic Analyzer, Hitachi, Japan).

Effects of Cyclotides on Escherichia coli Growth

Antibacterial activity was assayed using a previously described method with some modifications.²² Stored E. coli (DH5α) cultures were inoculated into 5 ml of LB broth and incubated overnight at 37 °C with shaking at 150 rpm. Subsequently, 4 ml of the overnight culture was transferred into 200 ml of fresh LB medium and incubated under the same conditions until the optical density at 600 nm (OD₆₀₀) reached 0.5–0.6. Aliquots (5 ml) of the culture were transferred into test tubes containing purified cyclotides. Bacterial growth was monitored by measuring OD₆₀₀ at 30 min intervals. The assay duration was limited to 9 h to maintain cells in the exponential growth phase and to avoid secondary effects associated with prolonged culture.

Growth Inhibition Assay of HL-60 Cells

The human promyelocytic leukemia cell line HL-60 was cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Hyclone), and 1% (v/v) penicillin–streptomycin solution (Nacalai Tesque). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and sub-cultured every three days.²³

For the assay, HL-60 cells were seeded at a density of 1 × 10⁶ cells/ml in 90 mm dishes. Two treatment groups were prepared: control and cyclotide-treated groups were treated with sterilized water or 1 µM cyclotides, respectivery. After three days of incubation, viable cells were counted, and the cell growth rate was calculated.

Statistical Analysis

Error bars represent standard deviation from six independent experiments. The significance of the differences was verified using a Student’s t-test compared with the control, and asterisks indicate statistical significance at P<0.05.

Results

Cloning of Cyclotide Genes

To elucidate the gene structures of cyclotides in C. ternatea cv. “Theparatpailin 63,” total RNA was extracted from petals, and first-strand cDNA was synthesized. PCR amplification was then performed using gene-specific primer sets targeting cT1, cT2, cT3, cT4, cT12, and Cter A. Amplification products of approximately 400 bp were obtained using primer sets for cT2, cT3, cT4, cT12, and Cter A. The amplified fragments were cloned into plasmid vectors and subjected to nucleotide sequencing. These sequences were identified based on homology searches using NCBI nucleotide Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Sequence analysis of the PCR product obtained with the cT2 primer set revealed a 424 bp cDNA sequence. Translation of this sequence yielded a putative amino acid sequence showing high identity to the reported cT2 peptide. Similarly, the cT3 primer set produced a 383 bp cDNA fragment, whose deduced amino acid sequence corresponded to the known cT3 sequence. PCR amplification using the cT4 primer set generated products of 436, 439, and 382 bp. Sequence analysis identified these as cT1 (436 bp), cT4 (439 bp), and cT5 (382 bp). Likewise, PCR amplification with the Cter A primer set yielded a 436 bp fragment identical to cT1 and a 439 bp fragment identical to cT4 (Figure 1, Table 1). These results indicate that cT1, cT2, cT3, cT4, and cT5 are expressed in the petals of C. ternatea cv. “Theparatpailin 63”.

Figure 1.

Alignment of amino acid sequences of cyclotides deduced from cDNA sequences.

Table 1.

Deduced Cyclotides Amino Acid Sequence Encoded by the PCR Fragments

Primer set	Deduced amino acid sequence	Identified cyclotide
cT2	GEFLKCGESCVQGECYTPGCSCDWPICKKN	cT2
cT3	GLPTCGETCTLGTCYVPDCSCSWPICMKN	cT3
cT4	GIPCGESCVFIPCITGAIGCSCKSKVCYRN	cT1
cT4	GIPCGESCVFIPCITAAIGCSCKSKVCYRN	cT4
cT4	GIPCGESCVFIPCISTVIGCSCKNKVCYRN	cT5
cT12	GIPCGESCVFIPCITGAIGCSCKSKVCYRN	cT1
cT12	GIPCGESCVFIPCITAAIGCSCKSKVCYRN	cT4
Cter A	GIPCGESCVFIPCITGAIGCSCKSKVCYRN	cT1
Cter A	GIPCGESCVFIPCITAAIGCSCKSKVCYRN	cT4

To further characterize their genomic organization, genomic DNA was amplified using the same primer sets. Sequence analysis revealed that all cyclotide genes contain an intron located 55 bp downstream of the start codon. The introns varied in both length and sequence among cyclotides (Supplemental Figure 1). Notably, two distinct introns were identified in cT1, suggesting the presence of at least two genomic copies of this gene.

Purification of Cyclotides From C. ternatea cv “Theparatpailin 63”

Petal and sepal extracts were subjected to column chromatography using Amberlite XAD7-100G resin, followed by sequential elution with 20%, 40%, 60%, and 80% acetonitrile (CH₃CN). MALDI-TOF mass spectrometry analysis revealed that the 60% CH₃CN fraction contained compounds with molecular weights in the range of 2,500–4,000 Da, consistent with the expected molecular masses of cyclotides. Each 60% fraction derived from petal and sepal extracts was further purified using OASIS HLB 1 cc extraction cartridges (Waters, Milford, MA, USA) and eluted with 1 ml of 40% CH₃CN.

Aliquots (10 µl) of each eluate were analyzed by HPLC with UV detection at 215 nm. Four distinct peaks were observed at approximately 40 min retention time in both petal and sepal 40% CH₃CN fractions, suggesting the presence of cyclotides. To determine whether these peaks corresponded to cyclotides, each was subjected to MALDI-TOF-MS analysis. The four peaks were designated as Peak A, Peak B, Peak C, and Peak D, respectively (Figure 2).

Figure 2.

HPLC profile of the petal 40% CH₃CN elution fraction.

Identification of Peak A, Peak B, Peak C, and Peak D

MALDI-TOF-MS analysis of Peak A revealed an (M + H)⁺ ion at m/z 3169.20, which matches the calculated molecular mass of cT5. Given that the cDNA sequence of cT5 had previously been identified from C. ternatea cv. “Theparatpailin 63”, Peak A was assigned as cT5.

Peak C exhibited an (M + H)⁺ ion at m/z 3098.11, corresponding precisely to the molecular weight of cT4. Similarly, MALDI-TOF-MS analysis of Peak D showed an (M + H)⁺ ion at m/z 3057.92, consistent with the expected molecular weight of cT3, as determined from its cDNA sequence (Table 2).

Table 2.

Cyclotides Isolated at Protein Level From C. Tematea. Values for Relative Molecular Mass are Reported as Experimental Monoisotopic

Peak	(M+H)⁺	Cyclotide	Sequence	Me
A	3169.20	cT5	GIPCGESCVFIPCISTVIGCSCKNKVCYRN	3168.48
B	3073.98	cT3(O)	GLPTCGETCTLGTCYVPDCSCSWPICM(O)KN	3073.26
C	3098.11	cT4	GIPCGESCVFIPCITAAIGCSCKSKVCYRN	3097.44
D	3057.92	cT3	GLPTCGETCTLGTCYVPDCSCSWPICMKN	3057.26

In contrast, MALDI-TOF-MS analysis of Peak B yielded a molecular ion at m/z 3073.98, which did not match any known cyclotides, including those previously identified from C. ternatea cv. “Theparatpailin 63”. To further characterize this compound, proteins in Peak B were subjected to reductive carboxymethylation followed by tryptic digestion. Mass spectrometric analysis of the digested peptides indicated that Peak B corresponded to cT3 containing an oxidized methionine residue. Therefore, Peak B was identified as cT3(O), the methionine-oxidized form of cT3 (Table 2).

Additional peaks detected in the HPLC chromatograms were also analyzed by MALDI-TOF-MS; however, no other molecular ion peaks corresponding to cyclotides were observed. These results indicate that the four cyclotides identified in this study—cT3, cT3(O), cT4, and cT5—are the predominant cyclotides present in the petals of C. ternatea cv. “Theparatpailin 63”.

Quantification of Cyclotides in C. ternatea cv. “Theparatpailin 63”

To quantify the cyclotides present in C. ternatea cv. “Theparatpailin 63”, 300 μl of the 40% petal elution fraction was analyzed by HPLC, with 50 μl injected per run. Four distinct peaks (Peaks 1–4) were collected, concentrated using a vacuum centrifugal evaporator, and quantified for total protein content using the Qubit Protein Assay Kit (Invitrogen). The measured protein concentrations were 0.468 μg/μl for Peak A (cT5), 0.092 μg/μl for Peak B (cT3(O)), 0.181 μg/μl for Peak C (cT4), and 0.151 μg/μl for Peak D (cT3) (Table 3).

Table 3.

Concentration in cT5, cT3(O), cT4, cT3

Cyclotide	Protein conc. (μg/μl)
Peak A (cT5)	0.468
Peak B (cT3(O))	0.092
Peak C (cT4)	0.181
Peak D (cT3)	0.151

Because cyclotide cT5 was obtained in the highest yield, it was selected to construct a calibration curve correlating the amount of cT5 (Peak A) with the corresponding HPLC peak area at 215 nm. A strong linear relationship was observed between the cT5 amount (2–9 μg) and its HPLC peak area, described by the equation (Figure 3):

x (cT 5, μ g) = (y (peak area) - 778, 770) / 1, 345, 946

To estimate the cyclotide content in C. ternatea cv. “Theparatpailin 63”, 10 μl of the 40% petal elution fraction was injected into HPLC. Using the calibration curve (Figure 3), the quantities of each cyclotide in the 10 μl sample were calculated as follows: 2.69 μg for Peak A (cT5), 2.31 μg for Peak B (cT3(O)), 2.91 μg for Peak C (cT4), and 3.27 μg for Peak D (cT3) (Table 4). Given that the concentration of the 40% petal elution fraction corresponded to 0.86 g dry weight per ml, the cyclotide contents per gram of petal dry weight were calculated as 312.8 μg for cT5, 268.6 μg for cT3(O), 338.4 μg for cT4, and 380.2 μg for cT3 (Table 5).

Figure 3.

Cyclotide standard curve using cT5. The vertical and horizontal axes represent peak area at 215 nm and weight of cT5, respectively. The R² value is 0.9887

Table 4.

Amount of Cyclotide in Each Injected 10 μl Petal Fraction

Cyclotide	Amount of cyclotide (μg-cT5 equivalent)
Peak A (cT5)	2.69
Peak B (cT3(O))	2.31
Peak C (cT4)	2.91
Peak D (cT3)	3.27

Table 5.

Amount of Each Cyclotide in Petals

Cyclotide	Cyclotide contents (μg/g-dry weight)
cT5	312.8
cT3(O)	268.6
cT4	338.4
cT3	380.2

In total, approximately 1,300 μg of cyclotides were detected per gram of dried petals, indicating that cyclotides comprise roughly 0.13% of the petal dry weight (Table 5). Cyclotide content in sepals was estimated in the same manner. One gram of dry sepals contained 171.4 μg of cyclotide cT3(O) and 147.6 μg of cT3, whereas cT5 and cT4 were not detected (Table 6). The presence of both cT3 and cT3(O) in sepals suggests that only cT3 is expressed in this tissue, and that a portion of its methionine residues undergo post-translational oxidation.

Table 6.

Amount of Each Cyclotide in Sepals

Cyclotide	Amount of each cyclotide (μg/g-dry weight)
cT5	Not determine
cT3(O)	171.4
cT4	Not determine
cT3	147.6

It should be noted that in this study, quantification was performed directly from the HPLC peak areas at 215 nm using a calibration curve based on purified cT5. This approach ensures that the cyclotide concentrations determined for C. ternatea cv. “Theparatpailin 63” flowers are accurate and reliable. To our knowledge, no previous studies have quantified the cyclotide content in the petals and sepals of butterfly pea. Our findings thus provide the first comprehensive quantification of individual cyclotides in this plant.

Effects of Cyclotides on the Growth of Escherichia coli

Cyclotides are plant-derived cyclic peptides characterized by a unique cystine knot motif and diverse biological activities. To evaluate their potential bioactivity, the effects of four cyclotides—cT5, cT3(O), cT4, and cT3—identified from the petals of C. ternatea cv. “Theparatpailin 63” were examined on the growth of Escherichia coli. E. coli cultures were grown in liquid medium supplemented with each cyclotide at final concentrations of 1 µM and 10 µM. Bacterial growth was monitored at various time points by measuring the optical density at 600 nm (OD₆₀₀).

In the control cultures supplemented with water, a linear increase in cell density was observed up to 9 hours of incubation. In contrast, cultures treated with cT5 and cT3(O) exhibited a significantly higher growth rate than the control beginning at 5 hours, with the greatest difference observed at 9 hours. The growth-promoting effect was dose-dependent, with a greater increase observed at 10 µM compared to 1 µM. Conversely, the addition of cT4 or cT3 did not result in any appreciable change in E. coli growth, and the OD₆₀₀ values remained comparable to those of the control even after 9 hours of incubation (Figure 4).

Figure 4.

Effects of cyclotides on the growth of Escherichia coli. Stored E. coli (DH5α) was added to 5 ml of LB liquid medium and incubated at 37 °C. Five ml of culture medium and cyclotide were placed in test tubes and absorbance at 600 nm was measured every 30 minutes. (A) solid triangle, 10 µM cT5; solid squares, 1 µM cT5; open circles, water (control). (B) solid triangle, 10 µM cT3(O); solid squares, 1 µM cT3(O); open circles, water (control). (C) solid triangle, 10 µM cT4; solid squares, 1 µM cT4; open circles, water (control). (D) Solid triangle, 10 µM cT3; solid squares, 1 µM cT3; open circles, water (control)

Effects of Cyclotides on the Growth of HL-60 Cells

HL-60 cells, a human promyelocytic leukemia cell line, are widely used as a model system for studies on hematopoiesis and leukocyte differentiation. To investigate the potential cytotoxic effects of cyclotides, the proliferation of HL-60 cells was assessed following treatment with cyclotides purified from petals of C. ternatea cv. “Theparatpailin 63”. A mixture of the four cyclotides (cT5, cT3(O), cT4, and cT3) was prepared in proportions corresponding to their natural abundance. HL-60 cells (1 × 10⁷) were treated with sterile water (control) or 1 μM of the cyclotide mixture and incubated for three days. Cell counts on day 3 revealed that the growth of HL-60 cells treated with cyclotides was approximately 80% of that of the control group, indicating that cyclotides purified from C. ternatea cv. “Theparatpailin 63” inhibit HL-60 cell proliferation (Figure 5).

Figure 5.

Effects of cyclotides on the growth of HL-60. HL-60 cells (1 x 10⁷) were treated with sterile water (Control) or 1 μM cyclotides. Cells were counted to calculate the rate of cell growth at day 3 after treatments. Cell growth rate indicates the fold compared to the number of cells at day 0 (1 x 10⁷ cells). Bars indicate the standard deviation of six experiments. Asterisks indicate a significant decrease (t test, P < 0.05)

Discussion

Previous transcriptomic analyses have revealed that C. ternatea expresses a large and diverse repertoire of cyclotide precursor genes. Out of 71 cyclotide precursor transcripts obtained, 51 sequences display unique cyclotide domains have been identified, indicating extensive diversification of cyclotides in this species.²⁴ These genes are expressed in multiple tissues, including leaves, flowers, and other organs; however, their expression levels vary markedly among tissues, suggesting tissue-specific regulation of cyclotide biosynthesis.²⁴ In addition, recent whole-genome sequencing has elucidated the genomic organization of C. ternatea, providing a comprehensive framework for understanding the evolution and expansion of cyclotide precursor genes.²⁵ Despite these advances at the genomic and transcriptomic levels, quantitative information on cyclotide accumulation in individual plant organs has remained limited.

Most previous studies on cyclotides in C. ternatea and other cyclotide-producing plants have relied on mass spectrometry–based analyses to demonstrate the presence, diversity, and relative abundance of cyclotides.^4,16 These approaches have been highly effective for profiling cyclotide repertoires and identifying novel sequences; however, they generally provide only relative comparisons based on peak intensities rather than absolute quantification. This limitation arises primarily from the large number of cyclotide isoforms present in a single plant, the difficulty of purifying individual cyclotides to homogeneity, and the lack of appropriate internal standards for quantitative mass spectrometry. As a result, the actual levels of cyclotides accumulated in specific tissues, such as petals or leaves, have remained largely unclear. In the present study, we determined the total cyclotide content in petals, the first quantitative estimate of cyclotide abundance in this organ. The ability to quantify total cyclotide content in petals represents an important advance for cyclotide research in C. ternatea. Absolute quantification provides a critical link between transcriptomic data, peptide-level accumulation, and biological function. Such information is essential for understanding the physiological roles of cyclotides in planta, including their potential involvement in defense and stress responses, as well as for evaluating their suitability as bioactive compounds for pharmaceutical or agricultural applications.

Cyclotide contents in the petals ranged from 268 to 380 µg/g dry weight, whereas levels in sepals were markedly lower or below the detection limit (Tables 5 and 6). Since we have only quantified cyclotides in petals and sepals, we have not determined the cyclotide content in the leaves of C. ternatea cv. “Theparatpailin 63”. On the other hand, Poth et al reported that leaf tissue of C. ternatea contains 5 μmol/g of Cter M. The cyclotide content detected in the petals in this study was approximately 0.1 µmol/g, which is about one-fiftieth of the reported value.¹⁹ In our analysis, a calibration curve was constructed using purified cT5 and corresponding peak areas obtained by HPLC at 215 nm, allowing direct quantification of cyclotides in flowers of C. ternatea cv. “Theparatpailin 63”. Crude purification using XAD-7 resin was employed prior to HPLC analysis, and potential losses of cyclotides during this step were evaluated. Comparison of peak areas at 215 nm before and after crude purification revealed no substantial differences, and the recovery of cyclotides using XAD-7 exceeded 95% (data not shown). These results indicate that cyclotide loss during sample preparation was minimal and that the cyclotide contents reported here accurately reflect their abundance in the flowers of C. ternatea cv. “Theparatpailin 63”, indicating that the lower levels observed are not attributable to sample loss but reflect genuinely lower accumulation than previously reported.¹⁹ Because the biological activity of cyclotides was not evaluated in this study, no conclusions can be drawn regarding their potential insecticidal or other bioactivities based solely on their concentrations.

Cyclotides are generally classified into two structural types: Möbius and bracelet.³ Möbius-type cyclotides contain a characteristic cis Xaa-Pro dipeptide in loop 5, whereas bracelet-type cyclotides lack this motif. Previous studies have reported that bracelet-type cyclotides such as cT1 and cT4 exhibit antibacterial activity at concentrations below 1 µM, while Möbius-type cyclotides show such activity only at concentrations exceeding 100 µM.¹⁸ The cT4 and cT5 identified in the present study are of the bracelet type and were therefore expected to display antibacterial activity. However, no reduction in bacterial cell numbers was observed under the conditions used in this study (Figure 4). It has been reported that the antibacterial activity of cyclotides is lost in nutrient-rich media such as LB medium; therefore, no antibacterial activity is observed under such conditions. Accordingly, the absence of antibacterial activity in our assay is likely attributable to the use of LB medium rather than to an intrinsic lack of activity of the cyclotides tested. These results are consistent with previous findings.²⁶ Therefore, no definitive conclusion regarding the intrinsic antibacterial activity of these cyclotides can be drawn from the present assay conditions.

In this study, a slight but reproducible enhancement of E. coli growth was observed in liquid cultures supplemented with cT5 and the methionine-oxidized form of cT3(O). This effect contrasts with the antimicrobial activity reported for many cyclotides One possible explanation is that cT5 and cT3(O) may interact with bacterial cells in a manner that differs from the membrane-disruptive mechanisms commonly described for other cyclotides. Rather than causing membrane permeabilization, these peptides may weakly associate with the bacterial surface or alter membrane properties in a way that facilitates nutrient uptake or metabolic activity. Such subtle modulation of membrane function, insufficient to induce cytotoxicity, could result in a modest enhancement of bacterial growth under certain culture conditions. Alternatively, the observed growth promotion may be an indirect effect. Cyclotides are rich in amino acids and, following partial degradation or modification in the culture medium, could serve as a supplementary nutrient source for E. coli. Although cyclotides are generally considered highly stable, limited proteolytic processing or chemical modification under the experimental conditions cannot be excluded. In addition, the presence of cyclotides may influence the physicochemical properties of the culture medium, such as ion availability or redox balance, thereby indirectly affecting bacterial growth. It is also conceivable that cT5 and cT3(O) interfere with stress responses in E. coli. Low-level interactions with cellular membranes or regulatory proteins could reduce basal stress, leading to a slight increase in growth rate. Similar hormetic effects, in which low concentrations of bioactive compounds stimulate growth while higher concentrations are inhibitory, have been reported for various antimicrobial peptides and secondary metabolites.^27-29 The growth-promoting activity of cyclotides in E. coli observed in this experiment, including elucidating their function, will be a topic of future research.

Cyclotides purified from C. ternatea cv. “Theparatpailin 63” were found to inhibit the proliferation of HL-60 cells. Several cyclotides have been reported to exhibit anticancer activity. Kalata B1 induces cell death by binding to and lysing cancer cell membranes,¹¹ and cT3 and cT4 have also been shown to possess anticancer properties.¹² However, the anti-cancer activity of cT5 has not yet been reported. Since cT5 is the major component in the cyclotide mixture used in this study, it is possible that cT5 contributes to the observed inhibitory effect on HL-60 cell proliferation.

Conclusion

C. ternatea cv “Theparatpailin 63″ was found to contain cT1, cT2, cT3, cT4 and cT5. Among these, cT3, cT4, cT5, and methionine-oxidized cT3(o) were identified by HPLC profiling and MS analysis. The cyclotides were present in petals at 268 to 380 maicrograms per gram of dry weight. In contrast, the contents of cyclotides in sepals were either lower than in the petals or below the detection limits. The major cyclotide in petals was cT1, whereas cT5 was predominant in sepals. Although the antibacterial activity of these cyclotides could not be confirmed, they significantly inhibited the growth of HL-60 cells. These results provide valuable insight into the cyclotides present in the petals and sepals of the new cultivar C. ternatea cv “Theparatpailin 63”.

Limitation of This Study

In this study, we analyzed the major cyclotides contained in C. ternatea cv “Theparatpailin 63,” which were observed as prominent peaks in the HPLC profile. While various cyclotides have previously been identified in C. ternatea and their bioactivities reported, this study may not have identified certain minor cyclotides present in the sample. Furthermore, their specific physiological roles have yet to be fully elucidated. Although whole-genome and transcriptome analyses have been conducted on C. ternatea,^24,25 those studies utilized different cultivars from the one used in this study. Therefore, it is essential to perform comparative genomic and transcriptomic analyses for a comprehensive comparison of cyclotide profiles between cultivars.

In this study, cyclotide levels in petals of C. ternatea cv. “Theparatpailin 63” were not compared with those in other plant tissues or in other cultivars. Therefore, it is not possible to determine whether the observed cyclotide levels are specific to petals or reflect overall accumulation in the plant. Furthermore, the biological activities of cyclotides, such as antimicrobial and insecticidal effects, were not systematically or quantitatively assessed. Therefore, it is not possible to establish a direct relationship between cyclotide concentration and their overall bioactivities.

Supplemental Material

Supplemental Material - Identification of Cyclotides From Butterfly Pea (Clitoria ternatea) Petals and Its Biological Activities

Supplemental Material for Identification of Cyclotides From Butterfly Pea (Clitoria ternatea) Petals and Its Biological Activities by Naoki Ikeda, Hikari Matsumura, Arisa Kajiwara, Yukihiko Nakagawa, Machiko Kondo, Kenichi Kubo, Akihisa Tsuji and Fang-Sik Che in Natural Product Communications.

Footnotes

Acknowledgments

The authors gratefully acknowledge the students who conducted this research and the university staff for their assistance and support.

ORCID iDs

Naoki Ikeda

Ken-ichi Kubo

Ethical Considerations

Ethical Approval is not applicable for this article.

Consent to Participate

There are no human subjects in this article and informed consent is not applicable.

Author Contributions

FC conceived and designed the study; NI, HM, AK, YM, MK, KK, and AT performed experiments; FC, NI, and AT drafted the manuscript. All authors read and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by Grant-in-Aid for Scientific Research (C) (24K08836) from Japanese Society for the Promotion of Science (JSPS).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

Data Availability Statement

The data underlying this article are available in the article.*

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online.

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