*2.3. Total Polyphenols and Flavonoids Contents in Origanum compactum Extract*

Total phenolic content for the ethanolic extract of *Origanum compactum* was 107.79 ± 5.39 mg GAE/g dm and flavonoid content was 14.98 ± 0.79 mg QE/g dm (Table 1). The total phenolic content recorded was good compared to the total phenolic content in other *Origanum* extracts. The hydro-methanolic extract of *Origanum vulgare* presented total phenolic content of 79–147 mg GAE/g DW [11]. Bower et al. cited total phenolic content of 430 μg of GAE/mg dm for methanolic extract of *Origanum vulgare* leaves [12]. A relatively low total phenolic content (38 mg GAE/200 mL) was reported for the infusion of the leaves and flowers from *Origanum microphyllum* [13].

**Table 1.** Total phenols content, flavonoids content and antioxidant activity of hydroethanolic extract from *Origanum compactum*.


#### *2.4. HPLC-PDA/ESI-MS Analysis*

The phenolic profile analysis was carried out by using high-performance liquid chromatography coupled to a photodiode array and electrospray ionization mass spectrometry (HPLC-PDA/ESI-MS) (Figure 1). As listed in Table 2, a total of sixteen phenolic compounds were detected in *Origanum* extract, according to standards, retention times, mass spectrometry and literature data. The compounds were assigned to phenolic acids (syringic acid, caffeic acid, lithospermic acid A isomer, salvianolic acid, rosmarinic acid and melitric acid) and to flavonoids (apigenin-6,8-di-C-glucoside, luteolin glucoside, luteolin glucuronide, diosmetin jaceosidin, apigenin and cirsilineol). In terms of quantification, peak No. 9, rosmarinic acid, turned out to be the most abundant one in the studied plant extract (48,128.62 mg/Kg extract).

**Figure 1.** Chromatographic profile of phenolic compounds in *Origanum compactum* extract (EtOH:H2O 7:3 *v*/*v*) acquired at 330 nm.


**Table 2.** Tentative characterization of phenolic compounds in (EtOH:H2O 7:3 *v*/*v*) extract of *Origanum compactum* by HPLC-PDA/MS.

As it has been already reported in previous studies, the primary classes of phenolic chemicals in oregano are phenolic acids and flavonoids [5]. The results achieved in this study are in agreement with such studies [5]. For phenolic acids, the majority of them were previously cited in *Origanum* composition. Syringic acid and caffeic acid were previously reported in plants of the Lamiaceae family, including *Oregano* [14], and caffeic acid plays an important role in the biochemistry of this family [23]. Lithospermic acid and caffeic acid were identified in 80% methanol extract of *Origanum vulgare* ssp. *Hirtum* [24]. Lithospermic acid A and B were isolated from the aerial parts of *Origanum. Vulgare* ssp. *Hirtum* by Koukoulitsa et al. [23,25,26]. Salvianolic acid was reported in *Origanum majorana* methanol extract [19], and several studies have reported rosmarinic acid in the *Origanum* phenolic composition [15,20,23,27–29]. Additionally, the obtained results showing the high presence of rosmarinic acid in *Origanum compactum* extract are similar to those reported by Boutahiri et al. for the same plant originating from another site in Morocco [29].

It has been reported that rosmarinic acid and derivatives appear to constitute the main phenolic acids in oregano [24]. This is applicable in our study. Rosmarinic acid derivatives combine one or more rosmarinic acids with additional aromatic groups, which include lithospermic acid, salvianolic acid and melitric acid [30].

Regarding melitric acid, it was cited in the phenolic profile of some plants such as *Satureja biflora* [31] and *Melissa officinalis* [30,32], and to our knowledge, no previous studies have reported it as an *Origanum* phenolic component.

Concerning flavonoids, apigenin and lutelin are among the most abundant individual flavonoids found in different extracts of oregano species [5,33]. Cirsilineol was identified in *sicilian oregano* from Italy by Tuttolomondo et al. [34], and diosmetin was cited in flavonoid components of *Origanum vulgare* [35,36]. Jaceosidin was cited in relation to other plants' phenolic composition; it was isolated from the ethanolic extract of *Centaurea nicaeensis* [37] and was identified as a major phenolic compound in *Artemisia argyi* [38].

#### *2.5. Antioxidant Activity*

2.5.1. Antioxidant Activity of Hydro-Ethanolic Fractions by Frap (Ferric Reducing Power Assay)

Ethanolic extract of *Origanum compactum* aerial parts (Figure 2) showed powerful antioxidant potential. The parameter EC50 (effective concentration), which corresponds to an absorbance equal to 0.5, was equal to 0.017 ± 0.00085 mg/mL (Table 1). For ascorbic acid tested under the same conditions, the EC50 was equal to 0.031 mg/mL. The antioxidant power of *Origanum compactum* extract was more powerful than ascorbic acid.

**Figure 2.** (**a**) Antioxidant activity of hydroethanolic extract of *Origanum compactum* by FRAP; (**b**) antioxidant activity of ascorbic acid by FRAP.

Lagouri et al. compared the ferric reducing antioxidant properties of the aqueous and methanol extracts of *Origanum dictamnus*; the obtained EC50 values were 0.028 and 0.038 mg/mL, respectively [39]. In another study, methanolic extract of *Origanum vulgare* showed the largest ferric reducing ability expressed by 1746.76 ± 45.11 μmol AAE/g extract [40].

The powerful ferric reducing power of *Origanum compactum* ethanol extract highlighted in this work is attributed to the phenolic contents of the extract, which are mainly represented by rosmarinic acid and its derivatives. It has been found that rosmarinic acid is the phenolic acid that provides the strongest antioxidant activity by ferric reducing power (FRAP) assay on hydromethanolic extract from *Origanum majorana* [41]. Similarly, Gonçalves et al. [10] partially attributed the high antioxidant capacity (FRAP) of the methanolic extract of *Origanum vulgare* to the large quantity of rosmarinic acid (23.53 mg/g of dry extract) [40].

#### 2.5.2. Total Antioxidant Capacity by Phosphomolybdate Method

The phosphomolybdate method is based on the ability of the extract to convert molybdenum molybdate ions MoO4 <sup>2</sup><sup>−</sup> into molybdenum molybdate ions MoO4 2+, and the consequent creation of a green phosphate/molybdenum (V) complex at acidic pH. The amount of ascorbic acid equivalents in one gram of dry extract (mg EAA/1 gE) was used to express the total antioxidant capability of the examined extract.

Total antioxidant capacity of *Origanum compactum* ethanol extract was found to be 470.905 mg EAA/1 gE (Table 1). According to a different study, the stem's methanolic extract and leaves' methanolic extract of Cuban oregano (*Plectranthus amboinicus*) showed important antioxidant potential, with TAC values of 634 μM AAE/g of extract and 782.56 μM AAE/g of extract, respectively [42]. Kolda¸s et al. noted that Kolda the antioxidant activities of water and methanol extracts of *Origanum vulgare* L. ssp. *Viride* were higher than those of ethyl acetate and hexane extracts in terms of phosphomolybdenum reduction potential [43]. Indeed, it has been reported that the antioxidant power of different *Origanum* extracts depends on the solvents used during their extraction, which has been correlated with the phenolic yield during the process [5].

The biological power of plant extracts is related to their content of polyphenols and flavonoids. Rosmarinic acid and its derivatives salvianolic acid and melitric acid are the main phenolic acids in *Origanum compactum* ethanol extract, while the main flavonoid is apigenin glucoside. The biological effects of rosmarinic acid and its derivatives, which include antioxidant, antibacterial, anti-inflammatory and anti-tumor actions, have been recently highlighted [30,44]. The antioxidant effect of rosmarinic acid is linked to its ability to scavenge free radicals, which increases membrane stability and protection against oxidative damage [45].
