Species of the genus Scrophularia have been applied for centuries in traditional medicine. The most important one among those, Scrophularia ningpoensis Hemsl., is widely used in traditional Chinese medicine for a broad spectrum of inflammatory diseases (De Santos Galíndez et al., 2002; Tong et al., 2006; Li et al., 2009) and listed in the Chinese Pharmacopeia against febrile diseases with excessive thirst or eruptions, constipation, cough due to exhaustion, conjunctivits, skin disorders, and others (Stöger, 2017). The use in the treatment of inflammatory disorders, wound healing, skin ailments, and health disturbances has been reported for numerous species such as Scrophularia auriculata L. or Scrophularia canina L. from the Mediterranean area (Giner et al., 2000; Guarrera and Lucia, 2007; Passalacqua et al., 2007) as well as for Scrophularia variegata M. Bieb. and Scrophularia striata Boiss. in traditional Iranian medicine (Azadmehr et al., 2013, 2015). A traditional use as an antipyretic and anti-inflammatory agent was described for Scrophularia oldhamii Oliv. and as a diuretic for Scrophularia grossheimii Schischkin (De Santos Galíndez et al., 2002). In Turkish traditional medicine, the species Scrophularia nodosa L. is well-known as a diuretic, to treat hemorrhoids, and also for wound healing, eruptive skin diseases, eczema, psoriasis, and pruritus (Baytop, 1999; Stevenson et al., 2002; Crisan et al., 2009). A use in these indications is also conveyed for the Turkish species Scrophularia depauperata Boiss., Scrophularia cryptophila Boiss. et. Heldr. Boiss., and Scrophularia floribunda Boiss. et. Bal. (Ozbilgin et al., 2014).

Recently, the activity of various extracts from several Scrophularia species has been studied in different cancer models: weak cytotoxic activity of an ethanolic extract from S. striata via induction of apoptosis and G₂/M phase arrest was shown in Jurkat human leukemia cells (Azadmehr et al., 2013). In WEHI 164 fibrosarcoma cells, a methanolic extract of this plant exhibited a significant and dose-dependent reduction of the activity of matrix metalloproteinases (Hajiaghaee et al., 2007), which play a role in tissue remodeling and tumor invasion. An apolar fraction from a methanolic extract of Scrophularia orientalis L. significantly reduced the viability of neuroblastoma cells due to a loss of mitochondrial membrane integrity (Lange et al., 2016). In MCF-7 breast cancer cells, weak cytotoxicity of an ethanolic extract from S. variegata was reported (Azadmehr et al., 2015). A dichloromethane extract from Scrophularia oxysepala caused significant cytotoxic and apoptotic effects in MCF-7 cells by influencing the expression of p53, caspase 3, and c-myc, and the cleavage of PARP (Valiyari et al., 2013). The anti-tumor activity of S. oxysepala in breast cancer was confirmed in vivo in an allograft model in mice inoculated with mammary carcinoma 4T1 cells. Animals treated with 50 or 100 mg/kg of an ethanolic extract developed significantly smaller tumors in size and weight (Baradaran et al., 2017). In a study of five Scrophularia species, S. floribunda and Scrophularia lucida L. displayed the highest cytotoxic activity against HL-60 promyeloic leukemia cells. These extracts were also tested in a three-dimensional co-culture cell model consisting of MCF-7 breast cancer spheroids placed on top of telomerase-immortalized human lymph endothelial cell (LEC) monolayers. There, tumor spheroids cause the formation of large cell-free areas in the lymph endothelial barrier, so-called “circular chemorepellent induced defects” (CCIDs), through which tumor cells penetrate the vasculature. This assay mimics intravasation which is an early step within the metastatic cascade resembling the pathological situation in rodents and humans (Madlener et al., 2010; Vonach et al., 2011; Viola et al., 2013). A detannified methanolic extract of S. lucida L. was the most active one in suppressing CCID formation (Giessrigl et al., 2012).

The lack of any information about the chemical composition of S. lucida prompted us to perform an activity-guided search for the active principle of this species contributing to the inhibitory effect in the CCID assay.

From the methanolic extract of the aerial parts from S. lucida, apolar compounds were depleted by extraction with petroleum ether. After subsequent partition with chloroform and ethyl acetate, the two organic fractions and the residue of the aqueous phase were tested in the CCID assay. The crude methanol extract significantly and linearly inhibited CCID formation between concentrations of 0.25–1 mg/mL (Figure 1). At a concentration of 0.25 mg/mL, the CF seemed to be even more effective than 0.25 mg/mL of the methanol extract; however, this difference was not significant. The EF was only active at 1 mg/mL and the AF was inactive at the tested concentrations. Therefore, the CF was chosen for further separation by CC on silica and five of the resulting sub-fractions (A–E) were active in the CCID assay with sub-fraction A exhibiting the strongest inhibition (Figure 2). The EF was separated by size exclusion chromatography followed by solid phase extraction or another subsequent size exclusion chromatography. Due to the lower activity of the EF in the CCID assay, these sub-fractions were submitted to chemical investigations only.

The processes resulted in the elucidation of 14 compounds, one of which is a new natural product (Figure 3). The 13 known compounds were identified by chromatographic, mass spectrometric and/or spectroscopic dereplication with authentic samples or data from literature. This led to the elucidation of seven flavonoids. The widespread natural compounds astragalin (1), luteolin-7-O-glucoside (8), kaempferol-3-O-rutinoside (6), and rutin (7) were confirmed by TLC- and LC-DAD comparison with the authentic substances. To the best of our knowledge, only the two latter have been reported in the genus Scrophularia until now, namely, in Scrophularia ilwensis C. Koch (Calis et al., 1993). Two further, less prevalent flavonoids, nepitrin (4) and homoplantaginin (2), had been isolated from S. ningpoensis (Li et al., 2009) and nepitrin also from S. striata (Monsef-Esfahani et al., 2010) before. ¹H-NMR- and ¹³C-NMR data of 2 correlated excellently with the data published earlier for homoplantaginin (Wang et al., 1998; Dawa et al., 2009). Hispidulin (9) was detected for the first time in a Scrophularia species and elucidated by LC-DAD comparison and LC–MS data.

A new flavonoid (3) was identified by thorough MS and NMR experiments: a molecular formula of C₂₄H₂₄O₁₂ was determined from the HRESIMS data. Fragmentation of the [M+H]⁺ ion at m/z 505.0 showed the loss of a 204 Da to yield a Y₀ ion at m/z 301.3, which again lost a CH3∙ in MS³, typical for a methoxy group bound to an aromatic system. The MS², MS³, and MS⁴ spectra were nearly identical to those of homoplantaginin. Fragmentation of the [M-H]^- ion at m/z 503.0 confirmed this information. In addition, the loss of 60 Da (CH₃COOH) in MS² indicated acetylation of the glucose of homoplantaginin. The signals of 3 in the ¹H-NMR spectrum excellently correlated with those of compound 2 (Table 1), despite a large downfield shift of the proton at C-2″ in the sugar for 1.55 ppm. The signals of the protons at C-1″ and C-3″ were shifted downfield for 0.24 and 0.22 ppm, respectively, as well. Additionally, from the singlet at δ = 2.038 ppm, linkage of an acetyl group was deduced. The connection of an additional acetyl group in position 2″ of homoplantaginin was confirmed by the ¹³C-NMR data. The signals of C-1″ and C-3″ were shifted upfield for 2.28 and 2.95 ppm, respectively. Signals of an acetyl group occurred at δ = 169.26 ppm (quarternary C) and δ = 20.82 ppm (methyl C). An HMBC crosspeak from the CH₃ protons to the C-2″ finally proofed the connectivity. Thus, compound 3 was unambiguously identified as 2″-O-acetyl-homoplantaginin.

The phenylpropanoid verbascoside, which had been detected in many species of the Scrophulariaceae family (see, e.g., Zhang et al., 2013; Pasdaran et al., 2016, 2017; Venditti et al., 2016), was isolated from S. lucida as well and identified by HPLC-DAD and MS.

The genus is well-known for synthesizing a broad range of iridoids (De Santos Galíndez et al., 2002) and two such compounds were isolated from and two further ones tentatively identified in the CF.

The major iridoid was scrovalentinoside (14). The compound was elucidated by MS and NMR experiments (see Supplementary Material). The spectroscopic data were in excellent accordance with those published after isolation of the compound from S. auriculata L. ssp. pseudoauriculata (Senn.) Bolós et Vigo (Giner et al., 1998) and from S. nodosa L. (Miyase and Mimatsu, 1999). A second iridoid was unambiguously elucidated as koelzioside (13) based on MS experiments and the analogy of the ¹H-NMR and ¹³C-NMR data as reported for the compound, until now only known from Scrophularia koelzii L. (Bhandari et al., 1992) and Scrophularia deserti Del. (Ahmed et al., 2003). Tentatively identified by thorough LC–MS experiments were two isomers of buergeriside C₁ (10 and 11), known from Scrophularia buergeriana (Miquel) (Kim and Kim, 2000), and 6-O-(2″-acetyl-3″,4″-di-O-trans-cinnamoyl)-α-L-rhamnopyranosyl-catalpol (scropolioside B, 12), reported in Scrophularia scopolii [Hoppe ex] Pers. var. scopolii (Calis et al., 1988). Compounds 10 and 11 both showed a UV spectrum typical for a para-substituted cinnamic acid derivative and yielded identical fragment ions, although the relative intensities were different in the MS² spectra of the [M+COOH]^- ion of 10 and 11. Fragmentation of the [M-H₂O+H]⁺ ion yielded the acylium ion of methoxycinnamic acid (Z₀), while CID of the [M+COOH]^- ion produced deprotonated methoxycinnamic acid (Y₀), dehydrated deoxyhexose (B₁), and typical cross-ring cleavage of the deoxyhexose. From the compounds described for the genus Scrophularia, this pattern only fits to buergeriside C₁, why we have tentatively identified 10 and 11 as buergeriside C₁ isomers. The UV spectrum of 12 points toward cinnamic acid. Fragmentation of the [M-H]^- ion (resulting from CID of the [M+COOH]^- ion) yielded the neutral loss of 60, 148, and 162 Da, fitting to an acetylated, cinnamoylated, and hexosylated compound. Scropolioside B is the only previously described compound in the genus Scrophularia being in accordance with these data. All iridoids detected in S. lucida are very rare natural compounds.

The quantitative determination by HPLC of the major compounds in the methanolic extract revealed 3% homoplantaginin, 1.4% scrovalentinoside, and 0.7% koelzioside.

In an earlier study, 20 μg/mL of the methanolic extract of S. lucida was reported to induce 70% apoptosis and to almost completely inhibit proliferation of HL-60 cells in S-phase within 48 and 72 h, respectively (Giessrigl et al., 2012). Here, we demonstrate that 250 μg/mL of the methanolic extract inhibits 60% CCID formation in the MCF-7/LEC model within 4 h and the CF of the methanolic extract inhibited CCIDs almost similarly (with insignificant difference; Figure 1). Sub-fraction A of the CF was the most active one and other four fractions (B–E) also exhibited significant CCID-inhibitory properties (Figure 2).

Hispidulin turned out to be the major component in sub-fraction A, whereas koelzioside and scrovalentinoside, the main components in sub-fractions C and D, respectively, showed significantly less activity. To evaluate the potential of hispidulin in suppressing CCID formation, the pure compound was tested as well-demonstrating an IC₅₀ of 88.7 μM confirming a contribution of hispidulin to the effects of the methanolic extract. This further implicated that other components present in sub-fraction A must have synergized strongly with hispidulin as only 250 μg/mL of sub-fraction A inhibited ∼90% CCID formation (compared to 26.6 mg/mL of purified hispidulin, which inhibited just 50%). Conceivably, residual amounts of sub-fraction B still been present in sub-fraction A caused this synergy. Hispidulin, in vitro and in vivo, had been shown to inhibit proliferation and to induce apoptosis of various cancer lines (Yu et al., 2013; Gao et al., 2015; Wang et al., 2015). The compound exhibited cytotoxicity toward HepG2 liver cancer cells but not normal liver cells (Gao et al., 2014). It also inhibited pancreas cancer cell mobility and invasion in vitro as well as neovascularization in C57/BL6 mice (He et al., 2011). Additionally, colon cancer cell intravasation through blood endothelial barriers was hindered in vitro (Holzner et al., 2017).

In the activity-guided search for components of S. lucida inhibitory in the CCID assay, we showed that intravasation of breast cancer cells through the lymph endothelial wall, which is the major route of breast cancer dissemination, is significantly inhibited by hispidulin and other compounds present in the CF of S. lucida methanolic extract.

In addition, this is the first study characterizing the chemical composition of S. lucida. It resulted in the proof or tentative identification of several rare iridoids and flavonoids for the first time in the genus Scrophularia and a new compound, 2″-O-acetyl-homoplantaginin, was unambiguously elucidated.

For a more profound assessment of the potential of S. lucida to inhibit metastasis in breast cancer, testing in an appropriate animal model is necessary as a next step.

VL, LS, and TL performed the phytochemical fractionation of the extract and isolation of secondary metabolites. JE performed the CCID assay of hispidulin. HK performed all NMR measurements and structure elucidation. MZ performed all MS and LC–MS measurements and structure elucidation. CN performed the CCID assays of the extracts and fractions. GK supervised all pharmacological experiments. AÖ collected the plant material, was responsible for correct identification, and prepared the extract. LK supervised all phytochemical work. LK, HK, MZ, CN, and GK compiled the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.