*3.3. HPTLC Analysis*

HPTLC phenolic profiles obtained with two mobile phase systems as well as two different derivatizing agents (Figure 4) are similar for most tested samples, allowing the specification of a typical profile for goldenrod honey and its authentication. The characteristic, repeating pattern of bands is visible, especially in the case of derivatization with *p*-anisaldehyde. In the case of samples 3, 6 and 10, the profiles are significantly different. In the case of sample 10, it is possible to infer an admixture of honeydew, which is manifested by characteristic additional pink bands at Rf approx. 0.48 and 0.6. This pattern is similar to the one we observed earlier for honeydew honey samples [16]. These pink bands are also present, although less intense, in sample 6. The obtained profiles seem to correlate with the higher content of phenolic compounds in the three above-mentioned samples and their stronger antioxidant properties which was particularly observed in sample 10 where the admixture of honeydew could explain the strongest antioxidant properties (see Section 3.4). In honey number 3, additional intense bands are visible. Figure 4A shows blue ones at Rf = 0.26 and a series of blue-green between 0.45 and 0.7. A similar series of bands are clearly visible in sample 6. Figure 4B shows an additional blue band at Rf = 0.11 in sample 3, observed by us earlier in the rape honey sample [16].

**Figure 4.** HPTLC plates images: (**A**)—developed with eluent system A, derivatized with NP/PEG reagent, (**B**)—developed with eluent system B, derivatized with *p*-anisaldehyde reagent, Track 1–10: goldenrod honey, track 11: *S. gigantea*, track 12: *S. canadensis*, track 13: *S. virgaurea*.

Since three of the samples (3, 6 and 10) differ from the others in terms of polyphenol profiles, we decided to evaluate the flower extract profiles of three species of goldenrod to check whether the differentiation of the honey profiles is not related to another species of *Solidago* as a honeybee flow. The extracts of the individual species differ in particular in the Rf region between 0.30 and 0.55 (Figure 4A), where the bands derived from flavonoid glycosides, mainly derivatives of quercetin and/or luteolin, were identified. *Solidago gigantea* flowers contain four distinct glycosides, whereas in *S. canadensis*, only one with the highest Rf was present in large amounts, and in *S. virgaurea*, two with lower Rf (0.34 and 0.38), were identified as quercitrin and hyperoside. Rutin was present in all three species (Rf = 0.18). In addition, in *S. canadensis*, extracts from the bands of flavonoid aglycones, quercetin and kaempferol (Rf = 0.82 and 0.84, respectively) were detected. Derivatization with *p*-anisaldehyde revealed an additional gray-green band in the *S. virgaurea* extract at Rf = 0.13, possibly from some phenolic acid. Based on the comparison of the tracks from honey and goldenrod flower extracts, it can be theorized that the two most intense bands present in all three species of flowers (Rf = 0.28 and 0.69, Figure 4A) are most likely derived from certain phenolic acids, and are also present in most of the analyzed honey samples.

Among the polyphenols mentioned as present in goldenrod honey are mainly phenolic acids: gallic, 4-hydroxybenzoic, *p*-coumaric and ferulic [1]. Flavonoids have also been identified, mainly chrysin, galanagin, pinocembrin, kaempferol, quercetin and luteolin [22]. In flowers of various species of goldenrod, flavonoids were identified: mainly quercetin glycosides, and also aglycones: quercetin, kaempferol andisoramnetin [23–25]. In addition, in *S. virgaurea* flowers, myricetin, naringenin, genistein and pinocembrin were identified [1]. Among phenolic acids, chlorogenic, caffeic and ferulic acids were mainly found in the flowers of *S. gigantea* and *S. canadensis* [23,25] and gallic, 3,4-dihydroxybenzoic and vanillic acids were found in the flowers of *S. virgaurea* [1]. The quoted literature data indicate a certain variability in the polyphenol profile between individual goldenrod species, with *S. gigantea* and *S. canadensis* being more similar to each other than to *S. virgaurea*. Similar polyphenol profiles were obtained by HPLC for *S. canadensis* and *S. gigantea* by Zekiˇc et al. [26].

To confirm the richest polyphenol profile obtained for *S. gigantea*, the total content of phenols, flavonoids and antioxidant activity of the extracts of three species of goldenrod (flowers and leaves) were analyzed (Table S2). The obtained results also indicate that this species is the richest in phenolic compounds, which is reflected in its antioxidant capacity. Moreover, the data shows a high correlation (TPC vs. FRAP: r = 0.991, TPC vs. DPPH: r = 0.972, TPC vs. CUPRAC: r = 0.948). Literature data also indicate this goldenrod species as richer in polyphenols and antioxidants [27–29]. Based on provided literature data and own results, some species–specific differences were found; however, no obvious polyphenol markers were identified that could indicate which species of goldenrod is the source nectar for the analyzed honeys.
