Flavonoids of the Caryophyllaceae

The plant family Caryophyllaceae, commonly known as the pink family, is divided into 3 subfamilies and contains over 80 genera with more than 2600 species that are widely distributed in temperate climate zones. Plants belonging to this family produce a variety of secondary metabolites important in an ecological context; however, some of these metabolites also show health-promoting activities. The most important classes of phytochemicals include saponins, phytoecdysteroids, other sterols, flavonoids, lignans, other polyphenols, essential oils, and N-containing compounds such as vitamins, alkaloids or cyclopeptides. Flavonoids are polyphenolic compounds that remain one of the most extensively studied constituents of the Caryophyllaceae family. Numerous structurally diverse aglycones, including flavones, flavonols, flavonones (dihydroflavones), flavonols, isoflavones, and their O- or C-glycosides, exhibit multiple interesting biological and pharmacological activities, such as antioxidant, anti-inflammatory, anti-oedemic, antimicrobial, and immunomodulatory effects. Thus, this review analysed the flavonoid composition of 26 different genera and more than 120 species of Caryophyllaceae for the first time.


Introduction
The Caryophyllaceae family, commonly known as the pink family, contains over 80 genera with more than 2600 species. The pink family is divided into 3 subfamilies, Paronychioideae, Alsinoideae, and Caryophylloideae, according to the presence or absence of stipules as well as the type of calyx and corolla. Plants of the Caryophyllaceae family are erect, prostrate, annual or perennial herbs or shrubs with simple cross-opposite leaves and swollen nodes. Tetramerous or pentamerous flowers are frequently gathered in panicle, raceme, or capitulum inflorescences (Hegnauer 1964;Kubitzki 1993;Schweingruber 2007).
The subfamily Paronychoideae, containing the genera Spergula L., Spergularia Presl., Polycarpon L. Herniaria L., and Paronychia Mill., occurs mostly in warm and tropical parts of the world. The characteristic attributes of these plants are leaves with stipules and visible separation of calyx from the corolla. The lack of stipules and the unique corolla are typical for members of the subfamily Alsinoideae: The genera Scleranthus L., Arenaria L., Sagina L., Cerastium L., Minuartia L., Stellaria L., and Colobanthus Bartl. are widespread on all continents and are even present in Antarctica. Several species of the subfamily Caryophylloideae are field weeds that inhabit northern temperate climate regions. The specific structures of this subfamily are long calyx tubes that occur in Agrostemma L., Maleandrium Roehl., Silene Mill., Gypsophila L., and Dianthus L. (the largest genus). A great number of Caryophyllaceae species are grown as decorative landscape plants. Furthermore, many members of this family produce secondary metabolites with medicinal properties (Brockington et al. 2011;Volodin and Volodina 2015).

Diversity of phytochemicals in Caryophyllaceae
Caryophyllaceae are known to be a rich source of pharmacologically active secondary metabolites spanning several structural chemical classes. Secondary metabolites are important for plants as protective chemicals against herbivores (insects, molluscs, vertebrates) and microbial pathogens (fungi, bacteria, viruses), UV light, and other plants competing for light, water, and nutrients. In addition, many secondary metabolites serve as signalling compounds to attract pollinating and seed-dispersing animals and provide communication signals among plants and symbiotic microbes (Wink 2011).
The main secondary metabolites of Caryophyllaceae are saponins, phytoecdysteroids, other sterols, flavonoids, lignans, other polyphenols, essential oils, and N-containing compounds such as vitamins, alkaloids and cyclic peptides.

Essential oils and volatile compounds
Essential oils are widely distributed in the plant kingdom. This finding suggests that essential oils are also produced in flowering parts of taxa in the pink family. As essential oils are isolated by distillation, they contain a variety of volatile molecules-terpenes and terpenoids, phenol-derived aromatic components, and aliphatic constituents. Components of volatile oils isolated from Dianthus acicularis are chiefly 2-pentadecanone (Fig. 3) and 2-tridecanone, which are presumed to be responsible for the insect repellent activity of this plant (Kirillov et al. 2017). According to analyses of the major constituents of Dianthus calocephalus and Dianthus carmelitarum essential oils, the presence of heneicosane, docosane, tetracosane, phytol, 4,4-dimethyl-2-pentene, pentacosane, and hexahydrofarnesyl acetone (Yücel and Yayli 2018). Additionally, floral fragrance compounds were also established in other Dianthus L. species and Saponaria officinalis with the largest amounts of benzenoids, phenyl propanoids, and isoprenoids (Jürgens et al. 2003). Gas chromatography and gas chromatography combined with mass spectrometry (GLC-MS) examinations of aerial parts of Silene morganae revealed the presence of over 30 compounds with the highest content of monoterpene hydrocarbons being of terpenoids (Azadi and Sohrabi 2014). Furthermore, benzenoids followed by FADs seems to be the dominating compound classes of aromatic compounds in night-blooming or mothpollinated flowers of Silene Mill. species (Jürgens et al. 2002;Jürgens 2004). Essential oils and their volatile components were also observed in Minuartia recurva (Jovanović et al. 2009), Dianthus caryophyllus (Nerio et al. 2010), Dianthus cruentus (Radulović et al. 2018), some Silene species (Dötterl and Jürgens 2005;Mamadalieva et al. 2014;Mihaylova et al. 2018), Gypsophila bicolor (Shafaghat and Shafaghatlonbar 2011), and two hermaphroditic Schiedea species (Powers et al. 2020).

Cyclic peptides
Cyclic peptides, consisting of a maximum of 14 amino acid residues, are typical N-containing secondary metabolites from Caryophyllaceae (Ma et al. 2006). Genera containing cyclopeptides as major phytochemicals among all plants from this family seem to be Dianthus L., Gypsophila L., Stellaria L., and Vaccaria Mill. For example, the cyclic peptides gypsophins A-F were isolated from the roots of Gypsophila oldhamiana ; the hexapeptides dianthins E, G, and H were found in the aerial parts of Dianthus superbus (Tong et al. 2012); and diandrines A-D (Fig. 5) and drymarins A-B occur in Drymaria diandra (Hsieh et al. 2004a, b;Ding et al. 2000). According to available data, seeds of Vaccaria segetalis are a valuable source of the penta-and hexapeptides segetalin B and segetalin A, respectively (Itokawa et al. 1995;Wang et al. 2011). It is worth mentioning that this group of compounds is present in taxa of the subfamily Alsinoidae, which grow in Antarctica (Jia et al. 2004).

Alkaloids
Another group of nitrogen-containing secondary metabolites are alkaloids, which also occur in Caryophyllaceae to some degree. In particular, alkaloids belonging to the b-carboline group have been described (Dai et al. 2018). For instance, siliendines A-D were isolated from the aerial parts of Silene seoulensis (Seo et al. 2020), drymaritin from the whole plant material of Drymaria diandra (Hsieh et al. 2004a, b), oldhamiaines A and B from the roots of Gypsophila oldhamiana , and arenarines A-D from Arenaria kansuensis (Wu et al. 1989;Bracher and Puzik 2004). Phytochemical investigation of the roots of Stellaria dichotoma led to the isolation of 23 various b-carboline-type alkaloids, including stellarines A-B, dichotomides I-XIV, dichotomines A, B, E (Fig. 6), and K, L, glucodichotomine B and 1-acetyl-3-methoxycarbonyl-b-carboline (Chen et al. 2010a, b;Luo et al. 2012). Brachystemma calycinum also produces alkaloids: Brachystemidines A-E were isolated from the roots of this plant (Cheng et al. 2002). Superbusines A and B, which are quinolone alkaloids, were detected in Dianthus superbus (Sun et al. 2019).

Vitamins
Analysis of plant-derived vitamins showed the presence of four tocopherols (a, b, c, d) with a different number of methyl substitutions in Silene vulgaris as well as vitamin C and phylloquinone, known as vitamin K1 (Fig. 7). Upon examination of S. vulgaris, the presence of the antioxidant b-carotene, a provitamin of vitamin A, was also reported (Vardavas et al. 2006;Morales et al. 2012;Mamadalieva et al. 2014). Moreover, b-carotene was reported in other Caryophyllaceae plants, e.g., in Stellaria media whose seeds contain vitamin B2 (riboflavin), vitamin B3 (niacin) and vitamin E (Slavokhotova et al. 2011;Taskin and Bitis 2013).

Phenolic compounds
Phenolic compounds constitute a large proportion of secondary metabolites in Caryophyllaceae plants. Phenolic acids are the main polyphenols produced by plants. However, only a few publications report on phenolic compound isolation and identification in Caryophyllaceae. For instance, caffeic acid was obtained from aerial parts of Silene (syn. Lychnis) flos-cuculi (Tomczyk 2008), p-coumaric acid, dihydroferulic acid, and syringic acid were identified in the ground roots, stems, leaves, and flowers of Gypsophila paniculata (Chou et al. 2008); and Dianthus species are a source of gentisic acid, a commonly reported aromatic acid in green plants (Griffiths 1959). Fractionation of a Gypsophila sphaerocephala extract resulted in the isolation of 3,4-dihydroxybenzoic acid, syringic acid, p-hydroxybenzoic acid, and rosmarinic acid (Fig. 8) from the methanol extract and rosmarinic acid and syringic acid from the water extract (Altay et al. 2018). Additionally, the Silene Mill. genus is also known as a source of phenolic acids (Mamadalieva et al. 2014). Derivatives of cinnamic acid or benzoic acid and aromatic amino acids (anthranilic acid), socalled anthranilamides with phytoalexin-related activity, are commonly found in parts of Dianthus caryophyllus infected by pathogens (Niemann 1993).
Catechins (flavanol derivatives) are similar in structure to flavonols, except for the lack of a carbonyl  group in the pyran ring (Heim et al. 2002). Plants of the Caryophyllaceae family were also screened for flavanols, but only a few species, including Herniaria fontanessii (Mbark et al. 1999) and Arenaria kansuensis , contained this group of compounds. The major flavanols, catechin, and epicatechin, act as strong antioxidant agents similar to other polyphenols (Iacopini et al. 2008).
However, flavonoid compounds remain one of the most extensive groups of polyphenols in Caryophyllaceae, and novel compounds are yet to be identified. The aglycones and their glycosides are probably present in almost all plants.

Flavonoids of the Caryophyllaceae and their main biological activities
Flavonoids are low-molecular-weight secondary plant metabolites composed of two benzene rings and one heterocyclic pyran ring that are chemically divided into groups according to their chemical substitutions. Flavonoid moieties can be modified by glycosylation, hydrogenation, hydroxylation, and methylation as well as malonylation and sulfatation. The chemical and biological activities of flavonoids and their derivatives are connected with their structure and the position of various substitutions on the molecule. The general activity of polyphenols concerns the reactivity of their phenolic OH groups. The hydroxyl groups can dissociate under physiological conditions to negatively charged phenolate ions. Thus, polyphenols can interact with proteins by forming hydrogen bridges and, more importantly, ionic bonds with positively charged amino groups. As a consequence, the bioactivity of proteins can be directly changed when a polyphenol binds to a receptor side or active centre of an enzyme. Polyphenols, especially those with several phenolic OH groups (such as rosmarinic acid or tannins), can change the 3D structure of proteins and impair their bioactivity. Because of these interactions, polyphenols affect many proteins in the human body and in microbes that are medicinally relevant. This is the mechanism by which plant polyphenols are medicinally active (Wink 2015;van Wyk and Wink 2017).
The biological activities of flavonoids may be also connected with their metabolites, which are produced in vivo. The gastrointestinal tract reveals primary role in the absorption, distribution, metabolism and excretion of flavonoids, which are substrates for conjugating and hydrolyzing enzymes in the small intestine, liver, and colon to O-glucuronides, O-methyl and sulfate esters. Firstly, metabolism of flavonoids take place in the small intestine followed by the liver where they are transformed and then produced glucuronides and sulfate derivatives. Flavonoid compounds that reach the colon are catabolize to low molecular weight phenolic acids by the intestinal microflora (Thilakarathna and Rupasinghe 2013). An anaerobic bacteria found in human gastrointestinal tract (e.g. Eubacterium ramulus) splits the ring structures of several flavonols and flavones leading to the formation of aglycones and common phenolics intermediates consisting of hydroxyphenylacetic, hydroxyphenylpropionic, acetate, and butyrate acids with varying degrees of hydroxylation (Blaut et al. 2003;Serra et al. 2012;Pei et al. 2020). The amount of urinary excretion demonstrates that the colonic catabolites are absorbed into the portal vein and this way run over the body in the circulatory system (Crozier et al. 2010). The flavonoid glucuronides and sulfate derivatives facilitate their excretion through urine and bile (Thilakarathna and Rupasinghe 2013). Urinary excretion of \ 1.0% confirms that C-flavones are poorly absorbed, and 10-88% recovery from feces indicates that they may be resistant to degradation by gut bacteria in rats . As with flavone Oglycosides, the C-glycosides are less bioavailable in humans than in rats. Nevertheless, it is known that the absorption of dietary flavonoids may be affected by the food matrix, the metabolic processes mediated by the liver, intestine, kidneys, as well as colon microbiota (Hollman 2004;Viskupičová et al. 2008;Hostetler et al. 2017;Cosme et al. 2020;Di Lorenzo et al. 2021).
To the best of our knowledge, apigenin, found in 28 species, is the major flavone in Caryophyllaceae plants. The apigenin exhibits cancer chemopreventive activity such as antiproliferative effects on human breast cancer cells, inhibition of cell growth by apoptosis in cervical carcinoma, or selective apoptotic effects in monocytic and lymphocytic leukaemias (Shukla and Gupta 2010;Imran et al. 2020). A similar number of species contain another widely distributed aglycone-luteolin. As with many other polyphenols, luteolin is a powerful antioxidant that can prevent inflammation and allergies and suppress the expression of cancer-promoting proteins (Imran et al. 2019a, b). Other important flavones are the luteolin 8-C-glucoside and apigenin 8-C-glucoside, orientin and vitexin, respectively. Plants rich in orientin are often used in traditional medicine for the treatment of respiratory disorders, pharyngitis, skin disorders, common cold, and mild anxiety (Grundmann et al. 2008;Lam et al. 2016). In addition, luteolin 8-Cglucoside acts as an antioxidant, antiaging, antiinflammatory, cardioprotective, radioprotective, and neuroprotective agent (Uma Devi et al. 2000;Praveena et al. 2014;Lam et al. 2016). Vitexin, successfully isolated from Caryophyllaceae, exhibits various medicinal properties, such as fat reduction, improved glucose metabolism, hepatoprotection, neuroprotection, cardioprotection, and even anticancer activity (Ganesan and Xu 2017;Peng et al. 2020).
Kaempferol exhibits multiple biological effects, such as antioxidant, anti-inflammatory, antidiabetic, antiaging, and antimicrobial effects, and is being applied in the chemotherapy of skin, liver, and colon tumours (Zhu et al. 2018;Cho and Park 2013;Imran et al. 2019a, b). Furthermore, kaempferol can be used in the treatment of cardiovascular diseases, degenerative disorders, diabetes, and microbial contamination diseases (Imran et al. 2018(Imran et al. , 2019a. The flavonol aglycone, quercetin can function as an antioxidant as well as a blood pressure-lowering and anticancer agent (Kukongviriyapan et al. 2012;Egert et al. 2009). Moreover, quercetin can decrease the levels of proinflammatory cytokines, e.g., interleukin 6, 8, 1b, and TNFa ). Rutin, a 3-O-rutinoside derivative of quercetin established in 18 different species of the Caryophyllaceae, is also often used in studies due to its extensive therapeutic properties: The health-promoting effects of rutin are linked with antioxidant, cytoprotective, neuroprotective, vasoprotective, and cardioprotective activities (Kim et al. 2009;Ganeshpurkar and Saluja 2017). The results from some studies also indicated a positive effect of rutin on Parkinson's and Alzheimer's diseases (Gullón et al. 2017). The main strategies for a neurodegenerative disease therapy involves the reduction of reactive oxygen species and amyloid beta-protein production, and the activation of mechanisms of neuronal death (de Andrade Teles et al. 2018).
The main biological activities of hesperidin isolated from Herniaria hemistemon (Elhagali et al. 2019) are chemotherapeutic, antiallergic, anti-inflammatory, endocrine, cardiovascular, and organ-protective effects (Kumar et al. 2008;Zanotti et al. 2013;Ganeshpurkar and Saluja 2019). The aglycone naringenin exhibited multiple therapeutic effects associated with its free radical-scavenging properties. Depending on the concentration and method of administration, naringenin can be useful in the treatment of viral, bacterial, and inflammatory diseases and obesity (Ke et al. 2016;Kozlowska et al. 2017;Salehi et al. 2019).
In addition, naringenin was tested for its potential anticancer activity and as a cardioprotective agent (Salehi et al. 2019). A wide range of therapeutic properties of naringin, a 7-hesperidoside derivative of naringenin, include the treatment of metabolic syndrome, oxidative stress, and conditions of the central nervous system (Sachdeva et al. 2014;Dhanya et al. 2015;Chen et al. 2016).
A medically useful group of flavonoids are isoflavones, which are also known as phytoestrogens (Heim et al. 2002). These compounds can bind to receptors of oestrogen and oestrogen hormone binding protein and inhibit an important enzyme of angiogenesis and tumour formation, tyrosinase (Wink 2015). It was concluded that plants rich in isoflavones are effective in treating cardiovascular and osteoporosis disorders as well as in reducing postmenopausal symptoms (Clarkson 2002;Atkinson et al. 2004;Vitale et al. 2013). To date, the distribution of genistein and daidzein is common in several legumes of the Fabaceae family, such as soybean (Bustamante-Rangel et al. 2018). However, there are reports of the presence of genistein in a species of the Caryophyllaceae family, e.g., Stellaria dichotoma or Stellaria holostea (Mikšátková et al. 2014).
Our approach included screening for flavonoid aglycones and their highly glycosylated derivatives within Caryophyllaceae family (Cook and Samman 1996). The flavonoid aglycone and glycoside group remains one of the most extensive groups of polyphenols in Caryophyllaceae. Most of these compounds occur in Silene L., Dianthus L. (Obmann et al. 2011a, b;Boguslavskaya et al. 1983), Gypsophila L. (Zhang et al. 2011a, b;Zheleva-Dimitrova et al. 2018), Stellaria L. (Mikšátková et al. 2014), Spergularia Presl.  and Herniaria L. (Elhagali et al. 2019;El Mabruki et al. 2014). Nevertheless, flavonoid compounds are probably present in almost all plants. We assembled information regarding their presence in 26 genera and over 120 species of the Caryophyllaceae family (see Table 1).

Flavones
One of the most pharmacologically valuable flavonoid classes present is that comprising flavones, which can be synthesized by various pathways, depending on whether they contain C-or O-glycosylation, Omethylation acylation, and hydroxylated B-ring. These compounds undergo characteristic reactions ascribed to three functional structures-hydroxyl and carbonyl groups and a double bond (Singh et al. 2014;Panche et al. 2016). Their natural distribution is demonstrated for almost all plant tissues (Figs. 10,11,12).

Flavonones (dihydroflavones)
Flavonones (dihydroflavones) differ from flavones by the lack of a double bond in the pyran ring. Hesperidin, naringenin, and its glycoside naringin (naringenin 7-hesperidoside) are commonly found in citrus fruits (Panche et al. 2016), but they can also be found in certain species of the pink family.

Conclusions
The Caryophyllaceae family contains a large number of genera and species that are widely distributed over different climate zones. It is evident that the plants from this family produce a wide range of    Fig. 11 The chemical structures of the apigenin and its derivatives identified in species of Caryophyllaceae family flavonoid-containing species, including those containing aglycones and their glycoside derivatives, which could be identified in 26 genera and more than 120 species within the Caryophyllaceae.
To the best of our knowledge, apigenin is the most common aglycone in this family and can be found in 28 different species, such as Vaccaria segetalis (Baeva et al. 1975), Stellaria media (Melnyk et al. 2018), Silene saxatilis (Zemtsova et al. 1975), Pteranthus dichotomus (Allaoua et al. 2016), Silene (Lychnis) flos-cuculi (Tomczyk 2008), Herniaria glabra (El Mabruki et al. 2014) and others. Furthermore, the C-bonded apigenin glucoside isovitexin has been isolated from more than 70 plants, making it the predominant flavonoid within this family. On the basis of the data collected in Table 1, it was concluded that the highly glycosylated C-and O-flavonoids (apigenin, luteolin, chrysoeriol, kaempferol, quercetin, formononetin, genistein, myricetin, tectorigenin) with either one, two or three sugar moieties, as presented in Fig. 12 The chemical structures of the chrysoeriol (a), acacetin (b), diosmetin (c), tricin (d), and their derivatives identified in species of Caryophyllaceae family this review, are commonly found in the Caryophyllaceae family. The genera Silene Mill., Dianthus L., Stellaria L., Herniaria L., Spergularia Presl., Gypsophila L. and Cerastium L. appear to contain high abundances of flavonoid compounds.
In summary, the structural diversity of flavonoids established in the Caryophyllaceae family makes them an interesting object of phytochemical and pharmacological investigations.