Evaluation of antibacterial activity of compounds isolated from the peel of Newhall navel orange

Abstract Fifteen compounds including one flavanone hesperitin (1), two cinnamic acid derivatives as (E)-4-hydroxycinnamic acid (2) and (E)-ferulic acid (3), eight polymethoxyflavones (PMFs) (4-11), and four hydroxylated PMFs (12-15) isolated from orange peel were subjected to antibacterial evaluation. Compounds 1-3 exhibited wide-spectrum antibacterial effects against four test strains involving Bacillus subtilis, Staphylococcus aureus, Xanthomonas citri subsp. citri (Xcc), and Meticillin-resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentrations (MICs) ranging from 0.0312 to 0.125 mg/mL. In contrast, all PMFs showed weak antibacterial activity against these four strains with MICs being equal to or more than 0.125 mg/mL. Hydroxylated PMFs demonstrated better antibacterial effect against Xcc relative to PMFs. In addition, the synergistic effect against Xcc was obtained when compounds 1 and 3 were combined. Furthermore, the scanning electron microscopy (SEM) results of Xcc treated with both compounds 2 and 3 showed shrunken and rough surface morphologies, indicative of the cell membrane damage. Graphical Abstract


Introduction
The by-products of citrus fruits such as peel, seed and leaf are important resources of nutraceuticals since they are rich in biologically active compounds and exhibit versatile biological effects (Uysal et al. 2016;Russo et al. 2021). Newhall navel orange is a kind of seedless sweet orange widely planted in Ganzhou, Jiangxi province, China. The average annual output of Newhall navel orange produced in Ganzou is more than 1 million tons in recent years, occupying almost half of the total annual output of navel oranges in China (Guo et al. 2020). Previous investigations have indicated that orange peel contains essential oils, flavonoids, limonoids, phenolics, and pectins as its major chemical compositions, and has various biological effects including anti-inflammatory, antioxidant, and anticancer (M 0 hiri et al. 2017). The chemical compositions and biological activities of orange peel vary a lot depending on the orange species, cultivars, geography, and the growing conditions (Cano et al. 2008). To the best of our knowledge, despite the peels from other orange cultivars are extensively investigated, the chemical compositions and biological activities of peel from Newhall navel orange planted in Ganzhou are largely unknown.
Currently, the increasing prevalence of drug-resistant bacteria represents an alarming global threat to public health, therefore prompting the search for effective antibacterial agents with novel modes of action (Benameur et al. 2021). The antibacterial potential of phytochemicals has been increasingly recognised in recent years. It is found that plenty of phytochemicals significantly suppress or eradicate the pathogenic bacteria (S anchez-Maldonado et al. 2011;Aghraz et al. 2020). As part of our continuous efforts on the value-added utilisation of Newhall navel orange peel, its ethyl acetate extract was found to exhibit wide-spectrum antibacterial activity (Guo et al. 2020). However, the compounds underlined this activity remain unknown. Therefore, the present study reported the antibacterial potential of compounds isolated from the Newhall navel orange peel by using Bacillus subtilis, Staphylococcus aureus, Xanthomonas citri subsp. citri (Xcc), and Meticillin-resistant Staphylococcus aureus (MRSA) as test bacterial strains, along with a preliminary structure-activity relationship (SAR) study. In addition, the combined effects of the three most potent compounds (1-3) on Xcc were investigated. Moreover, SEM was applied to explore the possible action mechanism of both compounds 2 and 3 against Xcc.

Antibacterial effect of compounds isolated
As shown in Figure S1 (Supplemental material), fifteen compounds involving one flavanone (1), two cinnamic acid derivatives (2-3), eight PMFs (4-11), and four hydroxylated PMFs (12-15) were isolated from the peel of Newhall navel orange. The biological assays revealed that compounds 1-3 exhibited good and wide-spectrum antibacterial effects against all four test strains with minimum inhibitory concentrations (MICs) ranging from 0.0312 to 0.125 mg/mL (Table S1). In particularly, when compared to other compounds isolated, both compounds 1 (MIC 0.0625 mg/mL) and 3 (MIC 0.0625 mg/mL) were found to be the most potent bactericides against B. subtilis, and both compounds 2 (MIC 0.0312 mg/mL) and 3 (MIC 0.0312 mg/mL) exhibited the lowest MIC against S. aureus. Moreover, compound 3 (MIC 0.0312 mg/mL) was superior to all other compounds isolated in the antibacterial assays against both Xcc and MRSA. As for the PMFs, however, all these compounds exhibited weak antibacterial activity against all test strains with MICs being or over 0.25, 0.125, 0.125, and 0.25 mg/mL for B. subtilis, S. aureus, Xcc and MRSA, respectively. It was reported that PMFs such as tangeritin and 5,6,7,4 0 -tetramethoxyflavone showed low capacity in inhibiting the growth of Escherichia coli (Wu et al. 2013), which was in line with our current observations. In comparison to the PMFs, some hydroxylated PMFs demonstrated more potent antibacterial capacity as shown in the cases of compound 13 against Xcc (MIC 0.0625 mg/mL), compound 12 against S. aureus (MIC 0.0625 mg/mL), and compound 15 against Xcc (MIC 0.0625 mg/mL). A preliminary SAR study was then carried out, suggesting the following information: (1) the presence of a hydroxyl group only at C7 position increased the antibacterial potential of PMFs against B. subtilis as evidenced by the comparisons between compounds 12 with 5, and 13 with 8.
(2) as for the strain of Xcc, the replacement of methoxy with hydroxyl group was beneficial to the antibacterial activity of flavones, which was supported by the higher MIC values of PMFs relative to their corresponding hydroxylated PMFs, as shown in the comparison pairs of 5/12, 8/13, 5/ 14, and 9/15. Being consistent with the present study, a recent review by Shamsudin et al. (2022) stated that the presence of a hydroxyl group at either C5 or C7 tended to increase the antibacterial capacity of flavonoids, whereas the methoxylation was inclined to retard this intended effect. The phenolic acids 2 and 3 have been reported to show antibacterial activity against B. subtilis with a MIC value being 0.25 and 0.38 mg/mL, respectively (S anchez-Maldonado et al. 2011), generally agreeing with the present result. In addition, it was worth note that the present study firstly identified the antibacterial activity of compounds 1-3 against Xcc, and especially the compound 3, which was comparable to the positive drug CuSO 4 (MIC 0.0312 mg/mL), making it a lead compound for the development of novel bactericide against Xcc.

Combined effect of compounds 1-3 against xcc
The present study investigated the combined antibacterial effect of compounds 1-3 by using Xcc as an indicator strain since all these three compounds displayed excellent inhibitory effect on the growth of Xcc. As shown in Table S2, When the compounds 1-3 were combined with each other at an amount ratio of 1MIC:1MIC, only the combination of compounds 1 with 3 demonstrated synergistic effect with fractional inhibitory concentration index (FICI) value of 0.5, while the other two combinations showed additive effects with FICI value of 1 for each combination. The synergism against Xcc observed between hesperetin and (E)-ferulic acid could be due to their combined interactions with the same possible primary target site but using different mode of inhibitory action, or because of their simultaneous reactions with two relevant target sites on Xcc, similar to the antibacterial mode of action of some essential oils and their constituents (Hyldgaard et al. 2012). In addition, the direct interaction via intermolecular hydrogen bonds between these two compounds may lead to the changes of structural conformation, facilitating the bonding with Xcc, thus resulting in the enhancement of inhibitory activity. However, more studies need to be performed to elucidate the precise mechanisms responsible for this interaction.

Possible action mechanism investigated by SEM
The possible mechanism of action of both compounds 2 and 3 against Xcc was preliminarily investigated by scanning electron microscopy (SEM). As shown in Figure S2, SEM micrographs revealed that rough surface morphology and shrinkage of cell were apparent in the cells treated with compounds 2 and 3, when compared to the untreated ones (control), supporting that the bactericidal effect of these two compounds against Xcc might be associated with the cell membrane disruption and further blocking the growth of cells. The loss of membrane integrity resulted in the increment of membrane permeability, the disturbance of membrane-embedded proteins, and the alteration of ion transport processes of bacteria (Devi et al. 2010). Similar to the present observation, it was reported that ferulic acid led to irreversible changes in membrane properties of E. coli through hydrophobicity changes and the occurrence of local rupture in the cell membrane, thus causing inhibitory effect on its growth (Borges et al. 2013).

Conclusion
The present study evaluated the antibacterial effect of fifteen compounds isolated from the peel of Newhall navel orange by using B. subtilis, S. aureus, Xcc, and MRSA as test strains. Three compounds including hesperitin, (E)-4-hydroxycinnamic acid, and (E)-ferulic acid showed good and wide-spectrum bactericidal activity against all test strains. In contrast, all eight PMFs demonstrated weak inhibitory effect on the growth of these four strains. A preliminary SAR study suggested that the replacement of methoxy group with hydroxyl group was favorable to the antibacterial activity of PMFs against Xcc. In addition, a synergistic antibacterial effect against Xcc was found when hesperitin and (E)-ferulic acid were combined. Furth more, SEM revealed that the possible mechanism of antibacterial action of both (E)-4-hydroxycinnamic acid and (E)-ferulic acid against Xcc might be associated with the disruption of cell membrane. The present results provided useful information for the development of novel antibiotics and were beneficial to the valorisation of Newhall navel orange peel.

Disclosure statement
No potential conflict of interest was reported by the authors.