3.1. Content in TP, Hydroxycinnamic Acid Derivatives and Flavonols of Herbal Preparations
Appraisal of the phenol content of a plant material is amongst the most popular subjects of study in favor of their health beneficial actions [23
]. The TP content of the prepared herbal formulations was assessed, and results are depicted in Figure 1
Material to solvent ratio affected the TP content of the examined MOL infusions (0.5, 2 and 4% w/v) in a linear manner. I(A) MOL 4% 10’ W held a double TP content (1949 ± 163 mg TP as CAF per 250 mL cup) in comparison to I(A) MOL 2% 10’ W and I(A)MOL 0.5% 10’ W. 2% MOL decoctions in comparison to the rest 2% MOL preparations possessed a richer content (1297–1471 mg TP as CAF per 250 mL cup). Initially, TP slightly increased with prolonged time of decoction process (from 5 to 10 min), no further increase was noticed when time was further extended (at 15 min). Similar were the observations for MOL preparations studied by Sentkowska, et al. [25
]. Apart from I(A) MOL 4% 10’ W and MOL decoctions the rest aqueous MOL preparations had similar TP content.
The aqueous MOL preparations were followed by the hydroethanolic ones regarding phenol content and that by the ethanol ones. The inferior performance of ethanol to extract phenolics from MOL was expected since it has been repeatedly published for MOL and other natural sources [12
Additionally, MOL infusion when compared to CHM and OLF had a considerably higher (correspondingly ~11 and ~5 times more) TP content. MOL infusions studied by [8
] also presented higher TP content than olive leaf ones. Considering that Folin Ciocalteu assay calibration curves for caffeic and gallic acid are similar [30
], findings of the present study were compared to literature and MOL preparations appeared remarkable TP sources [31
]. Jiménez-Zamora et al. [32
] also distinguished this material for its high TP content (1003 mg GAL/L) among 36 plants (6–1387 mg GAL/L).
Furthermore, the hydroethanolic MOL preparations of the present study have a richer TP content in respect to similar natural preparations such as Salvia fruticosa
, Origanum dictamnus
L., Olea europaeae
L. and Citrus sinensis
]. Similarly, ethanol MOL extracts of the present study had 13800 (S MOL 2% 10’ E) and 15130 (M MOL 2% 24 h E) mg CAF per 100 g dry extract, when TP content of other Lamiaceae ethanol extracts vary among 5000–15,100 mg CAF per 100 g dry extract [33
Values for MOL aqueous preparations of this study were higher to that reported by Triantaphyllou et al. [15
] for a relevant MOL preparation (5% w/v infusion of 25 min: 0.13 g CAF/100 mL) and respective ones of Komes and coworkers (2011) [8
] (1% w/v infusions of 5 and 15 min: ~791 and 929 mg GAL/L, respectively) and Skotti et al. [34
] (1% preparation of 15 min at 85 °C: 0.985mg CAF/mL, and ultrasounds at room temperature: 0.8 mg CAF/mL). Such differences are expected, due to discrepancies in biotic, abiotic parameters and postharvest treatment of the material [7
To further evaluate the bioactive potential of MOL a series of samples of same extract weight basis was assessed for content in total phenols, hydroxycinnamic acid derivatives, and flavonols. As shown in Figure 2
, all infusions and decoctions analysed had an almost similar profile, irrespectively to material to solvent ratio, time, and temperature employed regarding the above-mentioned bioactive classes (hydroxycinnamic acid derivatives and flavonols correspond respectively to ~2/3 and 1/3 of total phenols). Common tea preparation practices, such as infusions and decoctions, had higher content in the examined phenolic classes than those prepared upon maceration and ultrasounds. This finding was in line with data of İnce et al. [35
], according to which ultrasounds and maceration lead to extracts poorer in hydroxycinnamic acid derivatives and other phenolics in relation to the conventional (hot plate heating) extraction method employed. Hydroethanolic macerates had statistically similar content in the examined bioactives with the respective aqueous ones. The hydroethanolic extract examined upon ultrasound was statistically richer compared to its aqueous counterpart. Still, differences are not immense. Once more ethanol showed its inferior potency in comparison to water and the hydroethanolic mixture to extract MOL bioactives. The TP value (67 mg GAL/g dry extract) of the ethanolic MOL extract prepared upon ultrasounds was ~2.5 times lower compared to that of Lin et al. [36
]. The determined TP content values for the aqueous MOL preparations were in line with those reported by İnce et al. [35
] for aqueous MOL extracts (1:30 g per mL solid-to-solvent ratio) prepared conventionally, upon maceration, microwaves and ultrasounds (90–146 mg GAE per g dry material) and lower than that reported by Szabó et al. [37
] for aqueous extracts of different MOL origin (1% infused with hot water and let stand for 24 h; 359–427 mg GAE per g dry extract weight).
The high content of aqueous and hydroalcoholic extracts in phenolic bioactive classes was related to matters of solvents polarity and other solubility matters of the individual constituents. The latter has been well documented in literature for several natural sources and is associated with the nature of the material, nature (polarity and stereochemistry) of individual phenols, intermolecular forces among phenols and extraction solvent, easier penetration of solvent to solvent matrix due to swelling effect drawn by the presence of water among others [28
]. According to Arceusz and Wesolowski [39
] mixtures of ethanol with water are more effective for the extraction of phenolic acids from MOL.
Determination of the concentrations of the main individual phenolics could add some value to the evaluation of the effect of the extraction method on the phenolic dynamic of the herbal preparations. Still, this was beyond the scope of this study. The phenolic composition of MOL has been studied and is shown to bear qualitative similarities but quantitative variability in individual constituents according to many factors (e.g., nature, origin, postharvest treatment conditions of MOL) [25
]. Since in plant materials levels of bioactive ingredients may vary significantly, individual phenolics cannot be easily considered suitable quality markers for final formulations, when content in TP and other phenolic classes, as well as antioxidant activity data can be [41
The antioxidant activity of plant products has been widely accredited owning to its relation with treatment and prevention of diseases and disorders. Whereas, for safe antioxidant capacity evaluations more than one method based on different principles of action are needed [24
]. Therefore, in order to assess the antioxidant activity of the studied extracts, the latter were examined for their ability to inhibit the DPPH radical (on the same TP and dry extract basis) and their ferric reducing power capacity (on the same dry extract basis) (Figure 2
and Figure 3
Regarding results of DPPH findings on the same extract weight basis (Figure 3
a) it was shown that all aqueous and hydroethanolic preparations were potent radical scavengers. The inhibiting activity of the aqueous and hydroethanolic macerates and extracts upon ultrasounds presented statistically similar potency. Ethanol preparations showed a significantly lower activity in relation to the rest studied extracts. This was according to previous findings (Figure 1
and Figure 2
) as ethanol extracts were expected to contain significantly lower amounts of phenolic compounds. Our results were in line with those reported by others [26
] that found aqueous MOL extracts to be more antioxidant potent than ethanol counterparts.
All infusions and decoctions (of same extract weight basis) assessed for their ferric reducing ability showed an almost equal capacity (Figure 2
). These common tea preparation practices produce extracts of higher potency in relation to ones prepared upon ultrasounds or maceration. This was also observed from DPPH assay findings on same extract weight basis (Figure 3
a). Aqueous and hydroethanolic macerates were equally potent, whereas the hydroethanolic extract of ultrasounds was slightly better than its aqueous counterpart. Ethanol preparation was shown to be significantly inferior to relevant aqueous and hydroethanolic ones. The moderate linear correlation (R2
= 0.621) between FRAP and DPPH assay (on same extract weight basis) findings implied that the antioxidant activity as determined with the two assays could be not exclusively due to the action of the same phenolics.
On the other hand, DPPH radical scavenging activity data of same TP basis (Figure 3
b) revealed interesting information. The hydroethanolic preparations were now dominant in scavenging of the DPPH radical, followed by decoctions and I(B) MOL 2% 10’ W. The latter presented according to previous findings a moderate profile and a low TP content, statistically similar to hydroethanolic preparations (Figure 1
). Moreover, ethanol preparations presented comparable capacity to respective aqueous ones. These data were in accordance to Papoti and Tsimidou [41
] that presented natural compositional variability of olive leaf samples not to be reflected to DPPH•
inhibition values on same phenol basis.
The above-mentioned observations could be related with almost equivalent contribution of most of the main phenolic constituents [42
]. Additionally, synergistic effects among the individual components are not excluded. Moreover, the powerful antioxidants carnosic and gallic acids [28
], as well as the weaker scavengers of the DPPH radical oleanolic and ursolic acids [48
], are expected to be better extracted with ethanol from the material. Additionally, hydroalcoholic mixtures are expected to recover higher amounts of the main MOL constituents namely rosmarinic, caffeic, protocatehuic, vanillic and syringic acids. Moreover, rosmarinic and caffeic acid are known to be strong inhibitors of the DPPH radical, possessing better activity in comparison to cinnamic acids, protocatehuic and other benzoic acids [49
] expected in such MOL preparations. Additionally, glucoside form phenols such as luteolin 7-O
-glucoside (respected to polar MOL extracts) [13
] are also expected to be more efficiently extracted from MOL with hydroalcoholic solvents due to solubility matters [51
] and are dominant DPPH radical scavengers [42
Additionally, increase of material to solvent ratio and time of decoction preparation led to no or slightly enhancement of the radical scavenging ability (Figure 3
a,b) and ferric reducing capacity (Figure 2
) of the studied preparations (infusions and decoctions respectively). The latter is in line to Komes et al. [8
] that report prolongation of extraction time not to significantly affect the antioxidant activity of MOL extracts.
Finally, the concentration of an aqueous MOL dry extract capable to inhibit 50% DPPH radical formation (IC50 value) was found to be 309 μg dry MOL per extract mL, whereas for CAF 80 μg/mL. Considering that a cup (250 mL) of a 2% MOL infusion or decoction contains according to our findings ~1700–3300 mg dry extract it can be safely said that its consumption may effectively contribute to daily radical inhibitors intake.
The different conditions employed in the experimental parts of published antioxidant activity data of MOL preparations do not allow direct comparisons [35
]. Still, findings of the present study, in line with literature, indicate that MOL preparations show significant antioxidant activity.
3.2. Mineral Content of Herbal Preparations
Minerals are known to influence human metabolism, affect general health and be linked to physiological function of the human body and are therefore commonly examined in studies dealing with herbal issues. In the present study the Na, K, Ca and Li content of the examined preparations was determined, and data are presented in Table 1
. To better elucidate the effect of various herbal preparations on the mineral concentration a principal component analysis was employed (Table 2
, Figure 4
The content of the MOL preparations in the above-mentioned minerals was 0.8–2.9, 4.1–288.4, 50.1–176.1 and 0.1–0.4 mg per 250 mL respectively (Table 1
). Data for metal concentrations determined in the present study for 2% w/v MOL aqueous preparations were of the same size for K and Ca and higher for Na and Li to that of relevant MOL preparations examined by Özcan et. al. [56
Data for MOL herbal treatments shown in Figure 4
imply that high values of Na, K and Ca are found in MOL decoctions and secondarily in I(B) MOL 2% 10’ W, prepared at 80 °C. High temperature seems to enable the extraction of these macroelements from MOL. Concerning Ca this could be attributed to the fact that it is mainly accumulated into cells, making its extraction hard [57
] and higher temperatures could enhance its release. The intense conditions employed in decoction process, as well as the long extraction process (24 h) of the M MOL 2% 24 h W enhanced K extraction. Potassium is considered a highly extractable element from herbs in favor to its chemical properties and its abundance outside plant cells [57
Effect of material to solvent ratio in the prepared infusions influenced in a linear way the recovery of the studied minerals from MOL. Time of decoction preparation did not statistically affected mineral content. This was in accordance to Özcan et al. [9
] that found this parameter to be non-dependent to the mineral content of the studied infusions. Literature data indicate that this parameter depends on the nature of the material or the recovered metal [9
Ethanol was negatively correlated with Na, K and Ca extraction from MOL. However, ethanol was proved to be better concerning Li extraction for both studied practices. This could be linked to the chemical properties of Li, its lower density, in comparison to that of Na, K and Ca, and the fact that ethanol presents lower density in comparison to water. Table 2
demonstrates the correlation coefficients between minerals and axes 1 and 2. Both axes explain 79.7% (54.1 and 25.6) of the total variation of the analysis. Thus, Na, K and Ca are very important for the formation of axis 1 (coefficient greater than 0.800) and Li of axis 2 (r = 0.983). Concerning axis 2 it appears that I(A) MOL 4% 10’ W is the exclusive representative of high Li responses, therefore implying that the microelement is found in higher amounts in extracts of higher material concentration.
Differences among MOL, CHM and OLF infusions were not considered significant. The exception was the Na content of CHM that was considerably higher in respect to MOL and OLF infusions that possessed a similar content. Literature data confirm that MOL contains significant amounts of minerals (including Na, K, Ca, and Li) in respect to other plant materials [9
]. However, discrepancies among materials could be related to agricultural, climatic or raw material treatment variability.