*3.3. Phenolic Composition of Fillet, Mucilage, Rind, and Flower*

Data related to the phenolic compounds identification in the obtained *Aloe vera* extracts are presented in Table 5, namely the retention time, λmax in the UV-vis region, pseudomolecular ion, ions of major fragments in MS2, and tentative identification (the obtained extraction yields and the phenolic contents that can be found in fresh and dried samples are shown in Table S1). The chromatographic profiles recorded at 280 and 370 nm are shown in Figures 1 and 2. Up to 17 phenolic compounds were identified in the leaf extracts and eight in the flower extract, which were classified into four groups—phenolic acids, flavonoids, chromones, and anthrones. Most of these compounds have already been previously reported in *Aloe vera* [50–53], so that their identities were attributed by interpreting data acquired from HPLC-DAD-ESI/MS<sup>n</sup> with those of literature.



**Figure 1.** HPLC phenolic profiles of *Aloe vera* rind and mucilage extracts recorded at 280 nm (A1 and B1, respectively) and 370 nm (A2 and B2, respectively). See Table 5 for peak identification.

**Figure 2.** HPLC phenolic profile of *Aloe vera* flower extract recorded at 370 nm. See Table 5 for peak identification.

The chromones aloesin or aloeresin B (peak 1) and 2'-*p*-methoxycoumaroylaloresin (peak 17) and the anthrones 10-hydroxyaloin B (peak 9), 10-hydroxyaloin A (peak 10), aloin B or isobarbaloin (peak 14), aloin A or barbaloin (peak 15), malonyl aloin B (peak 16), and malonyl aloin A (peak 18) were detected in the three studied parts of the *Aloe vera* leaf (Table 6). The mucilage contained the highest content (131 ± 3 mg/g extract) of phenolic compounds, mostly anthrones (62.1%) and chromones (34.6%), followed by two luteolin glucosides (3.3%). This result is in accordance with the literature, which states that the vascularized layer that covers the inner fillet is rich in anthraquinone glycosides and anthrone derivatives [3]. The rind was ranked second, with 105 ± 3 mg/g extract of phenolic compounds, of which 44.9% anthrones and 43.8% chromones; it also contained luteolin and apigenin glucosides and the phenolic acid *p*-coumaroylquinic acid. However, the chromone levels found in the

rind did not differ statistically from those of the mucilage (Table 6). Although the phenolic profiles of the fillet and mucilage were similar, a significantly lower concentration (11.2 ± 0.2 mg/g extract) of these secondary metabolites was found in the fillet. In addition, this leaf part had an equal ratio of anthrones and chromones (Table 6).


**Table 6.** Content of phenolic compounds in *Aloe vera* leaf (fillet, mucilage, and rind) and flower extracts. See Table 5 for peak identification.

tr: traces. <sup>1</sup> The results are presented as mean ± standard deviation. <sup>2</sup> Homoscedasticity (H) was tested by the Levene's test: *p* > 0.05 indicates homoscedasticity and *p* < 0.05 indicates heteroscedasticity. <sup>3</sup> Statistically significant differences (*p* < 0.05) among two samples\* were assessed by a Student's *t*-test and among more than two samples were assessed by a one-way ANOVA (and indicated by different letters), using Tukey's honestly significant difference (HSD) or Tamhane's T2 multiple comparison tests, when homoscedasticity was verified or not, respectively.

Variations in the phenolic profiles of *Aloe* species have been reported. According to Fan et al. [50], aloesin is more abundant in *A. barbadensis* and *A. ferox* than in *A. chinensis* and *A. arborescens*. In these species, aloin A predominated over aloin B (according to our results), and lower concentrations were also found in *A. chinensis*. In general, higher contents and more complex phenolic compounds were reported in *A. barbadensis*. Kanama et al. [54] found minimal qualitative variations in the phenolic profiles of *A. ferox* exudate samples obtained from different regions of South Africa. Despite this, aloin B content varied from 18.4 to 149.7 mg/g, aloin A ranged from 21.3 to 133.4 mg/g, and aloesin from 111.8 to 561.8 mg/g of dried exudate. This result corroborates the data presented in Table 6, since aloesin predominated over both aloins, despite lower levels have been quantified in our samples.

6- -Malonylnataloin (peak 13), a malonylated derivative of the rare anthrone nataloin, was detected in the rind extract (Table 6). This anthrone *C*-glycoside is considered of great importance in systematic discrimination of different *Aloe* species and has been reported in *A. vera*, *A. arborescens*, *A. ellenbeckii*, *A. eru*, *A. grandidentata*, *A. brevifolia*, and *A. ferox* [52,55].

The quantification of aloins (as hydroxyanthracene derivatives) is recommended in routine quality control analyses of *Aloe* samples. These compounds are highly valorised in the pharmaceutical industry, allowed in dietary supplements, and used in small quantities as a bittering agent in alcoholic beverages. However, because of their laxative properties, levels of aloin A and B in *Aloe* leaf preparations intended for oral consumption were limited by the International Aloe Science Council to 10 ppm (10 mg/kg) or less [56]. These levels can be controlled and limited by adding purification steps in the manufacturing process.

The phenolic profile of *Aloe vera* flower (Figure 2) was different from that of leaf (Figure 1), being constituted mainly by the flavonoids apigenin-6,8-*C*-diglucoside, apigenin-2"-O-pentoxide-C -hexoside, apigenin-6-C-glucoside, and traces of luteolin glucoside derivatives (accounting for 93.4% of the extract), and by the phenolic acid 5-O-caffeoylquinic acid (Table 5). As shown in Table 6, this extract had the lowest levels (4.78 ± 0.05 mg/g extract) of phenolic compounds. As far as we know, it is the first time that some of these compounds are described in *Aloe vera* flower. No anthraquinone glycosides were detected in this part of the plant as previously stated by Keyhanian and Stahl-Biskup [51].
