Characterization of the Chemical Components of the Extracted Carduus Pycnocephalus L, and Assessment of Its Potential Novel Antioxidant, Antibacterial, and Anticancer Activities


 Carduus pycnocephalus L, which is related to the Astraceae family, was well-known as a privileged medicinal plant that has innumerable respected biological potency. The current research aims to identify the chemical constitutes of the essential oils of the extracted C. pycnocephalus by Gas Chromatography-Mass Spectroscopic analysis (GC-MS) and to assess the biological profiles of the plant and its botanical ingredients as a precise antioxidant, and anticancer, as well as, antimicrobial agents. The extraction process of the medicinal plant by methanol provided a possibility to extract and identify the polar chemical constitutes that have the most effective categories of components. The DPPH antioxidant potency of the botanical ingredients of the plant indicated that the flower extract is the most potent with IC50 = 30.69 mg/L followed by leaves (IC50= 32.78 mg/L), stem (IC50= 41.31 mg/L), and root (IC50= 46.84 mg/L). The antibacterial activities of the root, stem, leaf, and flower extracts of C. pycnocephalus exhibited remarkable potency to kill or inhibit the growth of the bacterial species. Leaf, and flower extracts revealed the most potent activities than the antibiotic standards against E. coli, S. typhimurium, and B. cereus species with inhibition zones ranged from 20-26 mm. Also, the extracted C. pycnocephalus revealed a moderate cytotoxic effect against hepatocellular carcinoma (HepG2) tumor cell line using MTT assay with IC50= 46.2 µg/mL. The experimental interpretations inveterate the potential of C. pycnocephalus extract indicated its biological impacts as antioxidant, antibacterial, and moderate cytotoxic agents that provided the ease of using it in cancer therapy.


Introduction
The Carduus genus is related to Astraceae family, which included roughly 100 species worldwide according to Chaudhary (2000) that are extensively distributed from place to place in the Mediterranean. Only three species of Carduus genus were recorded in the ora of Egypt, C. pycnocephalus, C. getulus and C. argentatus. The latter is a spiny annual herb growing wildly in the coastal desert of Egypt. The stem usually branches and has spiny wings, the plant reaches up to 60 cm with the taproot, which gives greyish-tomentose on the lower surface, with spinose lobes, and its owering time extends from March to May (Boulos 1983). Flu, stomach ache, and rheumatism of such human diseases were treated by Carduus genus in Chinese folk medicine (Esmaeili et al. 2005). In addition, the genus of Carduus revealed many biological characteristics, for example, antibacterial, antiviral, anticancer, antispasmodic, anti-in ammatory, and liver tonicity activities  Orhan et al. 2009). Rhinocyllus conicus (Coleoptera: Curculionidae), as a type of Carduus species (Asteraceae) that grown in Argentina, was applied as a control for thistles (de Briano et al. 2013). Many types of research since antiquity and in recent times have focused on the study of Carduus species such as the study of the reproductive system (Olivieri et al. 1983), ecology (Doing et al. 1969), the accumulation of heavy metals in plants growing in contaminated soils (Brunetti et al. 2009), recovery of the colonization of the rootstock of mycorrhizal fungi (Jaunatre et al., 2016), and comparative studies electrophoresis (Olivieri 1985).
The phytochemical analysis of several types of plants related to this Carduus genus indicated the characterization of diverse classes of chemical constitutes such as avonoids, and Coumarin (Jordon-Thaden & Louda, 2003), as well as earlier reports, reported the identi cation of other classes of secondary metabolites such as lignans, alkaloids, sterols, and triterpenes. In this due course, C. pycnocephalus was well-known in the literature as an anti-in ammatory, antispasmodic, and hypotensive agent, in which the isolated components from the different parts of this plant were characterized by spectroscopic analyses .
The literature survey in earlier reports has also identi ed the isolation of many other classes from C. pycnocephalus such as essential oils (Esmaeili et al. 2005), sterols, and triterpenes (Gallo & Sarachine 2009). Al-Shammari et al. (2015), have characterized ten compounds from the aerial parts of this plant that were related to avonoid class, and anthraquinone, anthraquinone-linked carbohydrate, steroids, and steroids-linked carbohydrate derivatives as subclasses including kaempferol-7-methoxy-3-O-α-L-rhamnoside, diosmetin-7-O-β-D-xylosyl-β-D-gluco-pyranoside, diosmetin-7-O-α-L-arabinosyl-β-D-gluco-pyranoside, and kaempferol-3-O-α-Lrhamnosyl-α-L-rhamnoside along with the structures that were depicted in Figure 1. Esmaeili et al. (2005), have reported the analysis of the essential oils isolated from C. pycnocephalus that were grown in Iran by Gas Chromatography-Mass Spectroscopic analysis (GC-MS) with the identi cation of twentynine components demonstrating hexadecanoic acid (23.3%) as the main constituent. Marengo et al. (2017), have reported the characterization of four Carduus species, namely (C. argyroa Biv., C. nutans subsp. macrocephalus (Desf.) Nyman, C. pycnocephalus, C. cephalanthus Viv) that grown in the region of the Mediterranean by phytochemical and biomolecular analyses. The current research aimed to investigate the chemical constitutes of the essential oils of the extracted C. pycnocephalus by Gas Chromatography-Mass Spectroscopic analysis (GC-MS), and to utilize the extracted sample to explore its biological aptitude as cytotoxic, and antimicrobial potency, as well as, the antioxidant characters of the extracted plant parts.

Plant material and extraction process
The plant materials of C. pycnocephalus were collected from the sandy habitat near Gamasa City, northern Mediterranean coast, Egypt (31°30′22.24″N 31°22′4.70″E). The plant materials were cleaned and divided into small pieces. A weighed 20 gm of the previously prepared plant materials were placed in a conical ask (250 mL) contained 150 mL methanol and extracted by a shaker after two hours at room temperature. The extract was concentrated to a xed volume after the complete extraction process. The produced methanol extract was ltered using qualitative Whatman lter paper no. 1 (125 mm, Cat No 1001 125, Germany) and stored at 4 o C (Abd-ElGawad et al. 2020; Souza et al. 2018). Gas Chromatography-mass Spectroscopy (Gc-ms) Analysis The chemical constitutes of the plant were characterized by implementing the plant extract on Trace GC-TSQ mass spectrometer (Thermo Scienti c, Austin, TX, USA) with a direct capillary column TG-5MS (30 m x 0.25 mm x 0.25 µm lm thickness). The temperature of the column oven was rstly held at 50 o C, raised subsequently by a rate of 5 o C per minute to reach 250 o C, and hold for 2 min, and then the temperature was raised to the nal temperature (300 o C) by 30 o C per minute and hold for 2 min. the injector and MS transfer line temperature were kept at 270, and 260 o C, respectively. Helium (He) was used as a carrier inert gas at a constant ow rate of 1 mL/min. The solvent was released after 4 min and the diluted samples of 1 µl were injected directly using Autosampler AS1300 coupled with GC in the split mode. EI mass spectroscopy was collected at 70 EV ionization voltage over a range of 50-500 for m/z in packed scan mode. The temperature of the ion source was xed at 200 o C. The chemical components of the individual extracted plant materials were interpreted by a comparison of their mass spectral data with those of WILEY 09, and NIST 14 mass spectroscopic database. Reagents 1,1-Diphenyl-2-picrylhydrazyl (DPPH • ), ascorbic acid, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), RPMI-1640 medium, and DMSO were purchased from Sigma Aldrich (St. Louis, USA). Fetal bovine serum (FBS; Gibco Life Technologies, Paisley, UK). Cephradin, Tetracycline, Azithromycin, and Ampicillin "antibiotics" were purchased from Merck (Darmstadt, Germany). Nutrient Agar (Contained Peptone, HM Peptone B #, Yeast extract, Sodium chloride, Agar, and Beef extract) were purchased from Himedia Laboratories Pvt. Ltd. LBS Marg, Mumbai-400086, India.

Antioxidant DPPH assay
The extracts of root, stem, leaves, and ower of C. pycnocephalus plant were in vitro screened as antioxidant agents by a colorimetric DPPH free radical assay. The method depended on measuring the intensity in the violet color of the DPPH solution. The assay was applied exactly as applied by the procedure reported by Kitts et al. (2000). In this course, different concentrations of each sample were prepared in serial dilutions (5-50 mg/L) in six testing tubes by mixing with methanol. 0.135 mM of DPPH • solution was prepared and subsequently add to each tube of the serial dilution of the investigated samples of the plant species. The tubes were next incubated in dark at room temperature for approximately 30 min. The intensity in the violet color was measured on Spectrophotometric apparatus at a maximum wavelength of 517 nm. The linear regression analysis was applied to calculate the inhibitive concentrations of each tested sample by plotting the exponential curve (Parejo et al. 2000)

Procedure Of Anticancer Activity
Tumor cell lines HePG-2 (Hepatocellular carcinoma) was selected as a human tumor cell line was purchased from ATCC via a holding company for biological products and vaccines (VACSERA), Cairo, Egypt.

Preparation of MTT solution
The MTT solution was prepared by mixing a solution of MTT in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and media (5 mg/mL). The mixture was mixed by vortex or sonication, then ltered, and stored at -20°C.
Protocol of MTT assay (Zhu et al. 2017) The cytotoxic activity of the investigated sample C. pycnocephalus was evaluated by MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) colorimetric assay utilizing an altered process termed by Terblanche et al. (2017). For the determination of the IC 50 of each particular drug, the adherent HepG2 cells were seeded onto 96-well plates at an initial density (3 × 10 3 cells/well suspended in 100 µL of complete medium). The crude plant extracts were prepared and used to stimulate the cells using ve concentrations (31.3, 62.5, 125, 500, and 1000 µg/mL) in culture media. The experimental techniques were performed in duplicates, then plates were incubated for 24 hours in a 5% CO 2 at 37 ºC for settle down and adherence. Next, a serial dilution of each prepared concentration of the drug was applied onto the cells for 48 hours after adherence. The medium was removed by aspiration and a weighed MTT (0.5 mg/mL) was dissolved in a culture fresh medium and practically applied onto cells and the plates were incubated at 37 ºC and 5% CO 2 for 4 hours. Ultimately SDS (100 µL) was added into each well. The Reduction at cell growth was measured at (λ max . = 570 nm) (BioTek, Elx800, US) and the results were expressed as a percentage of control . The IC 50 values of the drug, articulated the concentration that affects roughly 50% death of tumor cells, were estimated through a straight linear regression, type sigmoidal, analyzed using Origin 8.0® software (OriginLab Corporation). The IC 50 values were calculated by the t line i.e. Y= a*X + b, in which IC 50 = (0.57-b)/a. The percentage of inhibition in cell growth was calculated from the following equation (Eq. 2), in which A is the absorbance of each control, and the tested sample: The relative cell viability percentage was subsequently calculated from the following equation (Eq. 3), in which A expressed the control, and sample absorbance at λ max . = 570 nm.

Microbial Tendency
The antimicrobial aptitudes of C. pycnocephalus extract were estimated using the agar well diffusion assay ( standard antibiotics for comparison scales. Preparation of the culture media 28.0 grams of the nutrient agar were placed in a 2 L conical ask and suspended in 1 L distilled water. The conical ask contents were heated to boiling point to a completely dissolved medium. The mixture was sterilized by autoclave at 15 lbs pressure, 121 o C for 15 minutes. The medium was left to cool to 45-50 o C. The medium can be enriched with 5-10% blood or biological uids. The mixture was well-shaken and poured sterile Petri plates.

Microbial Susceptibility Testing
The antimicrobial activity of the investigated plant extract was estimated by lter paper disc technique (Murray

Results And Discussion
In this research, we aim to explore the reactive chemical components of the essential oil of the methanol extracted C. pycnocephalus that are considered as the major factors affected on the biological aptitudes such as antioxidant, cytotoxic, and antibacterial activities. Each type of biological character is affected by a signi cant class of compounds that control its biological behavior i.e. the phenolic contents are crucial for potent antioxidant characters.
Alternatively  Once upon a time, medicinal plant extracts have been applied in the treatment of various diseases, where the discovery of medicines lies in the presence of their active essential components in the contents of the plant extract, which led to an advance in pharmacology. The C. pycnocephalus plant has been widely used in traditional medicine for the treatment of many diseases.

Antioxidant Activity -Dpph Assay
As commonly reported, the antioxidant activities of C. pycnocephalus extracts were evaluated by DPPH • (2,2′diphenyl-1-picrylhydrazyl radical) colorimetric assay as the most commonly used assay for the extracted plants. The procedure is usually applied for the hydrophilic antioxidant constitutes, while their application for evaluating the antioxidant capacity of the hydrophobic components is limited. In this method, the reactive components that provided potent radical scavenging activities should contribute with a weak A-H bonding, which will increase the possibility for stabilizing or trapping the free radicals of DPPH • at a maximum wavelength 517 nm resulting in a discoloration of the DPPH molecule. The violet color of the DPPH radical will change subsequently to colorless by increasing the concentration of the scrutinized samples. The importance of antioxidants arises from the ability of these components to inhibit the oxidation of lipids. The process followed by scavenging or trapping the free radicals of DPPH, and hence determining the radical scavenging activity, as well as the assay in another route is an indication for reducing the capacity of the antioxidants in their reactions with DPPH radical.
The inexpensive reagents applied to enable to run of the investigated sample by this pro cient colorimetric assay (Blois 1958 Table 2 consistently also presented the radical scavenging activity (%) of the extracted root, stem, leaves, and ower of C. pycnocephalus plant. Figure 3 indicated the plotted percentages of radical scavenging activity against the various concentrations of each tested sample of C. pycnocephalus extract. A linear correlation between the scavenging activity % and their applicable concentrations (5-50 mg/L). The radical scavenging activity percent increased by increasing the sample concentration in a proportional relationship. Besides, the stem extract has the most potent scavenging activity for DPPH radicals at the lower concentration (5 mg/L) with % scavenging radical activity at 17.14±1.01%, however, the root extract located the second-order of activity with % scavenging radical activity at 12.58 ± 0.74% at the same concentration.

Potential Antibacterial Activity
The antibacterial activity of the extracted botanical ingredients of C. pycnocephalus from methanol was assessed by a disc diffusion technique as an in vitro antimicrobial susceptibility testing. The tested samples were prepared in a concentration of 10 mg/L from the root, stem, leaf, and ower extracts. The results are shown in Table 3, and Figure 5 revealed that the four samples are potent antibacterial agents against E. coli, P. aeruginosa, S. typhimurium, and B. cereus bacterial species. Particularly, potent antibacterial activities were recorded for leaf, and ower extracts against E. coli species with inhibition zones equivalent to that of antibiotic standards (20 mm). The root and stem extracts revealed remarkable antibacterial activities against P. aeruginosa species with inhibition zones 22, and 20 mm, respectively, with high potency than the antibiotic standard "Azithromycin" (13 mm), along with good activities of the leaf, and ower extracts with inhibition zones at 10 mm.
By studying the results of the antibacterial activities of samples as inhibitors of bacterial growth, we found that the four samples had distinct activities in the process of inhibiting the growth of bacterial species of the type S. typhimurium by 14, 13, 26, and 25 mm, respectively, relative to the results of the antibiotic "Tetracycline" (10 mm). Medium to high activities of the four samples was observed against S. epidermidis bacterial strains compared to the results of the four antibiotics, while the highest result of inhibiting the growth of those bacterial species was for the ower extract with an inhibition zone at 15 mm. Looking at the results of the four samples as components of C. pycnocephalus, we found that the activity of methanolic extracts against Grampositive bacterial species is not good compared to their results with Gram-negative bacteria. This does not indicate the lack of quality of those extracts to inhibit the growth of Gram-positive bacteria, as some extracts have higher results than antibiotics such as extracts of leaves and owers towards inhibiting the growth of bacterial species of type B. cereus by an amount of 23 and 25 mm, respectively, and these results are higher than that obtained by all the antibiotics (5-20 mm) ( Table 3). Among the good results are also a high activity of inhibiting the growth of S. aureus of the ower methanolic extract by an amount of 14 mm and the good activity of leaf and ower extracts as inhibitors of K. pneumonius growth with an amount of 13 mm compared to the highest inhibitory activity of the antibiotic "Tetracycline" with an inhibition e ciency of 20 mm. The results also demonstrated that the lowest e ciency to inhibit the growth of bacterial microorganisms was found against S. haemolyticus and S. xylosus using any of the four extracts with inhibition e ciency from inactive to 5 mm ( Table 3).
The mechanism of action for the bacterial infections (Sawa, et  Cytotoxic Activity The cytotoxic activity of C. pycnocephalus methanol extract was assessed using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay under distinct conditions. The method is identical to a cell number growth curve. The MTT reagent as a reliable indicator is so sensitive to the light, this engaged the run of the experiments in the dark. Hepatocellular carcinoma (HepG2) tumor cell line was selected to assess the anticancer potency of the investigated extracted medicinal plant. The method was applied for determining the cell metabolic activity based on the aptitude of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the MTT tetrazolium dye to its formazan "insoluble" that has a purple color. The number of viable cells should increase with growth, decrease with cytotoxic treatments, and remain the same (or plateau) with cytostatic treatments. The IC 50 values expressed the concentration that represented 50% of the inhibition of cell growth was calculated by applying the curves obtained from plotted the percentages of cell survival versus drug concentration (µM). Thus, the potency of cytotoxicity will rise by the decrease in the extract concentration and IC 50 values. The MTT solution may be affected by the results of cytotoxicity, so we have run a control sample "blank" that was a few "empty" wells containing MTT solution without any of the cell lines. The control sample is a bene t for calculating the cell viability percent, as it produces 100% viability of healthy cells. The experiments were run using ve concentrations of each plant extract (31.3, 62.5, 125, 500, and 1000 µg/mL) prepared in a serial dilution (Table  4).   EC 50 of C. pycnocephalus extract The dose-response relationship of the assessed C. pycnocephalus extract is plotted in Figure 6. The doseresponse curve in Fig. 6a was normalized in the X-axis direction by its EC 50 value (Fig. 6b). The value of EC 50 of the methanol extract of C. pycnocephalus was initially calculated by plotting the sample absorbance against the log of doses at different concentrations of the serial dilution (Fig. 6). The low concentrations of the extract are not enough to produce a response, while the high doses produce a maximal response, and the vertical point of the curve resembles an EC 50 value. The data analysis speci ed that the higher concentration (dose = 1000 µg/mL) as calculated for EC 50 value (2.82 µg/mL) has a cytotoxic effect on HepG2 cell lines.

Conclusion
C. pycnocephalus plant is related to Asteraceae family displayed a wide spectrum of biological activities (Rustaiyan & Faridchehr 2021;Conforti et al. 2008). In this work, we used the extract of C. pycnocephalus as a medicinal plant or any of its plant parts such as the stem, leaves, owers, and roots to identify the chemical components that were isolated from the essential oils of the methanolic extract to study the effect of these components on the different biological results. We found that the four parts of the plant have high antioxidant capacities compared to the results of ascorbic acid as a result of these extracts containing a high percentage of phenolic components. Higher than antibiotics. Through that study, we discovered the e ciency of the plant extract as a cytotoxic agent with a moderate potency against the growth of hepatocellular carcinoma (HepG2), which indicated that the drug will not affect the growth of normal cells. The results also speci ed that the extracts of different parts of the plant, especially the that of leaves and owers, have high and distinctive antibacterial activities to kill or inhibit the growth of the bacterial species such as E. coli, S. typhimurium, and B. cereus strains with higher e ciency than antibiotics. The effective chemical constitutes that were isolated from C. pycnocephalus.

Figure 2
Chromatogram and structures of basic components of the essential oil of the extracted C. pycnocephalus by GC-MS.  The inhibition zones were raised from the effects of the extracted root, stem, leaf, and ower of C.
pycnocephalus on the plates seeded with different bacterial species by disc diffusion technique. R≡ Root extract; S ≡ stem extract; L ≡ Leaf extract; F ≡ Flower extract. Figure 6