Herbal remedies are humanity’s earliest medical practices. Early civilizations heavily relied on them, and they remain the predominant form of treatment globally. Plants exhibit various health benefits such as combating microbes, reducing oxidation, fighting cancer, managing cholesterol, supporting heart and nerve functions, enhancing the immune system, reducing inflammation, and alleviating pain and fever, among others. These plants generate secondary compounds evolved from primary ones, serving as a vital foundation for numerous medicinal drugs [30].

Earlier research on C. arvensis identified its content of elements like alkaloids, phenolics, flavonoids, carbohydrates, sugars, mucilage, sterols, resin, tannins, unsaturated sterols/triterpenes, lactones, and proteins. Further studies indicated that C. arvensis has properties such as antioxidant, vasorelaxant, immune-boosting, antibacterial, anti-diarrheal, diuretic effects, and shows cytotoxicity against both human cancer cells (HELA) and Lymphoblastic Leukemia Jurkat cells [19, 24, 31, 32].

Herein, the main detected phytoconstituents using LC-ESI-MS in negative ion mode can be classified into phenolic acids, flavonoids, lipids, and terpenes (Table 1).

The first compound of phenolic acid (tR= 12.02 min) was putatively assigned as quinic acid, which afforded pseudo-molecular ion at m/z 191 [M-H]−, other fragments at m/z 173 and 97 [25]. Compound 2 (tR=16.16 min) afforded peaks at m/z 475 [M-H]−, 353 [M-H-122 (benzoyl)]−, 191 and 137 corresponding to benzoyl-caffeoylquinic acid. Compounds 3 and 4 (tR=18.83 and 19.10 min, respectively) were characterized as 5-O-caffeoylquinic acid and 4-O-caffeoylquinic acid, respectively because the identical [M-H]− ion is present at m/z 353 and same fragmentation pattern (m/z 191, 179, 135) but with different ion intensities [33]. Compounds 5, 7, 8 and 9 (tR= 20.70, 23.50, 25.23 and 26.04 min, respectively) were identified as a 3-O-caffeoylquinic acid dimer, 5-O-caffeoylquinic acid dimer, 5-O-caffeoylquinic acid dimer isomer and 4-O-caffeoylquinic acid dimer, respectively. They exhibited signal at m/z 707 [2 M-H]− combined with additional fragments at m/z 353, 191, 179, and 135 resulting in the typical fragmentation pattern of caffeoylquinic acid [34, 35]. Compound 6 (tR= 21.23 min) afforded molecular ion at m/z 705 [M-H]− and m/z 513 [M-H-192]− due to neutral loss of the quinic acid unit. Further signal at m/z 339 [M-H-192-174]− due to further loss of a dehydroquinic acid unit. Therefore, it was assigned as a 5-O-caffeoylquinic acid dehydrodimer [36].

Compound 11 (tR= 27.10 min) was pinpointed as caffeic acid based on its specific [M-H]- at m/z 179 and its fragmentation characteristics. While, Compound (tR= 2550.47 min) appeared to be a derivative of caffeic acid, given its [M-H]- at m/z 423. Compound 12, (tR= 28.30 min) was recognized as coumaroylquinic acid, evidenced by its [M-H]− ion at m/z 337 and fragments at m/z 191, 173, 93, and 85. Compound 14 (tR= 29.77 min) afforded a molecular ion signal at m/z 367 [M-H]− and other fragments at m/z 191 and 173, pinpointing as feruloylquinic acid. Lastly, Compound 22 (tR= 40.86 min) displayed a precursor ion at m/z 193 [M-H]−, which is typical for ferulic acid [25, 36, 37]. It was noted that caffeoylquinic acids are the most abudant phenolic acids group in C. arvensis 85% MeOH extract. These compounds can be classed into four major classes: mono, di, tri, and tetracaffeoylquinic acids. Also, It widely found in a variety of plants as well as in different food staff, including fruits, vegetables, coffee, and spices [38]. Further, It have a wide range of bioactivities, such as antioxidants, antibacterial, anticancer, antiparasitic, antiviral, antidiabetic, antiinflammatory, and neuroprotective effects as well as the reduction of some chronic and cardiovascular diseases [39, 40].

Most flavonoids detected in C. arvensis were flavonoid glycosides tentatively assigned as quercetin and kaempferol derivatives such as, compound 15 (tR= 33.25 min) displayed the [M-H]− at m/z 741, 609 [M-pentosyl-H]−, 301[M-pentosyl-2hexosyl units -H]−, 179 of quercetin aglycon. Thus, this compound characterized as quercetin-O-pentosyl-dihexosides. Compound 17 (tR= 36.58 min) represented a precursor ion at m/z 609 [M-H]− and produced fragments at m/z 463 and 301 revealing the loss of deoxyhexosyl and hexoside moieties, respectively. Therefore, it was assigned as quercetin-3-O-rutionside (rutin) which was detected in C. arvensis before [41]. Compound 18 (tR= 37.39 min) was identified as quercetin-O-hexoside due to presence of [M-H]− at m/z 463 and other peaks at m/z 301, 179 and 151. Compound 20 (tR= 39.65 min) revealed a molecular ion at m/z 607 [M-H]−, that gave fragment ions at m/z 505 [M-H-18-44-40]− revealed the loss of H2O, CO2, and C3H4, respectively. Followed by fragment ions at m/z 463 [M-H-18-44-40-144]−due to the release of 3-hydroxy-3-methylglutaryl moiety and m/z 301[M-H-18-44-40-144-162]− which reflect a further neutral loss of hexoside unit. Hence, it assigned as quercetin-7-O-[3-hydroxy-3-methylglutaroyl] hexoside. In addition, all quercetin derivatives were detected in C. arvensis and C. althaeoides leaf extracts before [24, 25, 42]. Compound 16 (tR= 34.18 min) was tentatively identified as apigenin-C-hexoside-O-pentoside due to the presence of [M-H]− at m/z 563 with distinctive fragments at 443, 413 and 293 [43]. Compound 21 (tR= 40.19 min) displayed the deprotonated molecular ion at m/z 593, and yielded a main fragment at m/z 285 due to loss of rutinoside moiety. Thus, this compound was assigned as kaempferol-3-O-rutinoside and detected in C. arvensis whole partsand C. dorycnium L. flowers extracts before [25, 44]. From the perivious data, it was observed that quercetin derivatives are the major flavonoid in this plant. Among them, quercetin-3-O-rutinoside (rutin) which the main constituent of C. arvensis as shown in Table 1; Fig. 1. Rutin is a low molecular weight flavonol-type flavonoid. It has various pharmacological properties such as antioxidant, antitumor, antiinflammatory, antibacterial, antiallergic, antiprotozoal, hypolipidaemic, cytoprotective, antispasmodic, antiviral, antiulcerogenic, and antihypertensive. Therefore, it can be found in a huge number of herbal multivitamin preparations and called as vitamin P [45].

Compound 10 (tR= 26.57 min) afforded [M-H]− at m/z 387 with a main fragment at m/z 161, which is characteristic of magastigmane glycoside derivative as reported by Abdul Khaliq et al. [25]. Megastigmanes compounds are oxygenated isonorterpenoids and frequently referred to as oxidative by-products from β-carotenoids. In addition, magastigmane glycosides have several bioactivities including antioxidant, antibacterial, antiinflammatory, anticancer, and hepatoprotective activities [46].

Compound 26 (tR= 58.61 min) gave [M-H]− at m/z 327and produced major fragments at m/z 291, 229, 211, 171 which lead to the tentative characterization of this compound as trihydroxy-10,15- octadecadienoic acid derivative which relative to glycolipids. For our knowledge, compounds 10 and 26 were detected before in C. arvensis extracts [25].

Likewise, the total area percent of identified components in the 85% MeOH ext. of C. arvensis was 99.99% as shown in Table 1. It was noted that the phenolic acids are the highest area percent, followed by flavonoids, glycolipids, unknown compounds and sesquiterpenes, respectively as shown in Fig. 2. Among them, the major constituent in the 85% MeOH extract was rutin (33.64%) followed by 5-O-caffeoylquinic acid dimer (29.42%), ferulic acid (7.74%), 3, 4-di-O-caffeoylquinic acid (6.91%), and 4, 5-di-O-caffeoylquinic acid (5.81%). Hence, the bioactivities of C. arvensis 85% MeOH extract and its fractions may be due to their richness in polyphenolic compounds.

The cytotoxic, anti-cancer, anti-inflammatory and anti-bacterial studies were conducted on both the C. arvensis MeOH ext. and n-BuOH fr. and their loaded Alg-Cs/NPs.

At first, alginate and chitosan are examples of natural polymers that are suitable for the assembly of NPs because of their many benefits such as fast gelation, enhanced biocompatibility, and reduced toxicity. Nevertheless, they are used in hydrogel for objectives including regulating medicine release and tissue engineering [47].

The small particle size C. arvensis extracts loaded with Alg-Cs/NPs were expected to be lower than 100 nm in this study as a result of increasing the stirring speed of Alg-C. arvensis mixture before and during the addition of CaCl2 and chitosan solutions. In the present study, the average diameters of the prepared MeOH-NPs and n-BuOH-NPs were 90 ± 4.8 nm and 92 ± 5.5 nm respectively. While that of unloaded Alg/Cs polyelectrolyte NPs was 55 ± 3.9 nm, the bigger size of C. arvensis-NPs compared to empty ones indicates the incorporation of the drug inside the NPs.

Katuwavila et al. [48] designed a natural, biodegradable, and biocompatible polysaccharide nano-delivery method for the medication doxorubicin, particularly chitosan and alginate. The sizes of Cs-Alg NPs and doxorubicin loaded Cs-Alg NPs were 100 ± 0.35 and 100 ± 28.0 nm. First, chitosan droplets were formed by mixing with Tween 80. Afterward, these droplets were solidified through ionic crosslinking using an alginate solution. This step may be the reason to the smaller particle size. The larger particle size obtained by DLS measurements compared to TEM could be due to the swell ability of polymeric hydrogels in the solution as it was previously mentioned by Bhattarai et al. [49]. In a study conducted by Yin et al. [50], the team sought to create composite nanoparticles smaller than 200 nm. They examined how different component contents and process parameters impacted the particle size, swelling behavior, and Cs release rate from the carrier materials. The specific mixture chosen for their research, at a chitosan to sodium alginate ratio of 2:1, resulted in a shimmering suspension with a positive zeta potential. This combination was found optimal for encapsulating doxorubicin, achieving an impressive 95% encapsulation efficiency. Their findings align with a prior study by Sohail and Abbas, [51], where they developed a drug delivery system for amygdalin using anionic Cs-Alg NPs. Their particle sizes ranged between 119 ± 19 nm and 261 ± 18 nm, primarily targeting anti-cancer applications.

The TGA results of this study may be due to the easy loss of anion and ammonium groups in the Alg/Cs polyelectrolyte complex (Hofmann elimination) [52]. However, the nanoparticles containing C. arvensis extracts show that the nanoparticles possessed improved thermal stability contrasting Polymers with its polymeric nanoparticles without C. arvensis. This phenomenon further illustrates that a portion of C. arvensis accumulated on the polymer shell of the nanoparticles, which prevents the polymer shell during thermal decomposition. The results of this report agreed with the previous publication [53], who assumed that the Prussian blue formed in the polymer shell hindered the thermal degradation of polymeric nanocapsules.

The previous studies on C. arvensis did not evaluate the hemolytic effect on the RBCs, whereas the anti-oxidant, anti-microbial and anti-cancer activities were discussed [19, 54,55,56]. The CS and Alg polymers were attributed to having low toxicity and hemocompatibility [57]. San et al. [58], suggested that both the unbound TO and its combination with TO-FA-Cs/Alg-NPs demonstrated minimal hemolytic activity (5%). However, when exposed to the maximum TO concentration (7.5–10 mg/ml), this behavior changed, suggesting that they might be suitable for human use. Further tests are recommended, especially for applications involving blood contact. In this study, both the MeOH ext. and n-BuOH fr. of C. arvensis and their loaded Alg-Cs/NPs exhibited non-hemolytic activities (percent hemolysis, 3–12%) even at maximum concentration of 1000 µg/ml. The work’s outcomes concurred with those of the earlier investigation reported by Wiya et al. [59]. They stated that the hemolytic effect of the both the aqueous and ethanolic slime extracts ranged from 7.01 ± 0.54 to 13.42 ± 0.28%.

The previous studies conducted to estimate the anti-inflammatory effect of C. arvensis, it was observed that in aseptic arthritis brought on by carrageenan, the gel containing the extract of C. arvensis has an expressed anti-oxidative action. Furthermore, when administered topically to the site of pain, the anti-oxidative activity of the product containing the extract of C. arvensis is comparable to that of ibuprofen [56]. Another study revealed that that EtOH ext. of C. pluricaulis Choisy possesses strong analgesic and anti-inflammatory properties [60]. In this study, both the MeOH ext. and n-BuOH fr. of C. arvensis and their loaded Alg-Cs/NPs showed excellent anti-inflammatory activity compared to the standard drug (diclofenac).

The previous studies conducted to estimate the cytotoxic activity of C. arvensis showed that, the chloroform extract of C. arvensis exhibited a cytotoxic impact akin to taxol when tested on Hela cells (15 vs. 12 µg/ml). This heightened cytotoxicity from the chloroform extract might be due to the extraction of lipophilic glycosides by chloroform, which are non-polar compounds [31]. Moreover, anti-bacterial, anti-cancer and toxic activities of C. arvensis, C. austro-aegyptiacus and C. pilosellifolius extracts were tested against clinical samples, various cell types, and specific lab animals. Notably, C. austro-aegyptiacus displayed significant antimicrobial action. In comparison to vinblastine sulphate’s anti-tumor activity (30.3 ± 1.4) against CACO (colorectal carcinoma), both C. arvensis and C. pilosellifolius presented anti-tumor effects of (6.1 ± 03) and (16.4 ± 0.3), respectively [52]. Additionally, C. arvensis’s EtOH extract had a strong cytotoxic effect on the cancer cell line Jurkat lymphoblastic leukaemia cells. Moreover, at lower extract doses, apoptotic effects were observed, while necrotic outcomes were prominent at higher concentrations [32].

In this study, it was observed that the MeOH extract and n-BuOH fraction of C. arvensis and their loaded Alg-Cs/NPs have admirable anti-cancer activity compared to the standard drug doxorubicin. Also, the encapsulation of both the MeOH ext. and n-BuOH fr. of C. arvensis has enhanced their anti- cancer activity and lowered the IC50 from 153.7and 141.1 µg/ml for methanolic and n-BuOH extracts to 93.11and 86.54 µg/ml, alternatively. Moreover, the encapsulation of the MeOH ext. and n-BuOH fraction thought Alg-Cs/NPs has enhanced the safety of C. arvensis on normal cells and actually increased the viability of Vero cell lines by 10%.

The findings align with prior research demonstrating the anti-microbial properties of C. arvensis against both gram-positive and gram-negative bacteria [19, 55, 61]. The assessment of anti-microbial efficacy was performed on clinically relevant pathogenic bacteria (E. coli and S. aureus) by using MICs tests and disc diffusion. Nearly the same inhibition zone (22 ± 0.82 mm) against S. aureus compared to 21.35 ± 0.76 mm. Furthermore, the encapsulation of the aqueous and ethanolic extracts thought Alg-Cs/NPs enhanced the anti-microbial effects of C. arvensis and actually decreased the MIC from 31.25 to 7.78 bacteria.

The treatment’s effectiveness and cellular absorption can be significantly impacted by the size, shape, and surface chemistry of NPs revealed that the obtained C. arvensis- Alg/Cs-NPs had a suitable size for use in therapeutic applications and could be useful for the treatment of liver cancers [62].