**2. Results and Discussion**

#### *2.1. Phenolic Composition of H. acetosella Leaf Extracts*

First, before measuring the content of phenolic compounds, the color characteristics of the *H. acetosella* leaves were investigated (Figure 1). The leaf color is usually green, but three accessions appeared red (PI500777, PI500801, and PI500805). The petiole colors could be divided into four types: 7 accessions were green, 5 accessions green-red, 2 accessions light-red, and 4 accessions red (Table 1). The phenolic compounds of the *H. acetosella* accessions were detected by UV-spectrophotometry and UPLC (Table 2). The total phenolic content (TPC) and total flavonoid content (TFC) levels ranged from 193.14 to 434.67, and 199.10 to 262.19 mg/100 g, respectively, with the highest level in PI500707. A previous study on *H. acetosella*, investigating a different variety using a different extraction method, reported TPC and TFC levels of 1730 and 775 mg/100 g, respectively, values more than three times those found in the present study [19]. Phenolic compounds play an important role in the adaptation of plants to the environment, and their content is determined by the origin, harvesting time, and cultivation conditions [3,20]. Previous data has shown that the TPC and TFC contents can vary by approximately three times according to the region where the plants are collected [21,22]. In the present study, the major polyphenols in *H. acetosella* were identified as caffeic acid (CA) and chlorogenic acid (CGA), while gallocatechin (GC) and gallic acid (GAL) were present in relatively small amounts (Table 2). In particular, CA is present in three Hibiscus plants, *H. cannabinus*, *H. sabdari*ff*<sup>a</sup>*, and *H. acetosella*, with the highest amounts in *H. acetosella* [12]. CGA is also a major phenolic compound in *H. sabdari*ff*a* aqueous extract [23]. The PI500707 accession also exhibited significantly higher levels of polyphenols such as GC (1.57 mg/100 g), GAL (1.97 mg/100 g), CGA (41.56 mg/100 g), and CA (42.93 mg/100 g) in the collected accessions. As expected, the TAC level was associated with the coloration of the leaf, as

well as with the anthocyanins content. In green leaves, the TAC content was less than 1 mg/100 g, but in three accessions with red leaves, the contents ranged from 17.25 to 19.98 mg/100 g (Table 2). Three anthocyanins, delphinidin-3-sambubioside (Dp3-Sam), delphinidin-3-glucoside (Dp3-Glu), and cyanidin-3-sambubioside (Cy3-Sam), were only detected in red leaves but also seen in UPLC 3D profiling (Figure S1). Similar findings have also been reported in a previous study of phenolic compounds in Hibiscus plants [4,6,24].

**Figure 1.** Leaf color of the 18 different *H. acetosella* accessions used in this study.

**Table 1.** Leaf and petiole color of the 18 different *H. acetosella* accessions used in this study.



**Table 2.** Contents of phenolic compounds in leaf extracts from 18 different *H. acetosella* accessions (mean value ± S.D., n = 3).

adjacent to mean value indicate the result of Duncan's multiple range test at the 5% probability level (n = 3); (1) not detectable.

#### *2.2. E*ff*ects of Phenolic Extracts on the Radical Cation Scavenging Activity*

To compare the antioxidant properties of the 18 *H. acetosella* accessions, the radical cation scavenging activity of their phenolic extracts (10 times diluted) was determined using the ABTS assay (Table 3). The ABTS assay mainly depends on hydrogen peroxide in the presence of ABTS to produce the radical cation and has been previously used for measuring the total antioxidant activity in a wide variety of plants rich in polyphenols [25]. The antioxidant activity varied from the highest value of 84.02% (PI500805) to the lowest value of 57.47% (PI500756) in the *H. acetosella* extracts, with that of the control ascorbic acid (500 μM) at 99.83%. These values of antioxidant activity were higher than those previously reported for *H. acetosella* [26]. The activity of the phenolic compounds from the ABTS assay increased with the contents of the related anthocyanins, TAC, Dp3-Sam, Dp3-Glu, and Cy3-Sam (Table 3). For example, PI500801 (74.71%), PI500777 (82.34%) and PI500805 (84.02%) exhibited the highest antioxidant activity, as well as abundant TAC and anthocyanins contents. In contrast, 15 *H. acetosella* accessions with no detectable anthocyanins contents, showed a slightly decreased antioxidant activity from 57.47% (PI500756) to 65.94% (PI500707). The antioxidant e ffect of the extracts was probably caused by flavonoids and anthocyanins, especially anthocyanins, which have been reported to exhibit excellent antioxidant activity in Hibiscus plants [27,28]. Interestingly, Maciel et al. [29] have purified anthocyanins such as Dp3-Sam, Dp3-Glu, Cy3-Sam, and cyanidin-3-glucoside (Cy3-Glu) from a crude extract of the *H. sabdari*ff*a* calyx, which exhibited a higher antioxidant activity from the DPPH assay than that of the crude extract. The present study attempted to make use of the DPPH radical activity assay, but an unknown precipitate was produced in the reaction mixture, which a ffected the measurements in the extracts from some of the accessions. This might have been caused by the reaction of unknown compounds with the organic solvent (Table S1). Nonetheless, all accessions, except for the four aberrant accessions, tended to exhibit a higher antioxidant activity measured by the DPPH assay than that measured by the ABTS activity assay. The antioxidant and bioactive properties of anthocyanins have also been linked to health benefits such as anti-cancer, anti-inflammatory, and anti-diabetic activities [30–34]. Consequently, these results are consistent with previous studies, indicating that anthocyanin compounds were the main contributors to the antioxidant activity of *H. acetosella*.


**Table 3.** Antioxidant and antibacterial activities of 18 di fferent *H. acetosella* accessions.

(1) Control used were: ascorbic acid (500 μM) in antioxidant assay and gentamicin (5 μg) (antibacterial assay). (2) Inhibition zone of *H. acetosella* leaf extract against *Staphylococcus aureus* (Gram-positive) and *Pseudomonas aeruginosa* (Gram-negative). The results are shown as the mean ± standard error of three replicates. Mean with the same letter are not significantly different at the 5% probability level (Duncan's multiple range test). nd; not detectable.

#### *2.3. Inhibitory E*ff*ects of Phenolic Extracts against Gram-Positive and Gram-Negative Bacteria*

The inhibitory e ffects of the phenolic extracts of *H. acetosella* leaves on the Gram-positive (*Staphylococcus aureus* ATCC 6538) and Gram-negative (*Pseudomonas aeruginosa* ATCC 9027) bacteria are shown in Table 3. Distilled water, used as the negative control, exhibited no inhibitory e ffect against the two bacteria (Figure S2), but gentamicin (5 μg), used as the positive control, exhibited inhibition zones against the two bacteria of 12.80 ± 0.34 mm (*S. aureus*) and 13.10 ± 0.42 mm (*P. aeruginosa*). The phenolic extracts of the 18 *H. acetosella* accessions showed antibacterial activity against two bacteria, the zones of inhibition ranging from 12.00 to 13.67 mm (*S. aureus*) and from 10.67 to 13.33 mm (*P. aeruginosa*). For the *S. aureus* bacteria, all accessions exhibited a basal antibacterial activity level (12 mm), with PI500758 and PI500764 exhibiting an increased level of antibacterial activity (13.67 mm), but the Gram-negative (*P. aeruginosa*) bacteria exhibited a wider range of levels of antibacterial activity (Table 3). These antibacterial activities were exhibited similar levels against both Gram-positive and Gram-negative bacteria in *H. sabdari*ff*a* [4,31,35]. The present study first confirmed the presence of antibacterial activity against two bacteria among the phenolic extracts of the 18 di fferent *H. acetosella* accessions as a basis for optimization in future research using a formal antibacterial measuring method.

#### *2.4. Relationship between Phenolic Extracts and Biofunctional Properties*

The Pearson correlation coe fficient and hierarchical clustering were used to assess the relationship between the contents of phenolic compounds in the extracts and the biofunctional properties (antioxidant and antibacterial activities) in the 18 *H. acetosella* accessions (Table 4, Figure 2). The antioxidant activity (ABST) was significantly correlated with TAC (0.933), Dp3-Sam (0.932), Dp3-Glu (0.924), and Cy3-Sam (0.913) contents (*p* < 0.001). The TPC, TFC, and GC contents also exhibited significant correlation coe fficients ranging between 0.526 and 0.567 (*p* < 0.05). Consequently, the antioxidant activity in *H. acetosella* was strongly correlated with its contents of phenolic compounds, with the related anthocyanin contents also being involved in the antioxidant activity. In contrast, the antibacterial activity against Gram-positive bacteria was not significantly correlated with the content of phenolic compounds but antibacterial activity against Gram-negative bacteria was negatively correlated with the GAL content (−0.433, *p* < 0.05). However, the content of GAL is too low in *H. acetosella* to produce an antibacterial response. Overall, the antibacterial activity varied between the accessions, suggesting the involvement of other specific phenolic compounds present in their extracts. Borrás-Linares et al. [4] have also reported that the antimicrobial assay revealed no significant or a negative correlation between phenolic contents and antibacterial activity in *H. sabdari*ff*<sup>a</sup>*, suggesting a similar response. Hierarchical clustering classified the accessions and measurements according to their chemical and biofunctional similarities. The accessions divided into two clusters: the first cluster contained high contents of anthocyanins and the second cluster according to the status of the phenolic compounds and biofunctional properties. The measurements were classified into three clusters: cluster I contained antibacterial activities, cluster II contained phenolic compounds without anthocyanins, and cluster III contained antioxidant activity with anthocyanins (Figure 2). These results, as mentioned earlier, indicated that the antioxidant activity was strongly associated with the levels of anthocyanins such as TAC, Dp3-Sam, Dp3-Glu, and Cy3-Sam.


**Table 4.** Correlation coefficients between the contents of phenolic compounds and antioxidant/bacterial activities.
