Introduction

Chitosan is a more useful form of chitin and the second most abundant biopolymer in nature that forms most of the exoskeleton of crustaceans [1]. It is one of the most utilized polymers in pharmaceutical research [2, 3]. Chitosan and its crosslinked grafts have obtained remarkable significance in the field of therapeutic applications like drug delivery, enzyme immobilization, tissue engineering, and pH-sensitive protein carriage [4, 5] due to its exclusive features such as biodegradability, biocompatibility, non-toxicity, and non-immunogenicity [6]. Furthermore, chitosan derivatives have exhibited antimicrobial activity [7] and anticancer activity with minimal toxicity on noncancer cells, such activity against various cancer cell lines significantly depends upon molecular weight and degree of deacetylation affected by the distribution pattern of β-(1,4)-linked N-acetylglucosamine and D-glucosamine units along the oligomeric chain [8]. Chitosan is informed as an important antioxidant agent and, therefore, could be used as an alternative to synthetic antioxidants such as butylated hydroxy-anisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) [9,10,11].

Otherwise, quinolines are distinct moieties existing in diverse biologically active natural or synthetic compounds. They have been widely utilized for the synthesis of various drugs depicted for several diseases. Quinoline skeleton executes numerous pharmaceutical applications such as antibacterial, antitumor, antifungal, anti-inflammatory, analgesic, and antimalarial [12, 13].

Pyranoquinolinone is of considerable interest as it is a core structure in a number of alkaloids that discloses cancer cell inhibitory activity and is regarded as a potential antitumor agent [14, 15]. The substances containing pyrano[3,2-c]quinoline moiety show powerful anti-proliferative effects against distinctive cell lines [16,17,18]. Additionally, many pyrano[3,2-c]quinolinone scaffolds possess good antioxidant activity and would be promising antioxidant agents [19, 20].

On the other hand, Schiff bases of heterocyclic rings are well known due to their broad range of applications in biological and industrial fields [21]. They are distinguished by the presence of the azomethine linkage which is responsible for a broad spectrum of bioactivities. Schiff bases compounds have been shown to have promising results on breast carcinoma cell line [22]. Furthermore, some Schiff base derivatives exhibit potential as anticancer drugs across five cell lines (HeLa, HCT-116, MCF-7, HepG-2, and HL-60 cells) [23, 24]. A series of compounds-based Schiff bases are reported to be excellent radical scavengers and act as antioxidant agents [25, 26]. Additionally, some pyranoquinolinone-based Schiff’s have received noticeable TOP2B inhibitory activity against breast cancer cell line (MCF-7) compared to the drug doxorubicin [27]. Moreover, Chitosan anchored Schiff bases have received intensive interest for their better-expanded bio-functional properties than the parent chitosan itself such as antibacterial, antitumor, and antioxidant activities [28, 29].

Meanwhile, several nanocomposites containing metal, and metal oxide nanoparticles, graphene, and inorganic clays manifest recent applications in industrial, biomedical engineering, wastewater treatment, and agricultural fields [30, 31] such as notable inhibitory activity against some bacterial strains like E.coli and S.aureus [32, 33] and high-performance capacitors in the field of energy storage [34]. More concretely, silver nanoparticles have obtained much interest in the Nanomedicine fields. Many mechanisms have been hypothetically proposed for their activity such as antioxidant protection, enhanced carcinogen detoxification, inhibitory activity of tumor cells, surveillance of immune enhancement, inhibition of angiogenesis, and modeling of cell proliferation [35]. Modern studies have also revealed that Ag NPs possess considerable anticancer activity and have significant ability to stimulate cytotoxicity in cancer cells through different mechanisms including DNA damage, oxidative stress, cell cycle arrest, necrosis, or apoptosis [36]. Interestingly, chitosan which is decorated with silver nanocomposites reveals promising antimicrobial, antitumor, antimycobacterial, electrical resistance, and flame retardancy [37,38,39,40,41].

The versatile chemotherapeutical activities of chitosan and Schiff bases as well as the quinolinones and pyranoquinolinones systems mentioned above inspired us to generate novel Schiff base holding quinolinone and chitosan in one framework and another Schiff base containing pyrano[3,2-c]quinolinone and chitosan analogs embedded in one skeleton which can be utilized as valuable substructures for improvement their pharmacological properties and exploration new anticancer chemotherapeutics and antioxidant drugs. Likewise, we have incorporated silver nanoparticles to the new synthesized Schiff bases, hoping that the produced compounds will be eco-friendly promising pharmaceutical candidates. As well, we describe their promising inhibitory activity against cancer cells proliferation and examine their antioxidant activity.

Results and discussion

Ag nanoparticles characterization

The crystal structure of synthesized silver nanoparticles was examined using XRD analysis. The four different diffraction peaks of 31, 46, 67, and 78 were pointing to planes (111), (200), (220), and (311), respectively. The planes signified that the Ag NPs are face-centered cubic structured (JCPDS 89-3722) [42, 43]. The results of the diffraction pattern are illustrated in Fig. 1 corresponding to pure silver metal powder. The crystallite size (d) was estimated by Debye–Scherer’s equation as follows:

$$ {\text{d }} = {\text{ k}}\lambda = \beta {\text{ cos}}\theta $$

where, K is the shape factor between 0.9 and 1.1, λ is the incident X-ray wavelength (Cu Kα = 0.154 nm), β is the full width at half maximum and Ɵ is the position of that line in the pattern [44]. The average crystallite size of Ag NBs is d = 14 nm. The TEM image (Fig. 1b) indicates that the shape and size of the Ag NPs are spherical and nanoscale, respectively [45].

Fig. 1
figure 1

Ag NPs characterization; a XRD and b TEM

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Synthesis route of the novel chitosan Schiff bases CSP and CSQ

Scheme 1 illustrates the reaction of chitosan with 3-formylquinolinone Q or 3-formylpyranoquinolinone P via Schiff base condensation reaction. Chitosan amino group and aldehydic group of Q or P reacted by nucleophilic attack forming azomethine group.

Scheme 1
scheme 1

Formation of chitosan Schiff bases CSP and CSQ

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Characterization of the prepared chitosan Schiff bases and their silver nanocomposites

FT-IR spectra

The chitosan Schiff base CSP was prepared and characterized by the method reported by Ibrahim et al. [46]. The comparative structural conformation of CS, CSQ, CSP, CSQ/Ag, and CSP/Ag membranes were verified from the characteristic transmittance peaks of the IR spectrum (Fig. 2). The spectrum of pure chitosan proved the appearance of overlapped characteristic absorption stretching vibrations between O–H and N–H at 3431 cm−1, in addition to the presence of two peaks at 2919 and 2866 cm−1 due to C–H. Three new stretching bands of NH, CH3, and the second hydroxyl group (OH) appear at 1619, 1466, and 1389 cm−1, respectively. In comparison to the IR spectrum of chitosan, the IR spectra of chitosan Schiff bases CSQ and CSP showed the appearance of new peaks at 1652 (C = O quinolone) for CSQ, and at 1748 (C = O α-pyrone), 1678 (C = O quinolone) for CSP, in addition to the broad band at 3400 cm−1 in CSQ, and at 3424 cm−1 in CSP corresponding to N–H and O–H. Besides, a strong new peak appeared at 1604 for CSQ and at 1634 cm−1 in CSP assigned to the characteristic absorbance of azomethine group (C = N), confirming the formation of chitosan Schiff bases [47]. The peaks observed around 1530 cm−1 were attributed to the N–H bending in secondary amides (amide II band) in CSQ and CSP. Furthermore, the IR spectra of CSQ and CSP displayed absorption bands around 1450 and 840 cm−1 due to OH in-plane bending, and the presence of symmetrical C-H bending in CH3 around 1161 cm−1. Two notable peaks around 1020 cm−1 and around 1221 cm−1 are characteristic for C–O–C bridge stretching, and C-O stretching vibration, respectively [48].

Fig. 2
figure 2

FT-IR spectra of pure chitosan CS, Schiff bases CSQ, CSP and Schiff basses capped AgNPs CSQ/Ag, CSP/Ag

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The FT-IR spectra of the prepared silver nanocomposites CSQ/Ag and CSP/Ag were also compared with that of CSQ and CSP, respectively, to investigate the possible functional groups of CSQ and CSP responsible for the reduction and stabilization of Ag NPs. The change of some IR signatures coupled with the appearance of new ones and energy-shifts for some stretching vibrations in the FTIR spectra of CSQ/Ag and CSP/Ag as compared with that of CSQ and CSP, respectively, confirms a successful capping of Ag NPs by CSQ and CSP (Fig. 2). Primarily, the broadness of the bands characteristic for the OH stretching vibrations along with their positive shift to 3428 and 3432 cm−1 for CSQ/Ag and CSP/Ag, respectively, in comparison to that of CSQ (3400 cm−1) and CSP (3424 cm−1), indicating the crucial role of OH groups in the reduction and stabilization of Ag NPs. Meanwhile, it was notable that the phenolic C–O group which appeared around 1221 cm−1 in CSQ and CSP disappeared completely in CSQ/Ag and CSP/Ag, confirming the participation of phenolic hydroxyl groups in the reduction and stabilization of Ag NPs. Interestingly, new peaks in the spectra of CSQ/Ag and CSP/Ag occurred at 1431 and 1434, respectively, distinctive for Ag NPs. All the previous differences prove the successful capping of Ag NPs with CSQ and CSP [49, 50].

X-Ray diffraction (XRD) study

The XRD spectra have been accomplished to analyze the crystalline structure of the synthesized membranes CS, CSQ, and CSP (Fig. 3). The chitosan sample exhibited a sharp peak at 2θ = 20.4◦ compatible with the previous studies [51]. This recommends the formation of intermolecular and intramolecular hydrogen bonds due to the presence of free amino groups in chitosan [52]. In the XRD patterns of CSQ, the intensity of the peak at 2θ of 20.4◦ decreased and becomes broader comparing with free chitosan; this reflects that the chitosan-quinolinone derivative has a less crystalline and more amorphous structure. This can be primarily due to the deformation of the strong hydrogen bonding in the chitosan backbone because of substitution of quinolinone on the N-atom of chitosan which causes the steric hindrance after Schiff base formation [53, 54]. According to the literature, the XRD of CSP showed depression in intensity and deformation of chitosan peak which at 2Ɵ of 20.4◦, confirming the deformation of chitosan crystallinity. In addition, there was higher semicrystalline nature for CSP designating that there was molecular miscibility and as a result of Schiff base crosslinking between chitosan and the pyranoquinolinone moiety [46]. A comparative figure for XRD patterns of CS, CSQ, and CSP is delineated in Fig. 4.

Fig. 3
figure 3

X-ray diffraction patterns of CS, CSQ and CSP

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Fig. 4
figure 4

Comparative figure of XRD patterns of CS, CSP, and CSQ

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Figure 5 reveals the X-ray diffraction of CSP/Ag and CSQ/Ag. The sharp peaks located at 2θ angles of 38°, 44.2°, 64.5°, and 77° in CSP/Ag were attributed to (111), (200), (220), and (311) planes of face-centered cubic Ag nanoparticles which confirms the silver reference file of JCPDF 03-065-287. While XRD patterns of CSQ/Ag disclosed the intense diffraction peaks at 2θ angles of 38°, 44.2o [55,56,57]. Such data approve the successful reduction in silver cations on the surface of chitosan Schiff bases CSQ and CSP.

Fig. 5
figure 5

X-Ray diffraction patterns of chitosan and CSBs with 1% silver nanoparticles

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Thermal gravimetric analysis (TGA)

Thermogravimetric analysis is employed to study the thermal stability behavior of the newly synthesized compounds as depicted in Fig. 6. Chitosan showed a total weight loss at 281 °C, corresponding to chitosan moiety, while in chitosan Schiff bases CSQ and CSP, the weight loss was observed at 322.55 °C and 343.05 °C, respectively. The observed temperature difference might be owing to the formation of chitosan Schiff bases. The outcomes verified the higher stability of chitosan Schiff bases at higher temperatures. The TGA curve of CSQ/Ag revealed a total loss in weight at 340 °C, illustrating the higher stability of CSQ/Ag as compared to CSQ. This displays the enhanced thermal stability owing to grafting on silver nanoparticles.

Fig. 6
figure 6

TG curves of CS, CSQ, CSP, and CSQ/Ag

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Scanning electron microscopy (SEM)

The SEM micrographs of CS, CS-Schiff’s bases, and their silver nanocomposites are displayed in Fig. 7. It is clearly seen that the pure chitosan had a nonporous and smooth membranous surface (Fig. 7a), whereas CSQ and CSP revealed a significant surface roughness and porous surface [58] (Fig. 7b, c). The roughness could be attributed to the loss of hydrogen bonding of chitosan chains, depicting the successful modification of chitosan surface [59]. Moreover, grafting of bulky group (quinolinone or pyranoquinolinone) into chitosan polymer matrices gives a surface roughness [60]. By comparing SEM images in Fig. 7b, c of CSQ and CSP with Fig. 7d, e of CSQ/Ag and CSP/Ag, it is obvious that Ag NPs were mostly incorporated uniformly into CSQ and CSP matrix. This was assured by EDX (Fig. 8) which demonstrated the existence of elemental silver peaks in region of 3 keV. In addition, we noticed that the intensity of EDX peak of Ag NPs of CSP/Ag is greater than the one of CSQ/Ag [29]. Figure 7d, e signifies the well-dispersion of spherical shape of Ag NPs on the surface of CSBs, confirming that successful preparation of the CSBs/Ag. Interestingly, there is a good interaction between Ag NPs and CSP, while dispersion of Ag NPs in case of CSQ was to lower extent. This may be due to the activity of 4-OH group of quinolinone that assist in reducing and stabilizing silver nanoparticles [29, 56].

Fig. 7
figure 7

SEM images of a CS, b CSQ, c CSP, d CSQ/Ag, e CSP/Ag

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Fig. 8
figure 8

EDX of a CSP/Ag and b CSQ/Ag

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Transmission electron microscopy (TEM) of CSP/Ag and CSQ/Ag

TEM analysis of CSP/Ag and CSQ/Ag was performed and graphically drawn through histograms presented in Fig. 9. They revealed a moderate dispersion of Ag NPs into CSP and CSQ matrix besides some aggregates. Both images contain transparent central areas with dense dots representing the loaded silver nanoparticles into the CSBs matrix. The continuous spreading of the black spots in the Schiff bases proved the successful deposition of silver nanoparticles into the polymeric structure. Semi-spherical Ag NPs are observed with an average size of 30–50 nm.

Fig. 9
figure 9

TEM images of a CSP/Ag and b CSQ/Ag with related histograms

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Cytotoxic evaluation

All the new Schiff bases and their nanocomposites CSQ, CSP, CSQ/Ag, and CSP/Ag have been investigated in terms of their cytotoxic effects in cancer cells, namely, liver (HepG-2), breast (MCF-7), and colon (HCT-116) by the colorimetric method as rendered in Figs. 10, 11 and 12. The inhibitory activities of the targetable compounds against tumor cell lines were compared with 5-fluorouracil as a reference drug which showed IC50 of 6.44, 28, and 21.5 μg/ml, respectively. Cytotoxic analysis of tested compounds was spotted and estimated by its ability to cause 50% inhibition of cell growth compared to the unprocessed control cells (IC50 value), as summarized in Table 1. The antitumor activity profile proposed that the examined compounds exhibited variable activities compared to the reference drug. The newly synthesized chitosan Schiff bases and their nanocomposites have good to moderate antiproliferative activity against the three cancer cell lines. From the displayed results, the IC50 values indicated that the compounds had potent growth inhibition against all tested cell lines. Particularly, the cited chitosan Schiff base CSQ/Ag, which is bearing with quinolinone ring and loaded with silver nanoparticles, revealed the highest antiproliferative activity toward three cancer cell lines HCT-116, MCF-7, and HePG-2 with minimum IC50 values in the range 41.9–55.1 μg/ml compared to chemotherapeutic agent 5-fluorouracil. Compounds CSQ and CSP have a moderate effect on cancer cells, while the compounds CSQ/Ag and CSP/Ag displayed good cytotoxic activities toward the three tested cancer cell lines, in addition, compound CS alone didn’t show significant activity. The presence of heterocyclic scaffolds in chitosan Schiff bases increased the anticancer activities. Chitosan Schiff base with pyranoquinolinone ring system CSP has a higher activity than compound CSQ with quinolinone ring, with IC50 equal to 86.3, 103 and 71.7 μg/ml against HepG-2, MCF-7, and HCT-116, respectively. It has been also noticed that insertion of Ag NPs onto the synthesized Schiff bases played a vital role in the enhancement of growth inhibitory activity. Investigations of the anti-proliferative activity toward HCT-116 cells showed that it is the most sensitive cell line to the influence of the target compounds. The prepared Schiff bases proved to be even more active than the starting chitosan. The IC50 outcomes are designed graphically in Fig. 13.

Fig. 10
figure 10

Effect of concentration (0–500 μg/ml) of compounds CS, CSQ, CSP, CSQ/Ag, CSP/Ag, and 5-fluorouracil on viability of human liver cancer (HepG-2) cell line

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Fig. 11
figure 11

Effect of concentration (0–500 μg/ml) of compounds CS, CSQ, CSP, CSQ/Ag, CSP/Ag, and 5-fluorouracil on viability of human breast cancer (MCF-7) cell line

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Fig. 12
figure 12

Effect of concentration (0–500 μg/ml) of compounds CS, CSQ, CSP, CSQ/Ag, CSP/Ag, and 5-fluorouracil on viability of human colon cancer (HCT-116) cell line

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Table 1 Cytotoxicity (IC50) of compounds CS, CSQ, CSP, CSQ/Ag, and CSP/Ag against different cancer cell lines
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Fig. 13
figure 13

Virtual IC50 values of the target compounds CS, CSQ, CSP, CSQ/Ag, CSP/Ag and 5-fluorouracil against HepG-2, MCF-7, and HCT-116 cell lines

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The effects of the investigational compounds on cell viability and proliferation

Liver cancer cell line HepG-2

The compounds CS, CSQ, CSP, CSQ/Ag, and CSP/Ag were investigated against human liver cancer cell line HepG-2 for 24 h and compared with a reference drug 5-fluorouracil, using the colorimetric assay. Estimation of viability % of HepG-2 cell line is shown in Fig. 10. Compound CSQ/Ag manifested significant growth inhibitory activity against HepG-2 which was more effective than standard drug at concentrations (125, 250, and 500 μg/ml). While titled compounds CS, CSQ, CSP, and CSP/Ag were found to be less active than CSQ/Ag.

Breast cancer cell line MCF-7

Assessment of viability % of breast cancer cell line (MCF-7) post-treatment with compounds CS, CSQ, CSP, CSQ/Ag, and CSP/Ag for 24 h related to standard control drug, 5-Fluorouracil, using the colorimetric assay is represented graphically in Fig. 11. Nearly, all the checked compounds exhibited notable inhibitory activity for MCF-7 cells. The results presented that prepared Schiff base CSQ/Ag induced remarkable inhibitory activity for MCF-7 cell line than the other tested compounds. In addition, the inhibition % of the cell’s viability of compound CSQ/Ag was higher than the reference drug at concentrations (125, 250, and 500 µg/ml). Meanwhile, Schiff bases CSQ, CSP, and CSP/Ag were found to be less active in the series.

Colon cancer cell line HCT-116

Exponentially growing colon cancer cells (HCT-116) were treated with CS, CSQ, CSP, CSQ/Ag, and CSP/Ag for 24 h compared with 5-Fluorouracil using the colorimetric assay (Fig. 12). The tested compounds showed activity comparable to the standard anticancer drug, 5-fluorouracil against HCT-116 cells. The results pointed out that compound CSQ/Ag executed remarkable results more than standard reference drug, 5-fluorouracil at concentrations (62.5, 125, 250, and 500 μg/ml). Moreover, the ability to inhibit viability of CSP/Ag was close to reference drug at concentrations (125, 250, and 500 µg/ml). In addition, the % inhibition of the cell’s viability of Schiff base CSQ was very close to positive control at concentrations (250 and 500 µg/ml).

Structure–activity relationship (SAR)

Noteworthy, the structure–activity relationship (SAR) of the aforementioned tested compounds indicates that these compounds mainly depend on their main structural features of presence chitosan attached to heterocyclic rings via azomethine linkage. From all results we got and what was found in the literature, conversion of amino group to azomethine group through Schiff base formation with heterocyclic carboxaldehyde scaffolds enhances the antitumor activity [61,62,63]. It has been noticed that Schiff base CSQ with quinolinone ring (IC50 ranged from 71 to 107 μg/ml) has a higher activity than CS chitosan itself (IC50 ranged from 104 to 137 μg/ml). This may be due to the presence of quinoline analogue which acts as pharmacophoric moiety and azomethine group. Also, chitosan Schiff base bearing pyranoquinolinone ring system CSP (IC50 ranged from 58 to 103 μg/ml) disclosed better potency against three tumor cells HepG-2, MCF-7, HCT-116 compared to Chitosan itself CS.

On the other hand, several prior studies proved that Ag NPs possess therapeutic cancer treatment potential because of their distinctive properties. In cancer cells, series of processes occurs starting with a loss of inner balance and redox state destabilization then Ag+ produced by oxidation of Ag NPs agglomerated in the cytoplasm and membranes of cells which affects cellular damage and lead to cell death; Oxidative stress induces DNA damage and resulting in cell destruction [64,65,66]. Regarding the previously mentioned, grafting of silver nanoparticles onto prepared chitosan Schiff bases caused a boosting in the antiproliferative activity against three treated cells. Compound CSQ/Ag (IC50 ranged from 41 to 55 μg/ml) disclosed better potency toward the cancer cell lines HepG-2, MCF-7, HCT-116 than compound CSQ (IC50 ranged from 71 to 107 μg/ml). Moreover, compound CSP/Ag (IC50 ranged from 56 to 102 μg/ml) was significantly more active toward all cell lines compared with CSP (IC50 ranged from 58 to 103 μg/ml). It was concluded that hybridizing of chitosan with quinolinone and pyranoquinolinone through Schiff bases formation led to enhancement the cytotoxic activity against most tested human cell lines. Interestingly, the acquired results claimed that the incorporation of silver nanoparticles into novel synthesized chitosan Schiff bases reduces the tumor cells.

Morphological changes in cells were featured on treatment with compounds and microscopic images after using the most powerful compound CSQ/Ag are presented in Fig. 14.

Fig. 14
figure 14

Morphological changes in HepG-2 cells incubated for 24 h a negative control (untreated) b with 250 µg/ml concentration of compound CSQ/Ag c with 500 µg/ml concentration of compound CSQ/Ag. Morphological changes in MCF-7 cells incubated for 24 h d negative control e with 250 µg/ml concentration of compound CSQ/Ag f with 500 µg/ml concentration of compound CSQ/Ag. Morphological changes in HCT-116 cells incubated for 24 h g negative control h with 250 µg/ml concentration of compound CSQ/Ag i with 500 µg/ml concentration of compound CSQ/Ag. The morphological alterations were microscopically detected at 100 × magnification using inverted microscope (CKX41; Olympus, Japan)

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Antioxidant activity

It is well known that free radical scavenging activity has received considerable attention owing to its importance in foods and in biological materials. In fact, the free radicals result in biological damages, generally by oxidative processes. This case occurs in autoxidation, lipid peroxidation, and possibly some aspects of aging, radiation damage, chemical carcinogenesis, or other pathological processes. On the other hand, increasing uncontrolled free radical activity might combine with other factors to cause some diseases such as neurodegenerative diseases, heart disease, cancers, heart diseases, acquired immunodeficiency syndrome, and neurological disorder [67]. An antioxidant agent is known as a material that shields a biological target against oxidative degradation. Vitamin C (ascorbic acid) and vitamin E (α‐tocopherol) are used as references in the detection of antioxidant effect of novel examined agents. Antioxidant activity of a compound is known as its ability to scavenge free radicals, which are recognized to be the major factor in the biological damage caused by oxidative stress.

The newly synthesized chitosan Schiff bases CSQ, CSP, CSQ/Ag, and CSP/Ag were investigated for their in vitro antioxidant activity showing the scavenging activity of chitosan and its novel Schiff bases for the DPPH radicals (Fig. 15). All samples exhibited DPPH scavenging ability and the concentration of sample influenced the percentage of DPPH. Comparing with the standard control ascorbic acid, all the chitosan Schiff bases CSP, CSQ, CSP/Ag, and CSQ/Ag demonstrated moderate DPPH scavenging ability. The compound that showed relatively high antioxidant activity was CSQ/Ag. A Significant association could be found between the concentration of the scavenging ability and the percentage of inhibition. This assay provided information that the scavenging activity of chitosan Schiff bases was enhanced compared with that of original chitosan. In our study, compound CSQ revealed higher antioxidant activity than chitosan CS. In addition, CSP is more active than chitosan alone CS. This might be due to the phenolic group of quinolinone and pyranoquinolinone rings which are condensed with chitosan to afford the corresponding Schiff base. In numerous studies, original chitosan was investigated as a potential antioxidant agent. The antioxidant activity of chitosan has been associated with its strong hydrogen-donating ability and it was reported that a low molecular weight and a higher concentration have a positive effect on the activity [68]. Moreover, many studies have been carried out aiming to increase the biological activities by chemical modification of chitosan with heterocyclic compounds. Schiff base derivatives of chitosan are one of the important modifications and the newly formed imine groups improve the pharmacological actions of chitosan as antioxidant activities [69, 70].

Fig. 15
figure 15

DPPH-radical scavenging ability of CSQ, CSP, CSQ/Ag, and CSP/Ag at different concentrations (31.25–4000 μg mL−1)

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An enhancement in the antioxidant activity was observed after the functionalization of chitosan Schiff bases CSQ and CSP with Ag NPs. It has been remarked that Compound CSQ/Ag is more active than CSQ. Compound CSP/Ag possesses higher antioxidant activity compared with CSP. The result obtained in this study was confirmed by the results given in the literature regarding the role of silver nanoparticles in increasing the DPPH radical-scavenging activity [71].

Conclusion

In this paper, novel chemically modified chitosan Schiff bases bearing quinolinone and pyranoquinolinone rings were synthesized. The decoration of new chitosan Schiff bases with silver nanoparticles was then happened to obtain novel nanocomposite. Characterization using spectral and surface analyses techniques was performed, showing evidence for their formation. The cytotoxic activity of the investigated compounds against the three tested cell lines indicated that chitosan Schiff bases disclosed better anticancer activities than chitosan itself. Furthermore, the effect of novel Schiff bases moieties on their antioxidant activity was examined by using the DPPH radical scavenging assay. Therefore, an improvement in the antioxidant activity was noticed after the functionalization of chitosan with quinolinone and pyranoquinolinone which are valuable scaffolds in medicinal chemistry. Interestingly, the incorporation of silver nanoparticles into the newly tested chitosan Schiff bases enhanced their anti-proliferative and antioxidant activity. As a result, Compound CSQ/Ag displayed to be the most potent in the two biological studies compared to the references.

Material and methods

Chemistry

Chitosan (Sigma Aldrich), glacial acetic acid (Fisher scientific), triethyl orthoformate (Merck), Remazol red (RB-133), sodium hydroxide, sodium chloride and hydrochloric acid were purchased from Sinopharm Chemical Reagent Company Limited. The used reagents were of analytical grade as received. Distilled water was consumed in all preparations.

Synthesis of chitosan Schiff bases

Compound P [72], Q [73], and CsP [46] was synthesized according to the reported literature methods. Then Chitosan (1 mol) was dissolved in 100 ml acetic acid 5% and stirred at 50 °C for 4 h, and the aldehyde (1 mol) was dissolved in the least amount of ethanol, then the aldehyde solution was added dropwise to the chitosan solution; the reaction mixture was stirred at 70 °C for 24 h. An Orange gel was formed and collected by filtration, washed several times with a mixture of ethanol and acetone to remove any unreacted materials, dried to give chitosan Schiff bases as powder compounds.

Synthesis of chitosan Schiff bases/Ag NPs composites

Firstly, Ag NPs were prepared as follows: the solution of AgNO3 (0.3 g/10 mL deionized H2O) was added dropwise to an aqueous solution of sodium borohydride (2.9 M, 6.6 mL) which contains 1 mL of NaOH (0.001 M) and kept under stirring at 70 °C for 60 min. The color of the colloidal solution was turned to yellow color by the addition of AgNO3. Secondly, chitosan Schiff bases loaded with Ag NPs were prepared using Ag NPs with a weight ratio of (1%) of nano-materials with respect to chitosan Schiff bases. Ag NPs were dispersed individually in ethanol by sonication for 20 min then it was added dropwise to the previously prepared Schiff bases solution under stirring for 6 h. The composite was precipitated in a mixture of acetone and ethanol, filtered, and finally dried in air.

Physicochemical characterization of chitosan Schiff bases.

FTIR analysis

Fourier transform infrared (FT-IR) measurements were accomplished using FT-IR (Thermo electron corporation Nicolet avatar 370 CSL) with the wavenumber range 500–4000 cm−1 in the Training section—ministry of military production.

X-ray diffraction

X-ray diffractograms of chitosan and chitosan Schiff base samples were carried out using ARL™ X'TRA Powder Diffractometer (Thermo Fisher Scientific Inc.) with Cu-K-alpha wavelength = 1.5405981 ͦA and current 44 mA–voltage 45 kV.

Scanning electron microscope (SEM)

The chitosan Schiff base sample was analyzed using an SEM model Quanta 250 field emission gun (FEG) attached to an energy dispersive X-ray (EDX) analysis unit with 30 K.V. accelerating voltage, 14 × up to 1,000,000 magnification and Gun.1n resolution).

Thermal gravimetric analysis (TGA)

The sample was carried out by Q500TA instruments.

Biological evaluation

Reagents and chemicals

Dimethyl sulfoxide (DMSO), (1%) crystal violet stain (It is composed of 0.5% (w/v) crystal violet and 50% methanol then made up to volume with dd H2O and was filtered using a Whatman No.1 filter paper) and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). Additionally, Fetal Bovine serum, RPMI 1640, DMEM, HEPES buffer solution, gentamycin, L-glutamine and 0.25% Trypsin-EDTA were acquired from Lonza.

Cell lines and Cell culture

Human Hepatocellular carcinoma cells (HepG-2), colon carcinoma cells (HCT-116) and breast carcinoma cells (MCF-7) were obtained from VACSERA Tissue Culture Unit. All cancer cell lines were preserved at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50 μg/ml gentamycin and were sub-cultured two times a week.

Cytotoxicity evaluation using viability assay

The cells were seeded in 96-well plate at concentration of 1 × 104 cells per well in 100 µl of growth medium. After 24 h of seeding freshly medium including several concentrations of the tested samples was added. Serial two-fold dilutions of the examined chemical compound were added to confluent cell monolayers operated into 96-well, flat-bottomed microtiter plates (Falcon NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37ºC in a humidified incubator with 5% CO2 for extent of 48 h. Three wells were utilized for each concentration of the test sample. Control cells were incubated without examined sample and with or without DMSO. The small percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment. After incubation of the cells for at 37 °C, various concentrations of samples were added, and the incubation remained for 24 h and viable cells yield was established by a colorimetric method [74, 75]. In brief, when incubation period was ended, media were extracted, and the crystal violet solution (1%) was added to each well for 30 min. The stain was isolated, and the plates were rinsed using tap water until all excess stain was removed. As well, 30% of Glacial acetic acid was added to all wells and perfectly mixed, then the absorbance of the plates was measured after shaking well on Microplate reader (TECAN, Inc.), with a wavelength of 490 nm. All outcomes were rectified for background absorbance recognized in wells without addition of stain. Treated samples were correlated with the cell control in the absence of the examined compounds. All experiments were carried out in triplicate. The cytotoxicity of each examined compound was calculated. Optical density was assessed with the microplate reader (SunRise, TECAN, Inc., USA) to decide the number of viable cells and the percentage of viability was calculated as [(ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the examined sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted in a graph to obtain the survival curve of each cancer cell line after treatment with the characteristic compound. The concentration necessary to induce toxic effects in 50% (IC50) of intact cells, was determined from graph of the dose response curve for each concentration by using Graph pad Prism software (San Diego, CA. USA).

Antioxidant assay

The antioxidant activity of extract was carried out at the Regional Center for Mycology and Biotechnology (RCMB) et al.-Azhar University by the DPPH free radical scavenging assay in triplicate and average values were measured.

DPPH radical scavenging activity

Freshly prepared (0.004%w/v) methanol solution of 2,2-diphenyl-1 picrylhydrazyl (DPPH) radical was prepared and stored at 10 °C in the dark. The methanol solution of the examined compound was prepared. An aliquot of 40 uL from the methanol solution was added to 3 ml of DPPH solution. Absorbance measurements were noted immediately with a UV–visible spectrophotometer (Milton Roy, Spectronic 1201). The absorbance decreasing at 515 nm was determined constantly, with data being noted at 1 min intervals until the absorbance stabilized (16 min). The DPPH radical absorbance without antioxidant (reference) and the control compound ascorbic acid were also measured. All the determinations were executed in three replicates and averaged. The percentage inhibition (PI) of the DPPH radical was worked out according to the formula:

$$ {\text{PI }} = \, \left[ {\left\{ {\left( {{\text{AC}} - {\text{ AT}}} \right)/{\text{ AC}}} \right\}{\text{ x 1}}00} \right] $$
(1)

where AC = Absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min [76].