Analysis of CE by GC/MS

The GC–MS is one of the best techniques for determining the presence of gaseous components, alcohols, acids, phenolic compounds, and esters [34]. The analysis of CE showed 25 peaks with 54 active chemicals (Fig. S2), with Linoleic acid esters being the most prevalent at 25.04%; followed by 6-octadecenoic acid and octadec-9-enoic acid with 21.08%; decanedioic acid, dibutyl ester, sebacic acid, butyl ethyl ester and hexadecanoic acid ethyl ester (7.71%); n-hexadecanoic acid, methyl (tetramethylene) silyl ester, α-D-galactopyranoside methyl-4-O-decyl- (4.37%); fluocinonide, cholestan-3-one, cyclic 1,2-ethanediyl acetal, (5α)- and D-glucopyranose (3.95%); geldaramycin, cyclopropaneoctanoic acid, 2-octyl-, cis-methyl ester and E,E,Z-1,3,12-nonadecatriene-5,14-diol (2.97%); ethyl 14-methyl-hexadecanoate and α-D-mannopyranose,1-O-(trimethylsilyl)-,2,3:4,6-dibutaneboronate (2.94%); methyl 5,13-docosadienoate butyl 9,12-octadecadienoate and n-propyl 9,12-octadecadienoate (2.85%); hexadecanoic acid, methyl ester and nonanedioic acid, bis (2-methylpropyl) ester (azelaic acid) (2.32%); 4-[5-(4-Chloro-2-cyclohexyl-phenoxymethyl)- 1,2,4 oxadiazol-3-yl]- furazan-3-ylamine and stigmasta −5,22-dien-3-ol, acetate, 4,4-dimethyl-5α-cholest-7-en-3β-ol with an area of 2.18%; aspidofractinine-1-carboxaldehyde, 17-methoxy-3-oxo-, (2à,5à), rhoifolin, and pregna-4,6-diene-3,20-dione, 17-hydroxy-6,16 α-dimethyl- (1.99%).

Collectively, these findings showed the existence of phytosterols, alkaloids, polyphenols, essential oils, and carbohydrates. All of these compounds are bioactive and have various antibacterial, anticancer, anti-inflammatory, and antioxidant capabilities, as presented in Table 1. According to reports by Soliman et al. (2021) [7] and Riadh et al. (2011) [35], GC–MS analysis of C. comosum isolated from the UAE and Tunisia displayed the presence of common organic compounds, including 9,12-octadecadienoic acid (linoleic acid), 6-octadecenoic acid, pentadecanoic acid, and hexadecanoic acid ethyl ester. Unsurprisingly, the Egyptian plant possessed other biological compounds that were not found in C. comosum collected from other regions. This could be explained by the fact that different climatic zones can have a substantial impact on the chemistry and activities of medicinal plants with the same phenotype.

Table 1 The major compounds identified in the ethanolic extract of C. comosum (CE), retention times (RT), percentage compositions, chemical structures, molecular formulas, molecular weights (Mwt), and their biological activities Table 2 Colloidal properties of chitosan nanoparticles formulations loaded with different concentrations of ethanolic extract of C. comosum (CE/CsNPs) and chitosan nanoparticles (CsNPs); showing their measured particle size, polydispersity Index (PDI), Zeta potential (ZP), entrapment efficiency % (EE) and loading capacity (%) data (n = 3)Synthesis and characterization of synthesized CE/CsNPs formulationsPS, PDI, ZP, and EE% measurements

The bioactive compounds in plant extracts, such as alkaloids, flavonoids, and terpenoids, are highly soluble in water but have limited bioavailability and efficacy due to their large molecular sizes, poor absorption, and inability to pass through lipid membranes [76]. Nanotechnological approaches have overcome these challenges, allowing the use of compounds with different properties in the same composition [77]. Furthermore, the latest studies are focusing on finding sustainable, eco-friendly, and creative techniques to synthesize nanomaterials that are less hazardous to the environment and lifeforms [78, 79].

This study focused on developing eco-friendly CsNPs using citrate as a cross-linking agent, loaded with the optimum concentration of CE. Citrate has been used as a sustainable cross-linker for bio-polymeric materials due to its affordability and being naturally present. It is also known for its tensile characteristics and antimicrobial capabilities through its cross-linking effects [80].

As illustrated in Table 2, the synthesized CsNPs have a PS of 42.54 ± 3.65 nm, a uniform size distribution with a PDI of 0.29 ± 0.03, and a ZP value of 69.2 ± 2.37 mV. These results align with previous studies showing NPs with a high positive charge due to the electrostatic repulsion between chitosan’s amino groups within the molecule [81]. A recent investigation conducted by Hadkar and Selvaraj (2025) [82] suggested that an electrokinetic potential over 30 mV is necessary to maintain stable colloidal characteristics in aqueous solutions with moderate ionic strength. Loading CsNPs with CE by different concentrations (CE/CsNPs 1–3) resulted in significant changes in colloidal properties, with an increase in PS ranging from 60.57 ± 3.25 nm to 130.5 ± 1.02 nm (Fig. 2A) due to the loading of the extract in CsNPs, as explained by Panwar et al. (2016) [83] and Nallamuthu et al. (2015) [84]. The increase in the PDI value, ranging from 0.24 ± 0.01 to 0.63 ± 0.12, indicates a decrease in the homogeneity in aqueous solution [24] by increasing the concentration of the extract loaded within the CsNPs. The coupling of CE compounds in the developed CE/CsNPs formulations reduced the surface charge (40.1 ± 2.56 mV to 24.5 ± 2.45 mV), possibly due to the neutralization of the positive charges of the chitosan's amino groups [85].

Fig. 2

A Colloidal properties of different chitosan nanoparticles formulations, B, C morphology examination of the synthesized CE-loaded chitosan nanoparticles (CE/CsNPs), D chitosan nanoparticles (CsNPs) by TEM, and (E) in vitro release of ethanolic extract of C. comosum (CE) compounds from chitosan nanoparticles (CsNPs) at pH 7.4 and pH 4.0. PDI: Polydispersity Index; ZP: Zeta potential

Interestingly, the estimated EE% values for the CE/CsNPs (1–3) formulations were 60.84 ± 2.97%, 70.15 ± 1.58%, and 55.75 ± 3.12%, respectively, validating their stability and suitability for anticancer and antioxidant applications [86]. According to the obtained data, the CE/CsNPs-2 formula possessed the highest entrapment efficiency (70.15 ± 1.58%). It displayed good particle size (71.52 ± 4.57 nm), ZP (37.7 ± 1.91 mV), and PDI (0.35 ± 0.04) among all the studied formulations; thus, it was selected as the best achieved CE/CsNPs formula for further investigation in the current research.

TEM analysis

TEM is an image-guided method that confirms the shape and estimated size of the selected formula (CE/CsNPs-2), calculated by the DLS [87]. Figures 2B-D shows the TEM micrographs of the developed CE/CsNPs (Fig. 2B & C) and CsNPs (Fig. 2D). Interestingly, CsNPs showed spherical NPs with a size range of 34.94 nm to 43.64 nm. As for the CE/CsNPs, the TEM image showed NPs with a spherical shape and a PS range of 49.69 nm to 71.93 nm, which is closely related to the Zeta-sizer values. This suggests that CE/CsNPs do not aggregate in aqueous media and have a narrow size distribution, allowing them to disperse uniformly throughout cancerous tissue [24, 88].

CE/CsNPs in vitro drug release

The percentage of cumulative drug concentration over time under various pH circumstances (pH 4.0 and pH 7.4) is displayed in Fig. 2E. Both acidic (pH 4.0) and alkaline (pH 7.4) conditions provoked a burst release of the CE. Afterwards, the release efficiency started to slow down in acidic medium (pH 4.0) with a maximum efficiency of 38% after 24 hr of dialysis. Nevertheless, the release of CE was obvious at pH 7.4, with an efficient release of 64% after 24 hr. This aligns with previous experimental studies, where CE/CsNPs-2 displayed a higher drug delivery efficiency after 24 hr in the physiological environment compared to the acidic environment [88, 89].

The behavior of CE release depends on the charging properties and how the Cs and citrate molecules interact with each other. At pH 4.0, the degree of protonation of the chitosan's amino groups is less affected, and the citrate molecules are suitably deprotonated, resulting in a strong connection between the two molecules'charges and thus preventing the release of CE compounds [90]. While raising the pH, which makes the citrate much more charged, it also neutralizes the charge density in the Cs, which causes the collapse of the NPs and the drug release. In Light of these results, the CsNPs showed a maximum release of CE at pH 7.4, which is comparable to intestinal pH. Thus, the CE/CsNPs can act effectively as an oral delivery agent, besides overcoming the stomach's acidic medium with significant delivery at physiological pH.

FTIR analysis

The spectrum of Cs (Fig. 3A) illustrated a characteristic peak of 2990 cm−1 representing the vibration of NH2, 3746 cm−1 for the stretching vibration of OH, and 2878 cm−1 representing the C–H bond in alkanes and carboxylic groups [91], and the C = O bond in the amide I molecules could be recognized at 1617 cm−1 [92]. The band shown at 1403 cm−1 is general for OH groups bending, 1230 cm−1 and 1137 cm−1 correspond to the stretching vibrations of C–O–C bonds, and the band at 604 cm−1 corresponds to the pyranoside rings, which are also indicated in the previously published results by Dalal et al. (2021) [93]. Accordingly, the cross-linking of Cs polymer with citrate is expected to shift the peaks related to amide groups. Thus, comparing FTIR spectra of Cs and CsNPs (Fig. 3A & B) illustrated that the peak at 2878 cm−1 of the –NH2 groups in Cs was shifted to 2850 cm−1 due to the acetylation of Chitosan and cross-linking with citrate molecules. In addition, the peak at 2990 cm−1 related to the C–H bond in alkanes and carboxylic groups was absent in CsNPs, indicating a change in chitosan structure upon nano-formulation [16, 20]. The existence of C–O–C and NH3+, respectively, was validated by the presence of two distinctive strong bands at 1020 cm−1 and 1159 cm−1, respectively, due to chitosan cross-linking with citrate during synthesis [94].

Fig. 3

FTIR analyses for (A) chitosan powder (Cs), B chitosan nanoparticles (CsNPs), C ethanolic extract of C. comosum (CE), and (D) CE chitosan nanoparticles (CE/CsNPs)

Furthermore, a distinct peak emerged at 1382 cm−1 (C − O bond) that could indicate that the protonated amine group of chitosan (− NH3+) interacted with the carboxyl group of the citrate (COO−) [16]. The absorption bands of CE functional groups are defined in Fig. 3C. The peak that appeared at 1760 cm−1 is assigned to the C = O group. The bands 3678 cm−1 and 3535 cm−1 are ascribed to O–H bands, which can be found in sebacic acid, hexadecanoic acid, and octadecenoic acid, along with the presence of potent phenolic compounds represented in rhoifolin [95], which has been confirmed in the previous GC–MS results. The band that appeared at 2850 cm−1 belongs to the N–H bond, possibly found in geldanamycin [96]. The 2920 cm−1 and 1646 cm−1 peaks were attributed to C–H and C = O stretching, respectively [97]. The relatively small bands that were visible at 1310 cm−1, 1267 cm−1, 1166 cm−1, and 1109 cm−1 are assigned to various types of esters that are present in CE. The peak at 1033 cm−1 is assigned to C-F, which is possibly present in fluocinonide. Furthermore, the bands 1520.4 cm−1 and 657.2 cm−1 are attributed to the N–O and C–Cl bonds, respectively, that perhaps exist in the milbemycin B, as confirmed in the GC–MS results. The peak at 663 cm−1 corresponds to the C = C group. Moreover, the peak at 666.7 cm−1 could be ascribed to the absorption band of the C–H bond bending in the benzene ring out of plane [98].

On the contrary, the CE/CsNPs spectrum shown in Fig. 3D displayed that the characteristic peaks of CE were absent. Since the O–H peak (3736.6 cm−1) becomes broader, it confirms that the reaction between CsNPs and CE enhances the bonding action [99]. In addition, a shift in the C–H stretching bands at 2924.2 cm−1, 1478 cm−1, and 1384 cm−1, along with the disappearance of the bands at 1760 cm−1, 1310 cm−1, 869 cm−1, and 819 cm−1, which reflect the encapsulation of CE within the CsNPs. Strong bands were observed at 1635 cm−1 and 1546 cm−1 for the amide C = O stretching [100].

CE and CE/CsNPs antioxidant activity

The antioxidant assay was conducted on CE, CsNPs, and CE/CsNPs to evaluate their ability to scavenge free radicals. DPPH is a stable free radical with a visible deep purple color that becomes decolorized. This occurs due to the transfer of a hydrogen atom donated by the antioxidant molecules to the proper hydrazine, which reduces the odd electrons of the N-atom in the DPPH [101]. CE/CsNPs showed the highest DPPH scavenging activity, ranging from 82.3% to 26.85%, followed by CE that exhibited reducing power ranging from 70.67% to 10.02%, however the CsNPs displayed limited antioxidant activity (Fig. 4A). Notably, the reduction capacity of each tested compound was apparent with the increase in the concentration of applied treatment [102]. Indeed, the antioxidant power of C. comosum could be ascribed to the presence of various antioxidant biological compounds, including unsaturated fatty acids, flavones, quinones, and alkaloids (Table 1).

Fig. 4

A The in vitro antioxidant capacity of CsNPs, CE/CsNPs, and CE; B Half-maximal inhibitory concentration (IC50) values of DPPH radical scavenging activity on CsNPs, CE/CsNPs, and CE solutions (ζ P < 0.0001). * p < 0.05 CE group vs. CE/CsNPs group, #p < 0.05 CE or CsNPs groups vs. ascorbic acid. In all panels, n = 3, results are expressed as mean ± standard deviation. CE/CsNPs: Ethanolic extract of C. comosum loaded in chitosan nanoparticles, CE: Ethanolic extract of C. comosum, CsNPs: Chitosan nanoparticles, and DPPH: 2,2-diphenyl-1-picryl-hydrazyl

Previous studies showed promising antioxidant results for these compounds, including β-carboline alkaloids (peak 21) [66]. Rhoifolin, which belongs to flavones, a subclass of flavonoids (peak 25) [95], as well as aspidofractinine (peak 25) [73], and various fatty acids and fatty acid esters [37,38,39, 42, 47]. Additionally, it was demonstrated that β-carotenoic acid is a strong free radical scavenger [57]. The smaller the IC50 value, the greater the antioxidant capacity of the samples [103]. Notably, CE/CsNPs exhibited the most potent antioxidant activity, with an IC50 value of 9.9 ± 0.12 µg/mL, comparable to ascorbic acid, as shown in Fig. 4B (p < 0.05). The CE results displayed moderate antioxidant activity with an IC50 value of 80.28 ± 1.2 µg/mL, while drug-free CsNPs showed weak antioxidant activity [11].

In vitro anti-inflammatory and anticancer activities

Medicinal plants are a largely unexplored source of bioactive compounds for cancer therapy [91]. However, they have significant limitations, including nonspecific targeting, low water solubility, and limited therapeutic efficacy [14]. The study aimed to demonstrate the antitumor and anti-inflammatory capabilities of the synthesized CE/CsNPs and the free CE on different tumor cells. To evaluate the cytotoxic effects of the treatments, the results were compared with those of doxorubicin. Notably, CE/CsNPs showed the lowest toxicity to normal cells (WI-38), as illustrated in Fig. 5A, indicating their biocompatibility for targeted cancer therapy while reducing the adverse effects on healthy cells [8]. The MTT results revealed a significant dose-dependent anti-proliferative response against cancer cell lines; as the amount of employed compounds increased, the viability of the cancer cells decreased.

Fig. 5

The anticancer effects of doxorubicin, CsNPs, CE/CsNPs, and CE solutions at different concentrations on tumor cell viability % of (A) normal fibroblast (WI38), B prostate cancer (PC3), C epitheliod carcinoma (HeLa), D liver cancer (Hep-G2), E colorectal cancer (HCT-116), F mammary gland (MCF-7), G lung cancer (A549) cells, and (H) half-maximal inhibitory concentration (IC50) values of DOX, Cs, CsNPs, CE/CsNPs, and CE solutions evaluated on different cancer cell lines in comparison to normal human cells (WI-38) 48 hr post-treatment (ζ P < 0.0001). *p < 0.05 CE/CsNPs vs. Cs group, #p < 0.05 CE/CsNPs vs. CsNPs group, ** p < 0.05 CE/CsNPs vs. CE. In all panels, n = 3, results are expressed as mean ± standard deviation. DOX: Doxorubicin, Cs: Chitosan powder, CE/CsNPs: Ethanolic extract of C. comosum loaded in chitosan nanoparticles, CE: Ethanolic extract of C. comosum, and CsNPs: Chitosan nanoparticles

Undoubtedly, the CE/CsNPs possessed strong antitumor efficacy against different cancer cells, especially lung, breast, and liver cancers, with cell viability % ranging from 10.2 ± 0.05% to 60.7 ± 0.04% for Hep-G2 cells, 11.9 ± 0.05% to 55.4 ± 0.01% for MCF-2, and 9.11 ± 0.39% to 70.66 ± 0.74% for A546 cells. Treatment of PC-3, HeLa, and HCT-116 cells with CE/CsNPs showed moderate antitumor activity ranging from 20.6 ± 0.02% to 82.5 ± 0.05%, 26.1 ± 0.94% to 79.5 ± 0.45%, and 21.4 ± 0.74% to 77.4 ± 0.57%, respectively (Fig. 5B-G). The data related to the safety assessment of CE/CsNPs and CE was further confirmed through studying the IC50 of the treatments on cancer cells, as presented in Fig. 5H. Both treatments displayed decreased cytotoxicity against WI-38 cells by 11.5 and 13.2 folds in comparison to doxorubicin, with recorded values of 77.68 ± 1.32 µg/mL and 87.75 ± 2.15 µg/mL, respectively. In contrast, the cytotoxicity IC50 values after treatment of cancer cells with CE/CsNPs were 15.49 ± 0.35 µg/mL, 50.68 ± 1.54 µg/mL, 9.46 ± 1.12 µg/mL, 12.87 ± 0.27 µg/mL, 8.5 ± 0.17 µg/mL, and 42.6 ± 1.65 µg/mL for PC-3, HeLa, Hep-G2, HCT-116, MCF-7, and A549 cells, respectively, which indicated high to moderate cytotoxic activity on cancer cells. Thus, we can conclude that CE/CsNPs were more effective against lung, breast, and liver cancers than prostate, epithelioid, and colorectal cancers.

Furthermore, the microscopical examination of WI-38 and A546 cells was done to validate the cytotoxic effect of the tested compounds, the cells that were incubated with various concentrations (1000, 500, 250, 125, 62.5, and 31.25 μg/mL) of CsNPs, CE/CsNPs, or CE were analyzed (Fig. 6); the A546 cells treated with CE/CsNPs revealed large-scale morphological cytoplasmic vacuolization, which accompanies cell death, as illustrated in Fig. 6E [104]. The CE/CsNPs had a non-toxic protective effect against WI-38 at doses less than 125 μg/mL, whereas CE showed reduced cytotoxicity at concentrations less than 62.5 μg/mL (Fig. 6B & C).

Fig. 6

Photos of WI-38 (A, B, C) and -A549 (D, E, F) cells incubated with different concentrations (1000, 500, 250, 125, 62.5, and 31.25 μg/mL) of CsNPs, CE/CsNPs, and CE. CE/CsNPs: Ethanolic extract of C. comosum loaded in chitosan nanoparticles, CE: Ethanolic extract of C. comosum, CsNPs: chitosan nanoparticles, WI38: human lung fibroblast, and A549: human lung cancer cell lines

Regarding inflammation, RT-qPCR was conducted to study the relative expression of cytokines after 24 hr of treatment. The treatment of cells with CE/CsNPs successfully down-regulated the TNF-α expression by 54.9% and 54.1% in A549 and Hep-G2 cells, respectively. Furthermore, the expression of IL-6 was remarkably decreased by 50.9% and 47.7% in A549 cells and Hep-G2 cells, respectively, compared to the untreated cells (p < 0.05). Treating the cells with CE revealed down-expression of the TNF-α in A549 and Hep-G2 cells by 34.1% and 45.9%, however IL-6 expression was down-regulated by 39.8% and 47.1%, respectively, compared to the untreated cells (P < 0.05) (Fig. 7). Thus, the collected data is suggesting the mitigation of inflammatory response in the treated cells.

Fig. 7

Relative expression of cytokines (A) interleukin 6 (IL-6) and (B) tumor necrosis factor α (TNFα) in A549 and Hep-G2 cell lysates 48 hr post-treatment with CsNPs, CE/CsNPs, or CE solutions at their corresponding IC50 values (ζ P < 0.0001). * p < 0.05 CE/CsNPs or CE groups vs. untreated group, # p < 0.05 CE/CsNPs vs. CE group. In all panels, n = 3, results are expressed as mean ± standard deviation. CE/CsNPs: Ethanolic extract of C. comosum loaded in chitosan nanoparticles, CE: Ethanolic extract of C. comosum, and CsNPs: chitosan nanoparticles

It could be speculated herein that the high anticancer and anti-inflammatory potencies of CE/CsNPs in comparison to free CE suitably could be related to the release of CsNPs loaded with CE directly into the cells, which will subsequently cause the nucleus to be directly affected and lead to the promotion of a high number of apoptotic genes, as argued by Alzahrani (2021) [