**3. Results and Discussion**

The statistical analysis of the data showed a significant interaction between plant parts and harvesting stage for all the recorded parameters. Therefore, the interpretation of the data refers to the combined effect of both factors. Moisture content and proximate analysis results are presented in Table 1. The moisture content of leaves was the highest at 29 and 43 DAS and decreased at 52 DAS with plant maturity, whereas stems contained more water at 43 DAS, followed by the harvesting stages of 52 and 29 DAS. For macronutrient content (fat, proteins, ash, and carbohydrates) and the energetic value of plant tissues, the highest content in leaves was observed at the last harvest (52 DAS), which could be probably attributed to a concentration effect. Moreover, the opposite trend was observed in the case of stems, where harvesting at 29 DAS resulted in the highest content of ash and carbohydrates and the highest energetic value. However, fat content did not differ significantly between the first (29 DAS) and the last harvest (52 DAS), whereas protein content was the highest at the last harvest (52 DAS). Similar trends regarding the macronutrient content at different harvesting stages have been previously reported by Mohamed and Hussein [5], who also suggested an increase in fat, protein and ash content with increasing maturity, whereas they also reported an opposite trend for carbohydrate content. The values for moisture and macronutrient content detected in our study were in the same range as those reported by Ezekwe et al. [17], who studied eight purslane accessions planted at different dates, as well with the study described by Teixeira and Carvalho [13], who evaluated the effect of salinity on the proximate composition of *Portulaca oleracea* cv. "Golden leaf" for two growing seasons. The only difference with the above-mentioned reports was observed in lipid content, which was lower in our study. This finding could be attributed to differences in genotype, harvesting stage and growth conditions. According to Jin et al. [10], stress conditions such as heat, drought and salinity may accelerate protein catabolism and result in lower protein content. In addition, Teixeira and Carvalho [13] suggested that apart from harvesting stage (49 and 57 DAS), growing season (spring and summer) may also have an effect on the proximate composition of purslane leaves.

Significant differences in moisture content were also observed between both plant parts, with leaves having a higher moisture content only at the first harvest stage (29 DAS), since developing plants gradually become succulent and contain more water in stems than leaves (Table 1). Similar results have been reported by Oliveira et al. [4], who also detected higher moisture content in leaves compared to stems, regardless of purslane genotype. Macronutrient content and energetic value also differed between plant parts at the studied harvesting stages. In particular, leaves contained more fat and proteins at the first harvest, whereas stems had a higher content of carbohydrates and ash and a higher energetic value at the same harvesting stage. Moreover, leaves had higher macronutrient content and energetic value than stems at the second and the last harvesting stages (apart from carbohydrate content at the second harvest), which could be partly attributed to the lower moisture content values and the resulting concentration effect. Oliveira et al. [4] also reported differences in fat content between leaves and stems of purslane plants from four different locations in Northern Portugal, whereas there were significant differences in the reported values compared to our study, probably because plants were collected in the wild instead of being cultivated. Moreover, no details regarding the harvesting stage were available in the study of Oliveira et al. [4] in order to make direct comparisons with our study. According to Ezeabara et al. [27], moisture, fat, protein and ash contents were higher in leaves than in stems, when plants were harvested at flowering stage, whereas the opposite trend was observed for carbohydrate content. These results are in agreement with the values of the last harvest of our study, indicating that late harvests increase the nutritional value of leaves in comparison to stems, due to maturation progress and the decrease in moisture content.

The contents of individual and total tocopherols in relation to the harvesting stage and plant part are presented in Table 2. Tocopherols, also known as vitamin E, are significant bioactive compounds with antioxidant properties against lipid peroxidation of biological membranes [28]. α-tocopherol was the main detected isoform in both leaves and stems, followed by γ-, β- and δ-tocopherols. Leaves contained significantly higher amounts of all isoforms and total tocopherols, regardless of the harvesting stage, while harvesting at later stages (52 DAS) had a beneficial effect on the content of most of the individual and total tocopherols. The only exception was observed for γ-tocopherol content, which was the highest at early growth stages (29 DAS). A contrasting trend was observed in the case of stems, where the highest content of individual and total tocopherols was recorded at the stage of 29 DAS, except for β-tocopherol where no significant differences were observed between harvesting at 29 and 43 DAS. Similarly to our study, Szalai et al. [7] reported that α- and γ-tocopherols were the main detected tocopherols in fully mature leaves of three purslane microspecies, although they did not detect the other two vitamin E isoforms (β- and δ-tocopherols) that were present in our study. Moreover, the highest content of α- and total tocopherols at the last harvesting stage (52 DAS) could be attributed to unfavorable growing conditions (namely, high temperatures and high transpiration), which could induce tocopherols' biosynthesis as a means of plants' antioxidant defense [29].



233

at *p* = 0.05. \*\* Comparison of means of different plant parts (stems and leaves) from the same harvest was performed with Student's *t*-test at *p* = 0.05.

 ± SD).

The free sugar composition of leaves and stems in relation to harvesting stage is presented in Table 2. The main detected sugars were glucose and fructose, followed by sucrose and trehalose, which were detected in lower amounts. Stems contained significantly higher amounts of fructose, glucose, sucrose and total free sugars than leaves, regardless of harvesting stage, whereas trehalose content was higher in leaves compared to stems when harvesting took place at late growth stages (43 and 52 DAS). In addition, an increase in glucose and fructose content was observed at late growth stages (43 and 52 DAS) for both leaves and stems, which was also depicted in the total sugar content of plant parts. Sugar composition was similar to our previously reported study, where six different purslane genotypes were evaluated in terms of chemical composition [15], whereas any differences in individual sugars' content could be explained mostly by genotypic differences and barely by the environmental conditions and cultivation practices, which were identical in both studies. Moreover, Mohamed and Hussein [5] suggested glucose as the main detected sugar, while fluctuating trends were observed in sugar content between plant parts at different growth stages (30, 49 and 59 days after planting). The lower amounts of sucrose in leaves compared to fructose and glucose could be attributed to the use of fixed carbon for the biosynthesis of fructose and glucose and the concurrent export of sucrose from leaves to be used as a biosynthetic substrate [30].

The organic acid content of plant parts in relation to harvesting stage is presented in Table 3. Four organic acids were detected in leaves and stems, namely oxalic, malic, quinic and citric acids, regardless of the harvesting stage. However, the composition of individual organic acids differed significantly between plant parts and harvesting stages. In particular, leaves contained mostly quinic and oxalic acids at all the harvesting stages, although quinic acid content was significantly higher at the last growth stage (52 DAS). On the other hand, stems contained oxalic, quinic and malic acids at the earliest growth stage (29 DAS), whereas quinic acid decreased significantly with plant maturity. Similarly, Oliveira et al. [4], who studied organic acid content in the leaves and stems of different purslane genotypes, detected significant differences between plant parts, although the magnitude of these differences varied depending on the genotype. In contrast to our study, Szalai et al. [7] detected oxalic, malic and ascorbic acids in the leaves of three purslane microspecies, a difference that could be attributed mostly to the effect of harvesting stage. However, it is worth mentioning the effect of genotype on organic acid profile and content, which is also important and has been already confirmed in previous studies [4,15].


**Table 3.** Composition in organic acids (g/100 g fw) of purslane stems and leaves in relation to harvesting stage (mean ± SD).

nd: not detected; tr: traces; \* DAS: days after sowing; ¥: no significant difference was observed between plant parts. Different Latin letters (a–c) in the same column refer to significant differences between harvest stages for the same plant part (stems or leaves) at *p* = 0.05. \*\* Comparison of means of different plant parts (stems and leaves) from the same harvest was performed with Student's *t*-test at *p* = 0.05.

Purslane is considered as one of the richest plant sources of omega-3 fatty acids. The composition of leaves and stems in relation to harvesting stage is presented in Table 4. The main detected fatty acids in both stems and leaves were linoleic, palmitic, α-linolenic, behenic, oleic and lignoceric acids, although the abundance of individual compounds varied among the studied plant parts and harvesting

stages. The major compound in stems was palmitic acid, followed by linoleic and α-linolenic acids. In addition, the highest content of palmitic and linoleic acids was detected at 29 DAS, whereas α-linolenic acid content was the highest at 43 DAS. In contrast, the fatty acid profile of leaves differed significantly from that of stems, with α-linolenic acid contributing the most to the overall fatty acid content, especially at the first two harvesting stages (29 and 43 DAS). The same fatty acids have been previously reported by Oliveira et al. [4], who also suggested α-linolenic, palmitic, oleic, stearic and behenic acids as the main fatty acids in leaves. However, the reported profile of individual fatty acids differed from that in our study, without additional information regarding harvesting stage being available. Similarly, Guil-Guerrero and Rodríguez-García [31] suggested a different profile of fatty acids in phospholipid and neutral lipid fractions of purslane leaves, which contained higher amounts of omega-6 (n6) than omega-3 (n3) fatty acids. Moreover, in our study the overall α-linolenic acid content (the sum of the contents of stems and leaves) was the highest (0.1 g/100 g fw) at the earliest harvesting stage (29 DAS), despite the significantly higher fat content of leaves at the late harvesting stage (0.1 g/100 g fw at 52 DAS; see Table 1). Similar amounts of α-linolenic acid (1.06 g/100 g dw) were reported by Guil-Guerrero and Rodríguez-García [31], although they also suggested that purslane leaves contain higher amounts of linoleic than α-linolenic acid. The highest polyunsaturated fatty acids (PUFA)/saturated fatty acids (SFA) ratio was observed in stems and leaves at the middle (43 DAS) and late harvesting stages (52 DAS), respectively. In contrast, the lowest n6/n3 fatty acid ratio in stems and leaves was recorded at the earliest harvesting stage (29 DAS). In addition, for all the studied harvesting stages and plant parts, the PUFA/SFA ratio was higher than 0.45, while the n6/n3 ratio was lower than 4.0, indicating a high nutritional value of the edible plant parts [32]. Moreover, the detected values for the above-mentioned ratios of our study were within the same range as the ones recorded in the study of Fontana et al. [19].

The identified phenolic compounds and oleracein derivatives in both plant parts of purslane are presented in Table 5, while quantification data are presented in Table 6 and Figure 1. A total of five compounds were identified in the hydroethanolic extracts of purslane aerial plant parts (Table 5). These compounds included three phenolic acids (caffeic acid derivatives, peaks 2, 3 and 4) and two oleracein derivatives (phenolic alkaloids, peaks 1 and 5). Caffeic acid (peak 2) was identified in comparison with a commercial standard, whereas compound 4 was identified as a caffeic acid derivative, thus its pseudomolecular ion was not clearly identified due to the very small amounts present. Compound 3 ([M <sup>−</sup> H]<sup>−</sup> at *<sup>m</sup>*/*<sup>z</sup>* 385) presented a unique MS<sup>2</sup> fragment at *<sup>m</sup>*/*<sup>z</sup>* 223 (sinapic acid), with a loss of a hexosyl moiety (162 u), being tentatively identified as sinapic acid hexoside. Compounds 1 ([M − H]<sup>−</sup> at *m*/*z* 664) and 5 ([M − H]<sup>−</sup> at *m*/*z* 502) corresponded to oleracein derivatives, being identified as oleracein C and A, respectively, taking into account previous findings in the literature regarding *P. oleracea* [33,34]. Oleraceins are cyclodopa alkaloids, which have been previously reported by Xiang et al. [35], who identified five oleraceins in dried purslane plants, among other compounds. Moreover, Farag and Shakour [36] suggested that the presence of these compounds in purslane aerial parts may be used as a criterion for the classification of different *Portulaca* taxa. To the best of our knowledge, the rest of the identified compounds have not been previously reported in purslane aerial parts.



Different Latin letters (a–c) in the same row refer to significant differences between harvest stages for the same plant part (stems or leaves) at *p* = 0.05. \*\* Comparison of means of different

plant parts (stems and leaves) from the same harvest was performed with Student's *t*-test at *p* = 0.05.


**Table 5.** Retention time (Rt), wavelengths of maximum absorption in the UV-VIS region (λmax), mass spectral data, identification and quantification of phenoliccompounds and oleracein derivatives in purslane aerial plant parts (leaves and stems). **Table 6.** Quantification of phenolic compounds and oleracein derivatives in purslane stems and leaves (mg/100 g dried weight (dw)) in relation to harvesting stage(mean ± SD).


 DAS: days after sowing; nd: not detected; TPCOD: Total phenolic compounds and oleracein derivatives; tr: traces. Calibration curves used: A: *p*-coumaric acid (*<sup>y</sup>* = 301,950*x* + 6966.7; *R*2 = 0.999); B: caffeic acid (*<sup>y</sup>* = 388,345*x* + 406,369; *R*2 = 0.999); C: sinapic acid (*<sup>y</sup>* = 197,337*x* + 30,036; *R*2 = 0.999). Different Latin letters (a–c) in the same column refer to significant differences between harvest stages for the same plant part (stems and leaves) at *p* = 0.05. \*\* Comparison of means of different plant parts (stems and leaves) from the same harvest was performed with Student's *t*-test at *p* = 0.05.

**Figure 1.** Chromatographic profile of the hydroethanolic extract obtained from purslane stems, recorded at 330 nm. Peak numbers correspond to the compounds already mentioned in Table 5.

The composition of phenolic compounds and oleracein derivatives differed among the tested plant parts and harvesting stages (Table 6). Concerning the quantification of oleraceins, *p*-coumaric acid was the standard applied to perform the quantitative analysis (an available compound with a similar structure); therefore, these compounds were quantified as equivalents to this phenolic acid. Leaves contained significantly higher amounts of individual and total phenolic compounds and oleracein derivatives compared to stems, regardless of harvesting stage. Moreover, harvesting at early stages (29 DAS) resulted in significantly higher contents of phenolic compounds and oleracein derivatives, especially in the case of leaves. Meanwhile, in stems the content of oleracein C was the highest at the same harvesting stage (29 DAS), resulting in the highest total contents of phenolic compounds and oleracein derivatives, accordingly. As mentioned previously for the case of tocopherols (see Table 2), phenolic compounds and oleracein derivatives may also contribute to the overall defense mechanisms of purslane; therefore, the increased content at the earlier stages could be attributed to the protective purposes of developing leaves. Similar results regarding the effect of harvesting stage on the compositions of phenolic compounds and oleracein derivatives have been reported by Lim and Quah [37], who also observed a decrease in total phenolic compounds and oleracein derivatives in leaves with increasing maturity. The differences in the compositions of phenolic compounds and oleracein derivatives could be attributed to differences in the tested genotypes [35,37], to different cultivation regimes and growing conditions [14,18] and also to extraction protocols [38].

The cytotoxic effects on PLP2 non-tumor cell lines of the samples showed no hepatotoxicity, with GI50 values being higher than 400 μg/mL for all the harvesting stages and plant parts (data not shown). Concerning the evaluation of in vitro activity against the four tumor cells lines, the extracts did not present any activity at the tested concentrations (data not show), with GI50 values being higher than 400 μg/mL.

The hepatoprotective effects of ethanolic extracts of purslane aerial parts have been previously reported by Eidi et al. [39], whose findings are in agreement with the results of our study. Similarly, ethanolic and aqueous extracts of air-dried purslane leaves showed hepatoprotective properties against paracetamol-induced liver damage [40]. Moreover, previous studies have suggested weak to moderate inhibitory effects against two mutagens (benzo[a]pyrene (B[a]P) and 2-Amino-3-methyl-imidazo[4,5-f]quinolone (IQ)) and no mutagenic activity on Salmonella typhimurium [41], while Choi and Ryeom [42] and Zhao et al. [43] reported significant antitumor activity against leukaemia and cervical carcinoma cell lines, respectively.
