Comparative Estimation of the Cytotoxic Activity of Different Parts of Cynara scolymus L.: Crude Extracts versus Green Synthesized Silver Nanoparticles with Apoptotic Investigation

Different parts of Cynara scolymus L. and their green synthesized eco-friendly silver nanoparticles (AgNPs) were screened for their cytotoxicity and apoptotic activity. Results showed that flower extract AgNPs exhibited more potent cytotoxicity compared to the normal form against PC-3 and A549 cell lines with IC50 values of 2.47 μg/mL and 1.35 μg/mL, respectively. The results were compared to doxorubicin (IC50 = 5.13 and 6.19 μg/mL, respectively). For apoptosis-induction, AgNPs prepared from the flower extract induced cell death by apoptosis by 41.34-fold change and induced necrotic cell death by 10.2-fold. Additionally, they induced total prostate apoptotic cell death by a 16.18-fold change, and it slightly induced necrotic cell death by 2.7-fold. Hence, green synthesized flower extract AgNPs exhibited cytotoxicity in A549 and PC-3 through apoptosis-induction in both cells. Consequently, synthesized AgNPs were further tested for apoptosis and increased gene and protein expression of pro-apoptotic markers while decreasing expression of anti-apoptotic genes. As a result, this formula may serve as a promising source for anti-cancer candidates. Finally, liquid chromatography combined with electrospray mass spectrometry (LC-ESI-MS) analysis was assessed to identify the common bioactive metabolites in crude extracts of stem, flower, and bract.


Introduction
Cynara scolymus L. is a perennial plant belonging to the family Compositae, and it grows in a big head from edible buds with multiple triangular scales of 8-15 cm in diameter. It is cultivated in the Mediterranean region for the fresh, immature flower head. As a vegetable, it is consumed as fresh, tinned, or frozen [1,2]. The plant is of a great nutritional value as it accumulates a considerable amount of folic acid, essential amino acids, and fatty acids, of which the most plentiful are the n-6 linoleic and palmitic acids [3]. C. Scolymus extracts have long been used in folk medicine for their great choleretic and

Plant Extracts
The plant was collected from El Behera Governorate, which has the largest cultivated area of artichokes in Egypt, during December 2020. Then, it was taxonomically identified at Faculty of Science, Suez Canal University, and a voucher specimen was kept at the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Suez Canal University under registration code (CS-2020). Three different parts were detached from the plant to be investigated separately: bracts, flowers, and stems. Each part was air dried by placing it on a shallow tray lined with a layer of paper towels in a dry place with good air circulation for 7 to 10 days, and then they were finely ground using an electric grinder.
Pharmaceutics 2022, 14, 2185 3 of 20 This step was followed by cold maceration (I Kg of each part) at room temperature for 1 week using methanol. The extraction process was repeated three times to ensure complete extraction [16]. The three extracts were concentrated under reduced pressure using rotary evaporator and stored in the refrigerator.

Determination of the Total Phenolic Content in the Different Plant Extracts
The total phenolics in the three different extracts (bracts, flowers and stem) of Cynara scolymus L. were assessed spectrophotometrically using Folin-Ciocalteu method, as previously mentioned [17]. UV absorbance was measured at λ 630 nm using a Milton Roy, Spectronic 1201 (Houston, TX, USA). Gallic acid was utilized as a standard. The results were reported in terms of gallic acid equivalents (mg GAE/g dry extract).

Green Synthesis and Preparation of Silver Nanoparticles
The biogenic synthesis of AgNPs in the presence of the total extract of bracts, flowers, and stems were prepared using a modified method of that reported by Kim et al. [18]. and Ashour et al. [19]. Initially, 10 mg of the extract was dissolved in 1 mL ethanol, then added to 10 mL of 10 mM AgNO 3 . A few drops of 1 M NaOH were added, and the mixture was agitated for 1 h at 400 rpm at 60 • C in the dark. All prepared nanoparticles were purified by centrifugation at 15,000 rpm for 1 h at 4 • C. The AgNPs were re-dispersed in double-distilled water and sonicated for 30 s in sonicating water bath, then centrifuged under the same previous conditions. The washing procedures using double-distilled water were repeated three times.

Characterization of Silver Nanoparticles 2.4.1. UV-Vis Absorbance Spectroscopy
The reduction of Ag+ ions was confirmed by measuring the UV-vis spectrum using a double-beam spectrophotometer (V630, Jasco, Tokyo, Japan). The spectrum was recorded throughout a range of 300-600 nm.

Size Analysis and Surface Charge Determination
Average particle size (expressed as Z-average), zeta potential (ZP) and polydispersity index (PDI) were accurately measured by photon correlation spectroscopy (PCS) using Malvern Zetasizer (Nano ZS, Malvern Instruments Ltd., Malvern, UK). Each sample was diluted 20 times with distilled water before analysis. All measurements were performed at ambient temperature (25 • C) and in triplicates. Finally, the mean and standard deviation values were accurately calculated [20].

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was carried out to examine the size and surface morphology of the synthesized AgNPs. The sample preparations were further diluted 50 times with double-distilled water. Then the diluted samples were negatively stained with phosphotungstic acid and dried on carbon-coated copper grids. The thin film formed was air-dried at room temperature and observed using transmission electron microscope (JTEM model 1010, JEOL ® , Tokyo, Japan) with an accelerating voltage of about 100 kV [21].

Annexin V/PI Staining for Apoptosis/Necrosis Assessment
Treatment with flower extract AgNPs (IC 50 = 1.52 µM, 48 h) was applied to A549 cells that had been cultured in 6-well culture plates (3-5 × 10 5 cells/well) overnight. Following collection of cells and medium supernatants, the cells were suspended in 100 L of Annexinbinding buffer solution "25 mM CaCl 2 , 1.4 M NaCl, and 0.1 M Hepes/NaOH, pH 7.4" and incubation with "Annexin V-FITC solution (1:100) and propidium iodide (PI) at a concentration equal to 10 µg/mL in the dark for 30 min." Afterwards, the Cytoflex FACS machine was used to collect the labelled cells, and the cytExpert software was utilized to evaluate the results [24][25][26].

Gene Expression Analysis (RT-PCR) for the Selected Genes
Gene expression of P53, Bax, and Caspases-3,8,9 was identified as pro-apoptotic genes, while Bcl-2 was identified as an anti-apoptotic gene, and their gene expression was assessed using RT-PCR to investigate the apoptotic pathway; their sequences in forward and reverse direction are shown in Table 1. MCF-7 cells were treated with flower extract AgNPs (IC 50 = 1.52 µM, 48 h) following the usual procedures, and the RT-PCR reaction was carried out [27,28]. Table 1. Sequences of forward and reverse primers.

Protein Expression Using Western Blotting
Furthermore, untreated, and treated A549 cancer cells were treated with flower extract AgNPs (IC 50 = 1.52 µM, 48 h) were tested for protein expression using Western blotting assay for further validation of the apoptotic pathway at the protein expression level. Cells were washed in PBS and lysed in boiling sample buffer before being electrophoresed on a sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). After boiling the lysates for 5 min in lamellae buffer, the proteins were separated using SDS-PAGE and transferred to an Immobilon membrane (Millipore, Merck KGaA, Darmstadt, Germany). After blocking in 5% nonfat milk for 1 h, the membranes were probed overnight at 4 • C with primary antibodies against "P53, Bax, Bcl-2, caspase-3, and caspase-9". After conjugating appropriate secondary antibodies, immunoblots were densitometrically analyzed to quantify the amounts of tested proteins [29].

Liquid Chromatography-Electrospray Ionization Mass Spevtrometry (LC-ESI-MS) Analysis
The LC-ESI-MS analysis was performed using a UPLC instrument equipped with a reversed-phase C-18 column (ACQUITY UPLC BEH C18 column, 1.7 µm particle size, 2.1 × 50 mm column). Mobile phase elution was conducted at a flow rate of 0.2 mL/min using a gradient mobile phase (A: H 2 O, B: acetonitrile (Merck, Darmstadt, Germany), both acidified with 0.1% formic acid (Merck, Darmstadt, Germany)). The UPLC compartment was equipped with an electrospray ESI source (electrospray voltage, 3.0 kV; sheath gas, nitrogen; capillary temperature, 440 • C) in negative ionization mode. The ESI-MS range was set at m/z 100 and 1000, with starting collision-induced dissociation energy of 30 eV, and MassLynx 4.1 software was used to process the spectra. The compounds were identified Pharmaceutics 2022, 14, 2185 5 of 20 by comparison of their data, particularly accurate masses, with those reported in the literature [30].

Total Phenolic Content in the Different Plant Extracts
The total phenolic contents of the three different extracts of Cynara scolymus L. were assessed separately using the Folin-Ciocalteu colorimetric method. The results were presented as gallic acid equivalents and found as 74.29 ± 3.85, 60.94 ± 3.28 and 26.59 ± 1.37 (mg/gm) for the flower, bract, and stem extracts, respectively, where the flower extract revealed the highest content compared to the bracts and the stem extracts.

UV-Vis Absorbance Spectroscopy
All prepared AgNPs formulations showed brown color due to the characteristic surface-plasmon resonance absorption band in the range of 400-500 nm. Figure 1. It is known that metal nanoparticles have a surface plasmon resonance absorption in the UV-Visible region. The band of surface plasmon occurs due to the coherent existence of free electrons in the conduction band due to the small particle size [31,32]. This confirms the reduction of Ag+ ion to colloidal Ag. × 50 mm column). Mobile phase elution was conducted at a flow rate of 0.2 mL/min using a gradient mobile phase (A: H2O, B: acetonitrile (Merck, Darmstadt, Germany), both acidified with 0.1% formic acid (Merck, Darmstadt, Germany)). The UPLC compartment was equipped with an electrospray ESI source (electrospray voltage, 3.0 kV; sheath gas, nitrogen; capillary temperature, 440 °C) in negative ionization mode. The ESI-MS range was set at m/z 100 and 1000, with starting collision-induced dissociation energy of 30 eV, and MassLynx 4.1 software was used to process the spectra. The compounds were identified by comparison of their data, particularly accurate masses, with those reported in the literature [30].

Total Phenolic Content in the Different Plant Extracts
The total phenolic contents of the three different extracts of Cynara scolymus L. were assessed separately using the Folin-Ciocalteu colorimetric method. The results were presented as gallic acid equivalents and found as 74.29 ± 3.85, 60.94 ± 3.28 and 26.59 ± 1.37 (mg/gm) for the flower, bract, and stem extracts, respectively, where the flower extract revealed the highest content compared to the bracts and the stem extracts.

UV-Vis Absorbance Spectroscopy
All prepared AgNPs formulations showed brown color due to the characteristic surface-plasmon resonance absorption band in the range of 400-500 nm. Figure 1. It is known that metal nanoparticles have a surface plasmon resonance absorption in the UV-Visible region. The band of surface plasmon occurs due to the coherent existence of free electrons in the conduction band due to the small particle size [31,32]. This confirms the reduction of Ag+ ion to colloidal Ag.

Size Analysis and Surface Charge Determination
Measurement of mean particle size by DLS technique gives a comprehensive picture of the particle size of the whole sample as well as the homogeneity of size distribution throughout the sample. That is why it was performed in the current study, although it is reported that DLS measurement is mostly used for AgNPs synthesized from bio-polymers not from the plant extracts and microorganisms [11]. Results given in Table 2 show that particle size values of the prepared AgNPs ranged between 23.6 ± 1.08 of the LL extract AgNPs and 27.2 ± 0.91 nm for the AA extract AgNPs. From the obtained data, it is clear that the three prepared formulations show small difference in the mean particle size. This can be an indication to the consistency of the method of preparation and the absence of effect on different parts of the plant on the obtained AgNPs size. It is well proven that the particle size, surface charge, and NPs shape have a significant impact on pharmacokinetics, cell internalization, tissue distribution, cellular uptake, and clearance. Additionally, physiological processes such as hepatic uptake and tissue diffusion, tissue extravasation, and renal excretion mainly depend on average particle size [33]. Additionally, controlling the particle size, charge, and surface chemistry of the nanocarrier enables to avoid various limitations of conventional treatments such as administration of higher doses, low bioavailability, and the poor chemical stability of the administered drug [34]. It is also reported that the NPs size is one of the most important factors used to predict the circulation time inside living tissues. The interaction of macrophages with targets nanoparticles is dependent on particle size, as shown by Doshi et al. [35]. It was reported in the literature that this small range of particle sizes showed higher cytotoxicity compared to larger particles. Xu and colleagues studied the effect of particle size of the AgNPs on different glioma cell lines. They found that the AgNPs of size range (20-50 nm) was found to be cytotoxic to glioma cells compared to 100 nm particles [36]. Liu et al. investigated the effect of AgNPs of different particle sizes on four different human cell lines. They found that the smaller nanoparticles enter cells more easily than larger ones, which may be the cause of the higher toxic effects [37]. On the other hand, PDI measures size distribution in the formula and ranges usually from 0 to 1. Low PDI values ranged from 0.123 ± 0.006 to 0.283 ± 0.020 show a narrow size distribution and encourage long-term nano dispersion stability; however, values greater than 0.5 suggest that there is no uniform size distribution [38]. Lower values of PDI are desirable for a lower variation in AgNPs formulation. The PDI of all prepared AgNPs formulae show uniform size distribution and perfect homogeneity; see Table 2.
Zeta potential is the overall charge acquired by the particles. It is important to obtain precise judgments about nanoparticles dispersions stability. The colloidal dispersions are considered highly stable when the ZP value is around 30 mV or greater due to the presence of electrostatic repulsion between particles [25]. A high value of zeta potential will ensure system stability, which helps the nanocarrier to resist aggregation. When the zeta potential is very low, attractive forces exceed repulsion and the dispersion will be unstable. So, nanoparticles with higher zeta potentials are electrically stabilized [39]. In this investigation, the results obtained for zeta potential were in the range between −27.2 ± 1.417 and −34.2 ± 0.66 mV, as illustrated in Table 2. The ZP results clearly indicate that all prepared AgNPs have sufficient charges that would prevent their agglomeration and are considered highly stable.

Transmission Electron Microscopy (TEM)
The surface morphology, size and shape of the synthesized AgNPs was illustrated by TEM. The TEM image Figure 2 revealed that AgNPs are spherical in shape, fairly monodispersed, and effectively dispersed without agglomeration. The inset of Figure 2 shows the particle size distribution of AgNPs derived from TEM data. The barely perceptible variation in PS measurement between the two techniques (TEM vs. DLS) results can be illustrated by the difference in the measurements techniques. TEM measurement is a number-based particle size measuring method however DLS is an intensity-based method. Figure 3 shows the results of an MTT assay testing the cytotoxicity of crude extract samples from the brackets, flowers, and stems of Cynara scolymus L. against prostate (PC-3) and lung (A549) cancer cell lines. Based on the cytotoxicity data shown in Table 3, crude extracts of brackets, flowers, and stems revealed low to moderate cytotoxic activity against A549 and PC-3 cells (IC 50 = 36.57 to 165.3 g/mL). When compared to their non-nano counterparts, cytotoxicity results were significantly enhanced when nano formulations were used. Flower AgNPs displayed strong cytotoxicity against PC-3 and A549 cell lines, with IC 50 values of 2.47 g/mL and 1.35 g/mL, respectively, compared to doxorubicin

Transmission Electron Microscopy (TEM)
The surface morphology, size and shape of the synthesized AgNPs was illust by TEM. The TEM image Figure 2 revealed that AgNPs are spherical in shape, fairly odispersed, and effectively dispersed without agglomeration. The inset of Figure 2 sh the particle size distribution of AgNPs derived from TEM data. The barely percep variation in PS measurement between the two techniques (TEM vs. DLS) results ca illustrated by the difference in the measurements techniques. TEM measurement is a n ber-based particle size measuring method however DLS is an intensity-based metho  Figure 3 shows the results of an MTT assay testing the cytotoxicity of crude ex samples from the brackets, flowers, and stems of Cynara scolymus L. against prostate 3) and lung (A549) cancer cell lines. Based on the cytotoxicity data shown in Table 3, c extracts of brackets, flowers, and stems revealed low to moderate cytotoxic activity ag A549 and PC-3 cells (IC50 = 36.57 to 165.3 g/mL). When compared to their non-nano c terparts, cytotoxicity results were significantly enhanced when nano formulations used. Flower AgNPs displayed strong cytotoxicity against PC-3 and A549 cell lines, IC50 values of 2.47 g/mL and 1.35 g/mL, respectively, compared to doxorubicin and Ag with IC50 values of 5.13 and 3.75 μg/mL against PC-3 cells, and with IC50 values of 6.19 22 μg/mL against A549 cells, respectively. Additionally, AgNPs of the bracts sho promising cytotoxicity with IC50 values of 14.29 and 16.4 μg/mL, while AgNPs of the s showed weak cytotoxicity. These findings demonstrated that the floral extract's Ag possessed significant cytotoxicity, justifying additional investigation into the mecha of action in A549 and PC-3 cells.   3.6. Apoptosis-Induction Activity 3.6.1. Annexin V/PI Staining Cytotoxic activity of flower extract AgNPs against A549 and PC-3 cells was inv gated for its mechanism for apoptosis-induction using Annexin V/PI staining. As se  3.6. Apoptosis-Induction Activity 3.6.1. Annexin V/PI Staining Cytotoxic activity of flower extract AgNPs against A549 and PC-3 cells was investigated for its mechanism for apoptosis-induction using Annexin V/PI staining. As seen in Figure 4, flower extract AgNPs induced total lung apoptotic cell death by 27.7% compared to 0.67% in the untreated control cells. These findings showed that this extract induced cell death by apoptosis by a 41.34-fold change, and it induced necrotic cell death by 10.2-fold. Additionally, it induced total prostate apoptotic cell death by 17.8% compared to 1.1% in the untreated control cells. These findings showed that this extract induced cell death by apoptosis by a 16.18-fold change, and it slightly induced necrotic cell death by 2.7-fold. Hence, flower extract AgNPs exhibited cytotoxicity in A549 and PC-3 through apoptosis-induction in both cells.

Gene Expression Analysis using RT-PCR
Gene expression analysis for apoptosis-related genes in untreated and treated A549 cells was performed to examine apoptosis-induction by flower extract AgNPs treatment. As seen in Figure 5, flower extract AgNPs upregulated P53 gene by 7.9-fold, Bax gene by 6.98-fold, and caspases 3, 8, 9 by 10.76, 2.84, and 8.47-fold, respectively, while it downregulated the Bcl-2 gene by 0.19-fold, this treatment-induced apoptosis in A549 cells was consistent with expected behavior [24,40] of proving apoptosis-induction.

Protein Expression Using Western Blotting
Further confirmation of apoptosis-induction in A549 cells using Western blotting was employed to measure the levels of p53, Bax, caspase-3 and 9, and Bcl-2 proteins. P53, Bax, and caspase-3 and 9 proteins were found to be upregulated in the study results. Both thick and thin bands and their relative quantification data from Figure 6 show that flower extract AgNPs treatment resulted in a decrease in Bcl-2 protein expression. These findings are in line with the upregulation of pro-apoptotic genes and the downregulation of anti-apoptotic genes observed during RT-PCR.
Previous studies revealed that there is a statistically significant relationship between total polyphenol consumption and the risk of developing cancer [41]. Due to the fact that the phenolic sets in polyphenols can receive electron with formation of steady phenoxy radical, polyphenols disturb or disorganize series of oxidation reactions in constituents of cells, and this is responsible for their ability to prevent various degenerative diseases, including different cancer types [42,43]. Moreover, it was declared that phenolic acids exert a direct anti-proliferative action [44]. It was also declared that the flowers of Cynara scolymus L. accumulate several phenolic acids including quinic, chlorogenic, gallic, ferulic, cinnamic vanillic, 1,5-Dicaffeoylquinic, and 1,3-di-O-caffeoylquinic acid (cynarin) [45,46]. The flower of the plant also contains a number of flavonoids, mainly luteolin and apigenin (both aglycones) and glycosides [47,48]. Based on the data on our present study, the flower extract of Cynara scolymus L. exhibited the highest content of phenolic constituents exactly 74.29 ± 3.85 (mg/gm) compared to 60.94 ± 3.28 and 26.59 ± 1.37 (mg/gm) for the bract and stem extracts, respectively. This can explain the relatively higher cytotoxic activity of the flower extract compared to the other two extracts. Since the aim of our work is to optimize the cytotoxic activity and to discover the most efficient way to make use of this natural product, we selected the flower extract to be formulated as AgNPs. As mentioned in the discussion, this formula exhibited more potent cytotoxic effects, and these results make it recommended as an optimistic drug lead.

Gene Expression Analysis using RT-PCR
Gene expression analysis for apoptosis-related genes in untreated and treated A549 cells was performed to examine apoptosis-induction by flower extract AgNPs treatment. As seen in Figure 5, flower extract AgNPs upregulated P53 gene by 7.9-fold, Bax gene by 6.98-fold, and caspases 3, 8, 9 by 10.76, 2.84, and 8.47-fold, respectively, while it downregulated the Bcl-2 gene by 0.19-fold, this treatment-induced apoptosis in A549 cells was consistent with expected behavior [24,40] of proving apoptosis-induction.

UPLC-ESI-MS Analysis for Identifiction of Bioactive Metabolites
The three parts (flower, bract, and stem) of Cynara Scolymus were analyzed using UPLC-ESI-MS, resulting in a noticeable difference in their chemical composition, as shown in Figure 7 and Table 4. Previous studies revealed that there is a statistically significant relationship between total polyphenol consumption and the risk of developing cancer [41]. Due to the fact that the phenolic sets in polyphenols can receive electron with formation of steady phenoxy radical, polyphenols disturb or disorganize series of oxidation reactions in constituents of cells, and this is responsible for their ability to prevent various degenerative diseases, including different cancer types [42,43]. Moreover, it was declared that phenolic acids exert                   [41,51] apigenin [41,51]   chlorogenic acid [56]

Discussion
The crude extract of the floral portion of C. scolymus L. has the greatest levels phenolics and total flavonoids as reported in the literature by Palermo et al. [58]. P studies reported that the flower is especially rich in phenolic compounds such as tin and 1,3-di-O-Caffeoylquinic acid (cynarin) [45,49,54]. Bracts contain Apigen malonyl glucoside, hesperidin, and Cynaropicrin, as reported in [41].  [41,53,54] genic Acid, Luteolin, luteolin-7-O-rutinoside and Apigenin are existing in the thr with considerable difference in concentration as reported in [41,45,56], and this agrees with our study. These polyphenols exhibited cytotoxic effects and apop duction activity that help in the treatment of prostate and lung cancer. It is now why the crude extract of the C. scolymus L. flower portion showed a stronger c activity than those of the bracts or stem. Previous studies as those by Mileo

Discussion
The crude extract of the floral portion of C. scolymus L. has the greatest levels phenolics and total flavonoids as reported in the literature by Palermo et al. [58]. P studies reported that the flower is especially rich in phenolic compounds such as tin and 1,3-di-O-Caffeoylquinic acid (cynarin) [45,49,54]. Bracts contain Apigen malonyl glucoside, hesperidin, and Cynaropicrin, as reported in [41].  [41,53,54] genic Acid, Luteolin, luteolin-7-O-rutinoside and Apigenin are existing in the thr with considerable difference in concentration as reported in [41,45,56], and this agrees with our study. These polyphenols exhibited cytotoxic effects and apop duction activity that help in the treatment of prostate and lung cancer. It is now why the crude extract of the C. scolymus L. flower portion showed a stronger c activity than those of the bracts or stem. Previous studies as those by Mileo

Discussion
The crude extract of the floral portion of C. scolymus L. has the greatest levels phenolics and total flavonoids as reported in the literature by Palermo et al. [58]. P studies reported that the flower is especially rich in phenolic compounds such as tin and 1,3-di-O-Caffeoylquinic acid (cynarin) [45,49,54]. Bracts contain Apige malonyl glucoside, hesperidin, and Cynaropicrin, as reported in [41].  [41,53,54] genic Acid, Luteolin, luteolin-7-O-rutinoside and Apigenin are existing in the thr with considerable difference in concentration as reported in [41,45,56], and this agrees with our study. These polyphenols exhibited cytotoxic effects and apop duction activity that help in the treatment of prostate and lung cancer. It is now why the crude extract of the C. scolymus L. flower portion showed a stronger c activity than those of the bracts or stem. Previous studies as those by Mileo, Di V apigenin [56]

Discussion
The crude extract of the floral portion of C. scolymus L. has the greatest levels of total phenolics and total flavonoids as reported in the literature by Palermo et al. [58]. Previous studies reported that the flower is especially rich in phenolic compounds such as quercetin and 1,3-di-O-Caffeoylquinic acid (cynarin) [45,49,54]. Bracts contain Apigenin-7-O-malonyl glucoside, hesperidin, and Cynaropicrin, as reported in [41]. Flower and bracts contain Luteo-lin-7-O-glucoside (cynaroside), 3-O-Caffeoylquinic acid (Neo Chlorogenic acid). 5-O-Caffeoylquinic acid (Chlorogenic acid), Luteolin 7-O-malonylglucoside, 1,5-di-O-Caffeoylquinic acid, 3,5-Di-O-caffeoylquinic Acid, also reported in [41,53,54] Chlorogenic Acid, Luteolin, luteolin-7-O-rutinoside and Apigenin are existing in the three parts with considerable difference in concentration as reported in [41,45,56], and this finding agrees with our study. These polyphenols exhibited cytotoxic effects and apoptosis-induction activity that help in the treatment of prostate and lung cancer. It is now obvious why the crude extract of the C. scolymus L. flower portion showed a stronger cytotoxic activity than those of the bracts or stem. Previous studies as those by Mileo, Di Venere et al. conducted studies on the edible part (head) of fresh artichoke using breast cancer cell line MDA-MB231 and found that high dosages of polyphenolic extracts of artichoke extracts can initiate an apoptotic pathway and halt tumor development [59]. Additionally, Pulito, Mori et al. tested the edible parts (receptacles with inner and intermediate bracts) and leaves using cell cytotoxicity assay on human carcinoma cells MSTO-211H, MPP-89, and NCI-H28 mesothelioma cell lines and found that Cynara scolymus affects malignant pleural mesothelioma by promoting apoptosis and restraining invasion [60]. Through the use of intrinsic and extrinsic signalling pathways, luteolin and luteolin-7-O-glucoside (cynaroside) can slow or stop the growth of cancer cells both in vitro and in vivo by protecting against carcinogenic stimuli, inhibiting tumor cell proliferation, inducing cell cycle arrest, and inducing apoptosis [61,62]. Through several investigations, quercetin (QU) and quercetin glycoside (rutin), two polyphenolic flavonoids, stand out among the natural products. The biological characteristics of rutin (RU) and its aglycone (QU) include anti-oxidant, anti-inflammatory, and anti-carcinogenic actions [63]. Chlorogenic acid (CGA), also known as 5-O-caffeoylquinic acid, is polyphenol act as curative compounds against oxidative stress, which is a key player in the pathophysiology of many diseases, including cancer [64]. Moreover, 1,5-di-O-Caffeoylquinic acid, 3,5-Di-O-caffeoylquinic acid, and other phenolic compounds possess potent anti-RSV activity, anti-oxidant and anti-bacterial activity [65][66][67]. The primary caffeoylquinic acid derivative, cynarin, is present in the leaves and heads of artichokes. It may have cytotoxic, choleretic, anti-oxidative, hepatoprotective, anti-atherosclerotic, and antioxidative properties [68]. Extensive studies have demonstrated the significant anti-oxidant, anti-inflammatory, and anti-carcinogenic capabilities of apigenin. [69].
In this study, there are three mechanisms for evaluation of in vitro cytotoxic activity as Annexin V/PI staining, Gene expression analysis using RT-PCR [24,40] or protein expression using Western blotting. These methods provide simplicity, cost-management, and rapid technique. The comparison between the extract from Cynara scolymus' various sections and its nano form is the study's main objective. The potential for cytotoxicity increases with decreasing IC 50 values for the crude extracts under investigation. The biological findings showed that, in comparison to doxorubicin as a reference drug, the silver nano particles of polyphenolic fraction of flower extract (flower extract AgNPs) exhibited potent cytotoxicity against PC-3 and A549 cell lines. Using flow cytometry and gene expression tests to investigate apoptosis-induction, flower extract AgNPs demonstrated a greater ratio of apoptosis in PC-3 cells compared to A549 cells. Our results agreed with their reported anti-cancer activities Phuong Thuy et al. and Shallan et al., [70,71] either through apoptosis-induction or anti-oxidant activation. Erdogan et al. [72] previously created silver nanoparticles using Cynara scolymus leaf extracts and shown their anti-cancer potential by inducing apoptosis. Finally, these positive results suggest more in vivo research on the effectiveness of silver nanoparticles of the total phenolic fraction of C. scolymus L. flower extract against prostate cancer. The estimate of the polyphenolic fraction of floral extract's pharmacokinetics is another interesting area of research.

Conclusions
Cynara scolymus L. flower extract AgNPs exhibited potent cytotoxicity compared to the normal form against PC-3 and A549 cell lines with IC 50 values of 2.47 µg/mL and 1.35 µg/mL, respectively. It exhibited apoptosis activity in both A549 and PC-3 cells by 41.34 and 16.18-fold-changes. Additionally, apoptosis activity was further proved through upregulation of pro-apoptotic markers of P53, Bax, caspases-3, 8, 9, and downregulation of anti-apoptotic marker Bcl-2 in both gene and protein expression levels. So, this formula may serve as a promising source for anti-cancer candidates.