The ethnobotany, phytochemistry, and biological properties of genus Phagnalon (Asteraceae): a review

Abstract The genus Phagnalon Cass., included within the Asteraceae family, has a wide distribution, expanding from Macaronesia in the West to the Himalayas in the East, from S. France and N. Italy to Ethiopia and Arabian Peninsula. Various species of Phagnalon have been used in the popular medicine of several countries as medicinal herbs and food. This literature review, the first one of the Phagnalon genus, includes publications with the word ‘Phagnalon’, and considers the extracts and the single metabolites identified, characterized, and tested to evaluate their biological potential. The extracts and the secondary metabolites, have a varied application spectrum at a biological level, with antimicrobial, antioxidant, antidiabetic, antitumor, etc. properties having been reported. Unfortunately, in vitro tests have not always been accompanied by in vivo tests, and this is the major critical aspect that emerges from the study of the scientific aspects related to this genus. Graphical Abstract


Introduction
The genus Phagnalon Cass., belonging to the tribe Inuleae (Asteraceae), is distributed from Macaronesia in the West to the Himalayas in the East, from S. France and N. Italy in the North to Ethiopia in the South, but its greatest diversity is found in the Arabian Peninsula ( Figure 1). All species are perennial and grow in a variety of habitats ranging from rocky crevices in high mountains to sandy soils in coastal plains Lack 1985, 1986). It is important to underline that, at a phylogenetic level, analysing the sequences of the trnL intron and trnL-trnF spacer and ribosomal nrDNA together with the ycf3-trnS and trnT-trnL spacers of cpDNA, the relationship between the genus Aliella and the genus Phagnalon has been clarified. The monophyly of Aliella and Phagnalon is not statistically supported and Aliella is paraphyletic in most analyses. The resulting phylogeny suggests an African origin for Aliella and Phagnalon and identifies three main clades in Phagnalon, the Irano-Turanian clade, the Mediterranean-Macaronesian clade and the Yemenite-Ethiopian clade. Incongruence between the chloroplast and nuclear molecular data and the lack of resolution in some clades may indicate that hybridization could have played an important role in the evolution and diversification of Phagnalon and Aliella (Montes- Moreno et al. 2010). Actually, The Plant List (2022) shows 149 plant name records, of which only 31 have the rang of accepted species or sub-species whereas, according to another database (Plants of the Word Online 2022) more than forty have the rang of accepted taxa. Tables S1 and S2 list all the accepted taxa including the main synonymous. The first investigation on species of genus Phagnalon appeared in literature in 1975 (Bicchi et al. 1975) and in the following years many papers on the phytochemical composition, isolation of pure metabolites and biological properties of extracts and pure compounds have been published.
Since no previous review on this genus has been published, the aim of this paper is to provide a comprehensive update of ethnopharmacological, phytochemical and pharmacological aspects of crude extracts and isolated compounds from Phagnalon species. The review will also indicate perspectives and directions of future research on the genus in the course of discovery of novel active natural ingredients and their biological applications.

Review methodology
In this review, a complete survey of the chemical composition and biological properties of the essential oils, extracts and non-volatile compounds isolated from Phagnalon genus is provided. Moreover, the traditional uses of Phagnalon taxa are also reported. The available information on these genera was collected from scientific databases and cover from 1975 up to 2021. The following electronic databases were used: PubMed, SciFinder, Science Direct, Scopus, Web of Science, and Google Scholar. The search terms used for this review included Phagnalon, all the botanical names of the species, both accepted names or synonyms, essential oils, volatile components, traditional uses, activity, pharmacology, and toxicity. No limitations were set for languages. Tables S1 and S2 report the taxa of Phagnalon known so far, their synonyms, the accepted botanical names, and the geographical distribution.

Traditional uses
In the Palestinian area the aerial parts of P. rupestre, locally known as 'Qadeeh, Qadih, Sufan, Qray'i', were used in the past to treat asthma, as anaesthetic for toothache and internally to treat headache (Ali-Shtayeh et al. 1998). Furthermore, the infusion of the leaves was utilized against infection, kidney stones (Abu-Rabia 2005). The same species, in Jordan (vernacular name 'Kadha') is recommended for inflammation, rheumatism, migraine, depression, scalp infection. The dried herb is ignited and is used to cauterize the site of pain. Usually, skin cauterization is done in specific areas in the body like the hand; between fingers, or on the back of the leg and sometimes on certain areas in the head. Usually specialized persons are eligible to do this work Hudaib et al. 2008;Alzweiri et al. 2011). In Israel, the decoction of the whole plant is used to treat leprosy (Palevitch et al. 1986) Phagnalon saxatile (L.) Cass. in Spain, where it is known as 't e de piedra', it was used against mental-nervous diseases (Gonz alez-Tejero et al. 2008) and as carminative, analgesic, and to low the blood cholesterol levels (Pardo De Santayana et al. 2005), while in Sicily it has been reported the use of this plant as spice (Lentini and Venza 2007).
Phagnalon sordidum (L.) Reichenb. (Compositae) is a perennial weed widespread in the entire Mediterranean region. This plant is used alone or mixed with Lippia citriodora and/or Malva sylvestris to cure renal calculosis in the medicinal folk traditions of the Balearic Islands (Epifano et al. 2002). The occurrence of all these metabolites is reported in Table 1.

Compounds identified from Phagnalon
In spite of the large occurrence of flavonoids in other genera of the Astearaceae family (Formisano et al. 2012), the presence of such metabolites is quite limited in genus Phagnalon. In fact only few derivatives of apigenin, luteolin and quercetin were identified in P. rupestre (G ongora et al. 2001, 2002a, 2002b), P. saxatile (Conforti et al. 2010;Cherchar et al. 2018;Haddouchi et al. 2021) and P. sordidum (Cherchar 2018), whereas flavanone derivatives of eriodictyol were detected only in P. sordidum collected in Algeria (Cherchar 2018;Cherchar et al. 2019).
Benzofuran tremetone derivatives, identified in P. purpurescens from Canary Islands (11-13) (Zdero et al. 1991), P. rupestre from Spain (14) and P. sordidum from Algeria (15, 16) (Cherchar 2018;Cherchar et al. 2019) are very important compounds. In fact, this class of metabolites, largely and exclusively represented also in several other genus of plants of Asteraceae family, have been shown to possess very interesting biological properties. A complete review on their occurrence and on their biological activities has been recently published (Badalamenti et al. 2021).
Principal metabolites of genus Phagnalon are the derivatives of cinnamic acid such as ferulic acid (30) and caffeic acid (32), mainly present as mono-, di and tri-esters of quinic acid. In fact, with the exception of P. atlanticum (Hausen and Schulz 1977) and P. siniacum (El-Dahmy et al. 1994), they were identifies in all the other Phagnolon taxa studied so far (Table 1). Among the other metabolites it is noteworthy the absence of sesquiterpenes, largely represented in other genus of the Asteraceae family (Bruno et al. 2013); only two eudesmaniolides (46, 47) were isolated from the aerial parts of P. sordidum collected in Algeria (Cherchar 2018).

Essential oils
Regarding to the composition of the essential oils obtained from aerial parts of Phagnalon species of different geographic origin, only four articles have been published. Table 2 reports the essential oil compositions of the taxa studied so far. The essential oils, obtained by hydrodistillation were characterized by quite different profiles. P. graecum showed to be very rich in sesquiterpenes (64.7%) with germacrene    (23) (21), potassium 4hydroxy-3-methoxybenzoic acid methyl ester-5-sulphate (29)

Antibacterial and antifungal properties
The ethanolic and aqueous extracts of P. rupestre, collected in Palestinian territory, were shown to be particularly active against five bacterial species (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa)  (2), apigenin-7-O-b-Dglucopyranoside (5), luteolin-4 0 -Oglucopyranoside (7),  (8), eriodictyol-7-methyl ether (9) and one yeast (Candida albicans) (Ali-Shtayeh et al. 1998) whereas the ethanolic and aqueous extracts of P. sinaicum, collected in Sinai, Egypt, were tested in vitro and in vivo for its antifungal properties against Phytophthora infestans, the causal agent of late blight disease of tomato. The overall results suggested that the use of this Egyptian wild medicinal plant extract was promising, effective and environmentfriendly management measure against Phytophthora blight of tomato and thus, it could be used in the production of organically grown vegetables (Baka 2014a). Further investigations on the aqueous extract of P. sinaicum indicated good antifungal properties against Aspergillus flavus, A. niger, Curvularia lunata, Fusarium moniliforme and Penicillium chrysogenum (Baka 2014b) and a moderate one against Botrytis fabae, Fusarium oxysporum and Penicillium italicum (Baka 2015). The antimicrobial activities of the essential oil of aerial parts of Phagnalon sordidum (L.) Rchb, collected in Algeria, were evaluated against eleven bacteria. Staphylococcus aureus, Klebsiella pneumoniae and Salmonella typhimurium were the most susceptible microorganisms with a minimum inhibitory concentrations (MICs) of 0.01, 0.04, 0.04 mg/mL, respectively (Chikhi et al. 2019). A notable activity on a large panel of clinically significant microorganisms was also exhibited by P. sordidum essential oil from plants collected in Corsica. The best results were obtained against Staphylococcus aureus and Streptococcus dysgalactiae with a MIC value of 0.064 mg/mL, comparable or better than gentamicin, used as positive control (Brunel et al. 2016).

Antioxidant properties
The in vitro properties (inhibition of NO production and anticholinesterase) of Phagnalon saxatile (L.) Cass., collected in Sicily, were described. The methanolic extract showed antioxidant activity that was measured by DPPH assay and b-carotene bleaching test. The same extract inhibited NO production in the murine monocytic macrophage cell line RAW 264.7. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition was assessed by modifications of Ellman's method. Purification of the MeOH extract of P. saxatile allowed the isolation of phenolic compounds. Among them, the compounds that most effectively inhibited lipopolysaccharide-induced NO production were caffeic acid (32) and methylchlorogenic acid (35), with IC 50 values of 7 lg/mL and 12 lg/mL, respectively. Luteolin (2) and 3,5-dicaffeoylquinic acid (41) exhibited the most promising activity against AChE with an IC 50 of 25.2 and 54.5 lg/ mL, respectively, while caffeic acid (32) and luteolin (2) showed higher activity against BChE with an IC 50 of 32.2 and 37.2 lg/mL, respectively (Conforti et al. 2010). Four extracts, obtained by using different solvents, of the leafy stems of an Algerian accession of the same species were investigated for their content of phenolic compounds and their antioxidant activity. The extracts prepared with polar solvents (methanol and water) contained higher amounts of phenolic compounds and showed better antioxidant activity than the apolar solvents extracts (hexane, dichloromethane). The methanolic extract, richest in total phenolic and total flavonoid, had significant antioxidant activity as regarded by DPPH scavenging capacity (IC 50 of 5.5 lg/mL), ABTS þ scavenging capacity (IC 50 of 63.8 lg/mL) and inhibition of oxidation of linoleic acid (IC 50 of 22.7 lg/mL), when compared to synthetic antioxidants. Chlorogenic acids and several flavonoids were identified and quantified by UPLC-DAD-MSn. The di-O-caffeoylquinic acids isomers were the most concentrated phenolics (25.4 mg/g DW) in the methanolic extract (Haddouchi et al. 2014(Haddouchi et al. , 2021. Twelve metabolites were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. (Table 1), selected by using three complementary antioxidant activity assays. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (25) and nebrodenside A (17) were demonstrated by in vitro cell free model assays, and their protective effect against H 2 O 2 -induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established (Cherchar et al. 2019).
The n-hexane, chloroform, ethyl acetate, methanol, and water extracts of Phagnalon graecum Boiss., collected in Turkey, were investigated for their enzyme inhibitory activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase, tyrosinase, and antioxidant activities. Antioxidant activity of the extracts was determined by 2,2diphenyl-1-picrylhydrazyl radical scavenging, ferric ion-chelation activity, and ferric reducing antioxidant power tests. The extracts had insignificant inhibition against the tested enzymes, whereas they displayed a remarkable antioxidant activity. Due to its good biological properties, authors remarks that P. graecum could be utilized as potential antioxidant applicable for food preservation (Erdogan Orhan et al. 2013).
The caffeoyl conjugates of prenylhydroquinone glucoside and of quinic acid, either in the carboxyl-free or carboxymethyl forms (17)(18)(19)(41)(42)(43)(44), isolated from Phagnalon rupestre, showed inhibitory activity on lipid peroxidation induced by Fe 2þ/ascorbate and by CCl4/NADPH in rat liver microsomes, with IC 50 values ranging from 3 to 11 mM. After having demonstrated their effect on the xanthine oxidase-regulated superoxide production, the active compounds were tested for the direct inhibition of this enzyme. Methylated dicaffeoylquinic conjugates (42, 44) competitively inhibited the enzyme and the highest potency was obtained for the 4,5-diester (44), with an IC 50 value of 3.6 mM, nearly ten times lower than that of the 3,5-analogue (42). In conclusion, authors claimed that the presence of the caffeoyl moiety is essential for both the antiperoxidative and radical scavenging activities, and the methylation of the quinic carboxyl group enhances the potency on xanthine oxidase inhibitory activity (G ongora et al. 2003).
The same metabolites (17)(18)(19)(41)(42)(43)(44) were examined for their effect on tyrosine nitration, as well as on the oxidation of dihydrorhodamine (DHR) 123 and cytochrome c2þ induced by peroxynitrite. All the compounds were fairly active in preventing the oxidation of DHR 123, though inefficient in the cytochrome c test. The highest potency corresponded to 2-isoprenylhydroquinone-1-glucoside (17), with an IC 50 of 40 mM (Olmos et al. 2005(Olmos et al. , 2007. Two phenolic antioxidant and anti-inflammatory compounds: isoprenylhydroquinone glucoside (17), and 3.5-dicaffeoylquinic acid methyl ester (42), have been studied for their inhibitory activity against protein carbonylation, a harmful post-translational modification of peptide chains associated with degenerative diseases. Both compounds have proven to be effective, with 50% inhibitory concentration (IC 50 ) values in the micromolar range, against bovine serum albumin carbonylation caused by hypochlorite, peroxynitrite, and phorbol ester-induced leukocyte oxidative burst (Mar ın et al. 2011).

Antitumor activities
The ethanolic extracts of the aerial pats P. rupestre, collected in Jordan, showed a moderate antiproliferative activity against Hep-2, MCF-7, and Vero cancer cell lines (Talib and Mahasneh 2010).

Other biological properties
A study was carried out in order to assess the in vitro anti-diabetic potential of P. lowei leaves methanolic extract to inhibit the activity of digestive enzymes (a-amylase, a-, b-glucosidases and lipase) responsible for hydrolysis/digestion of sugar and lipids. The extract exhibited significant inhibitory activity against key digestive enzymes linked to type II diabetes and obesity. A strong inhibition was observed for glucosidases and mild activity towards amylase and lipase (compared to reference compounds).
Consequently, the caffeoylquinic acids (37,39,41,43,45), principal metabolites of the extract, were demonstrated to be the most relevant hypoglycemic and anti-glycation agents. From the obtained results Phagnalon lowei showed to be a good candidate for further development of phyto-pharmaceutical preparations as complementary therapy for diabetes and obesity control (Sp ınola and Castilho 2017).
Later, the same group tested in vivo three phenolic substances: 2-isoprenylhydroquinone-1-glucoside (17), 3,5-di-O-caffeoyl quinic acid (41), and 3,5-dicaffeoylquinic acid methyl ester (42), isolated from the same species, for their efficacy in two mouse models of dermatitis induced by single and repeated application of 2,4,6-trinitrochlorobenzene. The effect of these compounds on metalloproteinase-9 (MMP-9) expression in macrophages stimulated with lipopolysaccharide (LPS) was also investigated. The results indicated that these three phenolics modulated the immune response in the skin after exposure to the contact allergen TNCB, at least partly via reduction of ear swelling and cytokine production, although with different ranges of activity. Compound 41 was found to be particularly effective in inhibiting most of the inflammation parameters ).

Conclusion and future prospective
The genus Phagnalon Cass., belonging to the tribe Inuleae (Asteraceae), is distributed in the Northern hemisphere and it has been largely used in the popular medicine of several countries as well as spice due to its interesting biological properties. Up to April 2022, 50 metabolites have been isolated from the roots and aerial parts of the different species, identified using spectroscopic techniques, and evaluated for biological potential.
Among these, flavonoids, aromatic compounds, such as benzofuran derivatives and prenylhydroquinone derivatives, and cinnamic acid derivatives are the most characteristic. Especially in the last two decades, the extracts obtained from the different parts of the plants, and the pure isolated compounds, have been tested and evaluated for their biological activities, such as antitumor, anti-inflammatory, antimicrobial, and antioxidant. However, the few studies on biological properties of Phagnalon species, present in literature, do not allow to confirm and validate, the use of species of this genus in traditional medicine.
Nevertheless, major critical issues remain: most scientific studies have aimed to test the extracts, or compounds isolated, through in vitro studies; the biological supports obtainable in vivo, and biochemical investigations relating to the mechanism of action of the tested samples, are lacking. Only few works report the isolation of essential oils, which could be used for a chemotaxonomic distinction between dubious species.
This literature review aims to direct current researchers to work on a widely promising genus at the biological level, since of the 31 accepted species only few have been investigated.