1. Introduction
Natural antimicrobial peptides (AMPs), as a critical component in the fight against infectious pathogens [
1], are considered to be the ideal alternative agents to traditional antibiotics due to the fact of their broad-spectrum antimicrobial activity and low potential for inducing drug resistance [
2,
3]. Furthermore, in playing an essential role in their immune systems [
4], the skin secretions of amphibians are known as effective sources for AMPs [
5,
6].
Despite their diversity, AMPs have characteristic features, including conformation, net charge, hydrophobicity and amphipathicity, all of which are associated with their ability to kill microorganisms [
7]. The membrane permeability caused by these factors is a widely accepted mechanism of AMP actions [
8,
9]. Briefly, the cationic AMP molecules initially associate with the anionic phospholipids and acidic polymers of the cell membrane through electrostatic interaction [
10]. Then, these AMPs adopt amphiphilic secondary α-helix conformations. The hydrophobicity of AMPs promotes the interaction between peptides and fatty acyl chains to form pores or align parallel to the surface on the cell membrane and to disrupt the cell membrane [
11,
12,
13]. Thus, it can be seen that the interaction with AMPs and the anionic surface of the membrane is a prerequisite for their membrane permeabilising actions [
14]. On this basis, Du et al. constructed two cationicity-enhanced analogues of novel AMPs, AcrAP1 and AcrAP2, by using positively charged lysine residues to substitute the neutrally charged serine residues. The modified products exhibited a more potent antimicrobial activity in killing
Staphylococcus aureus (
S. aureus),
Escherichia coli (
E. coli) and
Candida albicans (
C. albicans) [
15]. In addition, Chen et al. modified the natural peptide PAM-37 by replacing phenylalanine at position 34 with positively charged arginine. The modified peptide PAM-37 (F34-R) obtained one more net charge and displayed better antimicrobial activity against
S. aureus,
Listeria monocytogenes,
Salmonella typhimurium and
Pseudomonas aeruginosa (
P. aeruginosa) [
16]. In addition, some researchers tried to replace the cationic amino acids in AMPs, which mostly resulted in a significant decrease or complete loss of antimicrobial effect, indicating the high importance of cationic residues [
17,
18]. The natural peptide Thanatin, for example, showed excellent antimicrobial activity against a variety of pathogens including drug-resistance; however, when the positively charged R13 and R14 were replaced by alanine, the recognition of the outer membrane lipopolysaccharide was affected, resulting in the loss of antimicrobial activity [
19]. That is to say, when taking novel peptides as templates for modification, increasing net charge is a feasible starting point to increase their bioactivity.
Dermaseptin peptides form a highly conserved family with the typical amphiphilic consensus motif (AAXKAALXK, X can be any amino acid), a conserved tryptophan residue at the third position and C-terminal amidation [
20]. Thus far, more than 100 peptides have been proved to belong to the dermaseptin family. Dermaseptin PS1 showed significant proliferative inhibition on more than 16 human cancer cell lines [
21]. In addition, dermaseptin S4 and its analogues were effective against nine reference and clinical
Neisseria gonorrhoeae strains [
22]. Dermaseptin PS4 displayed better inhibition of cell proliferation than melittin when acting against the U251MG, H157 and MDA-MB-435S cell lines [
23]. The amide group and positively charged amino acid provide the cationic of dermaseptins, and the consensus motif provides the amphiphilic domain [
24]. These are the structural bases for the excellent bioactivity of dermaseptin family peptides. Meanwhile, novel dermaseptins have great potential to be used as peptide modification templates to improve their bioactivity further.
Here, we identified a novel dermaseptin peptide named t-DPH1 from Phyllomedusa hypochondrialis (P. hypochondrialis) through shotgun cloning. A series of bioactivity assessments demonstrated that this peptide had broad antimicrobial activities and potential antiproliferative activity with a mechanism of action through destroying the cell membrane. In addition, four cationic-enhanced analogues, with net charges ranging from +5 to +7, were designed to optimise the bioactivity of t-DPH1 and to further investigate the net charge threshold on the bioactivity of the peptide.
3. Discussion
Amphibian skin-derived peptides have been closely studied for over 50 years, many of which have been proved to have considerable bioactivities including working as potential antimicrobial agents, protease inhibitors, smooth muscle relaxants, insulin-releasing promoters, and others [
23,
24,
25,
26,
27]. In addition, natural AMPs can serve as templates to be modified by single or multi-specific site amino acid substitution to achieve higher molecular diversity and enhanced properties [
15]. That was the strategy adopted in this study.
The novel peptide t-DPH1, first discovered in this study, is a linear cationic AMP belonging to the dermaseptin family. The extensive antimicrobial activity of t-DPH1 was demonstrated against the tested seven microorganisms. Although Gram-negative bacteria have a thicker cell wall (composed of lipopolysaccharide) than those of Gram-positive bacteria (composed of peptidoglycan) [
28], t-DPH1 still exhibited more potent abilities against Gram-negative bacteria. Meanwhile, t-DPH1 demonstrated an inhibitory effect on the growth of four tested human cancer cell lines. As previously stated [
29], cationicity is the initial factor in interacting AMPs with cell membranes. Thus, when modifying parent peptide t-DPH1, positively charged lysine residues were used to substitute the amino acids located at the hydrophilic face of the sequence of t-DPH1, and a series of analogues with enhanced cationicity were designed.
The secondary structure and physicochemical properties of t-DPH1 and its analogues were analysed by online bioinformatic tools, and the CD spectra confirmed that, like most AMPs (e.g., magainin 2, temporin), t-DPH1 and its analogues tended to form random coils in an aqueous environment while forming a stable alpha-helix structure in a membrane environment [
30,
31]. This amphipathic conformation is a vital factor needed by AMPs for their bioactivities [
32]. Therefore, based on the predicted helical wheel plots, replacing one or more amino acids with a hydrophilic positively charged lysine residue on the hydrophilic surface not only improved the net charge of the parent peptide but enhanced the amphiphilicity as well. Accordingly, the α-helix degree of all modified peptides was improved compared with that of the parent peptide.
The bioactivity of the analogues was observed and compared with that of the parent peptide, and it was found that t-DPH1-K4 and t-DPH1-5K, with net charges of +5 and +6, achieved overall promotion in antimicrobial activity, especially in killing Gram-negative bacteria. Furthermore, compared with the parent peptide, t-DPH1-K4 and t-DPH1-5K had higher net charges and stronger amphiphilicity, which made it easier for them to approach and penetrate negatively charged lipopolysaccharides in the outer membrane of Gram-negative bacteria and interact with relatively weak peptidoglycans in the cell wall, leading to cell rupture [
33]. As a previous study proved, the anti-biofilm effect of cationic AMPs can be exerted through the mechanisms of interacting with the cell membrane, including disrupting the membrane potential of cells embedded by degrading the biofilm matrix and polysaccharides, etc. [
34]. In the assessment of anti-biofilm activity, t-DPH1-K4 and t-DPH1-5K also showed an eradication effect on the formed Gram-negative bacterial biofilm, which was not observed in other tested peptides. These confirmed the contribution of the positively charged amino group on the side chain of lysine, enhancing the cationic peptide’s binding to the negatively charged cell membrane [
35].
The bacteria cell membrane permeability assay and time–killing assay showed that t-DPH1 and its analogues had concentration-dependent damage to bacterial cell membranes. This may be because peptides can only form a limited number of pores in the cell membrane at low concentrations. High concentrations of peptides tend to accumulate on the membrane, resulting in membrane rupture [
36]. At the same concentration (their corresponding 2*MICs), there was no significant difference in the membrane permeability rate of t-DPH1-K4 to
E. coli cell membrane compared with that of t-DPH1. But t-DPH1-K4 could kill
E. coli much faster than t-DPH1 at their corresponding 2*MICs. Similarly, at the MIC concentration, the membrane permeability rate of
S. aureus cell membrane caused by t-DPH1-5K was similar to that of t-DPH1 at their corresponding MICs, but it could kill bacteria within 30 min, which was much faster than that of t-DPH1. These phenomena indicate that increasing the net charge can make AMPs approach the cell membrane faster and, thus, quicken the bactericidal speed, but it cannot further improve the permeability effect of the peptide.
In addition to excellent antimicrobial activity, t-DPH1-K4 and t-DPH1-5K also had a stronger anti-proliferation effect on the tested non-small lung cancer cells than that of the parent peptide. Compared with normal cells, there are more anionic molecules on the surface of cancer cells such as O-glycosylated mucins, heparin sulphate, and sialylated gangliosides [
37]. The enhanced cationic properties promoted the interaction between peptides and cancer membranes. However, although t-DPH1-5K can kill cancer cells at low concentrations, it can also inhibit normal cell proliferation, limiting its further application. The cytotoxicity of t-DPH1-K4 to normal cells was higher than that of the parent peptide, but it was still in an acceptable range.
However, when the net charge was further improved to +7, the antimicrobial activity of t-DPH1-6K was not significantly improved compared with that of t-DPH1-5K, but the antiproliferative activity decreased dramatically. Keeping the same quantity of net charge, the hydrophobicity was adjusted by replacing the Glycine
1 residue with the hydrophobic tryptophan residue. Still, the product t-DPH1-6KW showed no other ideal bioactivity except the bactericidal activity against Gram-negative
E. coli. Given this result, it is necessary to discuss the relationship between the activity of AMPs and the range of net charge. Some studies believe that the charge range of AMPs with excellent activity is generally between +3 and +6 [
38]. However, the optimal amount has not been decided yet, which may also be related to the length of the peptide sequence, secondary structure and amino acid types of different peptides [
39]. A recent report showed that DFT503, an AMP containing one lysine, performed favourable activities both in in vitro and in vivo studies against antibiotic-resistant Gram-positive bacteria; however, DFT564 and DFT565, which contained three and four lysine residues, lost their antibacterial potency when additional basic amino acids were added, indicating that high cationic property was the main cause of its functional failure [
40]. On the one hand, when the net charge is higher than the upper limit, the bioactivities of AMPs may be reduced due to the mutual repulsion of the same charges so that the peptide could not bind with the cell membrane [
41]. On the other hand, the excessive hydrophilic residues may reduce the hydrophobicity of AMPs which then cannot be deeply inserted into the cell membrane, thus being unable to significantly improve the effect [
32]. These theories might explain the failure of t-DPH1-6K and t-DPH1-6KW. The balance between charge quantities and other properties, including hydrophobicity and amphiphilicity, is important in peptide modification. When the charge of AMPs reaches the threshold value, although the amphiphilicity and hydrophobicity of t-DPH1-6K and t-DPH1-6KW were within a reasonable range, their bioactivities were still affected by the excessive net charge.
It is worth mentioning that all the tested peptides kept consistent MICs against
E. coli in a resistance induction experiment over 12 cycles. At present, the drug resistance of
E. coli poses a serious threat to human health. The results showed that the resistance of
E. coli to ciprofloxacin, gentamicin, trimethoprime/sulfamethoxazole and third-generation cephalosporin increased significantly with the duration of drug administration [
42]. For the conventional antibiotic, ampicillin, resistance has increased to 50% or higher in high-risk populations [
43]. Therefore, when evaluating the antibacterial effect of new antibiotics, the characteristics of drug resistance should be taken into consideration. Although we cannot rely on these data to speculate the long-term effect, it does prove an advantage compared to traditional antibiotics. There is a low possibility for AMPs to induce drug resistance [
11], which is due to the mechanism of AMPs in that they bind with the cell membrane on multi-targets which disrupts the cell membrane rapidly [
9]. Furthermore, the designed analogues t-DPH1-K4 and t-DPH1-5K could kill the bacteria in 30 min at their corresponding MBCs, whereas the traditional antibiotics may take 6 to 12 h or more to achieve the same effect [
44].
In addition, to develop AMPs into alternative antibiotics and anticancer agents, haemolytic activity is an important index that must be paid attention to. Although t-DPH1-5K had the strongest antimicrobial and antiproliferative activities, it produced a nearly 40% haemolysis at the concentration of 10 μM. This may be due to the high amphiphilicity of the peptide, which induced a strong hydrophobic interaction with the cell membrane [
45]. The wide range of bioactivity makes t-DPH1-5K a great choice of antimicrobial drug lead, but it still needs further modification to decrease its haemolytic activity. By contrast, t-DPH1-K4 showed a wider therapeutic window and had the potential to become one of the potential drug candidates with great antimicrobial and anticancer dual effects, just like cecropins and magainins [
46].
Last but not least, this study used a lysine substitution strategy to enhance the cationicity of the parent peptide t-DPH1. However, in recent years, many studies have shown that the guanidinium moiety of arginine, which is also positively charged, has a stronger H-bonding capability and is more effective in mediating the peptide-membrane interaction [
47,
48,
49]. Thus, this may be one of the ideas to improve the bioactivities of t-DPH1-K4 and t-DPH1-5K further.
4. Materials and Methods
4.1. Skin Secretion Harvesting from P. hypochondrialis
The dorsal skin secretions of adult specimens of
P. hypochondrialis were obtained by electrical stimulation (5 V, 100 Hz, 140 ms width) [
50] through platinum electrodes for every 20 s. The skin secretions were rinsed off the skin with deionised water into a chilled beaker and stored at −20 °C, then snap-frozen in liquid nitrogen and lyophilised. The study was performed according to the guidelines in the UK Animal (Scientific Procedures) Act 1986, project license PPL 2694, issued by the Department of Health, Social Services and Public Safety, Northern Ireland. Procedures were vetted by the Institutional Animal Care and Use Committee (IACUC) of Queen’s University Belfast and approved on 1 March 2011.
4.2. Identification of Precursor-Encoding cDNAs from the Skin Secretion
Poly-A mRNA was extracted by Dynabeads mRNA Direct kit (Dynal Biotech, Wirral, UK) due to covalent pairing and was made into a first-strand cDNA library. Then a SMART-RACE Kit (Clontech, Oxford, UK) and a degenerate sense primer (S1; 5’-ACTTTCYGAWTTRYAAGMCCAAABATG-3’ (Y = C/T, W = A/T, R = A/G, M = A/C, B = T/C/G) were used to conduct the process of RACE-PCR so that full-length sequences of the mRNA could be obtained. The PCR products were subjected to purify and cloned by using a pGEM
®-T Easy Vector system (Promega, Southampton, UK) and sequenced by an ABI 3100 Automated Capillary Sequencer (Applied Biosystems, Forster City, CA, USA). Online APD3 antimicrobial peptide database (
https://aps.unmc.edu, accessed on 3 December 2021) was used to find out the sequence similarity between t-DPH1 and other reported AMPs.
4.3. Peptide Synthesis
Parent peptide t-DPH1 and its designed analogues were synthesised using a Tribute peptide synthesiser (Protein Technologies, Tucson, AZ, USA). The products were purified by reverse-phase HPLC (Phenomenex Aeris PEPTIDE 5 µm XB-C18 column, 250 × 21.2 mm, Macclesfield, Cheshire, UK) with a linear gradient formed from 80% buffer A (0.05/99.5 (v/v) TFA/water) and 20% buffer B (0.05/19.95/80.00 (v/v/v) TFA/water/acetonitrile) to 0% buffer A: 100% and buffer B in 60 min at a flow rate of 8 mL/min. The masses of purified products were verified by MALDI-TOF MS (matrix-assisted laser dissociation ionised-time of flight mass spectrometry) (Voyager DE, Perspective Biosystem, Foster City, CA, USA) in positive detection mode using CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix.
4.4. Physicochemical Properties Analyses, Secondary Structure Predictions and Determinations
The physicochemical properties of t-DPH1 and its analogues were determined via the Heliquest (
http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParamsV2.py, assessed on 5 November 2021), and helical wheel plots were constructed to predict their secondary structures [
51]. Then, circular dichroism (CD) analyses were used to determine the secondary structure of synthesised peptides, which was carried out on a JASCO J815 Spectropolarimeter (JASCO Inc., Easton, MD, USA). The peptide samples (100 µM) were prepared in 10 mM NH
4Ac solution and 50% TFE/NH
4Ac (
v/
v), respectively, and were measured in the range of 190–250 nm. K2D3 (
http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3/, accessed on 5 November 2021), the online analysis web server, was used to analyse the collected results.
4.5. MIC and MBC Determinations
To assess the antimicrobial activity of t-DPH1 and its designed analogues, MIC and MBC assays were conducted. Seven microorganism strains were used in the assay: Gram-positive bacteria S. aureus (ATCC 6538), E. faecalis (NCTC 12697) and MRSA (NCTC 12493); Gram-negative bacteria E. coli (ATCC 8739), P. aeruginosa (ATCC 9027) and K. pneumoniae (ATCC 43816); yeast, C. albicans (ATCC 10231).
For MIC assay, the microorganisms were initially incubated in Mueller-Hinton broth (MHB) (for bacteria) or yeast extract peptone dextrose broth (YPD-B) (for yeast) overnight and sub-cultured to achieve their respective logarithmic growth phases (5 × 10
5 CFU/mL). Then, 99 μL of each microorganism and 1 μL of the tested peptide solution were incubated in a 96-well plate. The final on-plate peptide concentrations were from 1 to 512 μM. Except for the peptide group, four other groups were set when conducting this experiment, including a negative control group (PBS), a vehicle control group (1% DMSO), a positive control group (bacteria: 20 μg/mL norfloxacin; yeast: amphotericin B) and a blank control group (MHB or YPD-B with no microorganism culture). Thus, the MIC value represents the lowest concentration of peptides at which no visible growth of the microorganism after 24 h incubation [
52]. For the MBC assay, 10 μL of the medium from each clear well was inoculated onto a Mueller–Hinton agar (MHA) (for bacteria) or yeast extract peptone dextrose agar (YPD-A) (for yeast) plate and incubated for 24 h for measurement MBC values.
4.6. MBIC and MBEC Determinations
To assess the antibiofilm activity of t-DPH1 and its designed analogues, the MBIC and MBEC assays were conducted. Bacteria types mentioned in
Section 4.5. were chosen to be tested in this assay. The bacteria were cultured in the Tryptic Soy Broth (TSB, for Gram-positive bacteria) or Lysogeny broth (LB, for Gram-negative bacteria). The final concentration of peptide solution was from 1–256 μM. Bacteria culture without any treatment served as growth control and sterile PBS served as negative control. For the determination of MBIC, cultures at 10
6 CFU/mL were incubated with the tested peptide in a 96-well plate (100 μL/well) for 24 h. After incubation, phosphate buffer saline (PBS) was used to wash every well. Then, the biofilm was fixed by 125 μL of methanol (90%,
v/
v) for 10 min, and the wells were dried and stained with 125 μL of crystal violet (0.1%,
w/
v) for 30 min, and then excess stain was removed. After drying, stained biofilm in each well was dissolved using 150 μL of acetic acid (30%,
v/
v). After dissolving, the glacial acetic acid was transferred to a new 96-well plate, and the absorbance at 595 nm was determined by the Synergy HT plate reader (Biotech, Minneapolis, MN, USA). MBIC was the minimum concentration of the peptide that displayed no biofilm formation.
For the MBEC assay, the bacteria were cultured in the 96-well plate for 24 h to form the mature biofilm. Then, the bacterial culture was removed, and the biofilm was further washed using PBS. Then, the fresh broth that contained different peptide concentrations (1–256 μM) was added to the plate for 24 h incubation at 37 °C. The subsequent steps were consistent with the MBIC assay, as mentioned above.
4.7. Sytox-Green Bacteria Cell Membrane Permeabilisation
SYTOX Green Nucleic Acid Stain (Life Technologies, Cramlington, UK) can quickly penetrate cells with compromised plasma membranes but does not cross the membranes of living cells, making it a useful indicator of dead cells.
S. aureus (ATCC 6538) were cultured in TSB, and E. coli (ATCC 8739) were cultured in LB at 37 °C overnight and then were subcultured to reach the logarithmic growth phase. Then, the growth medium was centrifuged at 1000× g for 10 min at 4 °C to collect the bacterial cells, where after, 5% TSB or 5% LB in 0.85% NaCl solution were used to wash the cells twice. Next, the pellet was resuspended. Next, the peptide solutions at the concentration of respective MIC and 2 × MIC were added in a black 96-well plate and incubated with bacteria cells at 37 °C for two hours. After incubation, the cells were stained with 5 μM SYTOXTM green nucleic acid stain and incubated for 5 min in the dark at 37 °C. The fluorescent intensity was measured with a Synergy HT plate reader (Biotech, Minneapolis, MN, USA) by an excitation and emission wavelength of 485 and 528 nm, respectively. Bacteria cells treated with 5% TSB or LB medium served as negative control; bacteria cells treated with 70% (v/v) isopropanol for one hour served as positive control; 5% TSB or LB medium without bacteria cells served as blank control.
4.8. Time–Killing Kinetics Determination
The kinetic time–killing assay was conducted to compare the killing rate of t-DPH1 and its analogues against
S. aureus (ATCC 6538) and
E. coli (ATCC8739). The bacteria culture was prepared the same way as for the MIC and MBC assay (mentioned in
Section 2.6). First, a suspension culture (10
6 CFU/mL) of bacteria was mixed with peptide solutions at the concentrations of respective MIC and two × MIC in sterile 1.5-mL tubes. Then, the aliquots were removed from culture tubes at 0, 5, 10, 15, 30, 60, 90, 120 and 180 min intervals. The bacteria at different time points were seeded onto MHA plates and incubated at 37 °C for 24 h before colony counting. The bacteria cultured without any treatment was employed as the growth control and bacteria culture treated with PBS served as negative control.
4.9. Resistance Induction by Serial Passages
Based on the results of MIC assay, synthesised peptides t-DPH1, t-DPH1-K4, t-DPH1-5K and t-DPH1-6K showed excellent ability against Gram-negative
E. coli. Therefore,
E. coli was selected to test whether the MIC value changed after consecutive passages. The drug resistance induction assay was conducted based on the reported methods [
53]. Briefly, MIC values of t-DPH1, t-DPH1-K4, t-DPH1-5K and t-DPH1-6K against
E. coli (ATCC 8739) were determined and recorded. After incubation, the bacterial cells growing at 1/2 MIC values were harvested and inoculated into fresh MHB for another MIC assay. After 20 h incubation, cells growing at 1/2 MIC from the previous passage were harvested and assayed for the MIC. The process was repeated for 12 cycles, and MIC values of each cycle were recorded.
4.10. Assessment of Mammalian Cell Proliferation Inhibitory Effect
The MTT assay was conducted to verify the proliferation inhibitory effect of t-DPH1 and its analogues. Human cancer cell lines (NCI-H838 (ATCC® CRL-5844, human non-small cell lung cancer)), (NCI-H157(ATCC® CRL-5802, human non-small cell lung cancer cell line)), (U251MG (ECACC 09063001, human neuronal glioblastoma cancer cell line)), (PC-3 (ATCC® CRL-1435TM, human prostate carcinoma cancer cell line)) and normal human cell lines (HMEC-1(ATCC®-CRL-3243, human microvascular endothelial cell line)) and (HaCaT (ATCC®-PCS-200-011, human keratinocyte cell)) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the European Collection of Cell Cultures (ECACC, Sallisbury, UK) for this assay. For the MTT assay, 8000 cells/per well were seeded in the 96-well plate for 24 h. Subsequently, experiment groups were dosed with fresh serum-free medium containing different concentrations of peptides (100 μM to 10 nM) in 3 replicates. Negative control groups and positive control groups were dosed with the fresh serum-free medium containing equal amounts of PBS, and 0.1% (w/v) Triton X-100, respectively. Then the 96-well plate was incubated at 37 °C with 5% CO2 for 24 h. After, ten microliters of MTT solution (5 mg/mL) (Sigma-Aldrich UK Ltd., Gillingham, UK) were added to each well and incubated in a dark environment for two hours. Then, the solution in each well was removed and 100 μL of DMSO were added and then the plate was shaken for 10 min on a shaking incubator before detecting the OD value by use of a Synergy HT plate reader (BioTek, Minneapolis, MN, USA) at λ = 570 nm.
4.11. Haemolysis Assays
The haemolysis assay was performed by mixing 2% (v/v) fresh defibrinated horse blood (TCS Biosciences Ltd., Buckingham, UK) with peptides in different concentrations. Before the test, horse blood was rinsed with PBS three to four times until the supernatant was clear. Next, peptide solutions (from 100 μM to 1 nM) were incubated with the suspension of red blood cells at 37 °C for two hours. The blood cells treated with 1% Triton-X 100 were used as the positive control, and the blood cells treated with PBS were used as the negative control. After incubation, 100 μL of the supernatant from each sample were transferred to a new 96-well plate, and the OD value of each well was measured with a Synergy HT plate reader (BioTek, Minneapolis, MN, USA) with the absorbance set to 570 nm.
4.12. Statistical Analyses
All the results were obtained from at least three replicates of experiments. Data were analysed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Data are shown as the mean values +/− SD. The p-value was calculated by multiple t-test and one-way ANOVA test from the mean values of the indicated data. Significant differences are demonstrated with asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).