Discovery of hyde C1 a broad spectrum antimicrobial peptide derived from chicory

Abstract

Antimicrobial peptides (AMPs) are emerging as promising alternatives to traditional antibiotics against multidrug-resistant (MDR) bacteria. In this study, we isolated and characterized a novel AMP, named Hyde C1, from the chicory plant (Cichorium intybus L.). Hyde C1 was purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and determined to have a molecular weight of 3686.4 Da. It exhibits strong antibacterial activity against both Gram-positive (S. aureus ATCC 29213) and Gram-negative (E. coli ATCC 25922, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606) bacteria, with minimum inhibitory concentrations (MICs) ranging from 2 to 16 µg/mL. Hyde C1 disrupts bacterial membranes, as evidenced by increased permeability, membrane depolarization, and scanning electron microscopy. Bioinformatic analysis revealed its amphipathic α-helical structure, with a high hydrophobic ratio (61%) and a net positive charge (+ 6), supporting its bactericidal mechanism. The peptide also demonstrated high stability under various pH, temperature, and salt conditions, along with low hemolytic and cytotoxic effects. These properties suggest that Hyde C1 is a strong candidate for development as a novel antimicrobial agent.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-19166-5.

Introduction

Antibiotics are widely utilized for the prevention and treatment of a wide range of diseases caused by pathogenic microorganisms. Despite the historical effectiveness of antibiotics in managing the majority of bacterial infections, the emergence of antimicrobial resistance has significantly reduced the efficacy of current antibiotic therapies^1–3. However, considerable evidence has shown that the widespread and excessive use of traditional antibiotics has led to the emergence of drug-resistant pathogens, especially antibiotic-resistant bacteria⁴. Public health is seriously threatened by the emergence of multidrug-resistant (MDR) bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), and Escherichia coli that produces extended-spectrum beta-lactamase (ESBL)⁵. The overuse of conventional antibiotics, combined with the slow pace of development of new antibiotic drugs, has contributed to the rise of antimicrobial resistance (AMR)⁶. In recent years, antimicrobial peptides (AMPs) have attracted considerable attention due to their promising clinical potential as a new class of antibiotics to combat the growing problem of antimicrobial resistance⁷. Antimicrobial peptides (AMPs) are amphipathic peptides with a net positive charge and contain 5 to 70 amino acid residues. These chemicals have been shown in several investigations to possess antibacterial, antifungal, antiviral, and even anti-cancer effects⁸.

Antimicrobial peptides are an important part of the innate immune system and act as a defense mechanism against microbial pathogens and are naturally found in various organisms, including animals, plants, and microorganisms. In plants, antimicrobial peptides play a crucial role in their survival, and plant-derived AMPs have been extensively investigated for their potential to inhibit a diverse range of pathogens⁹. Compared to other organisms, plants show a greater diversity of AMP isoforms, which can be attributed to gene duplication or polyploidy¹⁰. As a result, plants are a promising source of AMPs, and a large number of AMPs have been identified from various plant tissues, including leaves, roots, seeds, flowers, and stems, which show significant activity against human pathogens, phytopathogens, protozoans, insects, and even cancer cells^11,12.

The antimicrobial activity of AMPs is usually mediated by initial contact with the microbial cell membrane and disruption of the cell membrane integrity. While the primary mode of action of many AMPs is membrane disruption, some AMPs can cross the membrane barrier, become intracellular, and interact with intracellular components such as DNA, RNA, and various enzymes essential for microbial survival and replication^13,14. The relatively slower development of resistance by microbes to AMPs compared to conventional antibiotics makes them a promising alternative therapeutic approach in the long term. Unlike traditional single-target antibiotics, AMPs can kill pathogens at multiple targets, greatly reducing the emergence of drug-resistant bacteria^15,16.

Chicory with the plant name Cichorium intybus L. is a plant with significant medicinal value that belongs to the Asteraceae family. Its various components are used for medicinal benefits due to the presence of bioactive compounds such as alkaloids, flavonoids, inulin, caffeic acid derivatives, steroids, terpenoids, oils and coumarins. With a long history of use in traditional medicine, chicory has many healing properties and has antibacterial, antioxidant, anti-inflammatory, anti-tumor, anti-diabetic and many other health-promoting effects¹⁷. Research has shown its antimicrobial activity against several bacterial pathogens including S. aureus, Bacillus species, E. coli, Pseudomonas fluorescens and Pseudomonas aeruginosa¹⁸. The extraction of antimicrobial peptides from plants, which are natural sources, is regarded as a viable method¹⁹.

This research focuses on the isolation, purification and identification of a new antibacterial peptide from Cichorium intybus L. using in vitro and in silico methods. The antimicrobial potential of the peptide was evaluated by determining its minimum inhibitory concentration (MIC) against a wide range of harmful microorganisms, including Gram-negative and Gram-positive bacteria. In addition, the mechanism of action of the peptide was investigated considering bacterial membrane disruption, cell damage and internal reactions. Hemolytic activity and cytotoxicity were also evaluated, and molecular docking was used to provide deeper insight into the antibacterial mechanism of the peptide.

Results

Peptide isolation and purification

As shown in Figure S1a after fractionation using a C18 column, 19 fractions were obtained from the lyophilized protein extract of Cichorium intybus L. flowers. In order to evaluate antibacterial properties, each peak was collected manually from C18 RP-HPLC. Peak 11 has the highest level of antibacterial activity, as determined by the analysis of its antibacterial properties. This active peak was then subjected to further purification by re-injecting it into the same column under identical elution conditions. By monitoring at 220 nm, a single peak was observed in the eluted fraction (Figure S1b). This peak underwent additional analysis through SDS-PAGE, mass spectrometry, and N-terminal sequencing to confirm its identity.

Evaluation of purity of the peptide

The active fraction with antibacterial activity was analyzed by electrophoresis, as shown in Figure S1c. The electrophoretic results revealed a single band, suggesting a molecular weight of approximately 3500 Da.

Peptide identification

The peptide’s precise molecular weight, as seen in Figure S1d, was determined to be 3686.4 Da. This result closely matched the relative molecular weight that Tricine-SDS-PAGE revealed. The peptide was sequenced using Edman degradation in order to determine the amino acid sequence. From this analysis, a fragment of 33 amino acid residues was provided as LVSVWIISAALSLKNKVAYRNAKLVSKLFRIAL.

Antibacterial activity

Antibacterial activities of all peaks against Gram-positive (S. aureus ATCC 29213) and Gram-negative bacteria (E. coli ATCC 25922, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606) were evaluated using the RDA method, as previously mentioned (data not presented). The RDA test results in Figure S2 showed that peak NO. 11, which was derived from the flowers of Cichorium intybus L, exhibited significant antibacterial activity against E. coli, S. aureus, A. baumannii, and P. aeroginosa. The inhibition zones of peak 11 and gentamycin (a common antibacterial agent) that works well against bacterial species are shown in Figure S2. Inhibition zones of the peak 11 for S. aureus and Gram-negative bacteria (E. coli, P. aeruginosa, A. baumannii) were 18 and 16–21 mm, respectively, while the inhibition zones of gentamycin were 22 and 17–25 mm. The zone of inhibition has the largest diameter in E. coli and the lowest diameter in S. aureus. The minimum inhibitory concentration (MIC) was obtained using the micro-broth dilution technique, which was used to evaluate the inhibitory effects of peptide on both Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa, A. baumannii) bacteria. Comparing gentamycin as a control, it showed MIC ranges of 1 and 0.5–4 µg/mL for S. aureus and Gram-negative species, respectively. Table 1 showed that the extracted peptide has excellent activity against Gram-negative bacteria in the MIC range of 2–8 µg/mL and for S. aureus with a MIC of 16 µg/mL. The maximum activity was reported against E. coli at a significant minimum inhibitory concentration (MIC) of 2 µg/mL. For every bacterial strain, the MBC values were two times higher than the MIC values or equivalent to them.

Table 1.

MIC and MBC (µg/mL) of Hyde C1 and Gentamycin (Gen) against different bacterial strains.

Bacterial Strain	MIC (µg/mL)		MBC (µg/mL)
	Hyde C1	Gen	Hyde C1	Gen
E. coli ATCC 25,922	2	0.5	4	2
S. aureus ATCC 29,213	16	1	32	4
A.baumannii ATCC 19,606	8	4	16	16
P. aeruginosa ATCC 27,853	8	2	8	4

Sequence alignment and phylogenetic tree

According to BLAST findings, the obtained peptide sequence did not show any significant similarity with known AMPs. It was also discovered that this particular peptide, which comes from Cichorium intybus L, is unique among antibacterial peptides. Based on sequence alignment and phylogenetic tree analysis, the novel peptide showed the highest sequence similarity with Hyde A isolated from Australian Hybanthus debilissimus (Figures S3a, b). This new peptide was named Hyde C1 based on its similarity and the peptide purification source, which is Chironomus intybus L.

Bioinformatics analysis

The physicochemical properties and sequences of the peptide Hyde C1 are illustrated in Table 2. The molecular weight of the peptide Hyde C1 has been calculated to be 3686.524 Da. physicochemical parameters of the peptide Hyde C1 indicated that it possesses AMP characteristics, as evidenced by its high positive charge and hydrophobic ratio (+ 6 and 61%, respectively) and protein-binding potential (Boman index) of 0.12 kcal/mol. The instability and aliphatic index values for this peptide were 5.20 and 156.67, respectively. PSIPRED predicted the secondary structure of the peptide Hyde C1 as an α-helix (Fig. 1a). Figure 1b showed that the peptide Hyde C1 has amphipathic α-helix conformations with both hydrophilic and hydrophobic faces. I-TASSER predicted the peptide Hyde C1 to have an α-helix structure (Figs. 1c). The I-TASSER assessment revealed that peptide Hyde C1 has a C-score of 1.16, which a confidence value is used to measure models’ global correctness (ranging from 5.0 to 2.0).

Table 2.

The probability of antimicrobial activity and physicochemical features of Hyde C1.

Name of peptide	Sequence	Molecular weight (Da)	Net charge	% hydrophobicity	Boman index	Aliphatic index	Instability index
Hyde C1	LVSVWIISAALSLKNKVAYRNAKLVSKLFRIAL	3686.524	6+	61%	0.12	156.67	5.20

Fig. 1.

(a) Graphical result from secondary structure prediction of Hyde C1 using PSIPRED. (b) Helical wheel diagram of Hyde C1 and polar and non-polar amino acids, and their locations in peptide can be observed (c) The Hyde C1 3D structure contains α-helix, as predicted by I-TASSER. The 3D structure model visualized using Accelrys discovery studio visualizer software.

Molecular Docking

Evaluation of the quality of the predicted 3D structure through the Ramachandran plot and with the help of the PROCHECK module from the SAVES server showed that 87.1% of the residues of the peptide Hyde C1 are located in the most favored region (Figs. 2a). Docking of the peptide Hyde C1 with each of the two-layer membranes of Gram-positive and Gram-negative bacteria showed that the minimum free energy of peptide binding to Gram-negative and Gram-positive membranes is −58.2 and − 33.1 KJ/mol, respectively, which shows the binding affinity of the Hyde C1 peptide to the membrane of Gram negative is more than the Gram-positive membrane. However, the illustration of the interaction of the peptide with each of the membranes showed that the Hyde C1 peptide has completely entered the hydrophobic region of the Gram-positive bacterial membrane, but its N-terminal end remained outside the Gram-negative bacterial membrane and in contact with the hydrophilic region (Figs. 2b).

Fig. 2.

(a) Predicted structure of the AMPs along with the Ramachandran plot. Hyde C1 is an α-helical in 3D and further confirmed by the concentration of amino acid in the most favored region of the α-helical structure of the Ramachandran plot. (b) Interaction of Hyde C1 with Gram-negative and Gram-positive membranes, indicating that this peptide can enter the hydrophobic region of the bacterial membrane.

Time‑kill kinetic

After confirming that the Hyde C1 possessed excellent antimicrobial activity, we next assessed the killing kinetic of this peptide. Hyde C1 showed concentration-dependent killing of E. coli ATCC 25,922 (Fig. 3). Hyde C1 showed bactericidal activity and was capable of completely eliminating within 90 min and 180 min at 4 × MIC and 2 × MIC, respectively.

Fig. 3.

The killing kinetics of Hyde C1 against logarithmic phase of E. coli ATCC 25,922. Logarithmic phase of E. coli ATCC 25,922 exposed to Hyde C1 at 2 × and 4 × MIC. Hyde C1 showed bactericidal activity and was capable of completely eliminating within 90 min and 180 min at 2 × MIC and 4 × MIC, respectively. Untreated samples served as a control. The experiments were done in triplicate and the data are represented as the mean ± SD.

Propidium iodide uptake assay

Propidium iodide (PI) is a dye that enters cells after membrane integrity is compromised, where it binds to nucleic acids and produces fluorescence. In the control group, where no peptide was present, cells showed minimal PI fluorescence, indicating an intact cell membrane. In contrast, the addition of Hyde C1 to the culture resulted in an increase in PI fluorescence. As shown in Fig. 4a, after 60 min exposure to Hyde C1 at MIC and 2 × MIC concentrations, 41.9% and 83.5% of bacterial cells were stained with PI, respectively, while only 6.41% was without Hyde C1. These findings indicate that Hyde C1 disrupts the bacterial membrane, increasing its permeability and leading to greater uptake of PI by the cells.

Fig. 4.

(a) Effect of Hyde C1 on the cell membrane permeability of E. coli ATCC 25,922, measured by PI uptake. Flow cytometric analysis of membrane permeabilization assay by PI uptake at different concentrations (1 × MIC and 2 × MIC) in E. coli ATCC 25,922. (b) Uptake of NPN by E. coli ATCC 25,922 treated with Hyde C1 at different concentrations (1 × MIC and 2 × MIC). (c) Cytoplasmic membrane depolarization of E. coli ATCC 25,922 by Hyde C1, assessed by release of the membrane potential-sensitive dye DiSC₃-(5) measured spectroscopically at 620 nm to 670 nm excitation and emission wavelength. Data presented as means ± SD of three independent repeats in triplicate cells.

Outer membrane permeabilization

The NPN uptake assay was employed to evaluate how effectively Hyde C1 peptide disrupts bacterial outer membrane permeability. Figure 4b shows that Hyde C1 caused a dose-dependent increase in E. coli outer membrane permeability, as evidenced by increased fluorescence from 1-N-phenylnaphthylamine (NPN).

Cytoplasmic membrane electrical potential

When the cytoplasmic membrane is disrupted, its electrical potential is lost, causing diSC₃−5 to be released into the surrounding solution, leading to a detectable fluorescence increase. Figure 4c shows that membrane depolarization was assessed after the introduction of Hyde C1. At both MIC and 2 × MIC concentrations, Hyde C1 effectively permeabilized the membrane. The results show that Hyde C1 causes membrane depolarization in a dose-dependent manner.

Scanning electron microscopy (SEM)

To visualize the morphological changes induced by Hyde C1, E. coli and S. aureus cells treated with 2 × MIC concentration of Hyde C1 were observed using SEM. Figure 5b shows the SEM images of E. coli cells after treatment with Hyde C1 at 2 × MIC. Control cells not treated with peptide display clear, smooth surfaces (Fig. 5a), but Hyde C1 treatment shows significant surface shrinkage and pore formation. We observed similar effects of Hyde C1 on the membrane of S. aureus cells (Fig. 5d). Untreated Hyde C1 bacterial cells were intact with a nearly spherical shape and a smooth, intact surface (Fig. 5c). Cells treated with Hyde C1 (Fig. 5d) showed surface abnormalities, membrane distortion with surface indentations, and leakage of cellular contents.

Fig. 5.

Scanning electron microscopy of E. coli ATCC 25,922 cells: (a) control, without peptide; (b) Hyde C1 treated at 2 × MIC. Scanning electron microscopy of S. aureus ATCC 29,213: (c) control, without peptide; (d) Hyde C1 treated at 2 × MIC. Approximately 1 × 10⁶ bacterial cells were incubated without (a, c control) or with Hyde C1 (4 µg/mL, 2 × MIC) (b, d) for 3 h. (a, c) SEM images of the untreated cells showed cells with normal shapes and smooth surfaces. (b) Hyde C1 treated E. coli cells showed a series of characteristic alterations such as cell surface shrinkage, cell pores, and full cell lysis. (d) Hyde C1 treated S. aureus cells showed surface abnormalities, membrane distortion, and leakage of cellular contents.

Hemolytic activity and cytotoxicity

Based on the findings of the hemolytic activity of the peptide Hyde C1 on red blood cells (RBCs), Hyde C1 showed low (< 10%) hemolytic activity on human erythrocytes in the MIC range (2–16 µg/mL) (Fig. 6a). As the concentration of this antimicrobial peptide increases, the ratio of hemolysis also increases. It exhibited 19.2% hemolytic activity at the maximum concentration (128 µg/mL). Cytotoxicity of the peptide Hyde C1 against the HEK293 cell line was subsequently assessed using the MTT assay after 24 h of treatment (Fig. 6b). The cytotoxicity of Hyde C1 was moderate, with a range of 2.5–7% at MIC concentrations (2–16 µg/mL). In addition, the Hyde C1 cell death rate was 12.2% at a concentration of 128 µg/mL.

Fig. 6.

Hemolytic activity, cytotoxicity and stability of Hyde C1. (a) Hemolytic assay of Hyde C1 against human erythrocytes. Hyde C1 showed low (< 10%) hemolytic activity on human erythrocytes in the MIC range (2–16 µg/mL). (b) Cytotoxicity activity of Hyde C1 on HEK293 mammalian cell line using MTT assay It had a slight hemolytic activity in the range of 2.5–7% at MIC concentrations (2–16 µg/mL). (c) The effects of pH and (d) temperature on the antibacterial activity of Hyde C1 against E. coli ATCC 25,922 cells. Hyde C1 is stable at pH values between six and ten for an h and remained stable at a wide range of temperatures from 10 to 60 °C.

Effects of pH, temperature, and salt on antimicrobial activity

As shown in Fig. 6c, the stability of the peptide Hyde C1 was evaluated at different pH levels. The findings showed that Hyde C1 is stable at pH values between six and ten for one hour. However, when pH levels fell below 5, its effectiveness decreased significantly. The effect of temperature on Hyde C1, as shown in Fig. 6d, showed that this peptide remained stable when exposed to a wide range of temperatures from 10 to 60 °C.

For real-world use, antimicrobial peptides (AMPs) must retain their potency under physiological conditions. For this purpose, the antimicrobial effect of Hyde C1 was tested in the presence of various salts at physiological concentrations. The minimum inhibitory concentration (MIC) of Hyde C1 against E. coli remained constant at different salts. Notably, the MIC of Hyde C1 decreased from 2 µg/mL to 1 µg/mL and its antimicrobial activity increased when Zn ²⁺ was present. The MIC of the peptide Hyde C1 toward E. coli did not change in different salts at physiological concentrations Moreover, in the presence of Zn²⁺, not only was the MIC value of Hyde C1 was decreased, but the antimicrobial activity of Hyde C1 increased. Overall, Hyde C1 showed strong resistance to the effects of physiological salts.

Effects of serum on antimicrobial activity

The stability of Hyde C1 antimicrobial activity in the presence of serum was evaluated using a time-killing assay. As shown in Fig. 7, the bacterial count in the presence of Hyde C1 in PBS begins at around 10⁵ CFU/mL, and there is a rapid decrease in bacterial growth over time, reaching complete killing by approximately 180 h. Similar to the Hyde C1 in PBS condition, there is a reduction in bacterial growth in the presence of Hyde C1 with 25% serum, the killing rate is slightly slower compared to the PBS condition. Complete killing is still achieved around 180 h but with a slightly reduced initial effectiveness.

Fig. 7.

Effect of serum on killing kinetics of Hyde C1 at 2 × MIC against E. coli ATCC 25,922. Data are mean ± SD of three independent experiments.

Discussion

Currently, the widespread and indiscriminate use of traditional antibiotics, along with the reduction in the production of new antibiotic drugs, leads to the emergence of antimicrobial resistance (AMR). Considering the emergence of multidrug-resistant (MDR) microorganisms as an important public health concern, there is an urgent need for a new class of antibiotics.

AMPs have recently become a viable solution to combat multi-drug resistant (MDR) bacteria⁶. In this work, we successfully isolated a new antimicrobial peptide (AMP), Hyde C1, from Cichorium intybus L., offers valuable insights into plant-derived peptides as potential therapeutic agents against pathogenic bacteria. Hyde C1 demonstrates antibacterial properties against both Gram-positive and Gram-negative bacterial strains, which is a significant advance in the search for alternatives to traditional antibiotics. While the present study demonstrates the antibacterial potential of the isolated peptide against representative Gram-positive and Gram-negative bacterial strains, we recognize the critical need to evaluate its efficacy against multidrug-resistant (MDR) pathogens. Given the growing clinical challenge posed by MDR organisms, including methicillin-resistant S. aureus (MRSA) and other resistant species, future studies will focus on assessing the peptide’s activity against a broad panel of clinical and community-acquired MDR isolates. This will provide a more comprehensive understanding of the peptide’s therapeutic potential and its possible role as an alternative or adjunct to current antibiotics.

AMP effectiveness is influenced by factors like net charge, hydrophobicity, and the capacity to form amphipathic secondary structures. The net charge plays a crucial role in enabling electrostatic interactions with negatively charged components within bacterial cell membranes²⁰. Hydrophobicity allows AMPs to engage with the membrane, leading to disruptions in the bacterial membrane’s integrity²¹. Hyde C1 has 33 amino acids and exhibits a high net positive charge (+ 6) and a hydrophobic ratio of 61%, characteristics that promote membrane interaction, essential for antimicrobial efficacy. This profile aligns with studies by²² and²³, which showed that cationic and amphipathic structures in AMPs enhance their ability to disrupt bacterial cell membranes effectively. The α-helical conformation of Hyde C1 further supports its antimicrobial function, as helical structures facilitate interactions with bacterial membranes by penetrating lipid bilayers. In addition, the secondary structure of AMPs also had a significant effect. Using the Protein Structure Prediction (PSIPRED) server, Hyde C1 peptide was found to have an α-helical conformation in its secondary structure. Park et al. (2000) reported²³ that AMPs with α-helical structures, like Hyde C1, used this property for potent antibacterial action, because this structure increases the binding between antimicrobial peptides (AMPs) and the cell membrane, leading to an increase in the capacity of AMPs to cross the membrane. The Hyde C1 helical wheel represents the amphipathic α-helical conformation of this AMP, which is crucial for its antibacterial effect²⁴. Previous research has shown that α-helical AMPs with an amphipathic structure can interact with the membranes and their potential antibacterial effect can be greatly enhanced²⁵.

Hyde C1 demonstrated significant antibacterial activity against Gram-positive (S. aureus) with the MIC of 16 µg/mL and Gram-negative (E. coli, A. baumannii, and P. aeruginosa) bacteria at MIC values (2–8 µg/mL). In 2016 a research conducted by Srivastava et al. found an antibacterial peptide extracted from Paryosha flowers. This peptide demonstrated activity against both Gram-positive and Gram-negative bacteria, including E. coli and S. aureus²⁶. Angayarkanni et al. succeeded in extracting an antibacterial peptide from jasmine flowers in another similar work. This peptide exhibited antimicrobial activity against Gram-positive (streptococci) and Gram-negative (E. coli and Shigella) bacteria²⁷. In addition, the results of antimicrobial activity showed that Hyde C1 is more effective against Gram-negative bacteria than Gram-positive bacteria, and also the results of molecular docking showed that the binding affinity of Hyde C1 with Gram-negative membranes is higher than that of Gram-positive membranes, which may depend on the bacterial cell wall. These differences in antibacterial activity can be attributed to the distinct structures of Gram-positive and Gram-negative bacterial envelopes. Gram-negative bacteria possess an additional outer membrane rich in lipopolysaccharides (LPS), which provides a negatively charged surface that readily interacts with the highly cationic Hyde C1 peptide. This interaction facilitates the peptide’s insertion and destabilization of the outer membrane, leading to easier access to the cytoplasmic membrane and rapid bactericidal action. In contrast, Gram-positive bacteria lack this outer membrane but have a much thicker peptidoglycan layer, which can act as a barrier and slow the peptide’s access to the cytoplasmic membrane. Nevertheless, the amphipathic α-helical structure and hydrophobicity of Hyde C1 enable it to eventually penetrate the peptidoglycan and exert its membrane-disruptive effects. The observed MIC differences between Gram-positive and Gram-negative strains support this differential mechanism. These findings highlight the importance of bacterial envelope structure in determining AMP activity, and future molecular dynamics studies could further clarify the precise interaction patterns between Hyde C1 and various bacterial membranes. The findings of this study may also be attributed to the presence of a dense peptidoglycan layer within the membrane of Gram-positive bacteria, which could hinder the AMPs from destroying S. aureus. In contrast, Gram-negative bacteria possess a thinner peptidoglycan layer along with an additional outer membrane, making the tested strains (E. coli, A. baumannii, and P. aeruginosa) more susceptible to AMPs^28,29. While molecular docking provided useful preliminary insights into the peptide–membrane interactions, we acknowledge that molecular dynamics (MD) simulations represent a more robust approach for exploring such complex biological systems. Future studies will aim to incorporate MD simulations to achieve a more detailed and dynamic understanding of these interactions.

Most researchers agree that the bactericidal action of AMPs mainly involves pore formation on the bacterial surface, disruption of membrane structure, and subsequent release of cellular contents, ultimately leading to bacterial death^30,31. Our study shows that Hyde C1 exhibits bactericidal effects against bacterial strains by disrupting cell membrane structure, increasing membrane permeability, and damaging the bacterial membrane. This process is evidenced by heightened uptake of propidium iodide and NPN, markers of compromised membrane integrity, which ultimately result in bacterial cell death. This mode of action was further supported by the depolarization and SEM studies, where Hyde C1-induced membrane damage in E. coli cells was visible.

One of the key considerations in developing therapeutic AMPs is their selectivity towards microbial cells over mammalian cells. Hyde C1 exhibited limited hemolytic (7%) and cytotoxic (< 5.5%) effects within the MIC range. This low cytotoxicity is attributed to its hydrophobicity, supporting findings by Wiradharma et al., who correlated hydrophobicity levels in AMPs with toxicity in human cells³².

Beyond cytotoxicity and hemolysis, another key factor influencing AMP efficacy is stability under varied conditions. Hyde C1 retains activity across a broad pH range (6–10) and temperatures (10–60 °C). Additionally, we previously evaluated our AMP’s stability using the Antimicrobial Peptide Database (APD), which confirmed its acceptable stability (Table 2). Temperature stability is particularly important for AMPs intended for clinical use³³. However, salt sensitivity presents a major limitation for AMPs as therapeutic agents^34,35. The presence of cations can hinder AMP effectiveness by weakening the electrostatic interactions between positively charged peptides and negatively charged bacterial surfaces, thereby reducing their antimicrobial potency³⁶. To maintain its antimicrobial activity under such conditions, we tested its effectiveness at physiological salt concentrations. Our findings showed no change in Hyde C1’s minimum inhibitory concentration (MIC) in the presence of Na⁺, K⁺, Mg²⁺, Ca²⁺, and Fe³⁺ ions. Interestingly, Hyde C1 demonstrated increased activity in synergy with Zn²⁺. This strong salt resistance of Hyde C1 is likely due to its unique amino acid composition, including tryptophan and arginine residues, which are known to maintain antimicrobial activity in high salt environments³⁸.

Moreover, the serum killing assay demonstrated that Hyde C1 maintained its bactericidal effect even in the presence of 25% human serum, although a slight delay in bacterial elimination was observed compared to PBS alone. This finding highlights the peptide’s potential for systemic application, as many AMPs lose activity in serum due to enzymatic degradation or interactions with serum proteins³⁹. Hyde C1’s stability in serum likely stems from its amphipathic structure and favorable amino acid composition, particularly the presence of arginine and hydrophobic residues, which have been associated with improved serum tolerance³⁹.

Given the promising in vitro antibacterial activity and low cytotoxicity of Hyde C1, the next logical step is to evaluate its therapeutic efficacy in vivo. In particular, using a mouse model of MRSA infection would provide valuable insights into the peptide’s performance in a physiological environment and its potential for clinical translation. Such preclinical studies will help assess not only the antimicrobial efficacy but also the pharmacokinetics, safety, and stability of Hyde C1 under systemic conditions. These future directions are essential to move from laboratory findings toward real-world therapeutic applications.

Conclusion

Hyde C1 represents a probable candidate for the development of plant-derived AMPs, combining broad-spectrum activity, environmental stability, and low cytotoxicity. However, further in vivo studies are needed to assess its pharmacokinetics, stability in complex biological environments, and potential synergistic effects with existing antimicrobial agents. Future research could also explore structural analogs of Hyde C1 to enhance selectivity and reduce any residual cytotoxicity, thus advancing Hyde C1 towards clinical applicability.

Materials and methods

Ethics statemen

Approval for all experimental protocols and methodologies was obtained from the Ethics Committee at Shahid Beheshti University of Medical Sciences, Tehran, Iran (approval number: IR.SBMU.MSP.REC.13401.623). Informed consent was acquired from all participants. All research procedures complied with the Declaration of Helsinki and the institutional ethical guidelines.

Statement on experimental research and field studies on plants

The Cichorium intybus L. plants sampled comply with relevant institutional, national, and international guidelines and domestic legislation of Iran. All methods were performed in accordance with the relevant guidelines/regulations/legislation.

Test organisms and plant collection

Bacterial strains S. aureus ATCC 29,213, E. coli ATCC 25,922, P. aeruginosa ATCC 27,853, and A. baumannii ATCC 19,606 were sourced from the Department of Microbiology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. These strains were stored in Trypticase Soy Broth (Merck, Darmstadt, Germany) with 25% glycerol at − 70 °C. For testing, strains were grown on Nutrient Agar (Merck, Darmstadt, Germany) under aerobic conditions at 37 °C for 24 h. Chicory flowers were collected in May 2022 from Kashan, located in Isfahan Province.

Extraction and purification of the peptide

Antibacterial peptides were isolated from chicory flowers using the technique described by Seyedjavadi et al.³⁸. After freezing and grinding them into flour using liquid nitrogen, total protein was extracted with an extraction buffer (10 mM Na₂HPO₄, 100 mM KCl, 15 mM NaH₂PO₄, and 1.5% EDTA (Sigma-Aldrich, St. Louis, MO, USA). Then the crude extract was centrifuged at 4000 rpm for 20 min at 4 °C and the supernatant was filtered. A solution of 85% ammonium sulfate ((NH₄)₂SO₄) (Sigma-Aldrich, St. Louis, MO, USA) was added to the resulting supernatant for 24 h at 4 °C. The precipitate was extensively dialyzed against distilled water by using benzoylated membrane performance (MWCO 2000 Da) (Sigma Aldrich-USA) at 4 °C within 12 h to remove the residual (NH₄)₂SO₄. The protein extract was filtered using a 10 kDa cutoff ultracentrifuge (Millipore, Bedford, MA, USA) to separate low molecular weight peptides. These peptides were subsequently lyophilized in a freeze-dryer. The reversed-phase HPLC column (C18 column, 250 × 4.6 mm; Knauer, Berlin, Germany) with the gradient of 5–65% (v/v) solution B (0.05% TFA in acetonitrile) and A (0.05% TFA in water) at a flow rate of 1 ml/min for 85 min was employed for purifying the antibacterial peptides from lyophilized extracts. Each eluted peak was collected manually and subsequently concentrated by lyophilization to assess antibacterial activity, as indicated by absorbance at 220 nm. The peaks with the greatest activity were chosen for further purification. In order to confirm the purity of the active peak, it was re-chromatographed under identical conditions using the same column and solvent system.

SDS‑Page

The molecular weight and purity of the antibacterial active peak were determined using tricine-SDS-PAGE under decreasing conditions on a Bio-Rad electrophoresis system, as reported by Schagger and Von Jagow⁴³. Then, samples were run on a 16.5% Tris-tricine gel with tricine–SDS running buffer for overnight in 25 V tension. The samples were subjected to a 65 min cycle on a 12% Tris-tricine gel at 210 V. The molecular mass was estimated by using a protein ladder (2-250 kDa, Precision Plus Protein Standard from Bio Rad, 1610374) as the standard, and the protein bands were visualized by coomassie staining following electrophoresis.

Radial diffusion assay

The antibacterial activity of the collected fractions was assessed using the radial diffusion assay (RDA) protocol as outlined by Takemura et al.⁴⁰. Gentamycin (10 µg/mL) was employed as a positive control, which is a conventional antibacterial agent. In brief, a volume of 1 × 10⁶ CFU/mL of bacterial cells was added to 100 mL of Mueller–Hinton agar (Merck, Darmstadt, Germany) adjusted at 42 °C and then rapidly distributed in a Petri dish. Wells were punched and filled with the purified peptide, and after incubation at 37 °C for 24 h the clear zones surrounding the wells were studied. All the experiments were done in triplicates.

Minimum inhibitory concentrations and minimum bactericidal concentrations

The minimum inhibitory concentration (MIC) of peptide was evaluated by broth microdilution assay according to a modification described by the Clinical and Laboratory Standards Institute (CLSI)⁴¹. In summary, the peptide was serially attenuated in 96-well plates in a twofold manner to produce a final volume of 100 µL per well. Gentamycin, a conventional antibacterial agent, was employed as a control. The concentration range evaluated for the purified peptide was 1–256 µg/mL. Then, 100 µL of bacterial suspension (~ 10⁶ cell/mL) was added to every well followed by incubation at 37 °C for 24 h. The negative and positive controls were the purified broths alone and with the inoculum suspensions, respectively. The visual assay was used to determine the minimum inhibitory concentration (MIC), which was defined as 99% inhibition of bacterial growth in 96-well microplates. The minimal bactericidal concentration (MBC) of peptide was determined using a colony count assay. Twenty microliters of the mixture from each non-turbid well (no growth) from the MIC experiment was spread on Mueller-Hinton agar (Merck, Darmstadt, Germany) and incubated at 37 °C for 24 h. The minimum bactericidal concentration (MBC) is the lowest concentration of antimicrobial agents that there was no bacterial colony growth on the agar.

Mass spectrometry and n − terminal sequence analysis

The amino acid sequence analysis was performed on the freeze-dried active peptide. Electrospray ionization mass spectrometry (MS) was used to determine the molecular mass of the isolated antibacterial peptide, which gives the mass-to-charge ratio (m/z). Edman degradation was used to determine the amino acid sequence of the purified antibacterial peptide. This was accomplished by interfacing an ABI 140 C PTH Amino Acid Analyzer with an ABI Procise Edman Micro Sequencer (Model 492) to facilitate the sequential determination of the amino acid composition of the peptide.

Sequence alignment and phylogenetic tree

The antibacterial peptide database was searched to identify peptides that shared a high degree of similarity with our recently discovered peptide (https://www.aps.unmc.edu/AP/main.php). Consequently, the 12 peptides that exhibited the greatest similarity to the new peptide were identified. The Basic Local Alignment Search (BLAST) program (https://www.ncbi.nlm.nih.gov/BLAST) was used to align the identified peptides with our new peptide. The CLC main workbench software generated a phylogenetic tree after manually adjusting the alignment. In order to ascertain the reproducibility of tree topology, the phylogenetic tree’s reliability was evaluated using a bootstrap analysis that included 100 replications.

Bioinformatics and physicochemical analysis

The physicochemical properties of antibacterial peptide were predicted using ExPASy Proteomics server (http://www.expasy.org/tools/protparam.html). PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) was applied to predict the secondary structure and the helical wheel diagram was drawn utilizing online software to predict the position of amino acids in the peptide (http://lbqp.unb.br/NetWheels/). Besides, three-dimensional (3D) model of Peptide was generated via I-TASSER software (http://zhanglab.ccmb.med.umich.edu/ITASSER/), and the quality of model was assessed applying Accelrys, DS visualizer.

Molecular Docking

The three-dimensional structure of the peptide was predicted using the I-TASSER online server (http://zhanglab.ccmb.med.umich.edu/ITASSER/). The quality of the predicted 3D structure was done using SAVES online server (http://nihserver.mbi.ucla.edu/SAVES/) and PROCHECK module. The two-layer membrane structure of Gram-positive and Gram-negative bacteria according to the study of Hasannejad-asl. B et al.⁴² prepared with the help of CHARMM-GUI online server (http://www.charmm-gui.org). The abundance of different molecules in each of the two-layer membranes is shown in Table 3. Peptide molecule preparation for docking was done with Chimera software version 1.17.1 and Gasteiger charges and non-polar hydrogen atoms were added to it, and aromatic carbon rings were identified. Then, the peptide was energy-minimized with the help of Amber force field and saved in pdbqt format after 1000 steps. Autodock vina 1.12.1 tool was used for docking and each of the two-layer membranes was introduced as macromolecule receptor and peptide molecule as ligand, and the docking process was performed.

Table 3.

The structure of the 2-layer membrane of Gram-positive and Gram-negative bacteriad.

Membrane	Lipids
	POPE	POPG	TOCL1
Gram negative bacteria	62	12	4
Gram positive bacteria	0	46	32

Time-dependent killing kinetics

The time killing kinetics of Hyde C1 on E. coli ATCC 25,922 was determined using a colony count-based method⁴³. Bacterial cells of E. coli, during their logarithmic growth phase, were collected by centrifugation at 4500 rpm for 5 min, followed by two washes with PBS. The cells were then diluted to a concentration of 2–7 × 10⁵ CFU/mL. E. coli were exposed to the peptide at different concentrations, including the1 × MIC and 2 × MIC. At specific time points (30, 60, 90, 120, 180 and 240 min), 50 mL of the bacterial suspension was diluted and spread onto MHA plates. After 24 h of incubation at 37 °C, the bacterial colonies were counted. The data of three independent experiments are reported in this study.

Propidium iodide uptake assay

Flow cytometry was employed to assess the membrane-permeabilizing effect of Hyde C1 on E. coli ATCC 25,922 as the model, which allowed propidium iodide (PI) to enter cells with compromised membranes⁴⁴. E. coli cells in the logarithmic phase were centrifuged at 4500 rpm for 5 min and diluted to an OD₆₀₀ of 0.05. Various concentrations of peptide (1 × MIC and 2 × MIC) were introduced to the suspension, followed by 30 min incubation at 37 °C. Afterward, propidium iodide (50 µg/mL) was added and incubated for 15 min in darkness. The percentage of PI-stained bacterial cells was measured using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) at excitation and emission wavelengths of 580 and 620 nm, respectively. Sterile water served as the negative control, while 1% Triton X-100 was used as the positive control. Data from three separate experiments were recorded.

N-phenylnaphthylamine (NPN) uptake assay

N-phenylnaphthylamine (NPN) uptake assay was performed to assess the permeability of the outer membrane (OM) in response to Hyde C1⁴⁵. In summary, E. coli ATCC 25,922 cells in the logarithmic phase were diluted to an OD₆₀₀ of 0.05. NPN was added to achieve a final concentration of 10 µM, and the mixture was incubated for 20 min. Various concentrations of peptide (1 × MIC and 2 × MIC) were then introduced to the wells. Sterile water served as the negative control, while 1% Triton X-100 was used as the positive control. Fluorescence intensity was measured using a microplate reader to assess NPN uptake, with excitation and emission wavelengths set at 350 nm and 420 nm, respectively.

Cytoplasmic membrane depolarization assay

The ability of Hyde C1 to cause membrane depolarization in E. coli ATCC 25,922 was assessed using the fluorescent dye 3,3´-Dipropylthiadicarbocyanine iodide (DiSC₃−5) as previously described by Rasul et al.⁴⁶. E. coli ATCC 25,922 cells in the logarithmic phase were centrifuged, resuspended in sterile PBS, and adjusted to an OD600 of 0.05. A DiSC₃−5 solution was added at a final concentration of 1 µM, and the mixture was incubated in the dark for 20 min. Hyde C1 was then added at various concentrations (1 × MIC and 2 × MIC). Sterile water served as the negative control. Fluorescence intensity was measured using a microplate reader with excitation and emission wavelengths set at 622 nm and 670 nm, respectively.

Scanning electron microscopy

To examine the morphological alterations in bacterial cells when exposed to a peptide, scanning electron microscopy (SEM) was employed. E. coli and S. aureus suspension with a concentration of 1 × 10⁶ cells/mL was treated with 4 µg/mL of the peptide for a duration of 3 h and subsequently centrifuged for 10 min. Post-treatment, the cells were centrifuged three additional times, each for 5 min. The resulting pellet was preserved in a 2.5% (v/v) glutaraldehyde solution at 4 °C for 3 h. After fixing, the pellet underwent three washes in 0.1% PBS, followed by a secondary fixation in 1% osmium tetroxide solution for 1 h at room temperature. To prepare the sample, it was washed with PBS and then dehydrated through a graded ethanol series (25%, 50%, 75%, 95%, and 100%) for 10 min each, with a final dehydration in absolute ethanol for 45 min. Samples were then subjected to critical-point drying using CO₂, coated with a thin layer (20–30 nm) of gold-palladium, and visualized under an analytical SEM microscope (JEOL JSM-6510LA). For control purposes, bacterial cells cultured without peptide exposure were processed using the identical protocol.

Hemolytic assay

Human red blood cells (RBCs) were obtained by centrifugation at 4500 rpm for five min after heparinized whole blood was obtained. After three rounds of washing, the recovered erythrocytes were resuspended in PBS to reach a final concentration of 4% (v/v). Subsequently, erythrocytes were combined with the peptide Hyde C1 serial dilution (1–128 µg/mL) and incubated for one hour at 37 °C. Hemoglobin release was measured via the measurement of absorption at 567 nm using ELISA reader. Hemolysis (%) = [test OD − negative control OD)/(positive control OD – negative control OD)] × 100. In this experiment, RBCs treated with PBS were applied as negative control and 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, US) was utilized as the positive control in each run test. All experiments were performed in triplicate³⁸.

Cytotoxicity assay

In order to evaluate the impact of the peptide Hyde C1 on human embryonic kidney cells (HEK 293 T), 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide was used in the MTT test. Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS supplement was used to cultivate HEK 293 cells. Then, HEK 293 cells were treated with serially diluted peptide (1–128 µg/mL) after being seeded at a density of around 1 × 10⁵ cells/mL on 96-well plates. The plates were incubated in 5% CO₂ at 37 °C for 12 h. Following that, each well received 10 µL of MTT (0.5 mg/mL in phosphate-buffered saline, PBS) (Sigma-Aldrich, St. Louis, MO, USA) and was incubated for another 4 h under the same conditions. After removing the medium, 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The optical density of every well was measured at 570 nm applying an ELISA reader. Dead % = [1 = ((OD test)/(OD control)) × 100]. 1% Triton X-100 and PBS were utilized as positive and negative controls, respectively. All tests were performed three times³⁸.

Effects of serum on antimicrobial activity

Peptide stability in diluted serum was assayed as previously described by Lyu et al.²². Human serum was centrifuged at 4500 rpm for 10 min, and then inactivated at 56 °C for 30 min. E. coli 25,922 in exponential growth was washed three times with PBS at 4500 rpm for 10 min and diluted in PBS or in 25% serum to reach a final concentration of 10⁶ CFU/mL. The bacterial suspensions were incubated with 2 × MIC peptide at 37 °C for 6 h for the following time points:0, 0.5, 1, 2, 4, and 6 h. Aliquots of 50 µL were then taken, serially diluted in PBS, and plated on MHA plates. Bacterial colonies were counted after incubating the plates at 37 °C for 24 h.

Thermal, pH and salt stability determination

Chan et al.⁴⁷ developed the conventional approach used to assess the effects of temperature on the peptide’s antibacterial activity. In short, the peptide solution was incubated for one hour at several temperatures (20–100 °C). Using the RDA test, which Takemura et al.⁴⁰ had previously described, the antibacterial activity against the indicator strain, E. coli 25,922, was assessed. The control was the peptide that was not cooked to a temperature higher than that of the experiment. The peptide solution was incubated for one hour at 25 °C in buffers with several pH values (2.0–14.0) in order to determine pH stability. The RDA test was used to examine the antibacterial activity of the solutions against E. coli 25,922 after they had been brought to room temperature and pH adjusted to 7. The peptide was dissolved in 100 µL of pH-7 solution as a control. Each experiment was repeated three times.

To evaluate the effect of salts on the antibacterial activity of the peptide, it was incubated with salts at physiological concentrations (150 mM NaCl, 4.5 mM KCl, 8 mM ZnCl₂, 1 mM MgCl₂, 2 mM CaCl₂, and 4 mM FeCl₃). The minimum inhibitory concentration (MIC) of Hyde C1 was then determined against E. coli 25,922⁴⁸. Each experiment was conducted independently three times.

Statistical analysis

GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for graphical representation of the data. All experiments were performed in triplicate and repeated on three independent occasions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MG conceived and designed the study. MG, SGHA, FSH, BH, SSS, and ML contributed to comprehensive research. SGHA, MG, SSS and BH wrote the paper. BH, FSH, SSS, and ML analyzed data and prepared figures. MG, ML, FSH, and SGHA participated in manuscript editing. All authors reviewed the manuscript.

Data availability

The data sets supporting the results of the current research are available from the corresponding authors upon request Dr. Mehdi Goudarzi (gudarzim@yahoo.com). The peptide sequence data generated in this study are available in the antimicrobial peptide database (APD3) repository, accession number AP05412, accessible via the following link:https://aps.unmc.edu/database/peptide.

Declarations

Competing interests

The authors declare no competing interests.