Traditional Uses, Bioactive Compounds, and Pharmacological Investigations of Calendula arvensis L.: A Comprehensive Review

Calendula arvensis L. (Asteraceae) is a famous ornamental and medicinal plant widely distributed in Mediterranean countries and the southern region of Europe. This reputed species is widely used in traditional medicine in the treatment of many disorders and has various bioactivities, especially anti-inflammatory, antiviral, antimutagenic, antimicrobial, insecticidal, antioxidant, and immunomodulatory activities. The present review was conducted to provide a critical review of the comprehensive and current knowledge regarding C. arvensis species, in particular, its taxonomy and geographical distribution, botanical description, medicinal uses, phytochemical compounds, pharmacological properties, and toxicity investigations. The data collected on C. arvensis were obtained using different scientific research databases such as PubMed, SciFinder, SpringerLink, Web of Science, Science Direct, Google Scholar, Wiley Online, and Scopus. Phytochemical screening of different C. arvensis extracts and essential oils showed their richness in bioactive compounds, particularly in fatty acids, sterols, phenolics, flavonoids, saponins, tannins, alkaloids, and terpenoid compounds. The findings of this review showed that the pharmacological activities of C. arvensis confirm its importance and diversity as a traditional remedy for many diseases. This plant presents a wide range of bioactivities, namely, anti-inflammatory, antimicrobial, antitrypanosomial, antitumoral, antimutagenic, and immunomodulatory activities, as well as hemolytic properties and wound treatment. Nevertheless, pharmacokinetic validation and toxicological examinations are required to detect any possible toxicity for future clinical trials.


Introduction
C. arvensis, commonly known as "marigold," is one of the important medicinal herbs belonging to the Calenduleae tribe of the Asteraceae family [1]. It is widely distributed in Mediterranean countries as a native plant [2], usually indigenous to the southern region of Europe, namely Spain, Portugal, Portugal, Turkey, Greece, Malta, and Italy [3], and also cultivated in other parts of the globe such as California and Australia [4]. It is an annual herbaceous plant reaching 100 cm in height [5] and it grows in waste grounds, vineyards, and felds [4]. Its leaves are lance-shaped and have tectorial and secretory trichomes. Te inforescence consists of a single fower head reaching 4 cm with yellow or yellow-orange capitula (blossoms are synthesized throughout the year, but the fowering peaks are between March and July) [6,7]. Based on the polymorphism of stems, fowers, and achenes, several subspecies were assigned to C. arvensis [7], namely, C. arvensis var. parvifora, C. arvensis subsp. hydruntina, C. arvensis subsp. macroptera, and C. arvensis subsp. arvensis [8]. C. arvensis is distinguished from others by the presence of rostrate/bialate [4]. Te karyological evidence on C. arvensis revealed a 2n � 44 and a genome size of 5.41 pg [4,9]. Phytochemical screening of several essential oils (EOs) and extracts isolated from diferent parts of C. arvensis demonstrated the abundance of bioactive compounds in this plant. Tese include alkaloids, tannins, saponins, favonoids, phenolic compounds, sterols, and fatty acids [10][11][12][13]. Te phytochemical content of C. avensis fowers was characterized by a high content of linoleic acid, palmitic acid, and linolenic acid in the extract [10]. Te volatile compounds identifed from steam distillation extraction of C. arvensis were represented by aldehydes, esters, sulfur compounds, alkyl pyrazines, ketones (1-octen-3-one), lactones (δ-decalactone), and terpenoid compounds (α-terpineol and citronellol) [11]. In traditional herbal medicine, C. arvensis was reported to treat many disorders depending on the part used. It can be used as a cataplasm [14] and antidiabetic drug [15,16], and also against neurological disorders, microbial afections, ENT (Ear, Nose, Troat) diseases, typhoid diseases [17], skin diseases [18], digestive disorders [19], high blood pressure [20], and rheumatic conditions [21], as well as an antispasmodic [22], antiemetic [23], antihelmintic, diaphoretic [18], and antifungal [24] agent.
In Morocco, C. arvensis is among the famous plants used in folk medicine, the fowers are the main part used in several regions to treat infammation and infection in infusion [14,16]. In addition, fowers and leaves are used as antidiabetic drugs in the northern regions of Morocco [15,16,25]. However, in the southern regions, C. arvensis is used in traditional medicine to treat neurological, microbial afection, ENT, and typhoid conditions [17], as well as in wound healing [26]. In European countries such as Spain, Italy, and Greece ethnobotanical surveys showed that the aerial part of this plant is used as an emmenagogue, aperitive [27], and ecchymotic [28], and also has an antiseptic efect [29]. Using decoction, the inforescences have healing [30], emmenagogue, diaphoretic, diuretic, sedative, and antiinfammatory efects [31]. In Turkey, the aerial part of C. arvensis was used as an infusion, mash, lotion, maceration, or oil to treat wounds, burns, and skin cancer, and also for skin care and athlete's foot [32,33]. Te use of leaves and capitulum of this herbaceous plant can be used as a poultice to treat skin diseases. C. arvensis fower decoction was used for its hepatoprotective, germicide, and fungal efects [34].
In Asia, C. arvensis leaves and fowers in decoction oil form have traditional antirheumatic [21], astringent, and antispasmodic efects [35,36], with expectorant properties, and also relieve varicose veins [35]. Moreover, these parts have anthelmintic efects and are used as a tonic and diaphoretic agents [37]. C. arvensis is an ornamental, medicinal, and industrial plant. Pharmacological investigation of C. arvensis leaf EOs and extracts has revealed a broad range of biological properties. Its phytochemical diversity has demonstrated varying levels of antimicrobial efect on various microorganisms. Te antimicrobial efect of methanol/chloroform mixture extracts and aqueous extracts of C. arvensis leaves was assessed against six bacterial strains (Enterobacter aerogens, Bordetella bronchiseptica, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Bacillus subtilis) and fve fungal strains (Aspergillus niger, Mucor spp., Aspergillus fumigatus, Fusarium solani, and Aspergillus favus) [38][39][40]. Moreover, C. arvensis extract showed good antibacterial activity against Mycobacterium smegmatis with minimal inhibition concentration in the range of 13.2-62.5 μg [41]. Furthermore, C. arvensis aerial part EO showed antifungal activity against Aspergillus niger and Penicillium expansum [38] and did not show any interesting inhibitory efect against yeasts and dermatophyte strains [42]. Regarding the antiparasitic efect, the saponin substances arvensoside B and arvensoside D extracted from C. arvensis showed an impressive impact on the parasites of Trypanosoma brucei [43]. Additionally, several studies have proven that treatment with C. arvensis extracts has excellent anti-infammatory potential by inhibiting edema formation and managing pain conditions [44,45]. C. arvensis extracts and isolated molecules have also shown efective antioxidant activity in scavenging free radicals, as reported in several investigations [10,46,47]. On the other hand, various concentrations of the extracts of C. arvensis parts (leaves, 2 Advances in Pharmacological and Pharmaceutical Sciences stems, and fowers) using diferent solvents (methanol, ethyl acetate, chloroform, and hexane) recorded potent activities against the proliferation and growth of tumor cells, using a cancer cell model [10,47,48]. Enzymatic inhibitory efects of C. arvensis leaf and fower extracts have also been revealed and could be related to the presence of compounds derived from triterpenes and favonoids identifed in the diferent parts of this species [49]. Several other pharmacological activities of C. arvensis were also reported. As mentioned above, this medicinal herb could exhibit wound healing and immunomodulatory activities capable of activating lymphocytes and pronouncing blastogenesis and can be considered a potential immune booster [48].

Results and Discussion
3.1. Botanical Description. Calendula arvensis, as depicted in Figure 1, is an annual herb that can reach 100 cm in height [5]. Te leaves are lance-shaped and possess secretory and tector trichomes. Te inforescence consists of a single fower head up to 4 cm with yellow or yellow-orange capitula (blossoms appear throughout the year, but fowering peaks between March and July) [6,7]. Te peripheral fowers of the capitula (ligulate) extend over several rows (verticils) and are female; central fowers (tubular) are complete but functionally male [6]. Te achenes are only produced by the ligulate fowers. Te pollen is echinate, tetracolporate and monadic. Te shape of pollen in the equatorial view is circular to perprolate and the polar view is rectangular [50]. Tus, a complex polymorphism is related to the position of the fowers in the capitula, with three achene types (cymbiform, rostrate, and annula) (Figure 2), with a stem size of 5-60 cm and a leaf width of 5-20 mm [6]. Due to stem, fower, and achene polymorphism, diferent subspecies were assigned to C. arvensis [7], viz., C. arvensis var. parvifora, C. arvensis subsp. hydruntina, C. arvensis subsp. macroptera, and C. arvensis subsp. arvensis [8]. Te karyological data on C. arvensis showed a 2n � 44 and a genome size of 5.41 pg [4,9]. C. arvensis is diferencied from others by the presence of bialate/rostrate [4].

Taxonomy and Geographic Distribution. C. arvensis
belongs to the Calenduleae tribe of the Asteraceae family which includes about 25 annual and perennial species [1]. Te genus Calendula consists of about 15 species in the Mediterranean, Saharo-Arabian, and Irano-Turanian regions [51], native to the Mediterranean countries [2] from Macaronesia to Southeast Asia [4], and generally native to the southern region of Europe, including Spain, Portugal, Turkey, Greece, Malta, and Italy [3]. Te genus Calendula belongs to other parts of the globe such as California and Australia [4]. C. arvensis, a feld marigold, is abundant in spring, annually, and weeds. It is found gregarious in feld margins, graveyards, roadsides, wastelands, and open places [47]. It grows in waste grounds, vineyards, and felds [4].

Ethnomedicinal Use.
Several ethnobotanical studies have marked the importance of C. arvensis in traditional medicine to treat many disorders. Te traditional use of C. arvensis is related to the part used. Indeed, diferent parts of C. arvensis have traditionally been used to treat diferent diseases. Table 1 lists the application of C. arvensis in traditional pharmacopeia global systems. Te fowers represent the main used part in Moroccan traditional systems in several regions to heal various disorders. Te population of the Targuist region (Northern Morocco) used the fowers of C. arvensis as a cataplasm against infammation and infections [14]. Te population of Rabat-Sale-Kenitra regions (Morocco) used fowers and leaves as an antidiabetic drug [15]. Also, the same use of this  plant against diabetes was reported by an ethnobotanical study conducted in the Rif region (North of Morocco) [16] and in the Taza region (North-East of Morocco) [25]. Chaachouay et al. [16] reported the use of the fowers of C. arvensis in infusion, while Naceiri Mrabti et al. [25] reported the use of fowers and stems of C. arvensis in decoction. In the northwest of Morocco, the population of Oulad Daoud Zkhanine (Nador Province) used the decoction of whole plants against digestive disorders as well as for hair care [19]. Other ethnobotanical surveys, conducted in the Aguelmouss region (Khenifra Province, Morocco), have reported the importance of C. arvensis in traditional medicine, without mentioning the part used and the mode of preparation, in healing [26], and in the treatment of neurological and microbial conditions, and diseases related to ENT and typhoid [17]. Tese last two sections are important knowledge in the database ethnobotanical studies, which directs researchers to target a specifc part in the purpose to elaborate the phytochemical screening. Tus, a standard form of ethnobotanical surveys is primordial to conserve traditional herbal remedies.
In Western Algeria, C. arvensis fowers in infusion were used for depurative, emmenagogue, and antispasmodic effects as well as for stimulating hepatic activity and bile secretion. By maceration, the leaves were used to treat internal ulcers, warts, fstulas, frostbite, calluses body, and skin lesions and to calm vomiting [60]. Te fowers of the courant plant were used by the M'Zab Valley population (Algeria) to treat high blood pressure [20]. Another ethnobotanical survey in the Bissa region (north-east of the Dahra mountains in Algeria) indicated the therapeutic efects of C. arvensis leaves and roots on rheumatism [74]. In the mountains of Tlemcen (Western Algeria), this plant has been reported to exert disinfectant, anti-infuenza, antitranspiration, and antihemorrhagic efects, as well as to treat hemostatic and hepatic actions [63], thus this plant is traditionally known in the Tiaret mountains (Western Algeria) as a depurative, emmenagogues, and antispasmodic [59].
Te population of Alaşehir (Manisan, Turkey) and Sarigöl district (Manisa, Turkey) used the aerial part of C. arvensis as an infusion, mash, lotion, maceration, or oil to treat wounds, burns, skin cancer, and also for skin care and athlete's foot [32,33]. Uzun and Kaya [76] confrmed the use of leaves and capitulum of this plant by the population of Mihalgazi (Eskişehir, Turkey) as a poultice to treat skin diseases. Te decoction of C. arvensis fowers was used by the Erzincan population (Turkey) as a germicidal, hepatoprotective, and fungal agent [34]. A recent ethnobotanical study conducted by Aslan et al. [24] reported the use of C. arvensis fower decoction as an antifungal by the population of Yaslıca town and Arıkök neighborhood in the Bozova district of Şanlıurfa province (Turkey). In Syria's western region (Tartus and Latakia), the aerial part of C. arvensis was used to treat varicose veins, sore eyes, wounds, sprains, stings, bites, and skin problems [72].
In European countries, several authors have reported the traditional use of C. arvensis to treat diferent illnesses. In Spain, C. arvensis aerial part was used as an emmenagogue and aperitive by the population of the Canary Islands [27]. In the same country, the inforescence (in the form of a liniment) was efective against ecchymotic [28]. Gras et al. [29] showed that the aerial part of the C. arvensis bath exhibited an ocular antiseptic efect. As a poultice or infusion, the fowers of this plant were used in the villages of Lotzorai and Escolca (Sardinia, Italy) as antispasmodic and to treat burns [22]. In decoction, the inforescences have a healing efect [30] as well as an emmenagogue, diaphoretic, diuretic, sedative, and anti-infammatory efects [31]. In 2016, the population of the Mainarde Mountains (central-southern Apennine, Italy) used C. arvensis after maceration or as an ointment against burns, sunburn, chilblains, joint pain, and stings [62]. Te fowers of C. arvensis have been used by the population of Bosnia and Herzegovina against lung and liver cancer, and skin disorders [73]. Another ethnobotanical study conducted in Greece, [61] showed that the fowers, stems, and leaves of C. arvensis were used to treat swollen areas, acne, and diaper rash.
In Pakistan, the traditional use of C. arvensis was confrmed by several ethnobotanical studies ( Table 1). As a poultice, this species was consumed by the population of  Flowers -Strengthen eyesight, treat heart disease, and promote skin healing [55] Madyan Valley in district Swat (Pakistan) Leaves and fowers -Used as an anthelmintic, tonic, and diaphoretic [56] Jatlan Azad Jammu, Kashmir (Pakistan) -Decoction Used as a diaphoretic and antiemetic.

Aerial parts
Infusion, mash, and medical oil Treated skin cancer. Used in burn care [32,33] Erzincan (Turkey)

Decoction
Used as a hepatoprotector, germicide, and fungicide [34] Yaslıca Town and Arıkök neighborhood in the Bozova district of Şanlıurfa province (Turkey) Flowers Decoction Fungal treatment [24] Targuist area (North of Morocco)

Leaves and capitulum
Poultice Treat skin diseases [76] South-West of Pakistan Leaves and stem Infusion Treat diabetes [77] 6 Advances in Pharmacological and Pharmaceutical Sciences Tehsil Barawal, Upper Dir, Khyber Pakhtunkhwa (Pakistan) as a tonic and infammatory agent against eye troubles and oral sores [53]. Te fowers of C. arvensis were used by the population of Attock district (Pakistan) to strengthen eyesight, heart diseases and to heal the skin [55]. Te same efects were confrmed by Rehman et al. [52] and Jan et al. [69] in the Khattak tribe population of Chonthra Karak (Pakistan) and by the Kohistan Valley population, Khyber Pakhtoonkhwa (Pakistan). Another ethnobotanical study of this plant in the tribal communities of North-West Frontier Province (Pakistan) indicated that the leaves of C. arvensis were used for wound healing [36,54]. Khan et al. [70] confrmed the use of C. arvensis as a wound healer by the population of the Sawans Valley Mianwali, Punjab (Pakistan). Tis plant's fowers and leaves were used as stimulant, antispasmodic, astringent, healing wounds and injuries [67], as well as in the treatment of scrofula, diaphoretic, anthelmintic, discharge of mucus, and helminthic [64][65][66].
Other ethnobotanical studies showed that C. arvensis leaves and fowers in decoction oil form have traditional efects as antirheumatic (Tehsil Razzar District Swabi, Pakistan) [21], astringent, and antispasmodic [35,36], with expectorant properties, and also to relieve varicose veins [35]. Ali et al. [37] showed that the Malam Jabba Valley population of Swat (Pakistan) used the leaves and fowers of C. arvensis as an anthelmintic, tonic, and diaphoretic agents. Other ethnobotanical studies at Madyan Valley in district Swat (Pakistan) Malam Jabba, Swat (Pakistan), the Jatlan Azad Jammu, Kashmir region (Pakistan), and the Azad Jammu And Kashmir state (Pakistan) confrmed the anthelmintic, tonic, and diaphoretic actions of C. arvensis [18,23,56,57]. Leaves and fowers of C. arvensis have been known to be used in traditional pharmacopeia by the population of the Karak, Talash Valley of Lower Dir (Pakistan) and Khyber Pakhtunkhwa district (Pakistan) against toothache [58,71,78]. Te leaves and shoots of C. arvensis were used by the population of Chagharzai Valley, district Buner (Pakistan) against skin diseases [75]. Recently, this plant's stems and leaves were used against diabetes in the South-West of Pakistan [77]. In 2020, the population of the Gujranwala region, Punjab (Pakistan) used the leaves and fowers as extract preparation mode to treat severe pains [68].
Pizza and de Tommasi [84] isolated and determined the structure of four triterpenoid saponins from methanolic extract of C. arvensis aerial parts, these four compounds are oleanolic acid (4) (10 mg/2.2 g of MeOH extract). A similar study was elaborated on the same saponin separation pathway in order to identify four compounds 4-O-(β-Dfucopyranosyl)-4-alloaromadendrole (5), and three derivatives of arvoside B. In addition, De Tommasi et al. [87] have isolated the same compounds and two other saponins from the aerial parts of C. arvensi [87].
On the other hand, two new triterpenoid glycosides from C. arvensis aerial parts were identifed as arvensoside A (8) and B (9) by FAB, FAB-MIKE mass spectrometry, and 13 C NMR spectroscopy.
Te study by Vidal-Ollivier et al. on C. arvensis was based on the chemical and physicochemical evidence to isolate four compounds, (8), (9), calenduloside C (10), and D (11) from fresh-aerial parts of C. arvensis, two saponosides of these four compounds showed anti-infammatory activities.
Phytochemical determination of the aqueous and methanolic extracts detected their richness in phenolics and favonoids. Te total phenolic contents ranged from 47.89 ± 2.34 to 50.26 ± 0.18 mg GAE/g DW for the aqueous and methanolic extracts, respectively. Te total favonoid Advances in Pharmacological and Pharmaceutical Sciences Italy Aerial parts   α-terpineol, lactone-like, and δ-decalactone [11] Advances in Pharmacological and Pharmaceutical Sciences 9 France Aerial parts Hydro-ethanolic extract (60%)   Phytosterols β-sitosterol [94] Advances in Pharmacological and Pharmaceutical Sciences 11
Te phytochemical content of C. avensis leaves from Pakistan was studied by Akhtar et al. [49]. Qualitative analysis of phytochemicals showed that this plant contains several secondary metabolites such as alkaloids, saponins, tannins, and terpenoids. For the quantitative analysis of phytochemicals, the total phenolic content of the samples ranged from 20.2 to 85.6 mg/g DW in methanol/chloroform extracts and from 5.5 to 62.1 mg GAE/g DW in aqueous extracts. Te total favonoid content ranged from 2.9 to 44.5 mg QE/g DW of the sample for the methanol/chloroform extracts and from 2.4 to 37.1 mg QE/g DW for the aqueous extracts [38]. Te C. arvensis fower methanol extract was the richest in favonoids (74.14 ± 3.09 mg QE/g extract) and phenolics (118.18 ± 10.29 mg GAE/g extract) [49].
Phytochemical screening of all extracts (distilled water, 50% aqueous/ethanol, ethanol, chloroform, and petroleum ether) was carried out and showed the presence of proteins, alkaloids, terpenoids, and favonoids. Petroleum ether extract has been shown to be rich in favonoids, and the ethanolic extract contained alkaloids, favonoids, and proteins. Additionally, two phytochemical groups' favonoids and terpenoids were revealed in the aqueous/ethanolic extract, and the chloroform extract showed the presence of terpenoids, alkaloids, and proteins. Finally, these substances were not identifed in the aqueous extract [48].
Four saponins were isolated from the dried aerial parts of C. arvensis by Elias and Meo [89] and the identifcation was carried out by mass spectrometry, FAB, 1 H-and 13 C-NMR, and two-dimensional NMR studies. Tese compounds were C. arvensis hexane extracts were analyzed by GC-MS allowing the identifcation of 34 compounds, namely a 1,2saturated pyrrolizidine alkaloid derivative (27) (0.57 mg/g of the dry plant), sesquiterpene 8,14-cedranoxide (28) (0.70 mg/g of the dry plant), and (21) (0.78 mg/g of the dry plant) the only ones detected in this species were the three main compounds of their hexane extract. However, carbohydrates were the most abundant, together accounting for approximately 30.7%, followed by terpenoids and carboxylic acids with 27.9% and 27.4%, respectively [42].
3.5.1. Antimicrobial Activity. C. arvensis is one of the most interesting medicinal plants, its diverse chemical composition displays a varying degree of antimicrobial activity on diferent microorganisms. Jamal et al. [40] showed that C. arvensis leaf extract exhibits an important inhibition on some microorganisms including pathogens using the agar difusion method and the tube dilution method to defne the minimum inhibitory concentration (MIC). Te study showed that the potential of C. arvensis as a source of antibacterial agents can be utilized in the healthcare delivery process. Indeed, C. arvensis chloroform and petroleum ether extract exhibited, respectively, an inhibition at MIC � 2 μg/ mL against Klebsiella pneumoniae and Escherichia coli. Te greatest inhibition zone is produced by the chloroform extract at 512 mg/mL against E. coli. C. arvensis leaf extract was found to have better antibacterial activity in petroleum ether with a zone of inhibition of 1.9 cm. Likewise, the study of Jamal et al. [40] showed that the organic extracts of C. arvensis fowers have an important antibacterial potency evaluated by the agar well difusion method and by the microtitration technique. Except for Acinetobacter baumannii, Proteus mirabilis, and Listeria monocytogenes, the methanol extract of C. arvensis fowers had remarkable activity against the bacteria studied (Grampositive and Gram-negative). C. arvensis fower hexanolic extract inhibited all bacteria except Acinetobacter baumannii, Streptococcus agalactiae, and Staphylococcus aureus MRSA. Te MIC value of the methanol extract was between 12.5 and 25 μg/mL and the MIC value of the hexanolic extract was between 6.25 and 12.5 μg/mL. Terefore, the hexane extract was more potent than the methanol extract. Te hexanolic extracts of C. arvensis fowers obtained by soxhlet extraction were bacteriostatic for all the bacteria studied (E. coli MDR, E. coli, E. coli ATCC, E. coli enteropathogenes, Salmonella braenderup, Salmonella aequatoria, and Salmonella blockley), while aqueous and methanolic extracts obtained by maceration in cold water showed bactericidal activity. Moreover, the extracts studied did not show any activity on Candida species with the exception of the methanolic extract, which inhibited Candida famata and Candida tropicalis with inhibition diameters of 20 and 14 mm, respectively [47]. Zoufan et al. [39] showed the antibacterial efect of ethanolic and methanolic extracts of whole C. arvensis examined by the disc difusion method is greatest against P. aeruginosa, S. aureus, and E. coli.
Te antifungal activities of C. arvensis aerial part EOs and hydrosol extract were investigated against two phytopathogenic fungi; Penicillium expansum and Aspergillus niger using the agar well difusion method. Te obtained results in vitro showed that C. arvensis aerial part EO has an antifungal activity at high concentrations, while the hydrosol extract induced the best inhibition against A. niger and P. expansum. Te essential oil (60 mg/L) completely inhibited the growth of P. expansum with an important inhibitory activity against A. niger (86.6%). At a concentration of 30 mg/L, the hydrosol extract exerted a strong inhibition (100%) against both fungi.
Furthermore, the treatments of pears with C. arvensis L. aerial part hydrosol extract and EO showed a very marked protective activity on the severity of the infection caused by P. expansum. Against rot induced by P. expansum, the hydrosol extract (0.02 mg/L) showed a high protective efect (100%) for up to 7 days. At the highest EO concentration (0.2 mg/L), protective efects of 100% and 40% were observed up to the 7 th and 9 th day, respectively, and this was sufcient to reduce the disease severity [88]. According to Faustino et al. [42], methanol extracts of the C. arvensis sample did not show any interesting antimicrobial activity against Aspergillus, yeasts, and dermatophytes strains tested, and a weak activity was recorded against Microsporum canis and Trichophyton rubrum with both MLC and MIC of 400 μg/mL. Izzo et al. [102] showed that among the 68 extracts tested for their antibacterial action against eight Gram-negative and Gram-positive bacteria, C. arvensis ethanolic extracts possessed activity against only two Grampositive microorganisms (Staphylococcus aureus and Bacillus subtilis), while they were completely inactive on other bacteria. Te aqueous extracts of C. arvensis foliar tissues were examined for their activity against F. oxysporum f.sp. lycopersici isolated from tomato roots using the agar difusion method. Te toxicity of C. arvensis extracts against the fungus was pronounced at 4 days of incubation, with an inhibition diameter of 43.8 mm compared to the control (60 mm), reaching 75.5 mm at 16 days of incubation [103].
Likewise, the antibacterial efect of C. arvensis L., aerial part EO obtained by HD using a Clevenger-type apparatus was investigated against E. coli, P. aeruginosa, Bacillus cereus, and S. aureus using broth microdilution. According to the results obtained, C. arvensis EO (8 mg/mL) showed weak inhibitory action against B. cereus and E. coli. While it showed no antibacterial efect against P. aeruginosa and S. aureus [96].
Te antimicrobial activities of EOs obtained by HD and MD, as well as methanolic, ether, and hexane extracts of C. arvensis were quantitatively studied in respective broths against nine microorganisms (S. cerevisiae, C. albicans, M. smegmatis, B. cereus, S. aureus, E. faecalis, P. aeruginosa, Y. pseudotuberculosis, and E. coli) using double dilution and the MIC values (μg/mL) were defned. Tis study indicates that only the EO, obtained by HD, and the methanolic extract showed moderate efects against B. cereus and S. aureus with MIC of the order of 105-210 μg, respectively. Interestingly, all extracts displayed good antituberculosis efects against M. smegmatis with MIC between 13.2 and 62.5 μg [41]. According to Kim [104], C. arvensis extract had excellent antibacterial activity, and after 12 hours of culture, it inhibited the growth of B. subtilis, E. coli, and C. albicans by 40, 7, 35.2, and 27.5%, respectively. Moreover, the growth inhibitory efect against the positive bacteria, B. subtilis, was the highest.

Antiparasitic Activity.
Te spectral and chemical studies on triterpenoid and sesquiterpene bioactive chemotypes of C. arvensis showed a signifcant inhibitory efect against vesicular stomatitis and rhinovirus multiplication in cell cultures, in which glycosides 1 and 2 were most efective [87,98]. Besides, arvensoside B and arvensoside D saponins extracted from C. arvensis also possess an impressive impact on Trypanosoma brucei brucei at a MIC value of 50 and 100 μg/mL, respectively [43].
Lemna minor, Artimia salina (Brine shrimps) larvae, Callosobruchus analis, and other parasites were tested for their susceptibility towards the methanolic extract of C. arvensis [99]. Te insecticidal potential of C. arvensis pronounced a variable degree of susceptibility of strains and tested doses, in which Callosobruchus analis was the most sensitive insect with LD 50 of 0.51 mg/mL. A moderate level of cytotoxicity was found with a LD 50 value of 9.23 μg/mL against brine shrimp larvae with a mortality percentage of 40, 60, and 67% at 10, 100, and 1000 μg/mL, respectively [99]. Nonetheless, low toxicity (% inhibition ≤40%) at 10 and 100 μg/mL and moderate efect (% inhibition � 40-50%) at 1000 μg/mL were observed against Lemna minor [99].

Anti-Infammatory Activity.
Some studies have shown that C. arvensis extracts exhibit anti-infammatory efects. Indeed, Mascolo and collaborators showed that the injection of 100 mg/kg BW of C. arvensis extracts in rats induces an interesting anti-infammatory efect by inhibiting 32% (2.68 ± 0.8) of edema formation [45]. In the same way, Abudunia et al. [44] demonstrated signifcant anti-infammatory results of C. arvensis extracts, in which fowers extracted by hexane solution had the best edema reduction yield of rat paw edema, with 51.08, 71.33, 63.38, and 67.33%, induced by carrageenan and experimental trauma, respectively. Tese outcomes indicated the contributory efects of using C. arvensis fower extracts in the management of infammatory and painful conditions [44].  by soxhlet extraction, while the aqueous extracts were obtained by maceration. Both aqueous and methanolic extracts have free radical scavenging efects. However, the free radical scavenging efect of the methanolic extract appears to be closer to 332 mg/mL gallic acid. At all concentrations, the extracts were ranked according to their free radical scavenging potency in the following order; methanol extract > aqueous extract > hexanol extract. Te EC 50 of the methanolic and aqueous extracts are, respectively, 20.9 and 33.2 mg/mL. Te reducing power using the ferric-reducing antioxidant power (FRAP) method, showed that C. arvensis fower extracts show signifcant diferences in the following order; methanol extract > aqueous extract > hexanol extract. Te reducing ability of C. arvensis methanol extract was the most potent among the three extracts (203.96 mg AAE/g Ext). Similarly, all fower extracts inhibit β-carotene bleaching by scavenging free radicals derived from linoleic acid. Te methanol extract inhibits β-carotene at a rate comparable to BHT [47]. Abutaha et al. [10] showed that the 1 mg/mL of C. arvensis fower ethyl acetate extract (CAF EtOAC) extracted using a Soxhlet extractor present a percentage of scavenging activity (DPPH%) moderate at 50%. In order to discover novel sources of antioxidants, aqueous and methanol/chloroform extracts of C. arvensis were investigated using several methods, namely, 2,2 diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, total antioxidant capacity (TAC), and reducing power (RP). Te results showed that the antioxidant activity varies greatly not only between the extracts but also between the assays used. Te reducing power values ranged from 12 Vit C equivalent mg/g DW in aqueous extracts to 100 Vit C equivalent mg/g DW in the methanol/chloroform extracts. Te samples tested presented several TAC values, expressed as the number of ascorbic acid equivalents, from 19 mg vit. C equivalent/g DW in the methanol/chloroform extracts at 25 mg vit. C equivalent/g DW in aqueous extract. It was noticed that the aqueous extracts of C. arvensis had the highest percentage of scavenging, i.e., 48.7% and 8.7% for the methanol/chloroform extracts [38].

Antioxidant Activities of
Belabbes et al. [88] showed that C. arvensis EO had the ability to reduce DPPH radical (IC 50 � 76.2 mg/L), and more interestingly, C. arvensis hydrosol extract indicated the highest DPPH quenching activity (IC 50 � 25.1 mg/L). It was found that the antioxidant activity of C. arvensis EO is proportional to the concentration. However, by the β-carotene bleaching test, the hydrosol extract (IC 50 � 32.4 mg/L) displayed the highest potency, while the EO showed the lowest (IC 50 � 100.1 mg/L). Te BR antioxidant activity of C. arvensis methanolic extracts was determined as 0.024 μg/mL. Te activities at pH � 7.4 (TEAC method, mM Trolox eq.) show a great variability determined as 0.46 mM Trolox. By the DPPH method in methanolic solution, the results also show a great variability determined as 80.9 μg/mL [31].
Ercetin et al. [49] examined the antioxidant activity of the water, methanol, ethyl acetate, acetone, dichloromethane, and n-hexane extracts of C. arvensis L., leaves and fowers using FRAP, ferric ion-chelating capacity, and DPPH assays at 250, 500, and 1000 μg/mL. By the FRAP assay, the studied extracts had a rather weak efect, while the C. arvensis fower methanol extract displayed the highest absorbance determined as 0.479 at 1000 μg/L. Moreover, the best scavenging activity was obtained by C. arvensis fower methanol extract (52.25 ± 1.34%), which was the only extract with an activity greater than 50%. Terefore, the studied extracts showed a relatively higher ferric ion-chelating activity compared to other tests. At 1000 μg/L, the leaves (53.36 ± 1.23%) and fowers (53.25 ± 0.15%) of C. arvensis ethyl acetate extracts were found to be the best in assays.
Te infuence of C. arvensis fower methanolic and aqueous extracts at concentrations of 0.10 to 0.90 mg/mL, scavenged all types of radicals studied depending on the concentration applied. For both C. arvensis extracts, all the concentrations selected had an antioxidant potential lower than that of the control. At the concentration of 0.90 mg/mL, the aqueous extract scavenged approximately 60% of DPPH radicals, while at concentrations below 0.45 mg/mL no signifcant scavenging activity (SA) was observed compared to the control. For hydroxyl radical scavenging activity, both C. arvensis extracts showed high scavenging activity, while the addition of 0.45 mg/mL aqueous extract clearly scavenged the hydroxyl radical, with a scavenging activity of 34.74%. Te elimination of the hydroxyl radical (SA 80-90%) was obtained with 0.9 mg/mL of the methanolic or aqueous extract. In addition, the highest antioxidant efect of C. arvensis aqueous extract (0.90 mg/mL) against peroxyl radical exhibited signifcantly lower activity (antioxidant activity, 69.28%). High concentrations of the methanolic and aqueous extracts were more efective in reducing the peroxyl radical [92].
Likewise, C. arvensis fowers showed efective antioxidant activity on linoleic acid and DPPH radical oxidation. Tis herb showed a DPPH radical scavenging activity of 409.4 μg/mL as SC 50 and an inhibitory activity of 61.1% on linoleic acid oxidation [46].
According to Messina et al. [105], six extracts of C. arvensis obtained by various solvents (distilled water, acetone 70%, ethanol 80%, and hexane) and by supercritical fuid extraction (SFE) showed a protective efect against superoxide anions, hydroxyl radicals, scavenging hydrogen peroxide, and oxidative damage. It was observed that the supercritical fuid extracts of C. arvensis presented a very remarkable antioxidant efect compared to the ethanol 80% extract (DPPH and reducing power method), in comparison to a synthetic antioxidant.
Te relative levels of antioxidant activity of methanolic and aqueous extracts of C. arvensis of Jordanian origin were determined using the ABTS + method. Tere was a low variation in the total antioxidant capacity of the extracts. Te total antioxidant capacity ranged from 42 to 48.4 μmol TE/g DW for the methanolic and aqueous extracts, respectively [97]. Kim [104] showed that the DPPH scavenging activity of C. arvensis extract samples at concentrations of 10, 50, and 100 μg/mL showed almost the same radical scavenging activity as the control (ascorbic acid) (88.9, 91.2, and 91.3%). Lee et al. [106] showed that with high DPPH free radical scavenging activity, the possibility of using C. arvensis fower extracts as a natural Advances in Pharmacological and Pharmaceutical Sciences 21 antioxidant was high. Te DPPH free radical scavenging activity was 66.99% and 77.63% in the hot water and 80% MeOH extracts, respectively.
3.5.5. Anticancer Activity. C. arvensis extracts have been shown to be a promising source of cytotoxic compounds.
Te efects of C. arvensis fower extracts from villages around the Rabat-Khemisset region (Morocco) were assessed on the growth of myeloid cancer cells using MTT assay and it was shown that methanol and hexane extracts (obtained by soxhlet) and aqueous extracts (obtained by maceration) exhibited important antimyeloid cancer activity (IC 50 � 31 mg/mL). Consequently, both methanolic and aqueous extracts have been shown to be efective candidates as cytotoxic agents. Indeed, their efcacy was more pronounced compared to the hexanolic extract. At the concentration of 100 mg/mL at 24 h, it was also deduced that methanolic extract shows a maximum inhibition of 89%, testifying that this extract is a very important antimyeloid cancer agent [47]. At diferent concentrations, the efect of C. arvensis part (leaves, stems, and fowers) extracts obtained by a Soxhlet extractor using various solvents (methanol, ethyl acetate, chloroform, and hexane) against MDA-MB-231 and MCF-7 cells were estimated. Among all extracts, the fower ethyl acetate (CAF EtOAC) extract showed the highest activity, it inhibited cell growth in a concentration-dependent manner. Tis extract displayed IC 50 values of 70 and 78 μg/mL in MCF-7 and MDA MB-231 cells, respectively. However, the fower hexane extract presented a moderate cytotoxic efect, but the methanol and chloroform extracts showed no activity [10].
Te study of Attard and Cuschieri [48] indicated that the petroleum ether extract of C. arvensis aerial part is relatively nontoxic to peripheral lymphocytes, suggesting its use as an immune stimulant. In fact, all the extracts studied presented a concentration-dependent efect. Phytohemagglutinin (PHA) (SC 50 < 0.001 μg/mL) and the petroleum ether extract (SC 50 0.089 μg/mL) of C. arvensis obtained by maceration demonstrated an increase in proliferation compared to the other extracts (IC 50 > 10 μg/ mL). However, the most active extract of C. arvensis was the petroleum ether extract. In contrast, it has been reported that C. arvensis extract is a safe natural product, as at least it did not induce any cytotoxicity at 100 μg/mL. Te cytotoxicity of this extract was 0, 0.1, 0.3, 15.1, and 28.0% at increasing concentrations of 10, 100, 250, and 500 μg/mL, respectively [104].
Quetin-Leclercq et al. [95], showed that the saponins isolated from C. arvensis are very active against certain cancer cells (human HeLa tumor cells, Flow 2002 noncancer human cells, mouse 313 noncancer fbroblasts, and mouse B16 melanoma cells) as much as the reference compound, strychnopentamine. At concentrations of 10 μg/mL and above, they exhibited some degree of cytotoxicity, while the concentration of 50 μg/mL was the most potent.
According to Ullah et al. [107], the toxic potential of the methanol crude extract of C. arvensis whole plant was evaluated by a Lemna minor bioassay. It was observed that C. arvensis exhibits dose-dependent toxicity towards Lemna minor, with low toxicity (% inhibition ≤40%) at 10 and 100 μg/mL, and moderate activity (% inhibition � 40-50%) at 1000 μg/mL. A moderate level of cytotoxicity was found to have an LD 50 value of 9.23 μg/mL for saltwater shrimp larvae. Zihlif et al. [108] found that the C. arvensis ethanol extract has a potential antiangiogenic activity. A selectivity was demonstrated against the proliferation of endothelial cells, indicating a direct inhibitory power on the tumor angiogenesis key step. Te activity of this extract shows more than 50% growth inhibition. After 72 h at 50 μg/mL, an important antiproliferative activity was also shown against the MCF7 cell line. Te extract reduced the growth of these cells to less than 30%. While it showed weak antiangiogenic activity on PFL cell proliferation at 50 μg/mL. Interestingly, it showed potent activity against HUVEC cell proliferation with IC 50 � 28.7 μg/mL. 3.5.6. Enzyme Inhibitory Activity. Te investigation of the enzymatic inhibitory efects in diferent solutions (distilled water, methanol, ethyl acetate, acetone, dichloromethane, and n-hexane) of C. arvensis leaf and fower extracts, collected from the vicinity of Bodrum town in the province of Mugla (Turkey), against butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) was performed by Ercetin et al. [49]. Teir outcomes highlighted the high efciency of C. arvensis fower ethyl acetate extract in AChE inhibition assay (31.24 ± 1.29%) compared to C. ofcinalis extracts. Tis efect has been attributed to the presence of compounds derived from favonoids and triterpenes (tannins, malic and salicylic acid, mucilages, etc.) found in the fower methanolic extract of this specie [49].
3.5.7. Antimutagenic Activity. Elias and Meo [89] used the microsuspension technique of the Salmonella/microsomal assay (Ames test) to investigate the antimutagenic potential of natural saponins isolated from the dried aerial parts of C. arvensis. Teir preliminary fndings demonstrated that the saponins were not toxic for the tested strains of Salmonella typhimurium (TA 100, TA 98, and TA 97) for doses up to 400 μg. Te use of benzo[a]pyrene (BaP) and a mutagenic urine concentrate from a healthy smoker (consumed 40 cigarettes per day), showed that the four products (D, C, B, and A) of the studied plant exert an antimutagenic efect against 1 μg of BaP and 5 μL of smoker's urine [89]. Probable antimutagenic mechanisms of saponins have been reported to be the modifcation of cell surface activity and cell membrane structure [89]. In their research paper, a group of Australian chemists concluded that the saponin arvensoside B sugar units (chemically named 1⟶3-Galactopyranosyl-D-glucopyranosyl-3-O-oleanolic acid) strongly infuence the hemolytic activity of oleanolic acid disaccharides [100]. Not only that but also the linkage positions 3 or 4 either arvensoside B or its analog, calenduloside found in C. ofcinalis are structural requirements for high potency [100].
3.5.8. Immunomodulatory Activity. Te immunomodulatory activity of various Maltese plant extracts (i.e., Carlina involucrata Poir, Galactites tomentosa Moench, Leontodon tuberosus L, Glebionis coronaria L. Tzvelev, Aster squamatus (Sprengel) Hieron, Reichardia picroides L. Roth, Sonchus oleraceus L, Calendula arvensis L, Inula crithmoides L, and Dittrichia viscosa L. Greuter) of the Asteraceae family was performed on human peripheral T-lymphocytesin vitro using macerated dried aerial parts in diferent solvents [48]. Te authors observed marked efects in lymphocytes treated with extracts, but only C. arvensis petroleum ether extract was able to activate the lymphocytes and pronounce blastogenesis similar to that of PHA, which can be used as a potential immune booster [48].
3.5.9. Wound Healing Activity. Te local application of a mixture of H. perforatum and C. arvensis EOs in the epithelial reconstruction of surgical wounds in childbirth with cesarean section in Italy on a group of 24 patients induced a wound size reduction of 37.6 ± 9.9% versus a reduction of 15.83 ± 4.64% in the control group [101]. Te above-mentioned results are very encouraging to shed more light on C. arvensis for further investigations on the extraction methods according to the diferent plant parts, isolation of the main biomolecules, and the elucidation of their mechanism of action.

Conclusion and Perspectives
In this paper, the sources and main pharmacological characteristics of C. arvensis have been investigated and presented. Te analysis of several published studies revealed that this natural compound has signifcant biological properties, especially its wide range of use in folk medicine by several ethnic groups around the world as a poultice, antidiabetic, antispasmodic, antiemetic, antihelmintic, antirheumatic, diaphoretic, and also as a treatment against neurological disorders, microbial disease, ENT and typhoid diseases, skin diseases, digestive disorders, and high blood pressure. Additionally, all C. arvensis fower extracts exhibit an inhibitory efect against most fungi and bacteria species. In addition, due to the phytochemical constituents of C. arvensis, including polyphenols, favonoids, terpenoids, tannins, and alkaloids, and also from its antimicrobial, antioxidant, and anti-infammatory properties, this medicinal plant can be considered an interesting bioactive compound to explore diferent molecular and cellular pathways in cancer prevention. However, a greater comprehension of its pharmacokinetics and pharmacodynamics is warranted for its incorporation as a drug in cancer therapy and other pathologies. In this context, the assessment of its safety potential is also required, through a more in-depth toxicological survey. Mass-analyzed ion kinetic energy MLC:

Data Availability
Te data used to support the fndings of this study are included in the article.