Tyrosinase Inhibitors from the Aerial Parts of Wulfenia carinthiaca jacq.

Activity guided isolation of a MeOH extract of the aerial plant parts of Wulfenia carinthiaca jacq. (Plantaginaceae), using a mushroom tyrosinase assay, resulted in the isolation of five phenylethanoid glucosides and four iridoid glycosides. Two of them, 2′‐O‐acetylisoplantamajoside and 2′,6″‐O‐diacetylisoplantamajoside, represent new natural products. Evaluation of the inhibitory activity of all isolated compounds revealed that the observed activity is not related to the isolated phenylethanoid glycosides but mainly due to the presence of the iridoid glycoside globularin (IC 50 41.94 μm;CI 95% ± 16.61/11.89 μm). Interestingly, structurally close related compounds (globularicisin, baldaccioside, and isoscrophularioside) showed no or only a weak tyrosinase inhibitory activity.


Introduction
The genus Wulfenia (Plantaginaceae) belongs to a group of genera with a disjunct distribution range and is currently classified into four species s.str.: Wulfenia carinthiaca s.l. (Carnic and the Dinaric Alps populations), W. baldacii (restricted to the Dinaric alps), W. orientalis (restricted to the southernmost Amanos Mts., Turkey), and W. glanduligera (central and northern Amanos Mts., Turkey). [1] Due to the limited local distribution and the attractive intensive blue color of the flowers, especially of Wulfenia carinthiaca, this species was selected as the national flower of the federal state of Carinthia/Austria. [2] Nowadays, also a white flowering cultivar, W. carinthiaca cv. 'Alba' is commercially available from different providers. Despite its popularity as ornamental plant, the phytochemical knowledge about the genus is limited. An investigation of the root material of W. carinthiaca resulted in the identification of the phenylethanoids plantamajoside, 2 0 -O-acetylplantamajoside, and 2 0 ,6″-O-diacetylplantamajoside, as well as the irdoides globularin, isoscrophularoside, and the dimeric iridoid wulfenoside. [3] In a comparative chemotaxonomic study of tribe Veroniceae (Plantaginaceae) [4] also other representatives (whole plant samples) were investigated: W. baldaccii afforded mannitol, plantamajoside, 2 0 ,6″-O-diacetylplantamajoside, catalpol and asystasioside E, epiloganic acid, aucubin, globularin, isoscrophularioside, picroside I, wulfenoside, and the new chlorine containing iridoid baldaccioside; W. orientalis contained mannitol and shikimic acid, catalpol, gardoside, aucubin, mussaenosidic acid, arborescosidic acid, globularin, isoscrophularioside, as well as the phenylethanoide 2 0 ,6″-O-diacetylplantamajoside; Wulfenia blechicii subsp. rohleanae, a 'species' which is distributed only in the Dinaric Alps and should be nowadays handled as W. carinthiaca, since the species status is neither supported by genetic nor by morphologic data. [1] Nevertheless, the investigation of the plant material afforded shikimic acid, catalpol, 6,7dihydromonotropein, aucubin, gardoside, epiloganic acid and arborescosidic acid, cis-globularin (= globularicisin), globularin, picroside, and isoscrophularioside. The MeOH extract of the aerial parts of W. carinthiaca (Carnic Alps population, cultivated material) showed in an HPTLC-based mushroom tyrosinase inhibition assay [5] a promising inhibitory effect, which was also evaluated in a 96 well based assay. In this assay, the MeOH extract showed an inhibitory effect of ca. 40% at a concentration of 500 lg/mL (highest soluble concentration). Tyrosinase, a copper-containing monooxygenase enzyme, catalyzes the first two steps of the melanogenesis, converting firstly L-tyrosine by hydroxylation to 3,4-dihydroxyphenylalanine and subsequently by oxidation to dopaquinone. [6] Inhibitors of this enzyme, especially those without side effects, are of interest in cosmetic industry to reduce undesired pigmentation like freckles and age spots.
In this connection, two questions arise: Is a differentiation of the two populations of W. carinthiaca (Carnic and Dinaric Alps) based on the secondary metabolite pattern possible and what compound(s) is/ are responsible for the observed tyrosinase inhibition?

Results and Discussion
In a first step, the obtained MeOH extract of the aerial plant parts of W. carinthiaca was investigated by LC/ MS (see Figure 1 and Table 1). Taking into consideration the constituents already described for the roots of W. carinthiaca and other species of this genus and analyzing the extracted ion chromatograms (ESI, positive-ion mode) of the corresponding sodium adduct ions ([M + Na] + ) the presence of the iridoid glucosides globularicisin (2), globularin (4), baldaccioside (7), and isoscrophularoside (8) (see Figure 2) could be suggested. [3] [4] This was confirmed by the isolation of the corresponding compounds and analysis of the recorded 1D-and 2D-NMR spectra. The LC/MS analysis revealed also the presence of several phenylethanoid glycosides in the extract of the aerial plant parts (see Figure 2) corresponding to plantamajoside and its derivatives (1, 3, and 5) known from the roots of W. carinthiaca. [3] Interestingly, all three compounds showed in the EIC an additional peak with an identical LC-online UV spectrum and identical m/z values corresponding to the sodium adduct ion in the positive-ion mode ESI-MS and the deprotonated molecule ion ([M À H] À ) in the negative-ion mode ESI-MS. Differences were only observed in the intensity of some of the detected fragments in the positive-ion mode ESI-MS (see Table 1). The MS spectra of compound 1 and 1a showed e.g. a significant difference in the relative intensity (9.7% rel. intensity (1) and 43.8% rel. intensity (1a)) of a fragment with an m/z of 478.8, suggesting a very similar, but not identical structure of both compounds. The compound pairs 3 and 6, as well as 5 and 9 showed an analogous behavior. In order to elucidate the chemical nature of the additional phenylethanoid glycosides, 6 and 9 were isolated together with 1, 3, and 5. All compounds could be obtained in a satisfying purity and quantity for NMR structure elucidation except compound 1a.
Comparison of the NMR spectra of compounds 3 and 6 with literature values enabled the identification of compound 3 as 2 0 -O-acetylplantamajoside. [3] Careful analysis of the HMBC contacts of the protons attached to the carbons 4 0 and 6 0 of the inner glucose moiety of compound 6 revealed a different connectivity of the caffeoyl moiety, which was found to be shifted from position 4 0 in compound 3 to position 6 0 of the inner glucose subunit in compound 6. The change of the ester position is therefore identical to the wellknown pair acteoside/isoacteoside and was already described for plantamajoside and the related compound isoplantamajoside, [7] also known as plantainoside D. [8] Therefore, compound 6 was identified as 2 0 -O-acetylisoplantamajoside. Comparison of the NMR spectra of compounds 5 and 9 resulted in an analogous result. Compound 5 could be identified as 2 0 ,6″-O-diacetylplantamajoside, while compound 9 was identified as 2 0 ,6″-O-diacetylisoplantamajoside. Both isolated acetylated isoplantmajoside derivatives are described here for the first time.
Due to the pairwise occurrence of plantamajoside and isoplantamajoside derivatives we tentative identified compound 1a as isoplantamajoside, which is supported by the observed LC/ESI-MS fragments showing slightly elevated levels of liberated caffeoyl units as well as the remaining molecule parts similar to the NMR-verified pairs 3 and 6 or 5 and 9. From a chemotaxonomic point of view it is interesting that a differentiation of the two populations of W. carinthiaca (Carnic and Dinaric Alps) seems to be possible, since W. carinthiaca from the Dinaric Alps (previous described as Wulfenia blechicii subsp. rohleanae) seems to contain no phenylethanoids at all. [4] For final confirmation of the chemotaxonomic relevance of these findings, results have to be verified with a larger number of samples which should also include wild type specimens.
In order to assess which compound(s) of W. carinthiaca is/are responsible for the observed inhibitory effect on mushroom tyrosinase, compounds 1 -9 were evaluated at a concentration of 500 lM in the 96-well plate assay. The results are summarized in Table 2. Since only the iridoid globularin (4, see Figure 2) showed a promising effect at this rather high test concentration, IC 50 value determination was limited to this compound together with free cinnamic acid, a known tyrosinase inhibitor, [9] and kojic acid as positive control (see Figure 3).
Globularin showed in the tyrosinase inhibition assay an IC 50 value of 41.94 lM (CI 95% AE 16.61/ 11.89 lM) and is therefore not as active as the positive control kojic acid (IC 50 of 5.40 lM (CI 95% AE 0.47/ 0.43 lM). Interestingly, free cinnamic acid shows significant higher IC 50 value of 353.99 lM (CI 95% AE 27.24/ 25.29 lM). Accordingly, it can be assumed that the iridoid part of globularin improves the interaction with the enzyme. Since the cis-isomer of globularin, globularicisin (2), showed almost no activity at 500 lM, the trans-configuration of the cinnamic acid unit appears to be essential for the inhibitory activity, but also the shape and substitution pattern of the iridoid part affects the activity. Replacement of the epoxide ring of 4 by a double bond (isoscrophularioside, 8) reduces the inhibitory effect from 79.59 AE 1.62% to 48.49 AE 2.08% at 500 lM. The activity reduction is even more evident when the epoxide ring is opened by the addition of hydrogen chloride to the molecule, which led to a drop of activity from 79.59 AE 1.62% (4) to 23.01 AE 3.16% (7) at 500 lM.

Conclusions
In summary, phytochemical investigation of the aerial parts of Wulfenia carinthiaca resulted in the isolation and identification of four iridoid glycosides and five phenylethanoid glycosides, of which two, 2 0 -O-acetylisoplantamajoside (6) and 2 0 ,6″-O-diacetylisoplantamajoside (9), are described for the first time. Detection of phenylethanoid glycosides in the investigated plant parts might be useful for the chemical differentiation of the two populations of W. carinthiaca (Carnic and Dinaric Alps). Furthermore, the pharmacological evaluation of isolated compounds led to the identification of the novel tyrosinase inhibitor globularin.