Dietary Plant Flavone Cynaroside and Its Biological Significance

Abstract

Flavonoids, the most diverse group of natural polyphenolics, are secondary plant metabolites that play a crucial role in human health protection. Two main classes—flavonols and flavones—comprise the main body of flavonoids with antioxidant properties and high biological activity, proven both in vitro and in vivo. Purified samples of flavones represent special interest. One of them, luteolin-7-glucoside (cynaroside), has attracted increasing attention as a potential agent possessing a number of biological activities. The current understanding of cynaroside bioactivities is outlined in this chapter, along with research gaps and potential future directions for this flavonoid’s study.

1. Introduction

Flavonoids assigned as low molecular weight secondary metabolism phytochemicals perform various biological properties both manifested in vitro and in vivo [1]. Extensive range of studies strongly suggest that long-term consumption of diets rich in plant flavonoids offers protection against development of cancers [2], cardiovascular diseases [3], diabetes [4], neurodegenerative diseases [5], and so on. By now, the mechanisms of flavonoids action have been proposed that include the influence of signaling processes, flowing in living systems, due to the specific interaction with regulatory proteins [6], the modification of eicosanoid biosynthesis [7], the prevention of platelet aggregation [8], the promotion of relaxation of cardiovascular smooth muscle cells [9], and so forth.

Flavones and flavone-derived compounds are a large group of flavonoids with a great number of properties and presented in vegetables, fruits, and aromatic plants [10]. The term “flavone” was used for the first time in 1895 by von Kostanecki and Tambor who were pioneers in the structural work of this particular class of flavonoids [11].

Cynaroside—which is 7-O-glucoside of luteolin (other names are luteolin-7-O-β-D-glucopyranoside or luteolin-7-O-β- Dglucopyranoside-5,3′,4′-trihydroxyflavone, Luteoloside [12], luteolin-7-glucoside, nephrocizin [13])—is a natural flavone, one of the bioactive compounds purified from various genuses of plant families: Apiaceae [14], Lamiaceae [15], Asteráceae [16], and so on (Figure 1 and Table 1).

Figure 1.

Chemical structure of cynaroside.

Family	Species
Apiaceae	Pimpinella anisum L. [10], Daucus carota [17], Angelica keiskei [18], Anthriscus sylvestris (L.) Hoffm. [19], Dystaenia takeshimana [20], Ferula varia [21]
Asteraceae	Cynara scolymus [22], Ixeris chinensis [22], Chrysanthemum morfolium Ramat [23], Glossogyne tenuifolia [24], Tanacetum parthenium [25], Taraxacum officinale [25], Scorzonera cana var. jacquiniana [26], Scorzonera cinerea [26], Scorzonera eriophora [26], Scorzonera incisa [26], Scorzonera parviflora [26], Dendranthema morifolium [27], Chrysanthemum indicum Linnén. [28], Chrysanthemum morifolium [28], Achillea millefolium L. [29], Artemisia rupestris [30], Carduus crispus [31], Bidens tripartite [32], Cirsium canum (L.) [32], Smallanthus sonchifolius [33]
Balsaminaceae	Impatiens textori [33]
Caprifoliaceae	Lonicera japonica [34], Lonicera maackii [35]
Fabaceae	Leucaena leucocephala [36], Vicia pannonica var. purpurascens [37]
Juncaceae	Juncus jerardi Lois., Luzula silvatica, Juncus ariculatus, Juncus conglameratus, Juncus filiformis, Juncus tenuis [38]
Lamiaceae	Salvia officinalis [39], Scutellaria immaculata and Scutellaria ramosissima [39], Lycopus europaeus L. [40], Elsholtzia blanda (Benth.) Benth [41], Phlomis younghusbandii [42], Melittis melissophyllum L. [43]
Myrtaceae	Pimenta racemosa [44]
Oleaceae	Ligustrum lucidum Ait [45], Phillyrea latifolia [46], Ligustrum delavayanum and L. vulgare [47]
Polygonaceae	Polygonum orientale [48], Rumex hastatus [15]
Pteridaceae	Pteris multifida [49]
Rosaceae	Crataegus monogyna [50], Agrimonia pilosa Ledeb [51], Prunus mume [52]
Rubiaceae	Ophiorrhiza mungos Linn [52]
Salicaceae	Salix matsudana [53]
Scrophulariaceae	Verbascum densiflorum and V. Phlomoides [54], Verbascum nubicum [55]

Table 1.

Natural sources of cynaroside.

The name “cynaroside” originates from the latin term Cynara denoting the name of the genus of artichokes where this flavone was firstly isolated in 1955 and defined to be glycoside in 1958 by Michaud (J.) and Masquelier (J.) correspondingly [56, 57]. Since that, interest to cynaroside has been growing considerably. For instance, due to electronic database PubMed, search for the term “cynaroside, luteolin-7-O-glucoside, nephrocizin” shows growing trend in publications (Figure 2).

Figure 2.

Trend of research in the field of cynaroside represented by PubMed database.

Several lines of evidence have emerged in recent years, suggesting that cynaroside, despite its nontoxic nature, can minimize hepatotoxicity [58]; repair the antioxidant system [59]; normalize energy, lipid, and carbohydrate metabolism; and enhance bile flow [60]. Therefore, scientific interest in flavones as therapeutic agents is rapidly increasing. The most interesting biological activities of cynaroside are summarized in Figure 3.

Figure 3.

Some biological activities of cynaroside.

The purpose of this chapter was to provide an overview of the distribution and biological properties of cynaroside. In this paper, we concentrate our attention on the most common knowledge about the biological effects of plant flavone cynaroside in the context of relevance to influencing human physiology and therefore interesting from a general perspective of improving human well-being.

The electronic databases included PubMed, and Google Scholar was initially used to find articles (published as early as 1955 up to manuscript submission). More than 100 articles were found in applied databases and manually screened sources. Reported publications focus on the cynaroside identification in plants, isolation, its biological and pharmacological properties in vivo and in vitro, but any articles summarizing or analyzing all this information were not observed. As was mentioned above, the search terms included “cynaroside” as well as “luteolin-7-glucoside” and “nephrocizin” (the mostly used alternative terms for “cynaroside”). Firstly, the search was limited to English-Language articles. Citation lists of retrieved articles were screened manually to ensure sensitivity of the search strategy. References of the included papers were then hand searched to identify in potential relevant studies in other languages (only abstract was used in Russian, French, and Chinese articles). Only published articles were included. No protocol was developed for this review.

2. Potential health benefits

Even though there is some data on LD50 of the plant extracts abundant on cynaroside [55], acute toxicity data on pure cynaroside are not available. In general, in vivo experiments carried on rats showed that the administration of cynaroside to six-week-old male Wistar rats for 1 week did not change animals’ food and beverage consumption, as well as animal body weights when compared to the control [1]. Thus, the rats treated with pure cynaroside up to a dose of 30 mg/kg did not demonstrate any signs of hepatotoxicity [61].

3. Hepatoprotective effect

The proposed hepatoprotective effect of cynaroside was due to the great importance of Cynara (artischocke) abundant in the flavonoid in folk medicine against liver complaints.

In vivo carbon tetrachloride (CCl₄)-induced toxicity model revealed that pretreatment of experimental rats with cynaroside suppressed the elevation of alanine aminotransferase (ALT, GPT, and glutamic-pyruvic transaminase), aspartate transaminase (AST, GOT, and glutamic oxaloacetic transaminase), malondialdehyde (MDA), and 8-Oxo-2′-deoxyguanosine (8-OHdG) and inhibited the reduction of glutathione (GSH) in a dose-dependent manner [61]. In vitro experiments with fresh hepatocytes, primary hepatocyte cultures, and immortalized cell lines also showed the protective effect of cynaroside from cell damage induced by carbon tetrachloride (CCl₄) [61], as well as brombenzene [62] and t-BHP [63]. Interestingly, Adzet et al. [64] observed specific GOT-recovering effect on isolated hepatocytes preincubated with cynaroside.

However, Quisheng et al. [60] in vivo demonstrated dose-dependent decline of both AST and ALT levels in blood serum. It is well accepted that increased enzyme levels are indicative of a tissue injury: the attenuated increase of serum AST and ALT indicates a direct protective role in hepatocytes; while AST is not specific to the liver, any elevation of the ALT is a direct indication of a liver injury. In the meanwhile, some authors mark that ALT and AST have relatively long half-lives (T1/2) (literature estimates approximately 17 and 47 hours, respectively) [65, 66] and thus do not reflect immediate changes in liver injury or recovery [67]. Therefore it could be worth considering that in the work of Adzet et al., the period of incubation of cynaroside with toxicated cells did not last more than 1 hour, while in the work of Quisheng et al. [60], all treated animals were sacrificed 24 hours after receiving the administration of the LUTG or hepatotoxin. Thus, it might be worth considering the preliminary incubation time as one important factor in cynaroside hepatoprotection.

Cynaroide in vitro also recovered significant (up to 81%) GSH level in tBHP-induced HepG2 cells after 5 hours of incubation [63], which was even higher than by luteolin (53%) and twice higher than quercetin (40%), and in vivo cynaroside was able to attenuate the CCl₄-induced reduction of GSH in a dose-dependent manner [61, 63]. On the other hand, cynaroside did not have any effect on the recovery level of GST either on cell lines in vitro or on cells isolated from rats that were subjected to brombenzene in vivo [62]; the effect of cynaroside on its own in normal conditions was not shown by the authors.

It is known that GSH conjugates can be formed directly or be catalyzed by GST [68]. All the toxins mentioned above (CCl₄, brombenzene, t-BHP) are known to initiate the formation of free reactive species. Thus, CCl₄ intoxication metabolized by CYP2E1 is forced by the prevention of lipid peroxidation by decreasing the level of LPO and reducing the level of MDA. Wang et al. [69] found that cynaroside affected some isoforms of P₄₅₀, namely, isoforms CYP2C9, 3A4, and 1A2; however, no effect was found on 2E1 isoform. That lets to propose that in the case of CCl₄ intoxication, cynaroside does not have an effect of toxin metabolization, but protects the lipids through its antiradical properties against CCl₃ metabolite.

The results of Park et al. [62] indicate a protective effect of cynaroside in reducing the degree of liver lipid peroxidation caused by the hepatotoxin bromobenzene in vivo in rats. Thus, cynaroside at a concentration of 10 mg/kg almost completely neutralized the process of lipid peroxidation caused by hepatoxin and restored the level of the enzyme epoxide hydrolase (an enzyme that deactivates bromobenzene). Bromobenzene is converted by P₄₅₀ enzymes to its reactive, toxic metabolite bromobenzene 3,4-epoxide that is metabolized to a nontoxic bromobenzene 3,4-dihydrodiol by either epoxide hydrolase or glutathione S-transferase. Cynaroside even not effecting GST was nevertheless shown to recover the level of epoxide hydrolase, suggesting that cynaroside might improve the level of hydrolase and also act as a radical scavenger.

There are two pathways by which tBHP is metabolized; both of them induce oxidative stress. The first, provided by cytochrome P450, leads to the production of peroxyl and alkoxyl radicals [70]. These radicals initiate lipoperoxidation of membrane phospholipids with subsequent alterations to membrane fluidity and permeability. The other pathway employs glutathione peroxidase. tBHP is detoxified to tert-butanol, and GSH is depleted by oxidation to its disulfide form (GSSG) [71].

Results showed that cynaroside protects from AST and ALT leakage and also strongly suggest the ability of cynaroside in protecting hepatocyte against membrane fragility. Recent findings have demonstrated that the carbon ring B is deeply immersed into lipid bilayer. A ring is adjusted toward the aqueous environment; the glycosylated form (in position 7) is dispersed over the lipid bilayer, thus providing the defense for double bonds from oxidation [72].

4. Cardio-protective effect

It has been shown that cynaroside in pharmacological concentrations has a positive inotropic and vasodilator effect. On the models of isolated hearts of rabbits and guinea pigs, perfused using the Langendorff technique, it was shown that cynaroside increases pressure in the left ventricle, accelerates the process of blood ejection into the arterial system, and enhances the total and relative coronary blood flow and, consequently, the supply oxygen for nonischemic myocardial damage [73, 74].

Also, in the last decade, the processes of apoptosis in cardiovascular pathologies have been intensively studied, and more and more researchers are assigning a leading role to apoptosis in the pathogenesis of various cardiovascular diseases. Xiao Sun et al. [70] demonstrated that cynaroside exhibits cytoprotective properties in relation to the H9C2 cardiac myocyte cell line against H₂O₂-induced apoptosis. Cardiomyocyte cells exposed to H₂O₂ suffered severe damage accompanied by apoptosis. Preincubation of cells with cynaroside reduced the degree of ROS generation and inhibition of caspase activation both in the mitochondrial pathway and through the so-called death receptors by enhancing the endogenous antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting the formation of intracellular ROS. In addition, cynaroside supported normal mitochondrial function by regulating the expression of Bcl-2 protein (B-cell lymphoma protein 2, apoptosis regulator protein Bcl-2), as well as the expression of protein kinases JNK (c-Jun N-terminal kinase, mitogen-activated protein kinase) and p53 (cell cycle regulating transcription factor). The results of microscopic studies showed that after incubation with cynaroside, there was a dose-dependent restoration of the morphological structures of cardiomyocytes damaged by H₂O₂, such as a dense membrane packing compared to a loose membrane layer, normal physiological spindle shape versus cell swelling, round shape of the nucleus in comparison with vacuolar degeneration, and so forth. An analysis of generalized experimental data also indicates some possible mechanisms of the protective modulating action of cynaroside in certain pathological conditions of the heart, which indicates an urgent need for further in-depth study of the mechanisms of the action of cynaroside and also to consider it as the basis for the creation of potentially effective and safe cardioprotective agents that can make a significant contribution to the treatment and prevention of a number of cardiovascular diseases.

5. Anticancer effect

Evidences are accumulating that pure cynaroside possesses antiproliferative and cytotoxic effects on the tested cancer cell lines with minimal toxicity toward the normal (VERO) cells, indicating cancer cell-specific cytotoxic (apoptotic) effect. Various researchers mentioned that cynaroside does not cause cytotoxicity in relation to normal cells (human neonatal epidermal keratinocytes (HEKn) [75], VERO [76], non-tumorigenic BEAS-2B [77], and Huh7 [78].

Luteolin-7-glucoside in more or less extent has been shown to inhibit a proliferation of a series of human cancer cell lines: breast (MCF-7 [76, 77, 79, 80], MDA-MB-231 [80], A549 [76, 80], H292 [81], renal [77], colorectal (COLO 320 DM [76], gastric (AGS [76], liver (HepG-2 [78, 79, 80], Hep 3B [80, 82], LO2 [83], SNU-449 [82], Huh-7 [82], SMMC-7721 [82], MHCCLM3 [82] and MHCC97-H [82], cervical (HeLa [79], melanoma (UACC-62 [77], and some others (mouse fibroblast cell line L929 [84]. No significant effect was observed on macrophage RAW264.7 cells, Ehrlich ascites carcinoma [36], and Rhabdomyosarcoma [12].

6. Anti-inflammatory activity

Palombo et al. [75] in their study on a model of psoriasis showed the ability of luteolin-7-glucoside to regulate proliferative responses, as well as differentiation of cultured human keratinocytes treated with interleukin 22 (IL-22). It was found that local administration of cynaroside leads to a noticeable reduction in acanthosis. Thus, treatment with cynaroside led to a decrease in the expression of proliferation markers, an increase in the production of differentiation markers, and, in general, to a phenotypic improvement.

Other in vivo tests have shown that luteolin-7-O-glucoside is responsible for wound healing activity. It has also been found to have significant anti-inflammatory, antioxidant, anti-hyaluronidase and anti-collagenase activities. Phases in the healing processes (inflammation, proliferation, and remodeling) were observed and recorded in the experimental groups to varying degrees. All tissues treated with cynaroside ointments showed good healing processes with the potential for faster reepithelialization and high collagen concentrations compared to other groups tested. The anti-inflammatory activity of cynaroside was mainly demonstrated by the inhibition of nuclear factor-kappa B (NF-kappa B), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and inducible NO synthase (iNOS) [85, 86].

7. Radical scavenging activity

An analysis of the scientific literature has shown that cynaroside has significant antiradical (ARA) and antioxidant (AOA) properties in relation to free radicals and ROS molecules (superoxide anion (O₂^•−) [80], hydroxyl radical (HO^•) [87], peroxyl radicals (ROO−) [23], and nitric oxide (NO) [85]. ARA of cynaroside using the DPPH method is quite widely presented in various works [23, 88]. It has been shown that cynaroside has a fairly high efficiency in the reduction of DPPH molecules (IC50 value varies in different studies: from 13.90 ± 1.46 μM to 277.3 ± 14.9 mmol/mol DPPH. In addition, it was found that cynaroside dose-dependently inhibits the formation of superoxide anion induced by various stimuli, such as N-formylmethionyl-leucyl-phenylalanine, human polymorphonuclear neutrophils and peripheral blood mononuclear cells, phorbol-12-myristate-13-acetate (PMA) and arachidonic acid (AA) in human neutrophils, superoxide anion produced by the xanthine/xanthine oxidase system, and so on [89]. Hsu et al. [80] showed that cynaroside prevents DNA dissociation caused by hydroxyl radical. Cynaroside at a maximum concentration of 1.8 μg/ml provided 64.0 (3.9%) and 83.1 (2.9%) protection of supercoiled DNA against nonspecific and site-specific oxidation caused by hydroxyl radical, respectively. In comparison, 4 μg/ml Trolox (a water-soluble vitamin E analog) provided 83.5 (0.5%) and 42.6 (2.5%) protection. Cynaroside also effectively slows down lipid peroxidation caused by peroxyl radical as well as reduces the concentration of nitric oxide (NO) free radicals produced by endothelial nitric oxide synthase (eNOS) under pathological conditions [85].

8. Antibacterial activity

Isolated cynaroside also exhibits antimicrobial activity, especially against gram-negative bacteria (Escherichia coli, Streptococcus pyogenes, and Micrococcus luteus with minimal inhibition zone of 0.5 mm), suppresses the formation of biofilm of gram-positive Pseudomonas aeruginosa and Staphylococcus aureus, and increases the frequency of mutations that reduce the resistance of Salmonella typhimurium to ciprofloxacin. Cynaroside also exhibits high activity against yeast fungi Candida albicans, Candida lusitaniae, Saccharomyces cerevisiae, and Saccharomyces carlsbergensis and molds Aspergillus niger, Penicillium oxalicum, Mucor mucedo, and Cladosporium cucumerinum [19].

9. Anti-coronavirus activity

The World Health Organization (WHO) welcomes the use of traditional medicines, including against COVID-19, assuming they must undergo the same serious testing for effectiveness and safety as any other new medicines. Moezzi [90] proposed cynaroside as a promising agent against SARS-nCoV2 by docking the flavonoid on the active site of the main peptidase.

10. Conclusion

The current chapter efforts to coordinate with current studies to identify more accurate biomarkers of the risks for nutrition-related diseases and should lead to dietary recommendations and the formulation of new food products. The food and nutritional supplement industry is very interested in the development and promotion of flavone-rich products as a result of the current evidence for flavones’ protective effects against diseases. An integration of the results of past and future experiments in various disciplines, including biochemistry, cell biology, physiology, pathophysiology, epidemiology, and food chemistry, will be needed to identify the most effective properties of cynaroside and to determine the optimal levels of intake for better health.

Acknowledgments

This study was supported by fund by the EU Erasmus Mundus Partnership (Action 2) CASIA project.

Conflict of interest

The authors declare no conflict of interest.