Synthesis, spectroscopic characterization, thermal analysis and in vitro bioactivity studies of the N-(cinnamylidene) tryptophan Schiff base

2.4.1 Antibacterial activity tests

The in vitro antibacterial activity of CinTrp was determined by screening against Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa, and K. pneumoniae) bacteria using the disk-diffusion method (Al-Qadsy et al., 2020; Azam et al., 2021b; Ommenya et al., 2020) in reference to ampicillin (Amp) and ceftriaxone (Cef). The test bacteria were kind gifts from Professor Fuad Al-Dubai (Thamar University), Thamar, Yemen. Nutrient agar (Hi-media, Mumbai, India) was prepared in accordance with the manufacturer’s directions and used to grow the bacteria. The test bacteria suspension was made to an inoculum density equivalent to 0.5 McFarland (approximately 1.5 × 10⁸ CFU (colony-forming unit)/mL). The plates were inoculated with the target bacteria using sterilized cotton swabs. Sterile Whatman filter paper (No. 1) disks with a diameter of 6 mm were impregnated with different concentrations (20, 40, 100, 250, 500, 750, and 1000 μg/mL) of the CinTrp and positive controls (Cef and Amp) in DMSO; DMSO was also used as a negative control (Rehman et al., 2013). Subsequently, the impregnated disks were placed aseptically on the surface of bacterially seeded Petri dishes and immediately incubated at 37 °C for 18–24 h. The susceptibility of bacteria to CinTrp was determined by measuring the diameter of the inhibition zones (ZOI) in millimeters. All experiments were carried out in duplicate.

2.4.2 Hemolytic assay

An in vitro hemolytic activity test was carried out as described in the literature (Kumar et al., 2011). Five milliliters of blood were taken from a healthy individual (a 37-year-old male volunteer with an O-positive blood group) after the provision of informed consent and transferred into an EDTA-containing tube. The erythrocytes were isolated based on the procedure described by Chiste et al. (Chisté et al., 2014). Briefly, the blood was centrifuged at 1500 rpm for 5 min at 4 °C, followed by careful removal of the supernatant, washing the pellet five times with sterile PBS, and centrifuging using the same conditions. The cells were re-suspended in PBS to obtain a 0.5% (v/v) erythrocyte suspension, which was subsequently used in the hemolysis assay. To each of nine marked test tubes containing 0.5 mL of the cell suspension, 0.5 mL of the following test samples were added: CinTrp solutions in sterile PBS, TX100 (positive control), and PBS (negative control). The tubes were incubated for 35 min at 37 °C and then centrifuged at 1500 rpm for 10 min at 4 °C to precipitate the cells. The obtained supernatant containing free hemoglobin was measured on a UV–Vis spectrophotometer at 540 nm. Sterile PBS and TX100 were used as minimal and maximal hemolytic controls, respectively. The level of hemolysis caused by CinTrp was calculated according to Eq. (1), and the final hemolytic percentage was the average of three independent experiments.

3.1.1 FTIR analysis

The FTIR spectra of the CinTrp and its precursors are presented in Fig. 1, and the structural characteristic bands are listed in Table 1. The spectra of Cin and Trp typically accord with literature (Cao and Fischer, 1999; Wang et al., 2017). In the spectrum of Trp, the peaks at 3401 and 1356 cm⁻¹, assigned to the stretching and bending modes of N–H indole, are shifted to 3403 and 1371 cm⁻¹, respectively, in CinTrp. According to the literature (Cao and Fischer, 1999; Polfer et al., 2006), bands of NH₂ and NH³⁺, expected in the region 3330–3030 cm⁻¹ for stretching and at 1750–1650 cm⁻¹ for bending modes, are commonly weak. However, the weak bands at 3077 and 3037 cm⁻¹ and the strong ones at 1660 and 1144 cm⁻¹ were assigned, respectively, to the asymmetric stretching, symmetric stretching, bending, and rocking vibrating modes of NH³⁺ moiety in Trp (Wagner and Baran, 2004). These bands were not observed in the spectrum of CinTrp, confirming amine transformation into imine. Moreover, the bands at 1582, 1410, and 741 cm⁻¹ represented the carboxylic ion asymmetric stretching, symmetric stretching, and bending bands, respectively, which were observed at 1631, 1400, and 739 cm⁻¹ in CinTrp. However, bands of carboxylate may overlap with that of the imine observed at 1579 cm⁻¹; that is, the alkali metal salt of an acidic compound commonly shows a red-shift of about 200 cm⁻¹ due to transformation into carboxylate (Zhang et al., 2011). In the spectrum of Cin, peaks characterizing υ(C–H) of aldehyde at 2812 and 2742 cm⁻¹ completely disappeared, confirming the success of the condensation reaction into CinTrp. The other peaks were assigned as shown in Table 1.

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Fig. 1

FTIR spectra of Cin, Trp, and the CinTrp Schiff base.

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3.1.2 ¹H- and ¹³C NMR

The ¹H- and ¹³C NMR analyses of CinTrp are given in Fig. 2 and Table 2 (Trp and Cin spectra are provided in Figs. S2–S5). Inspection of the CinTrp ¹H NMR spectrum around the imine group revealed that the peak positions of –CH⚌N and –CH (No. 13 and 2, respectively) were shifted from 9.66 and 3.51 in, respectively, Cin and Trp to 7.55 and 3.80 ppm (Δppm are −2.11 and 0.29, respectively), because of the difference in the deshielding effect of oxygen and nitrogen. In addition, the integrated curves agreed with the number of hydrogens in the molecules (Fig. 2, S2–S5). The chemical shifts of most Trp-derived protons involved slight shifts downfield (+Δppm, see Table 2), but to upfield (−Δppm) for Cin-derived ones in CinTrp spectra. Likewise, in the ¹³C NMR spectra of CinTrp, all carbons in the molecule are clearly identified (Fig. 2 and Table 2) with remarkable shifts in the carbon peak positions compared with the precursors. In addition, there was a large effect on the imine’s adjacent carbons, namely, No. 13 and 2. Thus, the aldehyde-derived carbon (No. 13), which was observed at 194.54 ppm in Cin (C⚌O), was shifted to a lower frequency of 160.93 ppm (Δppm is −33.61) in the CinTrp spectrum (–CH⚌N) because of the variation in electronegativity of the oxygen and nitrogen atoms (Al-Odayni et al., 2019). For the same reason, Trp carbon No. 2 was shifted from 54.88 to 78.33 ppm (Δppm is 23.45) in CinTrp. Moreover, the chemical shifts corresponding to protons and 13-carbons of benzene rings are less affected, mostly due to their distance from the reaction center. All of the other protons and 13-carbons were assigned in Fig. 2, and S1–S4 and Table 2.

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Fig. 2

¹H NMR and ¹³C NMR spectra of the CinTrp Schiff base in DMSO‑d_6.

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3.1.3 Mass analysis

The mass spectrum of CinTrp is shown in Fig. 3. The molecular ion expected at m/z 356 was not observed because of the low stability of the Schiff base, as suggested by thermal analysis (Al-Odayni et al., 2020). The fragmentation route is also schematically illustrated in Fig. 3, showing a relatively stable peak with an m/z of 275.15 (peak-B) assigned to COOK-truncated CinTrp. In this fragment, the presence of a C⚌N group indicates that the condensation reaction has taken place. The fragment with m/z of 549.30 (peak B-dimer) was assigned to the dimer of peak-B, possibly developed under fragmentation conditions. Other dimer-based fragments, for example, peak-A and peak-C, were also suggested. Fragment-A may undergo further ionization (i.e., to z = +2), providing a fragment with m/z of 248.14 (peak-A/2), which then decomposed into peak-C (Fig. 6). Overall, despite the absence of the molecular ion [M⁺] in the fragmentation pattern, the results are consistent with the molecular structure of CinTrp as the observed peaks are all parts of its possible fragmentation.

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Fig. 3

Mass analysis of the CinTrp Schiff base.

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3.1.4 UV–Vis analysis

Spectra of electronic transitions (UV–Vis) were practically recorded at a concentration of 6.3 × 10⁻⁵ M in absolute ethanol between 200 and 800 nm, as shown in Fig. 4 (only the range 200–500 is presented). The lowest wavelengths of the measurement are generally solvent-dependent; the cut-off limit for EtOH is 205 nm. In EtOH, two bands of Cin were clear at 286 (λmax, molar absorptivity (ε) = 29,492 M⁻¹ cm⁻¹) and 219 nm. (Rind et al., 2011). Trp typically showed three overlapping peaks in the region between 240 and 310 nm, λmax of 280 nm and ε of 8206 M⁻¹ cm⁻¹ attributed to the π–π transition of aromatic rings; the peak at 272 nm was also assigned to the ring electronic transitions. The peak at 289 nm was attributed to the carboxylic functional group. The CinTrp profile was similar to that of Trp, with a small shift toward a higher wavelength (redshift, λ_max = 283 nm and ε = 26,539 M⁻¹.cm⁻¹). Moreover, peaks of the new azomethine (n–π*) could be detected in the lower-energy region, at about 304 and 340 nm (Al-Qadsy et al., 2021).

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Fig. 4

UV–Vis spectra of Cin, Trp, and CinTrp (6.3 × 10⁻⁵ M) in ethanol.

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3.3.1 Antibacterial activity

The antibacterial activity of CinTrp was determined using the paper disc method (Fig. S7). The data pertaining to the ZOI are listed in Table 3. CinTrp exhibited lower activity against all tested bacteria compared with the reference drugs, with no visible effect on S. aureus and P. aeruginosa. Conversely, moderate activity against E. coli and K. pneumoniae was observed, showing an almost equal ZOI value as that of Cef on the E. coli strain at 500 µg/mL. The ZOI value of Cef against K. pneumoniae was double that of CinTrp. However, in all cases, the inhibitory effect of Cef on the Gram-positive bacteria was more potent than that with CinTrp and Amp, while the effect of Amp on Gram-negative bacteria was stronger. Therefore, the lowest concentration of CinTrp that led to observable inhibition was 500 µg/mL, with no significant differences detected up to 1000 µg/mL. In a previous study evaluating the ZOI at the minimum inhibitory concentration (MIC) of 375 µg/mL of CinTrp and some other Cin-amino acid Schiff bases (Nanda et al., 2015), it was claimed that the ZOI of CinTrp against E. coli and K. pneumoniae was 2 and 0 mm, respectively. In the current work, both bacteria were found to be sensitive to CinTrp at 500 µg/mL, with higher activity against E. coli (Table 3). According to the literature, the resistance of bacteria to certain drugs is known to be associated with their surface chemistry (Manimaran et al., 2021), including hydrophobicity, as well as the type of transportation channels. Despite the lack of an outer cell membrane in Gram-positive bacteria, S. aureus has a more hydrophobic surface than E. coli (Lather et al., 2016; Mirani et al., 2018). The greater hydrophobicity of S. aureus as well as its thicker cell-wall and lack of an external surface membrane may contribute to its resistance to CinTrp (Oh et al., 2018). Furthermore, it seems that the structural properties of CinTrp enhance its interaction with Gram-negative bacteria, and thus increase its permeability, allowing its apparent activity against E. coli and K. pneumoniae (Wei et al., 2011); however, the mechanism of action involved requires further investigation.

3.3.2 Hemolytic activity

The hemolytic effect of the CinTrp assessed over the range of 20–1000 μg/mL on human red blood cells (hRBCs) under physiological conditions (pH 7.4 and 37 °C) was evaluated against PBS and TX100 as negative and positive controls, respectively (Fig. 6 and Table 4). Typically, erythrocytes provide a useful tool for toxicity studies as their membrane properties are well described and are easy to obtain. Practically, the results revealed a slight or no hemolytic effect of CinTrp below 250 µg/mL (1.50%) compared with the 100% lysis of the TX100, with no significant differences up to 750 µg/mL (6.50%). However, a cell lysis of 23.13% at 1000 µg/mL was observed. Below 500 µg/mL, the effect was lower than the permissible range (<4.5%) (Bahojb Noruzi et al., 2020). Although dose-dependent hemolysis is a common observation, the mechanism underlying this phenomenon remains unclear; thus, necessitating further investigation before its biological utilization (Rahimi et al., 2017).

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Fig. 6

Percentage of hemolysis of hRBCs caused by the CinTrp Schiff base under the test conditions. Data points are means, and bars represent the standard error of the mean (SeM) from triplicates.

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The biological activities of other CAASBs taken from literature are given in Table 5. The most relevant molecules with experimental conditions more closely resembling those of the CinTrp investigated here are presented for comparison. Wei et al. (Wei et al., 2011) studied the antibacterial activities of several amino acid-based Cin derivatives at concentrations of 200, 400, and 600 µg/mL against various bacterial strains, including E. coli. The reported ZOI values were the same (no inhibition against P. aeruginosa) or higher (against E. coli and K. pneumoniae) than those obtained by other researchers, such as Nanda et al. (Nanda et al., 2015); for instance, the ZOI value of CinTrp against E. coli obtained in this work (8.0 mm, 500 µg/mL) was in the acceptable region compared with the findings reported by Nanda (2.0 mm, 375 µg/mL. Wang et al. (Wang et al., 2016) reported the antibacterial activity of more than 24 Schiff base compounds of Cin and amino acids against E. coli and S. aureus; however, CinTrp was not included and the consistency with the results of other studies (Wei et al., 2011) is not clear, probably because of differences in the experimental conditions.

Conversely, because of the lack of reports on the hemolytic activity of Cin-amino acids in the literature, the comparison was difficult. Considering the Schiff base functional group, the hemolysis of 100 µg/mL of a curcumin-based Schiff base (Ali et al., 2013) yielded a hemolytic effect of about 21% (compared with letrazole, 100% lysis), while CinTrp at the same concentration yielded a hemolytic effect of 1.10% (compared with TX100, 100% lysis). However, the employed concentration range appeared to be high, resulting in an apparently toxic effect. Therefore, more extensive investigation may be important to draw a definitive conclusion on this issue.