The best antioxidant activity of myrtle extracts amongst two sets of sixteen and four plant extracts was also previously reported by [127,128], regardless the extraction solvent (hexane or methanol). β-Carotene-bleaching test was used for determining the antioxidant activity and the results were presented as antioxidant activity coefficients. In the set of sixteen samples, the antioxidant activity coefficients for hexane and methanol extracts obtained from fresh leaves were 641 and 260, respectively, whereas for dried leaves, the values were 12.2 and 51.5, respectively [127]. In both fresh and dried material, myrtle extracts were the most active, nevertheless in the second set of extracts [128], the highest antioxidant activity coefficient was observed for fresh extract of *Myrtus communis* (AAC = 635), whereas in dried material, *Thymus vulgaris* was the most active (antioxidant activity coefficient = 34). The good antioxidant activity of hexane extracts can be partly or wholy assigned to the presence of nonpolar phenolic compounds such as tocopherols. Demo et al. [129] detected and quantified α-tocopherol in hexane extracts of myrtle leaves (2.144%).

Mothana et al. [35] studied the antimicrobial, anticancer, and antioxidant activity of 32 Yemeni plants. Concerning the antixoxidant activity and within this set of samples, only six had high DPPH free radical scavenging activity: methanolic extracts of *Achillea biebersteinii*, *Chrozophora oblongifolia*, *Myrtus communis*, *Oxalis corniculata*, *Phragmanthera regularis,* and *Tecoma stans* at 50 μg/mL. In contrast, Özcan et al. [130] did not find similar results for Turkish myrtle, although the methods (ABTS radical scavenging activity and capacity for oxidizing ferrous ion to ferric ion by various types of peroxides within the plasma) and the species used were different (anise, bitter fennel fruits and flowers, basil, laurel, oregano, and pickling herb). The peroxide value and the Trolox equivalent of methanolic extracts were 0.6866 μmol H2O2 and 0.3189 Trolox equivalent/g, respectively, although presenting higher amounts of total phenols (9.9761 mg gallic acid equivalent/g) than the other species (1.3175–10.5832 mg gallic acid equivalent/g). Only oregano extracts presented higher concentration of total phenols than myrtle leaves. The authors [130] also evaluated the antioxidant activity of the essential oils. These ones had better capacity for reducing ferrous ions, since they showed higher peroxide values, than the methanolic extracts, nevertheless poorer capacity for scavenging the ABTS free radicals.

Gião et al. [131] determined the antiradical activity (ABTS free radicals) of aqueous extracts of 32 plants from Portugal. Two types of extraction were used: boiling water added to the sample and left during 5 min at rom temperature (infusion), and water added to the sample and the mixture heated until boiling, which was maintained for 5 min. The authors detected that the antiradicalar activity was dependent on the species as well as of the method of extraction. The highest antioxidant activity was observed for avocado (0.157 mg equivalent ascorbic acid/g), followed by agrimony (0.067 g equivalent ascorbic acid/g), eucalyptus (0.149 g equivalent ascorbic acid/g), yarrow (0.118 g equivalent ascorbic acid/g), myrtle (0.141 g equivalent ascorbic acid/g), thyme (0.142 g equivalent ascorbic acid/g), and heath (0.065 g equivalent ascorbic acid/g). In addition, powder infusion was the best method for obtaining the most active extracts which also possessed the highest amounts of phenols. The authors found a positive correlation between the phenol content and the antiradicalr activity [131]. Later on, Gião et al. [132] studied the effect of different stages of processing (fresh, frozen, dehumidified/packed in two consecutive years, and storage after dehumidification under controlled relative humidity, maintained for one year in a dark room) on the antiradicalar activity of ten species (agrimony (*Agrimonia eupatoria*), eucalyptus (*Eucalyptus globulus*), walnut-tree (*Juglans regia*), myrtle (*Myrtus communis*), raspberry (*Rubus idaeus*), sage (*Salvia* sp.), savory (*Satureja montana*), sweet-amber (*Hypericum androsaemum*), thyme (*Thymus vulgaris*), and yarrow (*Achillea millefolium*)). The samples used were infusions and the antiradicalar method used was based on the abilty for scavenging ABTS free radicals. According to the authors [132], antioxidant activity and total phenolic content decreased by ca. 30–80%, between fresh and frozen forms, whereas from the frozen stage to the packaged form the variations observed were not statistically significant. The highest difference was observed for myrtle, which means that this species is sensitive to the technological processing and, therefore, for preserving their properties during processing conditions after harvest and throughout storage, other techniques must be thought and assayed in the near future.

Gonçalves et al. [133] compared the antioxidant activity of ten plant species from Portugal, including *M. communis*, measured through diverse methods (DPPH and hydroxyl radical scavenging activity, reducing and chelatin power, and inhibition of lipid peroxidation in mouse brain homogenates using thiobarbituric acid reactive substances). The extracts obtained were aqueous obtained by maceration at room temperature, for 2 h, or extraction in hot water (90 ◦C), for 5 min. *Pistacia lentiscus* L. and *M. communis* in cold and hot aqueous extracts were the most effective for scavenging the DPPH free radicals (377.30 and 319.81 mmol Trolox equivalent/g and 230.36 and 246.51 mmol Trolox equivalent/g, respectively). The same extracts were also the best ones for chelating iron ions without significant differences between the hot and cold extracts. However, the capacity for scavenging hydroxyl radicals was better in hot extracts, nevertheless never exceeding 50%, even at higher extract concentration (1.6 mg/mL extract). With the exception of this activity, the authors found a positive correlation between the total phenolic content and the antioxidant activity [133]. Concerning the capacity for preventing lipid peroxidation, all samples had activity, although those of *Centaurea erythraea*, *Paronychia argentea*, and *Ruscus aculeatus* were significantly less active than the other aqueous extracts.

#### *3.3. Effect of Extraction Method and Extraction Solvent on the Antioxidant Activity of Myrtle Leaves*

The antiradicalar activity of leaf extracts measured through the capacity for scavenging DPPH as well as the total antioxidant power were evaluated by Belmimoun et al. [134]. The authors used diverse methods and extraction solvents: decoction, maceration with ethanol, and extraction with solvents of increasing polarity by Soxhlet (dichloromethane and methanol). The IC50 value for the aqueous extract was 29 μg/mL, whereas the total antioxidant power was 68.05 mg/g, better when compared to the essential oils obtained by hydrodistillation (615 μg/mL and 36 mg/g, respectively), explained by the absence of phenol compounds in the essential oils [134].

Romani et al. [135] also evaluated the influence of different solvents on the antioxidant activity of leaf myrtle extracts obtained by liquid–liquid extraction. The authors also evaluated the role of pure compounds and group of compounds found in the myrtle extracts on the antioxidant activity found in the work. As expected, different solvents extracted diverse phenolic compounds. Hydroalcoholic extracts had galloylglucosides, ellagitannins, galloyl-quinic acids, and flavonol glycosides; whereas ethyl acetate extract and aqueous residues after liquid–liquid extraction were enriched in flavonol glycosides and hydrolysable tannins (galloyl-glucosides, ellagitannins, and galloyl-quinic acids), respectively (Table 3). The antioxidant activity of extracts was determined evaluating the capacity of extracts to prevent the formation of MDA and conjugated dienes after exposing human LDL to copper ions. Hydroalcoholic extract was mainly constituted by galloyl-glucosides and ellagitannins and was the most active in inhibiting LDL oxidation (IC50 = 0.36 μM) followed by the aqueous residue after liquid–liquid extraction (IC50 = 2.88 μM), also mainly constituted by galloyl-glucosides and ellagitannins, and ethyl acetate extract (IC50 = 2.27 μM), mainly constituted by myricetin glucosides and galloylquinic acids. The capacity for preventing MDA accumulation was also determined using pure compounds such as gallic acid, 3,5-di-*O*-galloylquinic acid, myricitrin, and rutin, and the IC50 values found were 20, 2.2,7.8, and 3.7 μM, respectively, showing that 3,5-di-*O*-galloylquinic acid was the most active. In addition, the authors [135] also determined the IC50 values considering the total polyphenol concentration and the concentration of the single compound present in each extract and it was possible to find that the copresence of different polyphenols increased the antioxidant activity. Two examples are the aqueous residue in which was practically constituted by galloyl-glucosides and had a IC50 value close to that of 3,5-di-*O*-galloylquinic acid, and the other example is that of the ethyl acetate extract that had a ratio between galloyl derivatives and flavonols of about 1:1, and the IC50 value is very similar to that of 3,5-di-*O*-galloylquinic acid. This last result indicates that flavonols do not play an important role in the antioxidant activity when they are mixed with hydrolysable tannins, but a ratio of 9:1, such as observed in the hydroalcoholic extract, the IC50 decreased drastically, that is, the activity increased. The authors [135] concluded that the antioxidant activity was dependent on the ratio between the sum of galloylglucosides, ellagitannins, and flavonols and also of the ratio between these galloyl derivatives vs. galloyl-quinic acids.

The effect of different solvents (water, hexane, chloroform, ethyl acetate, methanol, and a total flavonoids oligomer fraction) and essential oils on the capacity for scavenging DPPH free radicals revealed that the aqueous extract was the most active (IC50 = 1.9 μg/mL), even better than the total flavonoids oligomer fraction (IC50 = 3 μg/mL). Chloroform, hexane extracts, and essential oils were significantly less active than those extracts [66]. Later on, Hayder et al. [64] evaluated the capacity of myricetin-3-*O*-galactoside and myricetin-3-*O*-rhamnoside (flavonoids), isolated from the leaves of *Myrtus communis*, to inhibit xanthine oxidase activity, lipid peroxidation, and to scavenge the free radical DPPH. Both flavonoids were able to scavenge the free radicals with IC50 values of 1.4 μg/mL and 3.1 μg/mL for myricetin-3-*O*-rhamnoside and myricetin-3-*O*-galactoside, respectively, comparable than to that of the positive control (vitamin E) (IC50 = 3 μg/mL). Both flavonoids were able to inhibit xanthine oxidase (in the catalysis process there is production of superoxide radical anions). At 100 μg/mL, myricetin-3-*O*-rhamnoside and myricetin-3-*O*-galactoside showed percentages of inhibitory activities of 59% and 57%, respectively. However, when higher concentrations of myricetin-3-*O*-rhamnoside (300 μg/mL) and myricetin-3-*O*-galactoside (200 and 300 μg/mL) were used, there was an increase of xanthine oxidase activity, that is, a pro-oxidant activity. The inhibition of the malondialdehyde formation by K562 (human chronic myelogenous leukemia) cell line, induced by hydrogen peroxide, was also assayed, and the authors [64] found that the IC50 values for myricetin-3-*O*-galactoside and myricetin-3-*O*-rhamnoside were 160 and 220 μg/mL, respectively, measured through the thiobarbituric acid test. These concentrations did not involve a decrease of cell viability.

The evaluation of antioxidant activity of pure compounds isolated from myrtle leaves was also reported previously [65]. In this case, the compound studied was 3,5-*O*-digalloylquinic acid and the antioxidant activity was determined by its ability for inhibiting lipid peroxidation induced by hydrogen peroxide in the K562 cell line. The pure molecule displayed an important malondialdehyde formation inhibition percentage (82.2%) and low IC50 value = 180 μg/mL. This concentration did not induce a decrease of cell viability whereby the decrease of malondialdehyde amounts can only be attributed to the real antioxidant activity of 3,5-*O*-digalloylquinic acid [65] as reported for myricetin-3-*O*-galactoside and myricetin-3-*O*-rhamnoside [64].

Tumen et al. [136] studied the effect of dichloromethane, acetone, ethyl acetate, and methanol extracts of myrtle leaf and berries on the ability for scavenging DPPH and *N*,*N*-dimethyl*p*-phenylenediamine (DMPD) radicals, reducing power, and chelating activity. The authors reported that the polar extracts (acetone, ethyl acetate, and methanol extracts) exerted strong scavenging effect against DPPH and DMPD as well as good reducing power. However, the dichloromethane extract of the berries possessed the best metal chelation ability. Berry extracts were better antioxidants than leaf extracts, maybe to the highest concentrations of phenols in those extracts, according to the Tumen et al. [136].

Yoshimura et al. [137], using different solvents in the extraction of phenols from leaves of myrle from Japan, isolated, identified, and determined the capacity of every single compound for scavenging the DPPH free radicals. Among the compounds evaluated, the hydrolyzable tannins oenothein B, eugeniflorin D2, tellimagrandin I, and tellimagrandin II exhibited the best activity (IC50 = 6.12, 4.56, 8.00, and 7.62 μM, respectively).

Previously, Dairi et al. [138] studied the scavenging capacity of the ABTS and peroxyl radicals by Algerian myrtle extracts obtained by microwave assisted extraction and maceration. In addition, the authors also evaluated the antioxidant activity of leaf extracts in lipid system models oxidized *in vitro*: human LDL Cu2+-oxidation and AAPH-induced l-*α* phosphatidylcholine aqueous dispersion oxidation. The results showed that there were not differences in the amounts of phenols obtained by both methods and they presented strong ability for scavenging the ABTS free radicals, even better than BHA and α-tocopherol. The same extracts also exhibited higher capacity for scavenging the peroxyl free radicals than BHA but less effective activity than the references caffeic acid and myricetin 3-*O*-rhamnoside. In the lipid system (Cu2+-induced LDL system), both myrtle extracts, as well as myricetin 3-*O*-rhamnoside, were able to inhibit the production of conjugated dienes in a dose-dependent manner and to prolong the lag phase. When the AAPH-induced l-*α* phosphatidylcholine aqueous dispersion was used, both myrtle extracts were effective to prevent lipid oxidation, but less than myricetin 3-*O*-rhamnoside. The possible synergic effect of myrtle extracts, caffeic acid, and myricetin 3-*O*-rhamnoside on α-tocopherol-enriched phospholipid aqueous dispersions was also evaluated by the authors and they observed that no synergic or additive effect was observed between *α*-tocopherol and myrtle extracts or caffeic acid, but myricetin 3-*O*-rhamnoside had an additive effect. According to the authors [138], myrtle extracts, in which myricetin 3-*O*-rhamnoside can be found, can improve the antioxidant activity of complex lipid systems, stabilizing them [138].

The absence of significant differences in the amounts of total phenols in the extracts obtained by microwave-assisted extraction and maceration, reported by Dairi et al. [138], such was also verified by Dahmoune et al. [139] when compared the total phenol amounts of leaf myrtle extracts obtained by microwave-assisted extraction (162.49 mg gallic acid equivalent/g), ultrasound-assisted extraction (147.77 mg gallic acid equivalent/g), and maceration (128.00 mg gallic acid equivalent/g). However, the amounts of total flavonoids and tannins observed in the extracts were different depending on the extraction procedures. Microwave-assisted extraction was able to extract more flavonoids (5.02 mg quercetin equivalent/g) and tannins (32.65 mg quercetin equivalent/g) than ultrasound-assisted extraction (3.88 mg quercetin equivalent/g and 23.32 mg/g, respectively) and maceration (4.15 mg quercetin equivalent/g and 17.15 mg/g, respectively). The capacity for scavenging ABTS (IC50 = 38.20 mg gallic acid equivalent/mL), DPPH (IC50 = 16.80 mg gallic acid equivalent/mL), and peroxyl radicals (757.77 μmol Trolox equivalent/g) was also more effective when myrtle extracts were obtained by microwave-assisted extraction, than by other extraction processes. However, optimal microwave-assisted extraction conditions were needed in order to achieve these values of phenols and antioxidant activity. Such conditions were 42% ethanol concentration, 500 W microwave power, 62 s irradiation time, and 32 mL/g solvent to material ratio. Microwave-assisted extraction of

myrtle leaf allowed shortening the extraction time about 14 and 15 times when compared to the ultrasound-assisted extraction and maceration, respectively [139].

According to Pereira et al. [140], supercritical fluid extraction does not present disadvantages for extracting natural products, on the contrary, it can present the advantage to make selective extractions by varying pressure and temperature. Taking into account these premises, the authors used the response surface methodology to optimize the supercritical carbon dioxide fluid extraction conditions to obtain myrtle leaf extracts. The optimal conditions obtained were: 23 MPa, 45 ◦C, and CO2 flow rate of 0.3 kg/h agreeing to those predicted by the response surface methodology model. The capacity for scavenging ABTS was inferior when compared to the ethanolic extracts of the same plant. Keeping those parameters constant, the authors [140] used ethanol as cosolvent and at different percentages to know if the increase of polarity of solvent could improve the antioxidant activity. The authors observed that the increase of ethanol content increased the antioxidant activity that could be by around 4 and 5 times for scavenging ABTS and peroxyl radicals, respectively, when the flow rate of ethanol was 0.09 kg/h.

As leaves and branches of myrtle are frequently consumed as an infusion and decoction, Messaoud et al. [141] studied the chemical composition, volatiles and phenols, and the antibacterial and antioxidant activities of leaf infusions prepared during three different times (5, 10, and 15 min). The phenolic compounds and their amounts found during those periods are depicted in Table 3. Phenolic acids and flavonol glycosides were the major group of infusions (7.64 to 14.28 μmol/g and 7.05 to 12.11 μmol/g, respectively), which variations depended on the time of heating (Table 3). Longer heating periods (15 min), higher was the amounts of phenols found in the myrtle infusion. The antioxidant activity was measured using four in vitro methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) method, *β*-carotene bleaching test, chelating effect on ferrous ions, and ferric reducing power method. The results showed that the heating time influenced the antioxidant activity of myrtle infusions, independent on the method used. For DPPH method, the IC50 values found were: 356.14, 283.71, and 282.53 μg/mL in infusion samples after 5, 10, and 15 min of heating, respectively. For *β*-carotene bleaching test, the IC50 values found were: 247.91, 138.43, and 84.88 μg/mL in infusion samples after 5, 10, and 15 min of heating, respectively. For chelating effect on ferrous ions, the IC50 values found were: 223.56, 215.86, and 206.44 μg/mL in infusion samples after 5, 10, and 15 min of heating, respectively. For the ferric reducing power method, the values found were: 31.26, 35.81, and 38.93 mmol Fe2+/mL in infusion samples after 5, 10, and 15 min of heating, respectively. As observed for phenols, the antioxidant capacity of infusions increases as heating time also increases. Such results suggest that phenolic compounds are responsible for the activities found. In fact, the authors [141] found a linear correlation between the phenols content and DPPH radical scavenging activity (*r*<sup>2</sup> = 0.709), *β*-carotene bleaching test (*r*<sup>2</sup> = 0.831), and ferric reducing power method (*r*<sup>2</sup> = 0.858).

#### *3.4. Acylphloroglucinols on the Antioxidant Activity of Myrtle Leaves*

Generally, the antioxidant leaf extracts of myrtle are assigned to phenols, particularly hydrolizable tannins and flavonoids, nevertheless the acylphloroglucinols semimyrtucommulone and myrtucommulone A are also described as antioxidants. Rosa et al. [142] reported that these acylphloroglucinols had antioxidant activity because they were able to prevent the thermal (140 ◦C), solvent-free oxidation of cholesterol. Myrtucommulone A at 5 nmol and 10 nmol exerted a complete inhibition of cholesterol degradation after 1 h or 2 h, showing a 90% protection at 2.5 nmol and 50% protection at 5 nmol at 1 and 2 h, respectively. Semimyrtucommulone was less active than myrtucommulone A. A complete inhibition of the oxidative process of cholesterol was observed from 5 nmol at 1 h and 20 nmol at 2 h, showing a 70% protection at 10 nmol at 2 h [142]. The lipid peroxidation was determined having LDL as lipid substrate and the oxidation was induced by Cu2+. The results showed that both acylphloroglucinols preserved LDL from oxidative damage. In addition, a protective effect on the reduction of polyunsaturated fatty acids and cholesterol was also observed

by inhibiting the increase of their oxidative products (conjugated dienes fatty acids hydroperoxides, 7β-hydroxycholesterol, and 7-ketocholesterol). According to these results, semimyrtucommulone and myrtucommulone A can be seen as dietary antioxidants with antiatherogenicity.

From the myrtle leaves, it was possible to isolate and identify myrtucommuacetalone, myrtucommulone M, myricetin, isousnic acid, growth regulator G3 factor, and myrtucommulone E. Myricetin was able to inhibit reactive oxygen species production on zymosan-stimulated whole blood phagocytes (IC50 = 1.6 μg/mL), in a dose-dependent manner. The J774.2 cells treated with phorbol 12-myristate 13-acetate (stimulant used to distinguish the activity of the oxidative burst from zymosan activation that is involved in phagocytosis) alone (positive control) or in combination with myrtucommuacetalone and myricetin for 30 min, showed that these compounds were able to inhibit the production of reactive oxygen species. Myricetin was able to inhibit the production of these reactive species in both stimulant processes, suggesting that this flavonoid is able to inhibit reactive oxygen species, mainly superoxide, by a myeloperoxidase independent pathway [143]. Myrtucommuacetalone and growth regulator G3 factor also inhibited the production of nitric oxide in mouse macrophages (82.3% and 59.36%, respectively), at 25 μg/mL concentration [143].

#### *3.5. Complexity of Myrtle Extracts on the Antioxidant Activity*

As aforementioned, several methods have been used for evaluating the antiradicalar activity and distinct results were obtained due to diverse factors. The substrates were complex mixtures that could also contribute for the diversity of results. Sanna et al. [144] compared two spectroscopic methods (ultraviolet-visible spectroscopy and electron paramagnetic resonance) on the antioxidant ability of myrtle leaf exctracts. In both cases, DPPH free radicals were used for evaluating the antiradicalar activity of extracts, in which the depletion of DPPH in the presence of an antoxidant is measured. Since the samples used in both assays were the same, all differences in the results could only be attributed to the method itself [144]. The results showed that for ultraviolet-visible spectroscopy method there was not proportionality between the extract concentration and absorbance, because for increasing extract concentrations, the colour changed from dark purple (DPPH solution colour) to dark brown, although the extract had been depleted all the DPPH present in the solution. However, the decrease in DPPH signal intensities measured by electron paramagnetic resonance was concentration dependent. The authors concluded that the estimation of radical scavenging ability performed by electron paramagnetic resonance is more trustworthy than ultraviolet-visible spectroscopy measurements. Though this conclusion, the utilization of the ultraviolet-visible spectroscopy for DPPH method is still largely used.

The complexity of sample matrix makes difficult to assign the biological activity to a compound or to a set of compounds. However, Romani et al. [145] were able to assign the antiradical activity of myrtle leaf, from Italy, to gallotannins which were predominant in myrtle extracts as well as in the commercial extract of chestnut bark used as reference. However the amounts of gallotannins varied significantly from one harvesting year to another harvesting year (Table 3), which makes difficult to have a final product with identical magnitude of activity. The establishement of standardized extracts is required to prevent such variability. The chemical composition of tannin aqueous and hydro-alcoholic extracts of myrtle leaf (Table 3) was evaluated by HPLC/DAD/ESI-MS methods.

#### *3.6. Antioxidant Activity of the Leaf Foods*

The capacity of myrtle extracts to retard food oxidation was evaluated by Turhan et al. [146] when the brining process of anchovies with sodium chloride (15 g/100 mL) was done with myrtle, rosemary and nettle extracts and stored at 4 ◦C, for 28 days. The lipid oxidation was followed by determining the peroxide value, thiobarbituric acid reactive substance (TBARS), and oxidative rancidity score. Myrtle and rosemary extracts were the most effective in slowing down the lipid oxidation because they decreased the peroxide value from 37.77 meq O2/kg, in the control, to 11.48 meq O2/kg, in rosemary extract; this value is not significantly different from the myrtle extract. These values were found after

28 days of storage. These extracts were also able to decrease the TBARS values from 1.89 mg MDA/kg (control) to 0.59 mg MDA/kg and 0.50 mg MDA/kg, in the case of brined anchovies with myrtle and rosemary extracts, after 28 days of storage. These values can be attributed to the highest amounts of total phenols in the myrtle (72.4 mg/g) and rosemary (52.6 mg/g) extracts, when compared to the lowest amounts of phenols in the nettle extract (7.2 mg/g). The capacity for scavenging the DPPH free radicals and the reducing power were also better for those extracts, which may explain the best capacity of myrtle and rosemary extracts for retarding the lipid oxidation of brined anchovies when stored during 28 days [146].

The application of myrtle extracts in food to prevent oxidation was also evaluated by Dairi et al. [147]. The nutritional quality of extra virgin olive oil can be lost when lipids oxidation occur, by losing its phenolic compounds, particularly during heating procedures. Due to the antioxidant properties of myrtle extracts, Dairi et al. [147] studied the effect of myrtle extract, obtained by two different methods (microwave-assisted extraction and maceration) on the preservation of nutritional quality of extra virgin olive oil, particularly of the phenolic compounds after heating processes (butane-air flame, oven and microwave). The evolution of the phenolic compounds content was monitoring by reversed phase dispersive liquid–liquid microextraction (RP-DLLME)-HPLCDAD-FLD method. The results showed that the addition of myrtle extracts not only preserved the endogenous phenolic compound of extra virgin olive oil (hydroxytyrosol, tyrosol, luteolin, apigenin, and secoiridoid 1) when compared with the control, in which myrtle extract was not added, but also reduced the specific extinction coefficient (K232) values. This parameter checks the degree of a vegetal oil oxidation, being indicative of the formation of primary products of oxidation. However, these benefits induced by myrtle extract, mainly constituted by galloyl quinic acid, gallic acid, and myricitrin, were dependent on the type of heating of olive oil. In the phenol preservation, the most protective effect of the myrtle extract was found during flame and microwave heating, whereas in the prevention of primary oxidation products the most effective one occurred when sample oils were submitted to the flame heating. The myrtle extract did not exert any beneficial effect on the prevention of the formation of secondary products of oxidation, that is, it did not reduce the specific extinction coefficient (K270) [147]. According to the authors, myrtle extracts may be a tool for improving the oxidative stability of olive oil, by improving its phenol composition.

After knowing that the enriched olive oil with myrtle extract prevented its oxidation [147], later on, Dairi et al. [148] wanted to know if the enriched oil would have better antioxidant properties acting against free radical attacks that can occur during lipid digestion. To reach the objectives, the authors studied the effect of myrtle extract, obtained by two different methods (microwave-assisted extraction and maceration), on egg yolk phosphatidylcholine/bile salts aqueous dispersion oxidation under simulated intestinal conditions (pH 7.4), that is, a model that would permit to know if such extracts could prevent lipid peroxidation that might occur in small intestine during lipids digestion. AAPH (2,2 -azobis (2-aminopropane) dihydrochloride) or a Fe3+/ascorbic acid system were used to initialize the phospholipid peroxidation. In addition, the capacity for preventing DPPH and peroxyl radicals (ORAC) was also checked by the authors [148]. The phenolic composition of myrtle extracts is presented in Table 3, and the chemical composition was not greatly different in both extraction procedures. The extra virgin olive oil enriched with myrtle extract increased the neutralization of DPPH and peroxyl radicals, even better than the references α-tocopherol and butylated hydroxytoluene (BHT). When the lipidic model was used, the phospholipid stability increased by a factor of 33.6% and 34.8%, for myrtle microwave assisted extraction and maceration extraction when compared to the control (without myrtle extraction), when the lipid induction was performed with the Fe3+/ascorbic acid system. However, when the induction was made with AAPH, the effect was very poor. This work allowed the authors to conclude that the capacity of extra virgin olive oil enriched with myrtle extract to inhibit phospholipid peroxidation under simulated intestinal conditions can be seen as a potential functional food [148].

The antioxidant activity of sea salts flavoured with Mediterranean herbs (myrtle, rosemary, and mixtures) was evaluated by Rosa et al. [122] in chemical models of lipid peroxidation and in cell cultures. Simultaneously, the authors compared the antioxidant activity of these samples with those of normal salt. The flavoured myrtle added to salt was constituted by a mixture of extract of myrtle berry juice, leaves, and myrtle essential oil. These flavoured salts preserved liposomes from Cu2+-induced oxidation, decreasing the accumulation of malondialdehyde, by scavenging peroxyl radicals or chelating Cu2+ at the aqueous phase or at the liposome particle surface/core [122]. The methanolic extracts of flavoured salts also significantly reduced the reactive oxygen species generation in *tert*-butylhydroperoxide-induced intracellular Caco-2 cells. According to the authors this ability was correlated to the capacity of extract components to permeate cell membrane and scavenge reactive oxygen species inside cells [122].

Liposomes, resembling cell membranes, are lipid molecules that can encapsulate biologically both hydrophilic and lipophilic active substances or used as lipid substrate for evaluating the capacity of samples to preventing lipid peroxidation [54]. These authors evaluated the antioxidant ability of methanolic extracts obtained from the aerial parts of Greek myrtle before and after encapsulation. The activity was followed by three methods (Rancimat method, oxidative stability by DSC, and formation of malondialdehyde). The two first methods are based on the generation of volatiles and thermal release, respectively, indicating a terminal oxidation process, whereas the generation of malondialdehyde occurs at lower temperatures and at a different stage of oxidation. The encapsulation of the extract, for the same concentration, enhanced the antioxidant action more than the same extract in pure form [54]. Therefore, the encapsulation altered the activity of extract, improving it.

#### *3.7. Anti-Inflammatory Activity of Leaf Extracts*

Zaidi et al. [149] studied the effect of 24 selected Pakistani medicinal plants, including *M. communis*, which are traditionally prescribed for gastro-intestinal disorders. *Helicobacter pylori* infection is associated with gastritis, peptic ulcer, and gastric cancer. In these disorders oxidative stress is many times involved and, consequently, inflammatory processes. For this reason, the authors [149] evaluated the effect of plant extracts, such as aqueous myrtle extract, on the inhibition of secretion of IL-8 and inhibition or prevention of generation of reactive oxygen species (ROS) in clinically isolated *Helicobacter pylori* strain (193)-infected cells (human gastric cancer cell line AGS), in order to confirm the anti-inflammatory and cytoprotective effects in gastric epithelial cells attributed to those 24 plants. The authors observed that only four extracts (*Cinnamomum cassia*, *Myrtus communis*, *Syzygium aromaticum*, and *Terminalia chebula*) manifestly inhibited IL-8 secretion at both 50 and 100 μg/mL. In addition, only *Achillea millefolium*, *Berberis aristata*, *Coriandrum sativum*, *Foeniculum vulgare*, *Matricaria chamomilla*, and *Prunus domestica* were able to significantly suppression ROS generation (particularly superoxide anion radicals) from *Helicobacter pylori*-infected cells [149]. The ROS measurement generated was done by detecting the fluorescence emission, by flow cytometry, caused by the intercalation of oxidized hydroethidine, by superoxide, into DNA [59]. The chemical composition of the extracts was not determined by the authors and the results obtained were explained according to previous results. As so, they attributed the property for inhibiting IL-8 by myrtle extracts to the possible presence of myrtucommulone in the extract.

The anti-inflammatory activity of the aqueous and ethanolic extracts obtained from the aerial parts of *M. communis* was evaluated by Hosseinzadeh et al. [91] using xylene-induced ear oedema and a cotton pellet test, in mice. Antinociceptive activity was also performed using hot plate and writhing tests in mice. The ethanolic (0.05 g/kg) and aqueous extracts (0.005, 0.015, and 0.03 g/kg) demonstrated anti-inflammatory effects against chronic inflammation, whereas in the acute inflammatory activity (xylene-induced ear oedema study), the aqueous extract at doses 0.1, 0.2, and 0.03 g/kg showed significant anti-inflammatory activity. The ethanolic extract also had activity against acute inflammation in all doses (0.05, 0.15, and 0.35 g/kg), but was not dose-dependent. The aqueous and ethanolic extracts of the aerial parts of myrtle exhibited antinociceptive activity. The authors suggested that this activity might be mediated by opioid receptors [91]. The chemical composition of the extracts was not performed but the authors attributed the antinociceptive and anti-inflammatory activities to flavonoids and/or tannins, according to the references consulted by them. The highest concentrations of extracts with anti-inflammatory and antinociceptive activities were lower than the LD50 (lethal dose 50) values found for aqueous and ethanolic extracts (0.473 and 0.79 g/kg, respectively) [91].

Generally, the antioxidant and anti-inflammatory activities of myrtle extracts have been attributed to the phenolic compounds. The higher activity of leaf extracts than berry extracts were attributed by some authors to the presence of hydrolysable tannins in leaf extracts at higher concentrations than in berry extracts, which means the weak influence of anthocyanins, present in coloured berries, in the antioxidant activity [103,135]. However, Feisst et al. [150] reported, for the first time, that two nonprenylated acylphloroglucinols, myrtucommulone and semimyrtucommulone, isolated from leaf extracts of myrtle, potently suppressed the biosynthesis of eicosanoids by inhibiting cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LOX) *in vitro* and *in vivo* at IC50 values ranging from 1.8 to 29 μM. These enzymes are involved in the formation of the proinflammatory prostaglandins and leukotriens, respectively. At the same time, the authors showed that myrtucommulone and semimyrtucommulone were able to prevent the mobilization of Ca2+ in polymorphonuclear leukocytes at IC50 = 0.55 μM and 4.5 μM, respectively, mediated by G protein signaling pathways. This effect inhibited the generation of reactive oxygen species (peroxide) and the release of elastase at similar concentrations. However, the phenolic part of those acylphloroglucinols (isobutyrophenone) was much less effective or even not active [150]. However, the acylphloroglucinols only inhibited partially peroxide formation or failed to inhibit elastase release when ionomycin was added to polymorphonuclear leukocytes. According to these results, the authors suggest that the suppression of Ca2+ mobilization by the myrtle acylphloroglucinols is the main cause for the inhibition of peroxide formation and elastase release induced by fMLP (*N*-formylmethionyl-leucyl-phenilalanine), which is confirmed when ionomycin is added to the system that needs much higher concentrations of the acylphloroglucinols to produce biological effect. Ionomycin did not use the G protein signaling pathways for elevating internal Ca2+ [150].

The inhibition effect of myrtucommulone on COX-1 in human platelets, and 5-LOX in intact polymorphonuclear leukocytes, observed by Feisst et al. [150] led Koeberle et al. [151] to ascertain if this acylphloroglucinol was able to inhibit selectively prostaglandin E2 (PGE2) via microsomal PGE2 synthase (mPGES)-1. For this purpose, the authors measured the effect of myrtucommulone in diverse systems (cell-free assay using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1; intact A549 cells, and lipopolysaccharide stimulated human whole blood). The results observed by the authors were that myrtucommulone was able to inhibit the conversion of PGH2 to PGE2 (IC50 = 1 mmol/L) in the cell-free mPGES-1 system. In addition, the levels of PGE2 also diminished in intact A549 cells and in human whole blood at low micromolar concentrations, nevertheless the inhibition of COX-2 by myrtucommulone in A549 cells or isolated human recombinant COX-2 was only observed for higher concentrations (>30 mmol/L). Concerning COX-1 inhibition, the authors observed IC50 > 15 mmol/L values, both in cellular or cell-free systems, that is, only presenting moderate activity. According to these results the anti-inflammatory activity of myrtucommulone is due to the suppression of PGE2 formation and not so much to the inhibition of the COX enzymes [151].

The anti-inflammatory activity of myrtucommulone isolated from myrtle leaves was evaluated *in vivo* by Rossi et al. [152]. In this study, the authors induced inflammation in mice by the subplantar and intrapleural injection of carrageenan, respectively, that triggers the development paw oedema and pleurisy. The action of myrtucommulone was followed by administering the compound intraperitoneally. Myrtucommulone, at concentrations 0.5, 1.5, and 4.5 mg/kg i.p., reduced the development of mouse paw oedema and, at 4.5 mg/kg i.p., 30 min before and after carrageenan, showed anti-inflammatory activity in the pleurisy model. The mechanism involved in

the anti-inflammatory activity of myrtucommulone was determined by the authors and they observed that carrageenan injection in the pleurisy test reduced the exudate volume and leukocyte number, myeloperoxidase activity, the lung intercellular adhesion molecule-1 and P-selectin, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), leukotriene B4 (LTB4), lung peroxidation (thiobarbituric acid-reactant substance), and nitrotyrosine and poly (ADP-ribose). Such results permitted to the authors to suggest that the mechanisms involved in the protection effect of myrtucommulone in lung injury include suppression of adhesion molecules, inhibition of LTB4 generation and neutrophil infiltration [152].

The anti-inflammatory activity of natural compounds is sometimes attributed to their capacity for protecting tissues against oxidative damage. Chronic liver disease with fibrosis is a health problem that can be caused by duct obstruction which leads to cholestasis and liver damage. The oxidative stress seems to have an important role in the damage. Sen et al. [75] aimed at investigating the antioxidant and antifibrotic effect of *Myrtus communis* extracts against against liver injury and fibrosis occurring in rats with biliary obstruction in animal models. Glutathione and superoxide dismutase values that decreased in damaged liver, and malondialdehyde levels, myeloperoxidase activity, tissue luminol, lucigenin, transforming growth factor-beta (TGF-β), and hydroxyproline levels that increase in damaged liver, the treatment with myrtle extract reversed all of these parameters, that is, this extract protects the liver tissues against oxidative damage through its radical scavenging and antioxidant activities, decreasing the fibrotic activity by reducing the hepatic TGF-β and hydroxyproline contents [75]. Later on, Sen et al. [74] reported that ethanolic extracts obtained from myrtle leaves when supplemented to the diet of Wistar albino rats in which colitis was induced by acetic acid, on the fourth day, they decreased the levels of malondialdehyde, tissue luminol, lucigenin, nitric oxide, and peroxoxynitrite chemiluminescence, as well the myeloperoxidase activity, and increased the glutathione levels, when compared to those animals in which the disease was induced and any myrtle supplementation was given. The study showed that ethanol extract had significant antiiinflammatory activity protecting the tissues against oxidative damage [74].

Fekri et al. [89] evaluated the biochemical and histopathological effect of preventive and therapeutic doses of extracts of myrtle leaves against bleomycin-induced pulmonary fibrosis in an animal model. Inflammatory and oxidative processes are involved in the pulmonary fibrosis. For this reason, the authors [89] evaluated the effect of myrtle extract on the lipid peroxidation as well as on the activity of catalase in the animal submitted to bleomycin. The oxidation of lipids decreased and the activity of catalase increased, that is, the oxidative stress promoted by bleomycin was reversed with the myrtle treatment. Simultaneously, the methanolic extract of myrtle leaves decreased the hydroxyproline concentration in animals subjected to bleomycin-induced pulmonary fibrosis in early and late phases. Hydroxyproline is an indicator of collagen deposition in lungs in pulmonary fibrosis. The authors also reported the improvement in inflammation and fibrosis in myrtle group [89].

The anti-inflammatory activity of the aqueous and ethanolic extracts obtained from the aerial parts of *M. communis* was evaluated by Hosseinzadeh et al. [91] using xylene-induced ear oedema and a cotton pellet test, in mice. Antinociceptive activity was also performed using hot plate and writhing tests in mice. The ethanolic (0.05 g/kg) and aqueous extracts (0.005, 0.015, and 0.03 g/kg) demonstrated anti-inflammatory effects against chronic inflammation, whereas in the acute inflammatory activity (xylene-induced ear oedema study), the aqueous extract at doses 0.1, 0.2, and 0.03 g/kg showed significant anti-inflammatory activity. The ethanolic extract also had activity against acute inflammation in all doses (0.05, 0.15, and 0.35 g/kg), but was not dose-dependent. The aqueous and ethanolic extracts of the aerial parts of myrtle exhibited antinociceptive activity. The authors suggested that this activity might be mediated by opioid receptors [91]. The chemical composition of the extracts was not performed but the authors attributed the antinociceptive and anti-inflammatory activities to flavonoids and/or tannins, according to the references consulted by them. The highest concentrations of extracts with anti-inflammatory and antinociceptive activities

were lower than the LD50 (lethal dose 50) values found for aqueous and ethanolic extracts (0.473 and 0.79 g/kg, respectively) [91].

One problem of the plant extracts is the heterogeneity in the concentration of the bioactive compounds. This fact may originate biological responses with different strength. For overcoming this problem, there is the possibility to obtain standardized extracts with well defined concentrations of the bioactive compounds. Fiorini-Puybaret et al. [153] used a standardized ethanolic extract (0.75% of myrtucommulones) obtained from myrtle leaves, with the trade name Myrtacine® (Ducray Laboratory, Lavaur, France) with the objective to ascertain if this extract is able to treat acne in its diverse aspects: antibacterial activity against *Propionibacterium acnes*; antiproliferative activity on human keratinocytes and anti-inflammatory properties using a cellular model of inflammation. These biological properties were also compared with myrtucommulones A and B', which are present in the standardized extract. This approach was based on the fact that the nonprenylated acylphloroglucinols of myrtle possessed antimicrobial, antioxidant, and anti-inflammatory activities [30,150,154]. Anti-inflammatory effect was determined through the measurement of 6-keto-prostaglandin F1α and [3H]-arachidonic acid metabolite production by A23187-stimulated human keratinocytes. COX and lipoxygenase LOX metabolite production from ionomycin-stimulated human keratinocytes was also evaluated as well as the lipase activity [153]. The results showed that A23187-stimulated keratinocytes in the presence of Myrtacine® at 10 μg/mL inhibited in 23% the production of 6-keto PGF1α compared to the control. Preincubation of SVK14 keratinocytes with 0.1 and 0.5 μg/mL myrtucommulone A also significantly reduced 6-keto PGF1α production in 21% and 17%, respectively, whereas myrtucommulone B' only lowered the production of 6-keto PGF1α in 9%. At 3 and 10 μg/mL, Myrtacine® significantly decreased all metabolite production from cyclooxygenase (6-keto PGF1α, PGE2, PGF2α, PGD2, and PGA2) and lipoxygenase (LTB4 and 12-hydroxyeicosatetraenoic acid or HETE). Moreover, Myrtacine® also exhibited anti-lipase activity: at 100 μg/mL and 1 mg/mL was able to inhibit the activity of lipase by 53% and 100%, respectively [153]. This anti-inflammatory activity along with the anti-*P. acnes* and antiproliferative activities can lead Myrtacine® to be used in the treatment of comedones and inflammatory acne lesions [153].

The mechanisms involved in the anti-inflammatory activity of myrtucommulone isolated from myrtle leaves have been cleared by diverse research teams as aforementioned; nevertheless there is no information about its bioavailability. Gerbeth et al. [155] proposed to study the metabolic stability of that acylphloroglucinol, obtained by synthesis, using rat and human liver microsomes and its oral availability in a pilot rat study. The study started by using Caco-2 cells and the results showed a high absorption of myrtucommulone. In rat model, the authors [155] reported that after 1 h of administrating 4 mg/kg myrtucommulone, an average plasma level of 258.67 ng/mL was observed. Physiologically-based pharmacokinetic modelling of myrtucommulone in the rat, it was observed that it was rapid and extensively distributed by plasma, skin, muscle, and brain. Moreover, myrtucommulone undergoes phase I biotransformation in human and rat liver microsomes, resulting hydroxylated and demethylated metabolites [155].

**Table 3.** *Cont.*

*Flavonols—*8.58

**Table 3.** *Cont.*

#### **4.** *Myrtus nivellei*

The antioxidant and anti-inflammatory activities of berries and/or leaves of *M. nivellei* are much fewer reported than those found for *M. communis*. Rached et al. [156] collected fifty two plants in different regions of Algeria and evaluated their antioxidant activity through two methods: DPPH and β-carotene-linoleic acid bleaching. Forty-eight active extracts were found from 38 Algerian species and *M. nivellei* leaves were in this group with IC50 value = 4.90 μg/mL (DPPH assay), in an aqueous extract, obtained by decoction, with total phenol concentration of 242.68 mg/gallic acid equivalent/g and total flavonoid content of 28.53 mg catechin equivalent/g. The IC50 value was close to that found for BHA (IC50 = 4.15 μg/mL) but higher and, therefore, poorer than quercetin (IC50 = 1.66 μg/mL) and ascorbic acid (IC50 = 2.66 μg/mL). A liquid–liquid fractionation assay from the aqueous extract was performed using solvents with increasing polarity (chloroform, ethyl acetate and *n*-butanol). In the DPPH assay, the IC50 values found for these extracts were: 53.50, 3.08, and 4.40 μg/mL, respectively. The remaining aqueous extract also had capacity for scavenging the DPPH free radicals (IC50 = 64.84 μg/mL). A good linear correlation was found between the antioxidant activity of the diverse fractions and total phenols and total flavonoids' contents [156].

Different extracts of leaves of *M. nevellei* were obtained (aqueous, ethanolic, and methanolic) and their antioxidant activities were evaluated by [157]. Ethanolic extract revealed to be the most effective for scavenging DPPH free radicals (EC50 = 0.59 μg/mL), closest to the reference, ascorbic acid (EC50 = 0.39 μg/mL). However, the aqueous extract more easily reduced ferric ion (64.86%) than the ethanolic extract (35.14%). The ethanolic extract possessed higher amounts of phenols (734.3 μg gallic acid equivalent/mg) and flavonoids (181.1 μg quercetin equivalent/mg) than the aqueous extract (466.5 μg gallic acid equivalent/mg and 135.5 μg quercetin equivalent/mg, respectively) [157].

The anti-inflammatory activity of methanolic extract of the aerial parts of *M. nivellei* was evaluated for the first time by Touaibia and Chaouch [158] using the carrageenan-induced paw oedema test. The authors revealed that the dose of 400 mg/kg was able to reduce significantly the paw oedema (80.41%). This inhibition percentage was similar to that of diclofenac, but at 50 mg/kg. Oral lethal dose 50 (LD50) of the methanolic extract was higher than 1000 mg/kg, and therefore, the authors considered this dose as being highly safe [158].

*M. nivellei* is a Sahara-endemic plant used in folk medicine. In the absence of any preservation programmes can originate its disappearance very quickly. Touaibia and Chaouch [19] obtained in vitro calli from this species, evaluated their antioxidant activity and compared to those obtained from leaf extracts. Total phenol and total flavonoid' contents of calli extracts were inferior (73 μg gallic acid equivalent/g and 91 μg quercetin equivalent/g) when those obtained from leaf extracts (in situ): 348 μg gallic acid equivalent/g and 152.25 μg quercetin equivalent/g. These lower amounts of phenols might be responsible for the lowest capacity of calli for scavenging the DPPH free radicals (EC50 = 1.44 mg/mL). EC50 value for leaf samples (in situ) was 0.98 mg/mL. Nevertheless, calli extract exhibited higher capacity for preventing lipid peroxidation and reducing power than leaf extracts [19].

The chemical composition of Saharan myrtle only very recently was unravelled [20,22]. The biological properties of these extracts and their compounds were also reported [20]. From leaves of *M. nivellei*, Mansour et al. [20] obtained aqueous extracts by decoction and infusion that were analysed by ultrahigh-performance liquid chromatography photodiode array high-resolution mass spectrometry (UHPLC-PDA-HRMS) and then confirmed by nuclear magnetic resonance (NMR) spectroscopy. The phenolic compounds present in the infusion and decoction were also quantified by HPLC-UV-PDA. The fourteen compounds identified are depicted in Table 4. Myricetin 3-*O*-β-D-(6-galloyl)glucopyranoside, isomyricitrin, and myricitrin were the major compounds present in the aqueous extracts of *M. nivellei*. Decoction extracted more phenols (150.5 mg/g) than the infusion (102.6 mg/g), corresponding to 73.8 and 23.6 mg/100 mL of a single tea cup, respectively. The capacity of decoction, infusion, and isolated compounds for scavenging the DPPH free radicals were analysed by the authors [20] and compared with green and black teas. The EC50 values for decoction and infusion of black and green teas were:

EC50 = 10.2, 18.6, 22.9, and 18.0 μg/mL, respectively. The activities of isolated compounds were better than those of infusion and decoction. The EC50 values for 3,4,5-*tri*-*O*-galloyl-quinic acid, myricetin-3-*O*-β-D-(6"-galloyl)glucopyranoside, *iso*myricitrin, 1,2,3,6-tetra-*O*-galloyl glucose, myricitrin, quercetin-3-*O*-β-D-(6"-galloyl)glucopyranoside, myricetin-3-*O*-β-xyloside, myricetin, and quercitrin were: 3.8, 5.6, 6.7, 4.0, 4.3, 8.8, 7.0, 3.5, and 5.9 μg/mL, respectively. Myricetin, 3,4,5-tri-*O*-galloyl-quinic acid and 1,2,3,6-tetra-*O*-galloyl glucose had a similar capacity for scavenging the free radicals of that of the reference, ascorbic acid, that is a powerful natural soluble antioxidant. These results indicate that the effective antioxidant capacity of *M. nivellei* teas can be attributed to flavonoids, their glycosides and polygalloyl derivatives [20]. Nevertheless, it seems that when in combination with the other phenolic compounds, some antagonism may occur among them, since the antioxidant activity of the whole tea is lower than the isolated compounds.

The chemical composition of crude aqueous extract, ethyl acetate and butanol fractions of Saharan myrtle leaves conducted by liquid chromatography with diode array detection, coupled to mass spectrometry (ion trap) with electrospray ionization (HPLC-DAD−ESI/MS*n*) permitted to identify 17, 25, and 19 compounds, respectively (Table 4) [22]. The ethyl acetate fraction had the highest concentration of phenol compounds, followed by the butanol fraction and the crude aqueous extract. The antioxidant activities of the extracts were evaluated through three methods: DPPH, reducing power, inhibition of β-carotene bleaching, and thiobarbituric acid reactive substance methods. The results showed that the ethyl acetate fraction exhibited better activity than the remaining extracts or fraction. In the DPPH method, the EC50 values found for ethyl acetate, butanol fractions and crude aqueous extracts were: 3.27, 4.6, and 7.1 μg/mL, respectively; in the reducing power, the EC50 values were: 3.15, 3.93, and 6.23 μg/mL, respectively; in the inhibition of β-carotene bleaching, EC50 values were: 82, 92.9, and 112 μg/mL, respectively; and in the thiobarbituric acid reactive substance, the EC50 values were: 0.46, 0.74, and 0.87, respectively. According to the authors, the best activity of the ethyl fraction can be attributed to the presence of some compounds, such as myricetin-hexosyl-gallate, myricetin-3-*O*-rhamnoside, gallocatechin-gallate-dimer, digalloyl, trigalloyl-HHDP-glucoside, tetragalloylglucoside, and of the quercetin and kaempferol derivatives. Such results are in line with those already reported by Pereira et al. [111,112] in which the authors found a good correlation between antioxidant activity and flavonol glycosides in *M. communis* extracts. With the exception of the inhibition of β-carotene bleaching method, all samples had better activity than the reference used, Trolox. The anti-inflammatory activity was measured through the capacity of samples to suppress the NO production by LPS (lipopolysaccharide)-induced murine macrophage-like RAW 264.7 cells. Such as observed for the antioxidant activity, ethyl acetate was also the best sample (EC50 = 104 μg/mL) for suppressing NO production and, therefore, best anti-inflammatory activity, followed by the butanol fraction (EC50 = 127 μg/mL) and the crude aqueous sample (EC50 = 149 μg/mL), however all of them presenting much lower activity than the reference, dexamethasone (EC50 = 16 μg/mL). The authors also attributed the anti-inflammatory activity to the presence of flavonols, ellagitannins and phenolic acids. The authors [22] also evaluated the cytotoxic properties of Saharan myrtle extract and fraction against diverse tumor cell lines (breast cancer MCF-7, lung cancer NCI-H460, cervical cancer HeLa, and liver cancer HepG2 lines). The ethyl acetate fraction showed a significant higher potential against all cancer cell lines, followed by the butanol fraction and the crude aqueous extract, nevertheless the same fraction was also that exhibited the lowest cytotoxicity on non-tumor cells (porcin liver primary cells, PLP2). According to the authors [22], ellagic acid, ellagitannins, quercetin, and its derivatives may have a crucial role in the cytotoxicity activity.

**Table 4.** Phenols and flavonoids in Saharan myrtle extracts.

**Table 4.** *Cont.*

**Table 4.** *Cont.*

#### **5. Conclusions**

The myrtle berries are mainly used for doing liqueur; nevertheless berries can also be used for making jam, preserving their biological properties, such as the liposome oxidation. The antioxidant activity of berries, due to the presence of phenols, also seems to be a possibility to use the berries' pulp as prebiotics in some food formulations, such as probiotic-enriched ice-creams. For a myrtle liqueur of high quality it is also required that berries must be processed immediately after harvest. Two approaches can be followed: to store fruits in adequate conditions, such as controlled atmospheres; or to process the berries and store the hydro-alcoholic extracts. For the former case, the results showed that berries held at 80% O2 at 2 ◦C preserve their quality of phenolic and anthocyanins contents, and antioxidant activity, for at least 20 days. When fruits are immediately submitted to maceration, the extract obtained is stable for three months, being flavonoids and, particularly, anthocyanins the most instable compounds. The antioxidant activity of berry extracts can only be poorly attributed to the anthocyanins, since white berries, in some cases, exhibit stronger antioxidant activity than dark blue berries. Some type of flavonoids and gallic acid and their derivatives may be responsible for the antioxidant activities found in berry extracts. Beyond the maceration, several other methods of extraction (e.g., supercritical fluid extraction, ultrasound-assisted extraction, and decoction) may be used, nevertheless did not provide much better phenol content or antioxidant activity. The type of solvent of solvent mixtures used revealed also to be important on the capacity for extracting higher amounts of some types of phenol compounds and, therefore, on the antioxidant activity, nevertheless sometimes the results are dissimilar, depending on the research team and conditions of work. Seeds revealed to be best antioxidants than the remaining parts of the fruit, probably due to the presence of higher concentrations of galloyl derivatives. When leaf and berry extracts of myrtle were compared in terms of antioxidant capacity, leaf extracts revealed to be those that exhibited higher antioxidant activity, not only to the highest amounts of total phenols but also for the highest concentrations of hydroxybenzoic acids and flavonols and their derivatives although the most important factor in the relevant activity of leaf myrtle extracts is the ratio between the sum of galloylglucosides, ellagitannins, and flavonols and also of the ratio between these galloyl derivatives and galloyl-quinic acids. The utilization of leaf extracts for stabilizing complex lipid systems, olive oil, and brined anchovies revealed to be possible, particularly when myricetin 3-*O*-rhamnoside is present, due to its antioxidant activity. The anti-inflammatory activity was also reported for both berry and leaf extracts of myrtle. In some cases such activity was attributed to the flavonoids and/or hydrolysable tannins, nevertheless nonprenylated acylphloroglucinols (e.g., myrtucommulone and semimyrtucommulone) were revealed to have also a remarkable role in that activity.

The chemical composition and antioxidant activity of Saharan myrtle is much less studied, most likely to its restricted distribution, which only appears in specific places of the Sahara. Only very recently, a detailed chemical composition of their extracts was performed as well as their antioxidant, anti-inflammatory, cytotoxic, and antibacterial activities. In the aerial parts or leaves were possible to find compounds belonging to the galloyl derivatives, flavonols, and flavonols derivatives, and phenolic acids as reported for myrtle extracts, nevertheless, some new compounds were found, such as 2-hydroxy-1,8-cineole-β-D-glucopyranoside, 2-hydroxy-1,8-cineole 2-*O*-α-L-arabinofuranosyl (1→6)-β-D-glucopyranoside, rugosin A, rugosin B, and valoneic acid dilactone, which were not reported in myrtle extracts. The effective antioxidant capacity of *M. nivellei* teas can be attributed to flavonoids, their glycosides, and polygalloyl derivatives.

**Funding:** This study was partially funded by Fundação para a Ciência e a Tecnologia (FCT), under the projects UID/BIA/04325/2013—MEDTBIO.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*


*Edited by Vladimir Kutcherov*

*and Anton Kolesnikov*

Hydrocarbons provide our core energy resource. Information on their origin, properties and phase behavior is interesting from the point of view of physical chemistry. At the same time this information is of great value to the oil and gas industry. The book "Hydrocarbon" is comprised of 9 chapters, covering different topics: from origin of hydrocarbons to the method for hydrocarbon exploration. Distribution of polycyclic aromatic hydrocarbons in soil and their influence to environment are also discussed. This book should serve as a support to researchers and students as well as experts, both in academia and industry.

ISBN 978-953-51-0927-3

Hydrocarbon

Photo by Yulia-Images / iStock


Preface

Globally, there is a continuous concern for research and development of alloys for medical and biomedical applications. The goal is to improve both the classic technologies of implant implementation and the technologies of synthesis of the biomaterials from which they are executed, with a final aim to promote a new generation of multifunctional implants with long-term performances.

Biomaterials have been used for several thousand years by ancient cultures in Egypt, Babylon, Greece, Italy, China, and Central and South America to replace different parts of the human bone system. These materials were used and developed before coining of the modern term "biompatibility," which was introduced about 60 years ago when researchers began to study and develop this field.

Biomaterials must be **biocompatible**, meaning they must be non-toxic and not release ions so that they can be tolerated by the human body for long periods of time (decades). They must also have certain **mechanical properties**, including mechanical stability depending on the application in which they are used, and they must resist demands and not break or deform excessively over time. Finally, biomaterials require **efficiency**, that is, they must be reliable, accessible, cost-effective, easy to process, and sterile. Considering all this, when obtaining biomaterials, raw materials of high purity are selected and the influence of the alloying elements must be studied.

The global biomaterials market is constantly growing due to an increasing population that is susceptible to diseases, such as cardiovascular, dental, orthopedic, and neurological conditions. Expansion and improvement of the market are necessary to improve the health of patients and extend the duration of use of biomaterials in the human body.

This book provides an overview of biomaterials science with a focus on health and medical applications that can be improved with new biomaterials with non-allergenic elements. These materials are designed to meet functional requirements and overcome the disadvantages of classical alloys used as biomaterials in human tissue.

Over seven chapters, this volume explains the problems created by classical alloys and examines how the new generation of biomaterials helps both doctors and patients. What is remarkable for the domain of biomaterials is the evolution and substantial efforts that have been made every day for constant improvement.

This book is designed for students, doctors, patients, and researchers worldwide.

**Petrică Vizureanu (Academic editor)** Gheorghe Asachi Technical University of Iași, Romania

**Claudia Manuela da Cunha Ferreira Botelho (Co-editor)** University of Minho, Portugal

**1**

Section 1

Medical Devices