Phenolic Composition and Antioxidant Activity of Plants Belonging to the Cephalaria (Caprifoliaceae) Genus

The genus Cephalaria, belonging to the Caprifoliaceae family, is a rich source of interesting secondary metabolites, including mainly saponins which display a variety of biological activities, such as immunomodulatory, antimicrobial and hemolytic effects. Besides these compounds, flavonoids and phenolic acids were identified in Cephalaria species. Cephalaria is employed in traditional medicine e.g., to cure cardiac and lung diseases, rheumatism, and regulate menstruation. In this review we focus on the phenolic compound composition and antioxidative activity of Cephalaria species. The antioxidant effect can be explained by flavonoids present in all parts of these plants. However, future efforts should concentrate more on in vitro and in vivo studies and also on clinical trials in order to confirm the possibility of using these plants as natural antioxidants for the pharmacology, food or cosmetic industries.


Introduction
Phenolic compounds are plentiful and ubiquitous secondary metabolites of plants [1] of great interest due to the fact that they are capable of preventing many diseases due to their antioxidant potential [2]. It is worth underlining that a significant enhancement of interest in the antioxidant properties of plants traditionally used in folk medicine has been observed [3], including rare or native wild species on which literature data was lacking [4]. The current focus is toward antioxidants of natural origin, therefore the number of publications on the subject of the favorable effects on health of plant polyphenols has boosted significantly [5]. Perron and Brumaghim have reported that several publications on radical scavenging activity by polyphenols has been released, representing more than 700 papers from 1995 till 2009 alone [6].
Many experiments carried out by various methods have shown that most of the antioxidant potential of plants result from the redox properties of their phenolic constituents [2,3]. Many mechanisms of action of antioxidants have been observed. Phenolic compounds may do the following: inhibit the formation of free radicals, enhance cellular antioxidant defense mechanisms, impair the action of pro-oxidative enzymes, neutralize pro-oxidant ions and boost other antioxidants' action [7].
Bioactive phenolic compounds come from natural sources and simultaneously they are effective towards scavenging free radicals, which makes them very promising candidates for applications in health care, processed foods, the cosmetic industry and as auxiliary medicine remedies [3]. Thus, in recent years, the importance of the antioxidant activities of phenolic compounds and their potential usage in numerous kinds of industries as natural antioxidant compounds has reached a new level. Polyphenols are present in the human diet and are widely used for medical and cosmetic purposes [8]. The use of natural polyphenols in cosmetics is justified and worthwhile due to their capability to ameliorate cutaneous

Methodology of Evidence Aquisition
For a comprehensive literature overview, published phytochemical and antioxidant activity data were retrieved from the ISI ® Web of Science, Scopus ® , GoogleScholar ® , SciFinder ® , and Reaxys ® databases. Entries were considered until the end of March 2021. Exact spelling of scientific botanical names, including the abbreviations for botanical authors was brought in line with standard usage as recommended by "The International Plant Names Index" [47] and "The Plant List" [48]. Relevant original articles and books, with an unlimited time range and regardless of language were included in the review. Exclusion criteria were duplicate publications and non-relevant articles.

Phenolic Compounds in the Cephalaria Species
The investigations of Cephalaria species have led to the isolation and identification phenolic acids and different types of flavonoids, represented mostly by flavanone, flavonols, flavones, and anthocyanins. Table 1 summarizes such phenolic compounds (including the common/systematic name of constituent, species name and parts of the plant) mentioned in the surveyed literature.
In plants, flavonoids are responsible e.g., for the colour of flowers, the growth and development of seedlings. They also protect plants from various biotic and abiotic stresses and act as unique UV filters, allopathic compounds, and phytoalexins [49].
One of the first research on the occurrence of flavonoids in taxa of the Cephalaria genus dates from 1968, when Zemtsova and Bandyukova described the occurrence of quercetin 7-β-D-glucopyranoside (quercimeritrin) (6) in the aerial parts of Cephalaria balkharica E.A.Busch and in the flowers of. C. gigantea (Ledeb.) Bobrov. Moreover, luteolin 7β-D-glucopyranoside (cynaroside) (14) was isolated from the aerial parts of C. balkharica and C. gigantea [50].
Twenty five flavonoids were measured simultaneously in the aerial parts of nineteen Cephalaria species [56]. The authors found that the main flavonoids in the studied plants

Phenolic Acids
Phenolic acids are a large group of phenolic compounds in plants, that include two groups-hydroxybenzoic (C 6 -C 1 structures; e.g., gallic, p-hydroxybenzoic, protocatechuic, syringic) and hydroxycinnamic (C 6 -C 3 structures; e.g., caffeic, ferulic, synapic) acid derivatives with various number and position of methoxylation and hydroxylation in aromatic ring. In plants, these compounds exist in their free and bound forms, and more often bound forms occur as their glycosides and esters [59]. Phenolic acids have a crucial for plants growth and reproduction, and they are produced as a response to environmental factors (e.g., light) and to defend injured plants [61]. What is more, they are reported to have a wide spectrum of pharmacological activities including antioxidant [62], antibacterial [63], anti-inflammatory [64], and anticarcinogenic [59] activities.
To date, there are only a few reports regarding the occurrence of phenolic acids of the Cephalaria genus. The most frequently identified phenolic acid is caffeic acid (36), which was found in the roots of C. gigantea [42,59], aerial parts (0.84-1.27 µg/g of dry extract) and flowers (0.79-0.91 µg/g of dry extract) of C. uralensis [18] and in the aerial parts of eighteen species (0.01-4.27 mg/g) collected in the Anatolia area (Turkey) [46].

Antioxidant Activity
Most of the antioxidant potential in plants is caused by the redox properties of phenolic compounds that make it possible for them to act as hydrogen donors, reducing agents, and singlet oxygen quenchers. Their antioxidant activity is a result of different mechanisms such as free radicals scavenging, metal ion chelation, reduction, oxidase inhibition, as cofactors of enzymes catalyzing oxidative reactions, free radical stabilization and radical chain reaction termination [62,65,66].
The antioxidant activity of compounds isolated from the aerial parts of C. isaurica, C. paphlagonica, C. scoparia, and C. stellipilis was evaluated using the DPPH radical scavenging and CUPric Ion Reducing Antioxidant Capacity (CUPRAC) methods. The authors found that isoorientin was the most effective antioxidant compound in both the DPPH (IC 50 = 0.119 ± 0.0004 mg/mL, while for ascorbic acid was 0.01 ± 0.002 mg/mL) and CUPRAC (6.683 ± 0.636 mmol TRg −1 ) assyas, with a value comparable to Trolox and ascorbic acid used as the positive controls [43]. Isoorientin is well known antioxidant and its structure-activity relationship is well documented [43,67].
Kirmizigül et al., evaluated n-hexane extracts of C. davisiana, C. elazigensis, C. paphlagonica and C. stellipilis from different regions of Turkey, using the CUPRAC assay, for the cupric (II) reducing antioxidant capacity, were 0.334, 0.252, 0.136 and 0.120 mmol TR/g dry extract, respectively. It seems that antioxidant activity of these species resulted from synergistic effect of ALA and phytol. The extracts exhibited a high antioxidative activity of 0.334-0.120 mmol TR/g dry extract. C. davisiana was the most effective cupric (II) reducer [68].
Sarıkahya and co-authors tested also the hexane extracts of ten Cephalaria species (C. anatolica, C. aristata, C. aytachii, C. elazigensis var. elazigensis, C. hirsuta Stapf, C. taurica, C. tuteliana, C. procera, C. speciosa, C. tchihatchewii) for their antioxidant capacity using the DPPH radical scavenging and CUPRAC methods. The DPPH tests revealed that hexane extracts of C. tchihatchewii, C. hirsuta, C. anatolica, C. elazigensis var. elazigensis and C. speciosa have significant radical scavenging activity, with the IC 50 values of 3.77 ± 0.67, 5.13 ± 1.04, 5.20 ± 0.92, 5.28 ± 0.46 and 6.17 ± 3.13 mg/mL, respectively. The highest TEAC value (1.005 mmol ± 0.13 TE/g extract) they found for C. aristate and its reducing power was related to phenolic content (2.91 ± 0.15 mg GAE/g extract). The authors concluded that DPPH scavenging potential of Cephalaria extracts may be attributed to their phenolic compounds, that could donate electrons to DPPH. Because in the CUPRAC method, the reactive -OH groups of phenolic antioxidants are oxidized to the corresponding quinones and Cu(II)-bis(neocuproine) is reduced to the chelate, Cu(I)-bis(neocuproine), the correlation between CUPRAC values and phenolic contents of C. tchihatchewii, C. aristata and C. speciosa in this study is consistent with the above phenomenon [45].
Mbhele et al., evaluated various extracts (acetone, ethanol, methanol, hydroethanol and water) of the leaves and roots of C. gigantea by means of three different assays, including the DPPH radical test, 2,2 -azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS •+ ) decolorization test, and the ability to reduce FeCl 3 solution. Water extract from the leaves and roots possessed the lowest IC 50 (0.6 and 2.8 µg/mL, respectively) in the DPPH assay. Hydroethanolic extract from the leaves had the lowest IC 50 for both ABTS radical scavenging (1.0 µg/mL) and reducing activity (1.7 µg/mL). The water and hydroethanolic extracts of both leaves and roots of C. gigantea contained the highest amounts of phenolics and flavonoids and this suggest that these compounds could be responsible for their strong antioxidant activity [28].
The antioxidant activity of a C. jopponsis aqueous, ethanolic and ethyl acetate extracts were evaluated in vitro (phosphomolybdenum method) [38]. The studied extracts showed antioxidant activity ranging from 20.7 to 41.1 mg of ascorbic acid/g dry extract. Furthermore, Rahimi and co-authors [69] studied the effect of various fertilizers on the antioxidant activity of C. syriaca and they concluded that the antioxidant capacity (DPPH assay) of the studied samples was ranging from 47.10-60.16%.
Kavak and Baştürk [34] analyzed the antioxidant activity of the seeds of C. syriaca collected from different areas in Turkey. They found that studied extracts possessed DPPH inhibition activity ranging from 18.8 to 67.3%. Moreover, the ABTS results (TEAC values) were demonstrated values from 9.8 to 41.8 mmol Trolox eq/g DW.
The antioxidant activity of the oil extracted from the seeds of C. syriaca was evaluated by Atalan et al. [70]. The authors found that in the DPPH • test, plant extracts did not have a high activity. The highest value was observed at 70 µL/mL concentration and it was 9.27 µL/mL while the percent of DPPH inhibition by ascorbic acid (used as a standard substance) was 83.75 µL/mL.
The antioxidant effect of C. gigantea and C. uralensis extracts were evaluated in vitro using DPPH • , ABTS •+ and metal chelating assays. The higher DPPH • scavenging activity was found for the aerial parts of C. uralensis (IC 50 = 2.86 ± 0.12 mg/mL). The extract from the flowers of C. uralensis demonstrated the highest scavenging free radical effect in the ABTS •+ (IC 50 = 0.45 ± 0.21 mg/mL). The extracts from the aerial parts of C. uralensis were also the most active ones interfering with the formation of iron and ferrozine complexes, that suggest their high chelating capacity [27]. The main compounds identified in these extracts were chlorogenic acid (30), isoorientin (20) and swertiajaponin (15), the compounds which are well-known natural antioxidants showing strong effects in different tests [71].

Conclusions and Research Gaps/Future Investigations
This review summarizes the phenolics contain and antioxidant activity of species of the Cephalaria genus. According to literature information, only 29 species of the genus have been studied so far, and the available data are still fragmentary and insufficient. Moreover, the state of knowledge of Cephalaria species contains some gaps, which require more investigation.
So far, in the Cephalaria species, only 43 compounds belonging to the phenolic acids and flavonoids classes have been identified. Kaempferol, luteolin and quercetin and its derivatives have been the major constituents found in the investigated species. What is more, most of phenolic compounds they were detected using old, not very precise methods. Thus, it would be advisable to reexamine Cephalaria species for the presence of these compounds using modern analytical methods.
It seems to be interesting to combine these results with those of a chemotaxonomic study to see if there is any correlation between chemical profile and molecular and/or morphological features.
All the abovementioned findings suggest that an obvious gap in our knowledge about the Cephalaria genus also concerns their antioxidant activity. The research carried out so far has shown that these plants have a strong antioxidant potential. Thus, a focused investigation of the other species, and compounds isolated might be helpful to identify possible uses of these plants in the pharmacology, food or cosmetic industries.