*2.1. Polyphenol Composition*

The phenolic compounds present in the aerial parts and leaves of *C. vulgaris*, *E. multiflora*, and *E. scoparia* were identified by using an HPLC chromatogram at 330 nm (Figure 1). The main phenolic compounds were recognized by combining the retention times, UV spectra, and mass spectra of each peak with its standard, when available, and with literature data. The results revealed different quali-quantitative profiles among the studied parts, as shown in Figure 1. A total of 59 phenolic compounds were detected: 14 in *C. vulgaris*, 18 in *E. multiflora*, and 27 in *E. scoparia* (Table 1). Among them, 52 were positively identified (11 in *C. vulgaris*, 14 in *E. multiflora*, and 24 in *E. scoparia*). In terms of chemical classes, nine were phenolic acids and 43 were flavonoids, and among them, the majority belonged to the class of flavonols, mainly derivates of quercetin, myricetin, isorhamnetin, and kaempferol, while the rest of the compounds belonged to the class of flavanones, specifically eriodictyol and taxifolin. It is worth mentioning that, to the best of our knowledge, no previous studies have investigated the chemical composition of *E. scoparia*.

*Calluna vulgaris*leaves contained a total amount of phenolic compounds of 1567.78 mg/kg, comprising caffeoylquinic acid, which was the most abundant phenolic compound (1180 ± 8.18 mg/kg), followed by myricetin-*O*-rhamnoside (232.98 ± 0.30 mg/kg), myricetin-*O*-pentoside (48.81 ± 2.22 mg/Kg), and myricetin-*O*-hexoside (41.66 ± 1.88 mg/kg), whereas quercetin-*O*-hexoside (2.82 ± 3.24 mg/kg) was the lowest one. The results are in accordance with those presented by Mandim et al. [27] at the qualitative level, except for catechin, isorhamnetin-3-*O*-glucoside, and isorhmnetin-*O*-rhamnoside, which were absent in this studied species. However, a notable difference has been shown at the quantitative level, which could be, at least in part, attributed to the different organ of the plant used in this study, viz. leaves instead of inflorescences.

The leaves of *E. multiflora* contained 399.01 mg/kg of phenolic compounds, and were characterized by the presence of a quercetin derivative, myricetin-*O*-hexoside, and quercetin-*O*-(6"-cinnamoyl)-hexoside, while the aerial parts contained 227.6 mg/kg of phenolic compounds, and were distinguished by the presence of 4-caffeoylquinic acid, methyl-ellagic acid hexoside, and eriodictyol-*O*-hexoside, wherein 4-caffeoylquinic acid was the main compound in the aerial parts, with 83.75 ± 0.74 mg/kg, and where kaempferol was the least prevalent compound, with 0.95 ± 1.84 mg/kg. According to these results, it can be concluded that *E. multiflora* leaves presented higher phenolic compound content when compared to the aerial parts. The output of heat map analysis showed that the leaves and aerial parts of *E. multiflora* were clustered together into the same group and displayed the following main compounds in common: quercetin-*O*-hexoside, kaempferol-rhamnosylhexoside, rutin, caffeoylquinic acid, and kaempferol-hexoside. Moreover, in both parts, the presence of small amounts of three other compounds, quercetin, dimethylquercetin, and kaempferol, was noted. These results contradict those obtained by Mandim et al. [27], where quercetin was the most abundant compound, followed by kaempferol. This discordance could be partially related to the time and the location of the harvest, and/or the extraction method. *Erica scoparia* aerial parts presented a total amount of polyphenols of 9528.93 mg/kg. The most abundant compounds identified were myricetin-*O*-hexoside (2130.25 ± 0.78 mg/kg), myricetin-*O*-rhamnoside (1625.89 ± 0.39 mg/kg), and myricetin-*O*-pentoside (852.85 ± 1.97 mg/kg), whereas quercetin-*O*-(6"-p-hydroxybenzoyl)-hexoside (91.34 ± 1.22 mg/kg) was the least abundant one. Notably, myricetin-*O*-hexoside was shown to be the greatest phenolic compound in the leaves of *E. scoparia* (184.38 ± 0.26 mg/kg), while the smallest content was recorded for quercetin-*O*-(malonyl)-hexoside (18.52 ± 0.27 mg/kg). Thus, a remarkable discrepancy in the phenolic composition between the leaves and aerial parts of *E. scoparia* was observed. In addition, some phenolic compounds contained in the aerial parts seemed to be entirely absent in the leaves, such as taxifolin, digalloyl-quinic acid, and kaempferol.

A principal component analysis (PCA) alongside a heat map analysis were carried out on the phenolic compounds as variables to identify the connection between all the plant parts under observation (Figures 2 and 3). The PCA results presented two main components (F1 × F2) that determine 68.94%, whereas (F1 × F3) showed a contribution of 62.60%.


**Table 1.** Phenolic compounds detected in *C. vulgaris*, *E. multiflora*, and



#### *Molecules* **2022** , *27*, 3979


Nq: Not quantified.

**Figure 1.** Chromatographic profile of hydroalcoholic extracts from leaves and aerial parts of 3 different *Ericaceae* taxa at λ = 330 nm.

**Figure 2.** Heat map analysis of phenolic compounds (mean, N = 3) in leaves and aerial parts of 3 different *Ericaceae* taxa: *C. vulgaris* leaves (*Cv*-L), *E. scoparia* leaves (*Es*-L), *E. scoparia* aerial parts (*Es*-A), *E. multiflora* aerial parts (*Em*-A), *E. multiflora* leaves (*Em*-L).

**Figure 3.** The correlation between phenolic compounds (variables) and plant parts of *Ericaceae* taxa (observations) through PCA. (**A**) represents the first two factorials F1xF2. (**B**) represents the second two factorials F1xF3.

Both statistical analyses confirmed the presence of four different clusters: the first cluster regrouped both parts of *E. multiflora*, and the second and the third clusters were attributed to *E. scoparia* parts, while a completely distinguished fourth cluster was ascribed to *C. vulgaris* leaves. According to the principal components F1 and F2, the leaves of *E. scoparia* and *C. vulgaris* showed a false positive correlation, resulting in a unique cluster, whereas F1 and F3 led to the rejection of the previous correlation and the presence of two different clusters.

#### *2.2. Antioxidant and Cytotoxic Activities*

#### 2.2.1. Antioxidant Activity

The human body is constantly dealing with the formation of free radicals. When produced in excess, the latter trigger oxidative stress, causing serious tissue injuries. It is well known that many diseases are closely related to oxidative stress, mainly cancer and neurodegenerative disorders (Alzheimer's, Parkinson's, etc.). To cope with these health issues, plants provide a cheap and affordable source of natural antioxidants to prevent free radical-induced diseases, especially in countries with low incomes and limited healthcare resources [28]. Many primary antioxidant chemistry reactions can be grouped into the categories of hydrogen-atom transfer (HAT) and single-electron transfer (SET). The HAT mechanism occurs when an antioxidant compound scavenges free radicals by donating hydrogen atoms; the SET mechanism is based on the transfer of a single electron to reduce any compound, including metals, carbonyls, and free radicals [29,30]. It has been reported that, even if many antioxidant reactions are characterized as following either HAT or SET chemical processes, these reaction mechanisms can simultaneously occur [29,31,32].

Due to the complex nature of phytochemicals and their interactions, the importance of using various methods based on different mechanisms for a comprehensive study of the antioxidant properties of plant extracts has been argued. Therefore, the antioxidant activity of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was investigated by three different in vitro methods: in order to establish the primary antioxidant properties, the 1,1-diphenyl-1 picrylhydrazyl (DPPH) test, involving HAT and SET mechanisms, and the reducing power, a SET-based assay, were used. The secondary antioxidant properties were determined through the estimation of the ferrous ion (Fe2+) chelating activity.

The DPPH test is a rapid, simple, inexpensive, and widely used method to measure the free radical scavenging ability of pure compounds or phytocomplexes. Based on the results shown in Figure 4, all extracts, except for *Em*-A, demonstrated valuable radical scavenging activity, reaching approximately 90% of inhibition at the concentration of 0.5 mg/mL. Among the tested extracts, *Es*-A was the most active, as confirmed also by the lowest IC50 value (*p* < 0.001); at the concentration of 0.25 mg/mL, it showed activity higher than that of BHT, used as a standard drug, displaying radical scavenging activity superimposable to that of the standard (around 100%) at the concentrations of 1 and 2 mg/mL (Figure 4).

**Figure 4.** Free radical scavenging activity (DPPH test) of hydroalcoholic extracts from leaves and aerial parts of 3 different *Ericaceae* taxa: *C. vulgaris* leaves (*Cv*-L), *E. scoparia* leaves (*Es*-L), *E. scoparia* aerial parts (*Es*-A), *E. multiflora* leaves (*Em*-L), *E. multiflora* aerial parts (*Em*-A). Data are expressed as the mean ± SD of three independent experiments (*n* = 3) and were analyzed by one-way ANOVA followed by Dunnett's post-hoc test. \*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.05 vs. BHT.

Based on the IC50 values, the efficacy of the extracts and the standard decreases in the order *Es*-A > BHT > *Es*-L > *Em*-L > *Cv*-L > *Em*-A (Table 2); however, at 1 mg and 2 mg/mL, *Es*-L, *Em*-L and *Cv*-L exhibited radical scavenging activity close to that of BHT, while only *Em*-A reached about 80% of inhibition (Figure 4).


**Table 2.** Free radical scavenging activity (DPPH test), reducing power, and ferrous ion (Fe2+) chelating activity of hydroalcoholic extracts from leaves and aerial parts of 3 different *Ericaceae* taxa.

*C. vulgaris* leaves (*Cv*-L), *E. scoparia* leaves (*Es*-L), *E. scoparia* aerial parts (*Es*-A), *E. multiflora* leaves (*Em*-L), *E. multiflora* aerial parts (*Em*-A). NA: no activity. Data are expressed as the mean ± SD of three independent experiments (*n* = 3) and were analyzed by one-way ANOVA followed by Tukey–Kramer multiple comparisons test. a–e Different letters within the same column indicate significant differences between mean values (*p* < 0.001).

The reducing power reflects the ability to stop the radical chain reaction. In this assay, the presence of antioxidant compounds in the sample determines the reduction of Fe3+ to the ferrous form (Fe2+). As shown in Figure 5, all the extracts, except *Em*-A, displayed good reducing power, which was dose-dependent. Among the tested extracts, those of *E. scoparia* were the most active. In fact, at the concentration of 1 mg/mL, *Es*-A showed activity close to that of BHT; at 2 mg/mL, the reducing power of both *Es*-A and *Es*-L was higher than that of the standard. Based on the ASE/mL values, the efficacy of the extracts and the standard decreases in the order BHT > *Es*-A > *Es*-L > *Cv*-L > *Em*-L > *Em*-A (Table 2).

**Figure 5.** Reducing power of hydroalcoholic extracts from leaves and aerial parts of 3 different *Ericaceae* taxa evaluated by spectrophotometric detection of Fe3+-Fe2+ transformation method. *C. vulgaris* leaves (*Cv*-L), *E. scoparia* leaves (*Es*-L), *E. scoparia* aerial parts (*Es*-A), *E. multiflora* leaves (*Em*-L), *E. multiflora* aerial parts (*Em*-A). Data are expressed as the mean ± SD of three independent experiments (*n* = 3) and were analyzed by one-way ANOVA followed by Dunnett's post-hoc test. \*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, vs. BHT.

The Fe2+ chelating activity of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was estimated by monitoring the formation of the Fe2+-ferrozine complex. In this assay, only *Es*-A and *Em*-A displayed weak chelating properties, whereas all the other extracts were not active (Table 2).

From our findings, it is evident that all the extracts possess strong primary antioxidant properties; interestingly, that obtained from the aerial parts of *E. scoparia* is the most powerful. HPLC analysis revealed, for this extract, the highest content of flavonoid compounds, represented mainly by flavonols such as several myricetin glycosides, but also kaempferol, quercetin, and isorhamnetin glycosides. The flavonols, containing more hydroxyl groups (one to six OH groups), have a very strong ability to scavenge DPPH radicals and they are well-known, potent antioxidants. These compounds have a 3-hydroxyl group in the C-ring and *3 ,4* -dihydroxy groups (catechol structure) in the B-ring, but also possess the 2,3-double bond in conjugation with the 4-oxo function in the C-ring, which are the essential structural elements for potent radical scavenging activity [33].

*Erica scoparia* aerial part extract is rich in myricetin glycosides, which have been shown to possess strong primary antioxidant activity [34,35]. Thus, the best activity observed for *Es*-A could be correlated primarily to these compounds, but also to kaempferol, isorhamnetin, and quercetin glycosides.

#### 2.2.2. *Artemia salina* Lethality Bioassay

The toxicity of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was assessed by the *Artemia salina* lethality bioassay, extensively utilized as an alternative model for toxicity evaluation. This simple method offers numerous advantages, such as rapidity, low cost, continuous availability of cysts (eggs), and ease of maintenance under laboratory conditions [36]. It is a useful system for predicting the toxicity of plant extracts in order to consider their safety. The results of the bioassay showed that the median lethal concentration values were higher than 1000 μg/mL for all the tested extracts, thus indicating the lack of toxicity against brine shrimp larvae based on Clarkson's toxicity criterion [37].
