Introduction

Verbascum species, commonly known as “mulleins” (also named “koroviaks” in Russia and “siğirkuyruğu” in Anatolia), are well-known herbs with long-standing use in the folk medicine. Various applications of leaves, flowers and roots of Verbascum spp. for treatment of respiratory disorders, eczema, rheumatism, wounds and anal fistula in traditional Turkish medicine have been thoroughly descrbied (Baytop 1999; Tatli and Akdemir 2006). Moreover, mulleins are used in the European folk medicine as antiseptic, astringent and expectorant agents, and frequently applied in the treatment of inflammations, migraine, asthma and spasmodic coughs (Grieve 1995). Although mulleins have been used as remedy since ancient times, their popularity increased commercially in the past few years. Nowadays, the dried leaves and flowers, swallow capsules, alcoholic extracts and the flower oil of common mullein (Verbascum thapsus L.) can be found in the USA health stores (Turker and Gurel 2005). According to the assesment report of European Medicines Agency (EMEA; www.ema.europa.eu), written in 2008 on V. thapsus, V. densiflorum and V. phlomoides flos with traditional use, on the European market Verbascum flowers are included in combination or in mono-preaparation products—herbal substances for tea preparation, liquid exracts prepared with ethanol or syrup products.

The genus Verbascum L. belongs to the Scrophulariaceae family. The Latin name Verbascum is considered to be a corruption of barbascum from the Latin ‘barba’ (beard) in allusion to the shaggy foliage and was bestowed on the genus by Linnaeus. Mulleins are biennial or perennial, rarely annual plants, with deep tap roots (Turker and Gurel 2005). According to Heywood (1993) it comprises about 360 species, predominantly distributed in Asia, Europe and North America. West and Central Asia (especially Anatolia) are the main centres of diversity of the genus (Kaynak et al. 2006). In Turkey, Verbascum is represented by about 232 species, 84 % of whom are endemics (Huber-Morath 1978). The distribution of the species in the region has also been thoroughly described: 51 species in Russia, 49 in Flora Iranica and 20 in Flora Palaestina (Celebi et al. 2009), while in Flora Europaea 95 Verbascum species are included (Ferguson 1972). Bulgaria is situated in the zone of speciation of the genus, which has resulted in a considerable number of endemic species, as among the distributed in the country 46 species half are endemic (Stefanova-Gateva 1995). The genus Verbascum bears a very complicated taxonomy. To the best of our knowledge, untill now, there is no adequate taxonomic scheme, reflecting the relationships between the taxa. Huber-Morath (1971, 1978) divided the genus into 13 informal groups (from A to M), while Ferguson in Flora Europaea (1972) used two informal groups, named A and B. Recently, the taxonomic status of group A (Verbascum) in Turkey has been clarified (Celebi et al. 2009).

The extensive use of Verbascum spp. in traditional medicine and the interest from taxonomic point of view have resulted in-depth research on the genus in both phytochemical and pharmacological directions.

Chemical constituents of mulleins

The joint efforts of several groups globally resulted in the identification of over 200 compounds, which can be classified into several main groups: iridoids, phenylethanoids, flavonoids and neolignan glycosides along with saponins and spermine alkaloids.

Iridoids

The iridoids are widespread secondary metabolites in Scrophulariaceae family and, at present, the largest group of compounds found in Verbascum species. Over 70 iridoid glycosides (1–77) and several non-glycosidic compounds (78–87) have been isolated from flowers, leaves, roots and whole Verbascum plants (Figs. 1, 2, 3). The main group of these compounds and the most numerous representatives found in mulleins are C9-type iridoids: catalpol (1), aucubin (37) and their acylated derivatives (with variable position of the ester group). Ajugol (60) and harpagide (68) and their derivatives have been also identified in many of the investigated plant sources (Figs. 1, 2, 3). Harpagoside, among others, is a high-value molecule and the major constituent in pharmaceutical preparations of devil’s claw (Harpagophytum procumbens). Harpagoside is used for standartization of commercial devil’s claw products and according to the European Pharmacopoeia these products should contain at least 1.2 % harpagoside (Georgiev et al. 2013a). Therefore, Verbascum species, accumulating harpagoside, might serve as an alternative source of this pharmaceutically important molecule.

Fig. 1
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Structures of catalpol-type iridoid glycosides found in Verbascum spp

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Fig. 2
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Structures of aucubin-type iridoid glycosides reported in Verbascum spp

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Fig. 3
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Ajugol and harpagide-type iridoid glycosides found in Verbascum spp

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Only five C10-type iridoids: geniposidic acid (75), lychnitoside (76), genipin (79), α-gardiol (80) and β-gardiol (81) have been isolated from mulleins so far (Fig. 4). The isolation of rehmaglutin D (77) and glutinoside (78), possessing Δ3,4 saturated iridoid aglycone with chlorine atom and an unusual epoxy function, has been reported only for V. wiedemannianum (Abou Gazar et al. 2003a).

Fig. 4
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Structures of C10-type and unusual iridoids

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The distribution of iridoids in Verbascum spp. is presented in Table 1. Mulleins are widely studied species regarding their iridoid constituents, though the iridoids composition of some Verbascum species has been established only by thin-layer chromatography (TLC) (Grabias and Swiatek 1987). Several studies have been focused on V. phlomoides, V. thapsiforme, and V. thapsus, the sources of the drug Verbasci flos according to the European Phramacopoeia. Among these, Verbascum thapsus appeared to be best studied member of the genus—different parts of the taxon of different origin, extracted by various solvents, have been described. For instance, Warashina et al. (1991) reported on the isolation of 23 iridoids, mostly of catalpol type: 11–26, 30 and 32, from the water extract of the whole plant, while Pardo et al. (1998), focusing on the ethanolic extract of mullein roots, succeeded in isolation of four iridoid glycosides, among these aucubin (37). Furthermore, the methanolic extract of the whole plant (collected in Pakistan) was found to be a source of some minor constituents (79–81) along with previously reported iridoids (36, 60, 70 and 71; Hussain et al. 2009). Several rare non-glycosidic iridoids (82–86; Fig. 5) were identified from the 70 % aqueous acetone extract of the mulleins aerial parts (the samples were collected in Southwest China; Zhao et al. 2011).

Table 1 Distribution of iridoids in Verbascum spp
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Fig. 5
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Non-glycosidic iridoids from V. thapsus

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It should be, however, mentioned that the presence of catalpol (1), aucubin (37), ajugol (60), harpagide (66) and their derivatives in V. phlomoides and V. thapsiforme has been proved mainly by TLC analysis, while catalpol and aucubin type glycosides in extracts have been quantified spectrometrically (at 605 nm through forming a colored product with Ehrlich reagent) using aucuboside as a standart (Swiatek and Adamczyk 1983, 1985; Swiatek et al. 1984). Afterward, the isolation of specioside (4), phlomoidoside (49) (Klimek 1991a, 1996a), catalpol (1), saccatoside (11), aucubin (37) and 6-O-xylosylaucubin (40) from V. phlomoides (Gvazava and Kikoladze 2009) has been reported. The same authors identified catalpol (1), verbascoside A (16), aucubin (37), harpagide (68) and acetylharpagide (69) in V. densiflorum (Gvazava and Kikoladze 2009), a synomim of V. thapsiforme according to Flora Europaea.

The scientific interest on the iridoids distribution within Scrophulariaceae species has resulted to extensive investigations of several mulleins, besides the recognised sources of Verbasci flos. For instance, 12 iridoid glycosides from V. nigrum (Seifert et al. 1982, 1985; Vesper and Seifert 1994) and 11 iridoids from the aerial parts of V. sinuatum (Bianco et al. 1980, 1981a, b; Falsone et al. 1982; Eribekyan et al. 1987) have been reported. Moreover, 15 iridoids from the roots and flowers of V. lasianthum have been found (Akdemir et al. 2004a, b; Tatli et al. 2006), while systematic investigations of V. undulatum led to the isolation of nine iridoid glycosides mainly of aucubin type (Skaltsounis et al. 1996; Magiatis et al. 1998, 2000).

Phenolic compounds

The occurrence of three primary groups of phenolic compounds including phenylethanoids, flavonoids and neolignas in Verbascum spp. has been reported.

More than 20 phenylethanoid (C6–C2) and two phenylpropanoid (C6–C3) glycosides have been isolated from various mulleins so far, as most of these being triglycosides containing apiose, arabinose, glucose, rhamnose and xylose as a third glycosidic moiety in the molecules, attached to C-6 of the core glucose (Fig. 6). According to the published data verbascoside (=acteoside; 87) is widely distributed compund within the group, it has been isolated from nearly all Verbascum species studied, followed by poliumoside (91) and forsythoside B (101). Verbascoside was recently reported to possess an insecticidal activity against Drosophila melanogaster and Spodoptera frugiperda (Munoz et al. 2013), besides abundant biological properties (see below).

Fig. 6
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Phenylethanoid and phenylpropanoid glycosides found in Verbascum spp

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Evidently, V. thapsus and V. wiedemannianum are the most comprehensively investigated species regarding their phenolics content. The presence of the phenylpropanoid glucosides coniferin (112) and syringin (113) in V. letourneuxii has been only reported recently (Emam 2010). The distribution of phenylethanoid and phenylpropanoid glycosides within mulleins is summarized in Table 2.

Table 2 Distribution of phenylethanoid glycosides in Verbascum spp
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Verbascum species appear also to be a rich source of various flavonoids, as several flavanones (114 and 115), flavones (116–135) and flavonols (136–147) and their O-glycosides have been identified. In addition, four isoflavonoids in V. sinaiticum (148–151), two C-glycosides (152 and 153) in V. cheirantifolium and a bisflavonoid named amentoflavone (154) in V. thapsus have been found within the genus. Apigenin (116), luteolin (118) and their 7-O-glucosides (120) and (123) are common flavones in the genus. The chemical structures of the above mentioned compounds are presented in Figs. 7, 8, 9, 10, while their distribution within Verbascum species is given in Table 3.

Fig. 7
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Structures of flavanones

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Fig. 8
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Flavones reported in Verbascum spp

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Fig. 9
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Flavonols reported in Verbascum spp

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Fig. 10
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Isoflavonoids, C-glycosides and bisflavonoid

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Table 3 Distribution of flavonoids in Verbascum spp
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The presence of several neolignan glucosides (Fig. 11) with dehydroconiferyl alcohol sceleton has been reported only in V. thapsus (155–159; Warashina et al. 1992), V. salviifolium (160 and 161; Akdemir et al. 2004c) and V. letourneuxii (161 and 162; Emam 2010).

Fig. 11
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Structures of neolignan glucosides

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Saponins

The occurrence of triterpenic saponins mainly of oleanane type (163–184) in mulleins has been also reported. Up-to-date, the aerial parts of Turkish endemic V. wiedemannianum are the only source of ursane type saponins rosamutin (186) and niga-ichigoside F1 (187; Abou Gazar et al. 2003a). Ilwensisaponin A (166) and ilwesisaponin C (180) are the most frequently detected compounds from this group (Fig. 12a–c). The aerial parts of V. songaricum (Seifert et al. 1991; Hartleb and Seifert 1994, 1995), V . sinaiticum (Miyase et al. 1997) and V. thapsifrome (Miyase et al. 1997) appeared to be most abundant sources of saponins (see Table 4).

Fig. 12
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a–c Saponins found in Verbascum spp

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Table 4 Distribution of saponins in Verbascum spp
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Alkaloids

The distribution of alkaloids in mulleins is restricted to several species. The presence of alkaloids in V. nobile and V. songoricum has been reported for the first time in early 70es (Ninova et al. 1971; Ziyaev et al. 1971). Although, the hypotensive and spasmolitic effect of total alkaloid-containing extract V. pseudonobile Stoj et Stef has been established in the 1960s (Drandarov and Hais 1996) the isolation and structural elucidation of alkaloids from the species have been published much later (Koblicova et al. 1983), followed by isolation, separation of E-Z isomers and synthesis of macrocyclic spermine alkaloids from V. pseudonobile and V. phoeniceum (Drandarov 1995; Drandarov and Hais 1996; Drandarov 1997; Drandarov et al. 1999; Youhnovslki et al. 1999; Drandarov and Hesse 2002). The structures of the main—naturally occurring—alkaloids are given in Fig. 13.

Fig. 13
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Structures of spermine alkaloids

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Other compounds

Figure 14 summarizes the structures of sesquiterpenes buddlindeterpen A (196) and buddlindeterpen B (197) isolated from V. thapsus along with a diterpene buddlindeterpen C (198; Hussain et al. 2009). The same figure also bears the structures of a macrocyclic dimer lactone, verbalactone (199), isolated from V. undulatum (Magiatis et al. 2001) and picein (200) from V. dudleyanum (Tatli et al. 2008a).

Fig. 14
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Other compounds isolated from Verbascum spp

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Considering all reported data, several groups of bioactive metabolites from V. lychnitis, V. nigrum, V. phlomoides, V. thapsiforme and V. thapsus have been exhaustively studied. Other systematically examined species as V. salviifolium (Akdemir et al. 2004c, 2005; Tatli et al. 2008b), V. lasianthum (Akdemir et al. 2004a, b; Tatli et al. 2006; Kupeli et al. 2007), V. mucronatum (Akdemir et al. 2011) and V. wiedemannianum (Abou Gazar et al. 2003a, b) could be also considered as rather well studied. On the other hand, the knowledge on some species as V. songaricum is relatively limited. The taxon has been studied in details regarding its saponin content in aerial parts (Seifert et al. 1991; Hartleb and Seifert 1994, 1995) and flavonoids in the roots (Yuldashev 1996), while the iridoids presence has been proven only by TLC (Grabias and Swiatek 1987).

Metabolomics of Verbascum spp

Despite all applications (see below) the knowledge of the metabolites, accumulated in some Verbascum species, could be considered as still limited and based mainly on determination of the major compounds. Moreover to explore the chemodiversity of the genus, aiming to distinguish between species and to establish differences in metabolite profiles and chemical fingerprints, an application of emerging comprehensive analytical platforms (e.g. metabolomics) might be very useful. Metabolomics is a holistic approach, defined as systematic identification and quantification of all metabolites in an organism (Kim et al. 2010), at given conditions. Several platforms and techniques for high throughput analyses of targeted molecules have been developed (mainly mass spectrometry and nuclear magnetic resonance spectroscopy) during the past 15 years. Nuclear magnetic resonance (NMR) has been already proven as quite suitable and adequate method to perform metabolomics, as it allows simultaneous detection of abundant primary metabolites along with diverse groups of secondary metabolites (Verpoorte et al. 2007; Kim et al. 2010). Moreover 1H NMR-scpectroscopy possesses a great advantage over the other analytical platforms (e.g. mass spectrometry), as the signal intensity is only dependent on the molar concentration in the solution, which enables the direct comparison of concentrations of all compounds, which are present in a particular sample (Kim et al. 2010, 2011).

NMR-based metabolomics approach was applied to study metabolic differentiations of five Verbascum species (Georgiev et al. 2011a). 1H NMR fingerprinting in combination with multivariate data analysis (e.g. principal component analysis, PCA) allows classification of Verbascum species in two groups: group A (V. phlomoides varieties 7 and 33, and V. densiflorum) and group B (V. xanthophoeniceum, V. nigrum and V. phoeniceum). Further, it was found that the plants in group B synthesize higher amounts of bioactive iridoid glycosides [e.g. pharmaceutically important harpagoside (0.5 % on dry weight basis) 70] and phenylethanoid glycosides (in total about 6 % on dry weight basis)—verbascoside (87), forsythoside B (101) and leucosceptoside B (104). 1H NMR metabolomics data and hierarchical clustering analysis revealed that V. xanthophoeniceum and V. nigrum species have a similar leaf metabolome, which is quite different from the other mullein species, recognized by the European Pharmacopoeia (Georgiev et al. 2011a). It was suggested that NMR spectroscopy can be used for the rapid quantification of pharmaceutically important harpagoside in plant samples, e.g. for quality control of pharmaceutical products and/or herbal supplements (Georgiev et al. 2013a).

Pharmacology of Verbascum spp

Up-to-date, Verbascum species have been reported to possess various biological activities. Some of the best recorded pharmacological properties of mullein species are mentioned in this part as a result of a literature survey in Pubmed and Scopus databases.

Anti-inflammatory activity

Anti-inflammatory effect of different mullein species—traditionally used against inflammatory diseases, asthma, coughs, diarrhea, and pulmonary problems (Turker and Gurel 2005; Tatli and Akdemir 2006; Kupeli et al. 2007; Speranza et al. 2009)—has been studied extensively. An early study on V. thapsiforme showed that the aqueous extract of the plant exhibits strong anti-inflammatory effect, through inhibition of elongation stage of protein biosynthesis in rat liver, and the saponin fraction was found to be responsible for the anti-inflammatory effect of the species (Paszkiewicz-Gadek et al. 1990). The methanolic extract of the flowers of V. lasianthum, subjected to carrageenan-induced hind paw edema and to p-benzoquinone-induced writhings models in mice, demonstrated a significant anti-inflammatory and antinociceptive effect. Bioassay-guided fractionation of the extract resulted in the isolation of eight individual compounds, of whom aucubin (37) and ilwensisaponin A (166) were proven to posses remarkable anti-inflammatory and antinociceptive properties (Kupeli et al. 2007). The flower extract of V. pterocalycinum var. mutense, studied by the same research group (Kupeli Akkol et al. 2007) in carrageenan and PGE1-induced hind paw edema along with 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema models, was found to display activity only in carrageenan and PGE1-induced hind paw edema model, which led to a conclusion that the extract might act through inhibition of the cyclooxygenase (COX) activity. Ilwensisaponins A (166) and C (180) were proven to be the main active constituents in this plant species. The aerial parts of V. salviifolium were subjected to similar assays by the same research group and yielded β-hydroxyacteoside (89), apigenin 7-O-glucoside (120), luteolin 7-O-glucoside (123), luteolin and 3′-O-glucoside (125), as the active constituents in carrageenan and PGE1-induced hind paw edema models in mice (Tatli et al. 2008b). In another study, authors assigned a significant anti-inflammatory effect to the flower extract of V. mucronatum by applying the same experimental models (Akdemir et al. 2011). The methanolic extract, iridoid- and phenylethanoid-containing fractions, and isolated pure compounds of V. xanthophoeniceum (collected from Bulgaria) were tested for their anti-inflammatory effect by in vitro methods on nitric oxide and cytokine production by peritoneal macrophages accompanied by COX-1 and COX-2 expression and by in vivo method, using cobra venom factor (CVF)-induced edema in mice (Dimitrova et al. 2012). The extract was able to lessen paw-swelling at the doses of 40 and 200 mg/kg, while forsythoside B (101) showed the most pronounced inhibition of alternative pathway activity in CVF-induced edema model. Among the leaf and hairy root extracts obtained from several Verbascum species (V. nigrum, V. densiflorum, V. phoeniceum and V. phlomoides), grown in green sera conditions, Dimitrova et al. (2013) reported noteworthy anti-inflammatory feature of V. phoeniceum in in vivo carrageenan-induced edema model by inhibiting COX-1 and COX-2 enzymes. Pure harpagoside (70) ameliorated the development of zymosan-induced arthritis and reduced pathological changes in joints as shown by the decreased histological score for cell infiltration in synovial cavity, cartilage loss and bone resorption. Moreover, molecular docking simulations of harpagoside suggested that it may function with increased specific affinity towards COX-1 than COX-2 (Dimitrova et al. 2013).

In another study of Georgiev et al. (2012a) the crude methanolic extract, its fractions as well as a number of individual constituents—of iridoid and phenylethanoid origins—from V. xanthophoeniceum were subjected to primary cultures of normal human keratinocytes along with a thorough investigation of their effect on pro-inflammatory chemokines (IL-8, MCP-1 and IP-10) and gene expression for possible anti-inflammatory effect. Among the tested samples, verbascoside (87) and forsythoside B (101) were found to be effective, dose-dependent inhibitors of gene expression and de novo synthesis of above mentioned chemokines. Therefore, V. xanthophoeniceum-derived phenylethanoid glycosides could be considered as potential active components for topical compositions aimed at the regulation of chronic inflammatory skin disorders, such as psoriasis and atopic dermatitis (being characterized by over-expression of IL-8, MCP-1, and IP-10; Georgiev et al. 2012a).

The anti-inflammatory potential of aqueous flower extract of V. phlomoides was recently examined in in vitro and in vivo assays (Grigore et al. 2013). The findings from this study indicate that the extract was found to inhibit TNF-α-induced ICAM-1 expression significantly by 55–59 % on human umbilical vein endothelial cells at concentrations of 100 and 200 μg/mL, however, it did not display any effect in egg-white-induced rat paw edema model (Grigore et al. 2013). The authors concluded that in vitro anti-inflammatory effect of the extract could be corelated to its iridoid and phenylethanoid contents rather than polyphenolic constituents.

On the other hand, Speranza et al. (2009) examined the anti-inflammatory effect of verbascoside (the major phenylethanoid glycoside)-containing extract from V. mallophorum and reported that verbascoside per se exerted/had a significant anti-inflammatory action by causing a substantial diminution in the expression and activity of iNOS and extracellular O2. Strong anti-inflammatory activity of verbascoside was also reported in an early study by inhibition of carrageenan-induced edema and histamine and bradykinin-induced contractions in guinea pig ileum (Schapoval et al. 1998).

Wound-healing activity

Süntar et al. (2010) screened the methanolic extracts of thirteen Verbascum species (V. chionophyllum, V. cilicicum, V. dudleyanum, V. lasianthum, V. latisepalum, V. mucronatum, V. olympicum, V. pterocalycinum var. mutense, V. pycnostachyum, V. salviifolium, V. splendidum, V. stachydifolium, and V. uschackense), grown in Turkey, for their in vivo wound-healing effect by linear incision and circular excision experimental models accompanied by histopathological examination. Among them, V. olympicum, V. stachydifolium and V. uschackense were found to display promising wound-healing effect in the models used. In another study, Korkina et al. (2007) reported verbascoside (56 %)-containing extract to have remarkable wound-healing activity on full thickness excision wound model, which has been linked to its inhibitory capacity on release of reactive oxygen species (ROS) from the whole blood granulocytes and monocytes as well as its metal-chelation ability (with Fe+2 ions). In some more recent studies (Mehdinezhad et al. 2011, 2012), the flower extract of V. thapsus was shown to exert notable healing effect on experimental coetaneous and zinc oxide wound models in rabbits with topical application of the extract.

Antiviral and antimicrobial activity

Verbascum species have been widely screened for their antimicrobial activity. Amongst them, several species were demonstrated to possess remarkable antiviral activity. The lyophilized flower infusion of V. thapsus exerted significant inhibitory effect against Herpes simplex virus (type 1) using the yield reduction test (Slagowska et al. 1987; Zgórniak-Nowosielska et al. 1991). On the other hand, combination of adamantanamine glucuronide and the lyophilized flower infusion of V. thapsiforme exerted a discernible effect against influenza virus in chicken embryo fibroblast cell cultures (Serkedjieva 2000). In connection with these data, the methanolic extract of V. thapsus from Nepal was revealed to have a robust anti-influenza effect (Rajbhandari et al. 2009).

In a screening study on the mullein species from Argentina (Zanon et al. 1999), V. thapsus displayed the strongest inhibitory effect against Pseudorabies virus strain RC/79 (Herpes suis) in Vero cells (2 log) and in a follow-up study the same species was found to inhibit by 50 % plaque formation caused by Pseudorabies virus at concentration of 35 µg/mL, while incubation of the virus with the plant extract led to 99 % of inhibition during the adsorption phase (Escobar et al. 2012).

In addition, some early studies have revealed the antibacterial properties of some mullein species. For instance, verbalactone (199)—a macrocyclic dimer lactone derivative—isolated from V. undulatum was identified to display marked antibacterial effect (Magiatis et al. 2001). Turker and Camper (2002) showed strong antibacterial effect of the water extract prepared from the species against Klebsiella pneumonia, Staphylococcus aureus, S. epidermidis, and Escherichia coli, while the polar extracts of V. sinuatum (Senatore et al. 2007; Sener and Dulger 2009), V. gypsicola (Dulger and Gonuz 2004), V. georgicum (Sengul et al. 2005), V. antiochium (Ozcan et al. 2010), and V. pinetorum (Ozcan et al. 2011) exhibited significant activity towards a number of Gram (+) and Gram (−) bacteria. Several mullein species were tested against E. coli, Pseudomonas aeruginosa, S. aureus, Enterococcus faecalis, as well as the fungal strains of Candida albicans, C. parapsilosis, and C. krusei by disc diffusion methods, which led to identification of V. mucronatum and V. olympicum as demonstrating antibacterial activity against Gram (+) bacteria and S. aureus along with V. latisepalum, showing notable antifungal activity against C. krusei (Kahraman et al. 2011).

Anthelmintic activity

Up-to-date, only a few studies have been reported on anthelmintic activity of mulleins. The methanolic extract of V. thapsus was subjected to anthelmintic assays using adult roundworms (Ascaridia galli) and tapeworms (Raillietina spiralis) in which the time of paralysis and death was determined and compared to albendazole as reference drug. The results indicated that the methanolic extract has superior effect against R. spiralis than that of the reference drug (Ali et al. 2012). In a screening study, the extracts obtained from V. lasianthum, V. latisepalum, V. mucronatum, and V. salviifolum exerted a potent anthelmintic effect against Aspiculuris tetraptera at dose of 100 mg/kg in mice (Kozan et al. 2011).

Neuroprotective activity

Our literature survey pointed out that the knowledge on the neuroprotective effect of mulleins is still scarce. Inhibition of cholinesterases enzyme family has been one of the most accepted approaches for treatment of mild to moderate Alzheimer’s disease and in this sense, the phenylethanoid-enriched fraction and some pure compounds from V. xanthophoeniceum were exposed to in vitro testing of cholinesterase enzyme inhibition. It was found that both methanolic extract and phenylethanoid-enriched fraction, at 200 μg/mL, along with forsythoside B (101), at 100 μg/mL, exerted significant butyrylcholinesterase inhibition (75, 84.3 and 98.3 %, respectively; Georgiev et al. 2011b). In a similar study, cholinesterase inhibitory activities of the aqueous extract of V. mucronatum, its fractions and several individual constituents were examined and only verbascoside was shown to possess mild cholinesterase-inhibiting effect (Kahraman et al. 2010).

Biotechnology of mulleins

The high importance of Verbascum-derived bioactive molecules imposes the development of alternative way to supply them. Plant in vitro technologies are considered as an attractive and cost-effective alternative to classical approaches and possess an immense potential for sustainable supply of value-added plant-derived metabolites (“chemical factories” concept; Georgiev et al. 2012b, 2013b). An efficient protocol for the establishment of transformed root culture (=hairy roots) of V. xanthophoeniceum, using sonication-assisted Agrobacterium rhizogenes-mediated transformation, was reported (Georgiev et al. 2011c). Ten days after the inoculation with A. rhizogenes ATCC 15834 suspension, and 45 s of ultrasound exposure, hairy roots appeared on 75 % of the Verbascum leaf explants. Moreover, most vigorous V. xanthophoeniceum hairy root clones showed stable growth under submerged cultivation and accumulated high biomass amounts (13–14 g dry root mass/L). LC-APCI-MS metabolite profilling of the hairy roots revealed that verbascoside (87) was the most abundant secondary metabolite and its amounts were over 6-times higher than in the mother plant tissue (Georgiev et al. 2011c). Clearly, Verbascum transformed roots have an enormous biosynthetic potential and might therefore serve as an attractive source for bioproduction of pharmaceutically important metabolites (Fig. 15), though more detailed further research is pending.

Fig. 15
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Technological platform for Verbascum spp hairy roots induction and bioproduction of pharmaceutically important metabolites

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Conclusion and further perspectives

Mulleins have a long tradition of use in folk medicine. While the clinical efficacy is yet to be established, various Verbascum extracts, fractions and purified secondary metabolites were shown to posess valuable pharmacological properties (in vitro and in vivo), suggesting that mulleins may be beneficial in the treatment (and prevention eventually) of respiratory, inflammatory and infectious disorders, in addition to their neuroprotective and wound healing activity. It should be, however, pointed out that some of these activities are observed at relative high concentrations, and therefore these studies should be considered with caution.

Mulleins are found to be a rich source of diverse groups of secondary metabolites. Untill now more than 200 compounds are identified, including iridoids, phenylethanoids, flavonoids, and saponins among others.

The genus Verbascum provides a wide range of research possibilities for the future. Key tasks as the lack of adequate taxonomic scheme and the incomplete or medley phytochemical data could be eventually solved by applying the modern ‘omics’ platforms (inter alia metabolomics). Further intensive studies (using various animal models) are required to confirm Verbascum’s potential for treating various diseases, thereby enabling mulleins (or their bioactive principles) acceptance as therapeutic agents. For this, reliable standartization of mullein products is required and the chemical fingerprints need to be fully characterized.