Methods
Our review of documented medicinal and known traditional uses of C. benedictus and its constituents is based on the literature available in the electronic databases PubMed, Toxline, and the Cochrane Library. Publications were filtered by using the following terms: Cnicus benedictus, Centaurea benedicta , blessed thistle, cnicin, arctigenin, and arctiin.
To investigate the pharmacokinetic and toxicological profiles of the main active phytochemicals of C. benedictus , publicly available tools such as PubChem, SwissADME, and Chem Mine Tools were used. SwissADME is a freely accessible web tool designed to analyze the pharmacokinetics and drug-likeness of molecules based on in silico models and uses a variety of descriptors based on the rule-of-five (RO5) criteria compiled by Lipinski et al. (2001), such as, for example, molecular weight, logP, number of hydrogen bond donors and acceptors, and topological polar surface area (TPSA) [4 ], [5 ]. In order to qualify a compound as orally active and having suitable oral bioavailability, it has to fulfill the RO5 criteria. These criteria include the following characteristics: a molecular weight less than 500, a logP less than 5, hydrogen bond donors fewer than 5, and hydrogen bond acceptors fewer than 10. Rai et al.
(2023) presented the assumptions that guided the in silico study, from inputting the compound to interpreting the results through the SwissADME suit [6 ]. The Tanimoto similarity coefficient (Tsc) is a commonly employed measure of molecular similarity for pairwise comparison of molecules. According to its description, this value ranges from 0.0 (indicating full dissimilarity) to 1.0 (indicating equal molecules) [7 ].
Chemical structure of the tested molecules
Only the structure of arctigenin QOFJIF01 is deposited in the Cambridge Structural Database (CSD) [8 ]. As we can see from [Fig. 1 ], it is a molecule with a three-dimensional structure.
Fig. 1 Molecular structure, (a ) arctigenin (2D semi-structural scheme and 3D stick representation with indicated planes of rings [8 ] (b ) artiin; (c ) cnicin (2D semi-structural scheme).
Arctigenin and artiin are phenylpropanoid dibenzylbutyrolactone lignans [9 ]. The lignan dibenzylbutyrolactones are phenylpropanoid metabolites from plants. They are phenolic compounds consisting of two phenylpropane units (C6-C3) linked by the side chain C-8, C-8′ carbon atoms [10 ], [11 ]. The structural analogs of arctigenin and artiin exhibit a Tanimoto coefficient of 0.71. Trachelogenin, a rare natural plant-derived lignan, exhibits the highest structural similarity to arctigenin, as indicated by a Tanimoto coefficient of 0.96. The antiviral, anti-inflammatory, and analgesic actions of trachologenin can be attributed to this structural similarity, as indicated by IC50 values of 0.325 and 0.259 µg/ml in the HCVcc and HCVpp models, respectively [12 ].
Cnicin is classified as a sesquiterpene lactone of the guaianolide group. Sesquiterpene lactones are terpenes that have a uniform structure consisting of 15 carbon atoms. This structure is formed through the biosynthesis of three isoprene units, and a five-membered lactone rings consists of hydroxycarboxylic acid cyclic esters. The lactone component exhibits a α -methylene-γ -lactone structure, which is a ring combining oxygen with a carbonyl group in the β position (O=C…C=CH2) [13 ]. They are colorless, durable, and hydrophobic substances. The sesquiterpene lactones have a wide range of biological actions. The lactone is thought to be the primary cause of the biological effects of sesquiterpene lactones on organisms and cells. A Michael-type addition reaction occurs between unsaturated carbonyl structures and nucleophiles in biological systems, as exemplified by the sulfhydryl group (SH) present in the amino acid cysteine. It
is important to highlight the fact that more than 90% of human genome–encoded polypeptides contain the amino acid cysteine [14 ].
The active constituents of C. benedictus
C. benedictus contains a variety of chemical compounds, such as glycosides, triterpenoids, lignans, flavonoids, phenolic acids, steroid compounds, volatile oils (0.3%), tannins (8%), and mineral components ([Table 1 ]) [1 ], [3 ], [15 ], [16 ], [17 ], [18 ], [19 ], [20 ], [21 ]. The most active, well-known substances responsible for the bitter taste and pharmacological effects of C. benedictus are the glycosides cnicin, arctigenin, and arctiin [22 ].
Table 1 The active constituents of C. benedictus L.
sesquiterpene lactone
cnicin artemisiifolinβ -D-glucoside 11β ,13-dihydrosalonitenolide 15-O-β -D-glucoside
lignans
arctigenin
triterpenoids
α -amyrenon, α -amyrenon acetateα -amyrine, α -amyrin acetate
flavonoids
luteolin
steroids
phytosterol
phenolic acids
chlorogenic acid sinapic acid
volatile oils
citral
mineral contents
magnesium
Biological activity and use of C. benedictus in traditional medicine
Most contemporary recommendations regarding the therapeutic use of C. benedictus are based on the established position of this plant in traditional medicine ([Table 2 ]). There is a lack of controlled clinical studies supporting the beneficial effects of preparations containing C. benedictus in humans.
Table 2 The use of C. benedictus L. in traditional medicine (based on available literature).
internal
digestive disorders, indigestion, dyspepsia, diarrhea
external
wounds
In folk medicine, an infusion made from C. benedictus is considered a general tonic, expectorant, and beneficial for the digestive system [3 ]. Despite a limited amount of high-quality scientific data, the bitter components in C. benedictus make it traditionally used for gastrointestinal problems. It is believed to increase the secretion of gastric juice and saliva, counteract excessive intestinal fermentation, reduce bloating and indigestion, improve appetite, and influence liver function by promoting bile production and secretion [2 ], [23 ], [24 ], [25 ]. According to the Committee on Herbal Medicinal Products of the European Medicines Agency, C. benedictus products are used traditionally for temporary loss of appetite and symptomatic relief of dyspepsia and mild spasmodic disorders of the gastrointestinal tract
[26 ].
One of the plantʼs active components, cnicin, has demonstrated hepatoprotective properties, making it potentially valuable in the management of liver diseases. Both the British and German pharmacopoeias state that the active ingredients in C. benedictus act as cholagogues [27 ]. In the past, large doses were used as emetics, and the plantʼs seeds were employed as an emmenagogue.
It is also considered to be a galactagogue, meaning a substance that increases and stimulates lactation in women [2 ], [28 ], and an effective analgesic for menstrual pain [1 ]. In 2019, a questionnaire survey was conducted in Australia among breastfeeding mothers who used C. benedictus preparations to stimulate lactation. The majority of women participating in the study rated the effectiveness of C. benedictus as a lactogenic agent as low or moderate [29 ]. However, it should be emphasized that due to the lack of sufficient data from clinical trials, the product is not recommended by the European Medicines Agency for pregnant and lactating women [26 ].
The active ingredients present in the plant regulate metabolism and exhibit antibacterial and anti-inflammatory properties. Despite limited evidence for its effectiveness, the herbal remedy is traditionally used as a diuretic [30 ], [31 ].
Traditionally, an infusion of C. benedictus was used to treat infectious diseases such as tuberculosis, malaria, and plague [32 ]. The herb was also used topically for dressing wounds and ulcers [31 ], [33 ]. Due to its high mineral content, C. benedictus was used during reconvalescence, and the infusion is still used in South Africa in the context of cancer. However, the effectiveness of such applications has not been adequately confirmed in clinical trials [2 ], [27 ], [34 ].
Antimicrobial, antiviral, and antiparasitic activity
The antimicrobial activity of the plant has been demonstrated against Gram-positive cocci and Gram-negative bacillus. Vanhaelen-Fastré (1973) investigated the antibacterial properties of the essential oil obtained from the herb of C. benedictus and showed that 15 out of 30 chromatographically identified substances, including p-cymene, fenchone, citral, and citronellol, exhibited such properties [35 ]. The main contributors to the antibacterial activity of the plant are sesquiterpene lactones, primarily cynarin [2 ], [36 ], [37 ], [38 ]. Antibiotic properties are also exhibited by essential oils, aromatic aldehydes (benzaldehyde, cinnamaldehyde, and cuminaldehyde), and monoterpenes (citronellol, fenchone, and p-cymene) [27 ]. However, it should be noted that the antibacterial properties depend on the type
of extract tested and the origin of the plant raw material [16 ].
The extract of C. benedictus inhibited the growth of cultures of Bacillus subtilis, Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei, Staphylococcus aureus, Streptococcus pyogenes, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, Brucela species , and Enterococcus faecalis
[39 ], [40 ]. Yasin and Ibrahem (2017) demonstrated the antibacterial activity of the ethanolic extracts of the roots of this plant against bacteria such as Escherichia coli, Bacillus pumillus, Staphylococcus aureus , and Micrococcus . Additionally, the results of chemical analyses confirmed the presence of various active compounds in the roots of C. benedictus , including alkaloids, flavonoids, phenols, tannins, and terpenes. At the same time, no antibacterial activity was observed against the Klebsiella and Pseudomonas species, nor against S. aureus, S. typhi , and
yeast [2 ], [40 ].
These properties were also confirmed by studies conducted in animal models. In rats, the effect of the powdered root was investigated on the rate of healing of wounds that were experimentally induced by cutting a skin fragment on the back. After two weeks of application, the area of the wounds that had healed reached 95%, confirming the effectiveness of the C. benedictus formulation (a homogeneous mixture of 10 g of the plant root powder with 50 g vegetable jelly “vaseline” of unknown composition). The impact of this plant preparation on the rate of healing of the experimentally induced wound was comparable to the effectiveness of the antibiotic ointment Baneocin, a composite preparation containing the antibiotics bacitracin and neomycin, which are used topically to treat bacterial infections [31 ].
In particular, cnicin exhibits significant antimicrobial activity against various pathogenic microorganisms, including both Gram-positive and Gram-negative bacteria and fungi. Cnicinʼs antimicrobial action is mediated through the disruption of microbial cell membranes, inhibition of essential enzymes, and interference with microbial biofilm formation. Cell wall enzymes have consistently been a favored focus in drug exploration, owing to their crucial role in the survival of pathogens. According to Raina et al. (2021), the cytosolic enzyme MurA, also known as UDP-N-acetylglucosamine enolpyruvyl transferase (EC 2.5.1.7) [41 ], is essential for initiating the primary phase of bacterial cell wall production and has been identified as a target of numerous antibiotics. It has been shown that cnicin is a potential irreversible inhibitor of the bacterial enzyme MurA, which is crucial for bacterial survival and responsible for the initial step of the
cytoplasmic biosynthesis of the precursor molecules of peptidoglycan [42 ]. The antibacterial properties of cnicin are likely conditioned by the presence of α, β -unsaturated carbonyl groups within the molecular chain. Thanks to these groups, cnicin forms stable bonds with the MurA enzyme through the thiol group of Cys115, ultimately leading to the alkylation of this important site and the complete blocking of bacterial enzyme activity [43 ]. The X-ray crystallographic investigations carried out by Skarzynski et al. in 1996 revealed that this spherical protein is composed of two different domains, each containing an equal number of α helices and β sheets [44 ]. These domains are interconnected by a double-stranded linker. The catalytic domain consists of residues 22 – 229; the catalytic site is located at Cys-115, which participates in product formation ([Fig. 2 a ]) [44 ], [45 ]. The other domain, known as the C-terminal domain, consists of residues 1 – 21 and 230 – 419. The catalytic site is positioned in the cavity formed by these domains. [Fig. 2 b ] illustrates the hydrolysis of the ester function of cnicin, leading to the elimination of its macrocyclic component. The aforementioned mechanism is essential for the Cys115 loop to adopt the conformation evidenced in the MURA-fosfomycin complex [45 ]. Cnicin may also exert antibacterial effects through the formation of (−)-tulipalin B [46 ]. A study was conducted to evaluate the inhibitory effects of cnicin on the growth of B. subtilis, S. epidermidis, E. coli, K. pneumoniae, P. aeruginosa , and S. aureus . For S. aureus 25 923, 25 178, 6538, E. coli, K. pneumonia , and P. aeruginosa ,
the minimum inhibitory concentration (MIC) of the chloroform extract of cnicin is 124 µg/mL [1 ], [36 ], [47 ].
Fig. 2 Crystal structure of MURA inhibited by unag-cnicin adduct (a ) with the enlarged area showing the structural elements around the ligand-biding site (PDB ID: 2Z2C, 2.05 Å) [45 ]. Residues that form hydrogen bonds (dashed lines) with cnicin are shown in ball-and-stick representation with the interatomic distances shown in Å. Residues forming Van der Waals interactions with UDP are shown as labeled arcs with radial spokes that point toward the ligand atoms (b ).
Barrero and coworkers (2000) evaluated the microbiological activity of sesquiterpene lactones against the filamentous fungus Cunninghamella echinulata . Upon incubation of C. echinulata with cnicin, hydrolysis of the side chain of this compound occurred, resulting in the formation of salonitenolide. No hydrogenation products or inhibition of fungal growth was observed. The greater polarity of sesquiterpene lactones is responsible for the lower antifungal activity. This may be related to the degree of lipophilicity required for sesquiterpenoids to penetrate the fungal cell wall [47 ], [48 ].
Some data indicate the antiviral (anti-HIV) activity of lignans found in the herb of C. benedictus . There is also evidence of synergistic antiviral action between arctigenin, an important active compound in the plant, and oseltamivir (neuraminidase inhibitor) against oseltamivir-resistant H1N1 influenza viruses. The presence of arctigenin likely inhibits virus replication in host cells [49 ], [50 ]. The latest research demonstrates that arctigenin found in other plant species such as Forsythia viridissima fruit ethanol extract inhibits the replication of human coronavirus by suppressing viral protein expression and cytotoxicity [51 ].
C. benedictus is also considered a promising natural remedy in combating the SARS-CoV-2 virus, which, especially due to its mutations and the emergence of new variants, has been a global epidemic threat since 2019. In light of the danger of a resurgence of the pandemic, testing has begun to assess the efficacy of plant extracts, which are easily accessible sources for developing new antiviral drugs. Alhadrami et al. (2021) conducted in silico and in vitro experiments to investigate the potential of cnicin in inhibiting the replication of the SARS-CoV-2 virus. The results indicated that the inhibitory effect of cnicin was dose-dependent, with an IC50 value of 1.18 µg/ml [52 ], providing the impetus for conducting in vivo studies and clinical trials.
Queiroz and coworkers (2021) evaluated the anti-parasitic effect of cnicin on trematodes of the genus Schistosoma . Both in vitro and in vivo studies were conducted to assess the effectiveness of cnicin and its complexes with β -cyclodextrin (β CD) and 2-hydroxypropyl-β -cyclodextrin (HPβ CD) against Schistosoma mansoni . The research findings showed that intraperitoneal administration of cnicin in mice caused changes in the covering of adult forms and led to the death of the parasite. In a murine schistosomiasis model, it was found that oral administration of cnicin or its β CD complex was ineffective and did not result in a significant reduction in the number of parasites. In contrast, high anti-parasitic efficacy against S. mansoni was demonstrated in mice when cnicin complexes with HPβ CD were administered orally or intraperitoneally. These complexes reduced the number of adult parasitic forms and
significantly limited the number of produced eggs. Permeability studies using a fluorescent dye indicated that the HPβ CD complex could penetrate the covering of adult S. mansoni forms in vivo
[53 ].
Despite the promising results of preclinical studies, the antimicrobial, antiviral, and antiparasitic properties of C. benedictus have not yet been sufficiently confirmed in clinical conditions.
Antioxidant, anti-inflammatory, and analgesic activity and neuroprotective benefits
In folk medicine, the herb from C. benedictus was used to treat pain, including painful menstruation [2 ]. In Algeria, the plant is a well-known remedy used for the treatment of wounds and burns.
Many studies conducted on animals indicate anti-inflammatory and associated analgesic properties of the active ingredients of C. benedictus ; however, such research has not been carried out in humans. Tests were conducted on mice to assess the impact of the plant extract and cnicin on the intensity of pain induced by formalin injection. Both a methanol extract of the leaves of C. benedictus and cnicin itself demonstrated antinociceptive effects, which could involve various neuronal signaling pathways and modulations, including the pathway associated with the L-arginine/nitric oxide/cGMP/ATP-sensitive potassium channel (LNCaP) and the endogenous opioid system [54 ].
In a preclinical study involving mice, the anti-inflammatory effects of cnicin and extracts from C. benedictus were demonstrated in models of inflammatory skin diseases. The animals were divided into groups, and solutions of various compounds inducing local inflammation were applied to ear skin. The inflammatory agents included croton oil, phenol, capsaicin, arachidonic acid, and histamine. Tissue levels of inflammatory markers, swelling size, leukocyte levels, and vascularization extent were then assessed in the collected ear fragments. It was observed that both the application of cnicin and C. benedictus extract (leaves extracted with dichloromethane : ethanol 9 : 1 v/v) led to a reduction in swelling and levels of inflammatory markers such as myeloperoxidase, N-acetyl-β -D-glucosaminidase, nitric oxide, tumor necrosis factor α (TNF-α ), and interleukin-6 (IL-6). The study showed that cnicin interacts with cyclooxygenase-1 and nitric oxide
synthase through hydrogen bonding, leading to inhibition of these key inflammatory enzymes [55 ].
Demiroz and coworkers (2018) evaluated the effect of cnicin in a rat model of paw inflammation induced by the injection of venom from Ottoman vipers of the species Montivipera xanthina and Macrovipera lebetina obtusa . The venom of these snakes induces tissue swelling, leading to necrosis of the skin and muscles. Cnicin was administered orally at three different single doses (2.5, 5, and 10 mg/kg b. w.), and its effectiveness was compared with indomethacin, a nonsteroidal anti-inflammatory drug. The area affected by snake venom–induced inflammation was extensive in the first hours after administration and decreased in the following hours. The study demonstrated that the anti-inflammatory effect of cnicin, assessed half an hour after the onset of swelling, was comparable to the effectiveness of indomethacin. The most effective dose was considered to be 10 mg/kg b. w. [56 ].
An in vitro study demonstrated that cnicin also has a neuroprotective effect, effectively promotes axon regeneration, and accelerates functional recovery after nerve injury [57 ].
Published data suggest that lignans such as arctigenin and trachelogenin can reduce cyclic adenosine monophosphate (cAMP) levels and inhibit phosphodiesterase (PDE) activity and mast cell histamine release [2 ]. The recognition of drug discovery targeting PDE4 as a competitive and promising technique for the development of anti-inflammatory medicines has gained significant attention in recent decades [58 ], [59 ]. Patients with psoriasis, psoriatic arthritis, atopic dermatitis, inflammatory bowel disease, and asthma exhibit a notable increase in the activity and distribution of PDE4 in peripheral immune cells and skin biopsies. The available evidence suggests that inhibition of PDE4 leads to a notable elevation in cAMP concentration, which subsequently triggers the activation of protein kinase A (PKA). The regulation of inflammation and tissue homeostasis was found to be concurrently
influenced by the phosphorylation of nuclear transcription factors, including cAMP-responsive element binding protein (CREB), cAMP-responsive element modulator (CREM), and activated transcription factor 1 (ATF-1) [60 ].
Arctigenin has the ability to decrease the phosphorylation of Akt and PDE, resulting in a buildup of cAMP within cells and the activation of PKA. Crystallographic investigations have unveiled robust connections between arctigenin and the binding pocket of PDE4D, as depicted in [Fig. 3 ]. This finding provides evidence for the molecular-level inhibition of PDE4D by arctigenin. The experiments verified a moderate level of inhibitory action against PDE4D, as indicated by an IC50 value of 3.76 ± 0.28 µM [60 ].
Fig. 3 Crystal structure of PDE4D catalytic domain in complex with arctigenin (a ) with the enlarged area showing the structural elements around the ligand-biding site (PDB ID: RLRM, 1.45 Å) [60 ]. Residues that form hydrogen bonds (dashed lines) with arctigenin are shown in ball-and-stick representation with the interatomic distances shown in Å. Residues forming Van der Waals interactions with arctigenin are shown as labeled arcs with radial spokes that point toward the ligand atoms (b ).
Antagonistic effects of cnicin on calcium ions and platelet-activating factor (PAF) have also been observed in mast cells [61 ], [62 ].
The antioxidant effect of extracts from C. benedictus was confirmed in liver cells under conditions of oxidative stress. Oxidative stress was measured on the basis of the level of lipid peroxidation (LPO), glutathione content, total protein level, and enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) in the liver tissue homogenate. A significant improvement in most of the investigated variables was observed after the administration of the hydroethanolic extracts [63 ].
Arctigenin also exhibits anti-inflammatory and analgesic effects. This lignan demonstrates neuroprotective effects in vitro by reducing neurotoxicity induced by glutamate and kainic acid. Arctigenin inhibits the binding of kainic acid to many receptors, including glutamate receptors such as NMDA receptors, AMPA receptors, kainate receptors, and metabotropic glutamate receptors. Furthermore, there is empirical evidence for its efficacy in removing reactive oxygen species [64 ]. The mechanism of the antioxidant and anti-inflammatory action of arctigenin is complex and involves inhibiting the production and release of inflammatory mediators, such as arachidonic acid metabolites and free radicals. This leads to reduction in exudate formation and the inhibition of leukocyte migration into inflamed tissues [65 ]. In vitro , arctigenin also limits the production of nitric oxide (NO) and pro-inflammatory cytokines
such as tumor necrosis factor-alpha (TNF-α ) and interleukin-6 (IL-6) stimulated by lipopolysaccharide (LPS) in macrophage cell lines RAW 264.7 and THP-1. Arctigenin inhibits the expression and enzymatic activity of induced nitric oxide synthase (iNOS) caused by phorbol myristate acetate (PMA) and LPS [66 ], [67 ]. Additionally, it inhibits the phosphorylation of MAP kinases ERK1/2, p38 kinase, and JNK, and blocks the production of TNF-α
[9 ], [68 ]. These anti-inflammatory effects make cnicin a promising candidate for the treatment of inflammatory diseases.
The neuroprotective benefits of arctigenin have been observed both in vitro and in vivo , specifically in models of Parkinsonʼs and Alzheimerʼs disease. One of the pathological hallmarks of Alzheimerʼs disease is elevated phosphorylation and aggregation of tau, a neuronal microtubule-associated protein. In the hippocampus, arctigenin effectively reduced the phosphorylation of tau at Thr-181, Thr-231, and Ser-404. Additionally, arctigenin was found to enhance the phosphorylation levels of phosphatidylinositol 3-kinase, threonine/serine protein kinase B, and glycogen synthase-3-kinase β . Arctigenin has been found to be efficacious in safeguarding against cognitive impairments related to learning and memory, while also suppressing the development of hyperphosphorylated tau protein inside the hippocampus. In 2017, Qi et al. demonstrated that arctigenin treatment decreases tau hyperphosphorylation within the GSK-3β /PI3K/Akt- and
PI3K/Akt/GSK-3β -dependent signaling pathways [69 ], [70 ].
Anticancer activity
In South Africa, infusions made from the herb of C. benedictus are used to treat cancer-related health problems; however, their effectiveness has not been confirmed by clinical studies. C. benedictus is also one of multiple ingredients in the Flor-Essence preparation, which has clinically unconfirmed anticancer properties. In Canada, the preparation is used by patients with cancer, as well as by individuals suffering from other chronic disease [2 ], [27 ].
Preclinical studies suggest that certain compounds found in the herb, such as cnicin, arctigenin, or arctiin, may have anticancer properties.
Tumor cells in poorly vascularized, solid tumors are constantly or periodically exposed to unfavorable microenvironmental conditions, such as glucose deficiency or hypoxia. Glucose deficiency often activates the unfolded protein response (UPR) pathway, involving improperly folded proteins. This pathway increases the survival of cancer cells by inducing stress proteins. Arctigenin selectively inhibits the viability of tumor cells under glucose-deficient conditions. This compound inhibits the expression of UPR genes, such as PERK, ATF4, CHOP, and GRP78, leading to the initiation of apoptosis through the activation of caspases 9 and 3 [71 ]. Arctigenin at a concentration of 0.01 µg/ml has been identified as the primary compound exhibiting preferential cytotoxicity against nutrient-deprived cells. It inhibits the growth of pancreatic tumors, such as PANC-1, in mice, exhibiting preferred cytotoxicity under nutrient-deprived conditions [72 ].
Arctiin and arctigenin also demonstrated beneficial effects in skin tumors induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in mice. In rodents, these lignans caused an over 50% reduction in the number of papillomas over a period of 20 weeks. Additionally, arctigenin exhibited strong antitumor activity in a two-stage carcinogenesis test for mouse lung tumors induced by N-nitroquinoline oxide as an initiator and glycerol as a promoter of tumorigenesis. Oral administration of arctigenin significantly reduced the incidence of lung tumors, with approximately 50% inhibition in the total number of lung tumors and over 40% reduction in the percentage of mice with tumors in the lung lobe [73 ].
Other studies indicate that arctiin has a protective effect in the carcinogenesis induced by PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), especially within the mammary gland and colon. This is likely associated with the influence of this compound on the arachidonic acid metabolism pathway [74 ].
Studies have also highlighted the potential of cnicin as an anticancer agent. It exhibits cytotoxicity toward various cancer cell lines, including breast, lung, colon, and prostate cancers. Cnicin induces programmed cell death, inhibits cell proliferation, and modulates signaling pathways involved in cancer progression. Additionally, it has shown promising results in inhibiting angiogenesis, a process critical for tumor growth and metastasis. As part of the study on the anti-inflammatory properties of cnicin, the main active substance in C. benedictus , this compound was isolated using column chromatography, characterized through nuclear magnetic resonance (NMR) spectroscopy, and then subjected to cell tests to assess its inhibitory effect on nuclear factor κ B (NF-κ B), inducible nitric oxide synthase (iNOS) activity, generation of reactive oxygen species, and cytotoxicity in selected human solid tumor cell lines and in two non-tumor kidney cell lines
(Vero monkey kidney fibroblasts and LLC-PK11 pig kidney epithelial cells). The results demonstrated inhibition of NF-κ B and iNOS activity by cnicin. Cytotoxic effects were observed on LLC-PK11 pig kidney epithelial cells, human malignant melanoma cells (SK-MEL), and human breast adenocarcinoma cells (BT-549). Cnicin was not toxic to Vero cells but exhibited mild toxicity to LLC-PK11 cells [68 ]. Cnicin also showed fairly strong activity against a human breast carcinoma line (MCF-7), with an IC50 value of 3.25 µg/mL, possibly due to the a-methylene-c-lactone rings in its structure [75 ]. In addition, cnicin significantly inhibited NF-κ B (1.8 µM) and SP-1 (16.0 µM) in human chondrosarcoma cells (SW1353) induced with phorbol myristate acetate and also inhibited iNOS activity (6.5 µM) in mouse macrophages (RAW264.7 cells) induced with lipopolysaccharide [68 ].
Jöhrer and colleagues (2012) conducted research evaluating the potential cytotoxic effects of cnicin on multiple myeloma cells, which belong to one of the most common hematologic malignancies. The study demonstrated a strong antiproliferative effect of this compound and its ability to induce cell death in cell lines and primary multiple myeloma cells, even in the presence of cytokines that support survival and provide a suitable microenvironment for the tumor. Activation of caspases, accumulation of reactive oxygen species, and reduction in NF-κ B (nuclear factor kappa-light-chain-enhancer of activated B cells) levels contribute to the cytotoxic action of cnicin. Microarray analysis to detect genes involved in cnicin-induced cell death highlighted the pro-oncogene PIM2 . The Pim-2 protein is a novel kinase that conditions the survival of multiple myeloma cells in vitro and is strongly expressed in malignant cells but not in normal plasma cells in
vivo . The results established that cnicin induces multiple modes of cell death in multiple myeloma, suggesting Pim-2 as a new therapeutic target [76 ].
In 1997, Barrero and colleagues isolated malacytanolid, a product of the epoxidation and cyclization of cnicin. The cytotoxic activity of this compound was investigated in vitro in the mouse lymphoma cell line P-388, SCHABEL, and three human cell lines: A-549 (lung cancer), HT-29 (colon cancer), and MEL-28 (melanoma). The results indicate that this compound is 20 – 40-fold more cytotoxic than cnicin toward the P-388, A-549, and HT-29 cell lines [77 ].
It has been demonstrated that arctiin inhibits the growth of cancer cells by downregulating the expression of cyclin D1 and upregulating genes for MUC-1 (anti-adhesion mucin). Cyclin D1 is responsible for cell cycle regulation, and its overexpression is observed in many types of human cancers, such as breast cancer, prostate cancer, kidney cancer, and colorectal cancer. In vitro , arctiin inhibits the growth of PC-3 (human prostate cancer) and HaCaT (human immortalized keratinocyte) cell lines [78 ]. Arctigenin has been shown to block the activity of enzymes with tyrosine kinase activity (Akt) induced by glucose deficiency. Akt plays a crucial role in developing tolerance to starvation, increasing the survival of cancer cells under glucose deficiency conditions. Incubating cancer cells with arctigenin abolished the tolerance of cancer cells to nutrient-deficient conditions [72 ]. Inhibitory effects of arctigenin
have also been demonstrated in the human promyelocytic leukemia HL-60 cell line, likely through blocking DNA, RNA, and/or protein synthesis in leukemia cells [79 ].
In in vitro studies, it has been demonstrated that cnicin, arctigenin, and arctiin inhibit the proliferation of DU-145 prostate cancer cells, MCF-7 (breast cancer cells), and MCF-12A (non-malignant breast cells). In contrast, an extract from C. benedictus does not exhibit such activity. This is an interesting discovery that highlights the differences in anticancer activity between individual isolated active compounds and a whole-plant extract, which is a mixture of various pharmacologically active compounds [34 ].
Despite promising research results, the anticancer properties of individual components of the plant should be confirmed in clinical conditions.
Safety profile of C. benedictus
Side effects
The published data so far regarding the safety of using C. benedictus indicate its low toxicity, provided that proper dosages are observed, with values based on traditional medicine practices. The plant is used orally as an herbal tea or as herbal preparations in liquid or solid dosage forms [26 ]. There is not enough reliable information to definitively confirm whether preparations of C. benedictus are safe as medicines and what adverse effects they may induce. Consuming an infusion or decoction of the herb in large quantities (> 5 g per cup) may lead to gastric irritation, nausea, vomiting, colic, diarrhea, and hypertension. Allergic reactions to components present in the herb, especially in individuals sensitive to plants from the Asteraceae/Compositae family, have also been observed [26 ], [80 ].
Laboratory studies suggest that C. benedictus may increase the risk of bleeding; however, such an effect has not been observed in humans. The herb contains tannins (approximately 8%), which theoretically may exhibit hepatotoxic and nephrotoxic effects with prolonged use [2 ]. Prolonged use of plant materials containing tannins in amounts exceeding 10% has been shown to cause gastrointestinal discomfort, liver disease, and kidney damage and increase the risk of esophageal or nasal cavity cancer [1 ], [26 ]. For this reason, caution should be exercised in patients with gastrointestinal ulcers, reflux disease, hiatal hernia, and Barrettʼs esophagus.
In traditional medicine, C. benedictus is used to stimulate milk secretion. However, it should be emphasized that the available literature lacks sufficient data on the penetration of active ingredients from the plant into breast milk and any potential impact on the health of breastfed infants. In a randomized, double-blind clinical trial, the effectiveness of a specific tea called Motherʼs Milk Tea (Traditional Medicinals, Sebastopol, CA) was compared with lemon verbena tea in mothers experiencing breastfeeding issues. Each tea bag contained 35 mg of C. benedictus , along with several other herbs. Mothers were advised to drink 3 to 5 cups of tea per day. No differences were observed between the groups regarding adverse symptoms in mothers and infants related to the digestive, respiratory, or dermatological systems, etc. There were also no noted differences in the growth parameters of breastfed infants between the two groups [81 ].
To this day, there have been no reports of teratogenic effects of C. benedictus used in pregnant women. However, the lack of preclinical studies in animals and controlled clinical trials in humans does not rule out potential harm to the fetus, especially since, in traditional medicine, the plant is used as an abortifacient and menstruation-inducing agent [2 ]. For this reason, its use during pregnancy and lactation is not recommended [26 ].
Simultaneously, in the absence of sufficient data, the use of C. benedictus in children and adolescents under 18 years of age has not been established and is not recommended [26 ].
Interactions with other medications
There is a lack of confirmed clinical data in the available literature regarding the interactions of preparations containing C. benedictus with other medications. The herb is traditionally used to increase gastric secretion, and there is a risk of interactions between the active ingredients of this plant and antacids used in peptic ulcer disease. Laboratory studies suggest that the use of preparations containing C. benedictus may increase the risk of bleeding, especially when combined with anticoagulant medications such as warfarin, heparin, antiplatelet drugs, and nonsteroidal anti-inflammatory drugs [2 ]. C. benedictus contains components that act antagonistically to platelet-activating factor (PAF), which theoretically could reduce platelet aggregation stimulated by this factor and contribute to bleeding [1 ].
The lignans present in the plant exhibit antiviral activity. Synergistic antiviral activity has been demonstrated between arctigenin and oseltamivir, a neuraminidase inhibitor, against resistant influenza viruses (H1N1) [49 ], [50 ].
In silico analysis of the pharmacokinetic and toxicological profiles of the main active phytochemicals in C. benedictus
Safety pharmacology is a rapidly expanding field that focuses on evaluating the possible dangers of improper pharmacotherapy. Evaluating the safety of a chemical is a crucial aspect of introducing a new medicine to the market. The Organisation for Economic Co-operation and Development (OECD) has published several principles for conducting drug safety testing [82 ]. Contemporary technology provides a range of methods that, when utilized, should establish the circumstances necessary to implement safety pharmacology.
Without a doubt, in silico studies provide several opportunities to find answers in this field. Currently, the primary focus is on studying the pharmacokinetic and toxicological properties of potential medication candidates in order to better understand them. The in silico analysis described below contributes significantly to this understanding.
The bioavailability of cnicin, arctigenin, and arctiin
One of the goals of this study was to use SwissADME to forecast the physicochemical qualities, drug-likeness properties, ADME, and toxicity of three active constituents of C. benedictus to understand their pharmacokinetics and the traditional use of the plant.
The two-dimensional chemical structures of cnicin, arctigenin, and arctiin with canonical SMILES were extracted from Pubchem databases, and the bioavailability radar was constructed for each analyzed molecule ([Fig. 4 ]). The bioavailability radar allows for the initial assessment of the drug-likeness of the tested molecules based on six physicochemical properties: lipophilicity (LIPO) measured by XLOGP3, which ranges from − 0.7 to + 5.0, size (SIZE) determined on the basis of molecular weight (MW) ranging from 150 to 500 g/mol, polarity (POLAR) resulting from the topological polar surface area (TPSA) spanning from 20 to 130 Å2 , insolubility (INSOLU), insaturation (INSATU), and flexibility (FLEX). Solubility is determined on the basis of log S, which should not exceed 6. In turn, insaturation is characterized on the basis of the fraction of carbons in the sp3 hybridization (it should be no less than 0.25). Flexibility is
assessed on the basis of the number of rotatable bonds (which should not exceed 9) [6 ], [83 ], [84 ]. It should be noted that the grey area designates the optimum physicochemical space for properties predicting bioavailability by oral administration.
Fig. 4 The bioavailability radar of the main components of C. benedictus L.: arctigenin, arctiin and cnicin. The gray area represents the optimal range for each of the properties (lipophilicity: XLOGP3 between − 0.7 and + 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S no higher than 6, saturation: fraction of carbons in the sp3 hybridization no less than 0.25, and flexibility: no more than 9 rotatable bonds). The compounds names are indicated in the different font colors.
In [Tables 3 ]–[6 ], we present the molecular and physicochemical characteristics of cnicin, arctigenin, and arctiin, including their molecular formulas, molecular weights ([Table 3 ]), numbers of heavy atoms, numbers of aromatic heavy atoms, Csp3 fractions, numbers of rotatable bonds, numbers of H-bond acceptors, numbers of H-bond donors, molar refractivities, TPSAs ([Table 4 ]), lipophilicity characteristics ([Table 5 ]), and water solubility ([Table 6 ]).
Table 3 General characteristics of the phytoconstituents of C. benedictus L.
Small molecule
Pubchem ID
Molecular formula
Canonical SMILES
Molecular weight (g/mol)
cnicin
5 281 435
C20 H26 O7
CC1=CCCC(=CC2C(C(C1)OC(=O)C(=C)C(CO)O)C(=C)C(=O)O2)CO
378.4
arctigenin
64 981
C21 H24 O6
COC1=C(C=C(C=C1)C[C@H]2COC(=O)[C@@H]2CC3=CC(=C(C=C3)O)OC)OC
372.4
arctiin
100 528
C27 H34 O11
COC1=C(C=C(C=C1)C[C@H]2COC(=O)[C@@H]2CC3=CC(=C(C=C3)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)OC)OC
534.6
Table 6 Water solubility characteristics of the phytoconstituents of C. benedictus L. S–soluble, MS–moderately soluble, VS–very soluble.
Molecule
ESOL
Ali
SILICOS- IT
Log S (ESOL)
Solubility
Class
Log S (Ali)
Solubility
Class
Log S (SILICOS- IT)
Solubility
Class
mg/ml
mol/L
mg/ml
mol/L
mg/ml
mol/L
cnicin
− 1.95
4.27
1.13 × 10−2
VS
− 2.19
2.44
6.46 × 10−3
S
− 1.64
8.75
2.31 × 10−2
S
arctigenin
− 4.28
1.97 × 10−2
5.28 × 10−5
MS
− 4.84
5.44 × 10−3
1.46 × 10−5
MS
− 5.75
6.56 × 10−4
1.76 × 10−6
MS
arctiin
− 3.85
7.56 × 10−2
1.41 × 10−4
S
− 4.62
1.29 × 10−2
2.40 × 10−5
MS
− 3.99
5.51 × 10−2
1.03 × 10−4
S
Table 4 Physicochemical properties the phytoconstituents of C. benedictus L. Num.–number, arom. -aromatic, H-bond–hydrogen bond, TPSA–topological polar surface area.
Molecule
Num. heavy atoms
Num. arom. heavy atoms
Fraction Csp3
Num. rotatable bonds
Num. H-bond acceptors
Num. H-bond donors
Molar refractivity
TPSA (0A2)
cnicin
27
0
0.50
6
7
3
98.19
113.29
arctigenin
27
12
0.38
7
6
1
100.60
74.22
arctiin
38
12
0.52
10
11
4
132.72
153.37
Table 5 Lipophilicity characteristics of the phytoconstituents of C. benedictus L.
Molecule
iLOGP
XLOGP3
WLOGP
MLOGP
SILICOS-IT
Consensus Log Po/w
cnicin
1.97
0.25
0.95
0.92
1.82
1.18
arctigenin
2.64
3.59
2.99
2.12
3.99
3.07
arctiin
2.73
1.78
0.47
− 0.11
1.93
1.36
Lipophilicity is demonstrated as partition coefficients (log P) and distribution coefficients (log D). Larger values of a small moleculeʼs log P correspond to greater lipophilicity. To evaluate the lipophilic characteristics of C. benedictus compounds, we used five freely available models from SwissADME: XLOGP3 [85 ], WLOGP [86 ], MLOGP [87 ], [88 ], SILICOS-IT, and iLOGP [84 ].
Brain or intestinal estimated permeation method (BOILED-Egg) for blood–brain barrier (BBB) and gastrointestinal absorption prediction
To predict the brain penetration and gastrointestinal absorption of cnicin, arctigenin, and arctiin, we used the BOILED-Egg method [89 ], [90 ], [91 ]. The method was validated using a set of known compounds with experimentally determined permeabilities and involves estimating the partition coefficients of the tested molecules between an acetanol phase in the brain and intestines, which allows the calculation of predicted permeability across both the blood–brain barrier, and the gastrointestinal tract using the following equation:
Permeability across the blood–brain barrier (BBB)
Permeability across the gastrointestinal tract (GIT)
The pharmacokinetic and drug-likeness assessment performed by SwissADME confirmed a high level of GI absorption with arctigenin and cnicin and high BBB permeation by arctigenin. Only arctiin present in C. benedictus L. is a substrate for P-gp ([Fig. 5 ]).
Fig. 5 Boiled-EGG model for analyzing gastrointestinal absorption (GI) and blood brain barrier (BBB) permeant P-gp substrate. White region is for high probability for passive absorption by GI and the yellow area (yolk) is for high probability of brain penetration. Points colored in blue is predicted as actively effluxes by P-gp (PGP+) and in red if predicted as non-substrate of P-gp (PGP-). The names of the compounds are indicated in different font colors, i.e. black corresponds to the analyses active compound, and purple corresponds to the selected drugs for comparison.
More extensive SwissADME predictions of the pharmacokinetics and other properties of arctigenin, cnicin, and arctiin are presented in [Table 7 ]. Arctigenin is predicted with high probability to be an inhibitor of the cytochrome P 450 isoenzymes CYP2C9, CYP2D6, and CYP3A4. Almost all of the analyzed small molecules except arctiin returned as non-substrates of P-gp. Arctiin is also probably a CYP3A4 inhibitor.
Table 7 Pharmacokinetic parameters of the phytoconstituents of C. benedictus L.
Molecule
GI absorption
BBB permeant
P-gp substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Log Kp (Skin Permeation) (cm/s)
cnicin
high
no
no
no
no
no
no
no
− 8.43
arctigenin
high
yes
no
no
no
yes
yes
yes
− 6.02
arctiin
low
no
yes
no
no
no
no
yes
− 8.30
This study predicts that only arctigenin will be found in the yolk in the assay, indicating a significant level of brain penetration. On the other hand, cnicin is expected to be present in the white zone of the BOILED egg, indicating a high level of passive gastrointestinal absorption. Conversely, arctiin has the lowest probability of being absorbed by the BBB and hematoma. The comparative analysis presented here demonstrates a significant pharmacokinetic resemblance to NSAIDs such as ibuprofen and indomethacin. Cnicin exhibits a bioactivity profile similar to that of kainic acid. Kainic acid can be classified as a neurotoxin when consumed in high amounts, whereas low amounts of a diluted solution can chemically excite neurons [92 ]. Arctigenin is a competitive inhibitor of kainic acid binding.
Target predictions for cnicin, arctigenin, and arctiin
We identified the most probable macromolecular targets of the tested molecules cnicin ([Fig. 6 a ]), arctigenin ([Fig. 6 b ]), and arctiin ([Fig. 6 c ]) using the web tool SwissTargetPrediction. The identifications are based on a combination of 2D and 3D similarity with a library of 370 000 known activities on more than 3000 proteins from three different species: Homo sapiens, Mus musculus , and Rattus norvegicus
[93 ], [94 ]. A combined score above 0.5 indicates that the small molecules share a common protein target.
Fig. 6 Target prediction for cnicin (a ), arctigenin (b ) and arctiin (c ) cnicin made using SwissTargetPrediction..
The following comprised most of the arctigenin targets identified using the latest version of SwissTargetPrediction: cytochrome P450, oxidoreductase, family AG protein, nuclear receptor, and electrochemical transporter enzyme. Identified high-probability targets of arctigenin were secreted proteins such as sex hormone–binding globulin (SHBG), which is important for reproduction, and kinases such as mitogen-activated protein kinase kinase 1 (MAP2K1).
Cnicin was predicted to target the following molecular/biochemical pathways: phosphodiesterase, protease, cytochrome P450, oxidoreductase, enzyme, hydrolase, family AG protein, and transcription factor.
Arctiin targets included family AG protein, kinase, nuclear receptor, enzymes, electrochemical transporter, and ligand-gated ion channel.
