Isolation, Characterization and Antibacterial Activity of 4-Allylbenzene-1,2-diol from Piper austrosinense

Isolation for antibacterial compounds from natural plants is a promising approach to develop new pesticides. In this study, two compounds were obtained from the Chinese endemic plant Piper austrosinense using bioassay-guided fractionation. Based on analyses of 1H-NMR, 13C-NMR, and mass spectral data, the isolated compounds were identified as 4-allylbenzene-1,2-diol and (S)-4-allyl-5-(1-(3,4-dihydroxyphenyl)allyl)benzene-1,2-diol. 4-Allylbenzene-1,2-diol was shown to have strong antibacterial activity against four plant pathogens, including Xanthomonas oryzae pathovar oryzae (Xoo), X. axonopodis pv. citri (Xac), X. oryzae pv. oryzicola (Xoc) and X. campestris pv. mangiferaeindicae (Xcm). Further bioassay results exhibited that 4-allylbenzene-1,2-diol had a broad antibacterial spectrum, including Xoo, Xac, Xoc, Xcm, X. fragariae (Xf), X. campestris pv. campestris (Xcc), Pectobacterium carotovorum subspecies brasiliense (Pcb) and P. carotovorum subsp. carotovorum (Pcc), with minimum inhibitory concentration (MIC) values ranging from 333.75 to 1335 μmol/L. The pot experiment showed that 4-allylbenzene-1,2-diol exerted an excellent protective effect against Xoo, with a controlled efficacy reaching 72.73% at 4 MIC, which was superior to the positive control kasugamycin (53.03%) at 4 MIC. Further results demonstrated that the 4-allylbenzene-1,2-diol damaged the integrity of the cell membrane and increased cell membrane permeability. In addition, 4-allylbenzene-1,2-diol also prevented the pathogenicity-related biofilm formation in Xoo, thus limiting the movement of Xoo and reducing the production of extracellular polysaccharides (EPS) in Xoo. These findings suggest the value of 4-allylbenzene-1,2-diol and P. austrosinense could be as promising resources for developing novel antibacterial agents.


Introduction
The medicinal importance of plants has led to the exploration of plant extracts that are commonly used as antibacterial agents, because plant pathogenic bacteria are devastating to plants all over the world, which can cause various disease symptoms including spots, blights of leaf and soft rots of fruits [1][2][3]. Such events may greatly compromise the quality and output of crops [4,5]. In recent years, the occurrence of bacterial diseases in crops is increasing with the change in climate and planting structure. In some areas, bacterial diseases are becoming the predominant diseases and seriously limit the development of the agricultural industry. Currently, only a few varieties of pesticides, including kocide and thiadiazole copper

Growth Curve of the 4-Allylbenzene-1,2-diol against Xoo
The effects of various doses of 4-allylbenzene-1,2-diol on the growth curve of Xoo are shown in Figure 3. Compared with the control, the log periods were positively correlated with the treatment concentrations. Meanwhile, 4-allylbenzene-1,2-diol at MIC completely inhibited the growth of Xoo after treatment for 12 h.

Growth Curve of the 4-Allylbenzene-1,2-diol against Xoo
The effects of various doses of 4-allylbenzene-1,2-diol on the growth curve of Xoo are shown in Figure 3. Compared with the control, the log periods were positively correlated with the treatment concentrations. Meanwhile, 4-allylbenzene-1,2-diol at MIC completely inhibited the growth of Xoo after treatment for 12 h.

SEM Observation
The morphological changes of Xoo after treatments with various concentrations of 4allylbenzene-1,2-diol are presented in Figure 5. Untreated Xoo cells (control) were rodshaped with a relatively smooth surface and uniform in shape ( Figure 5A), while the 4allylbenzene-1,2-diol treatment for 5 h at MIC resulted in the appearance of ruffle in cells ( Figure 5B). More apparent cell deformation was observed when the concentration of 4allylbenzene-1,2-diol treatment increased to 2 MIC ( Figure 5C). Moreover, Xoo cells treated with 4 MIC 4-allylbenzene-1,2-diol were deformed, collapsed, and wrinkled, while irregularly shaped holes were observed. ( Figure 5D).

Experimental Materials and Reagents
Leaves and stems of P. austrosinense were randomly collected in Xinglong Tropical Botanical Garden, Wanning City, Hainan Province, China (18°44′ N, 110°11′ E). The obtained plants were identified by the Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences. Eight phytopathogenic bacterial strains used for in vitro antibacterial screening included Xoo (bacterial leaf blight of rice), X. oryzae pv. oryzicola (Xoc, bacterial leaf streak of rice), Xac (citrus bacterial canker), X. campestris pv. mangiferaeindicae (Xcm, bacterial black spot of mango), X. fragariae (Xf, bacterial angular leaf spot of strawberry), X. campestris pv. campestris (Xcc, black rot of cabbage), Pectobacterium carotovorum subsp. brasiliense (Pcb, bacterial soft rot of potato) and P. carotovorum subsp. carotovorum (Pcc, bacterial soft rot of Chinese cabbage). All of the strains were incubated in 20% glycerol and preserved at −80 °C for further use. Strains were cultured on Luria-Bertani (LB) agar plates (containing 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 16 g of agar and 1 L of distilled water) or in LB broth without agar at 28 °C in the dark. All the bacteria were obtained from the Environment and Plant Protection Institute of the Chinese Academy of Tropical Agricultural Science (Haikou, China).

Extraction and Isolation
The leaves and stems (2 kg) of P. austrosinense were air-dried and powdered, and then macerated in 10 L methanol for 7 days under room temperature. After filtration, the filtrate was evaporated under hypobaric condition to yield the crude extracts. The methanol extracts (320 g) were re-suspended in water (1.8 L) and partitioned with petroleum ether (3 × 1.8 L), followed by extraction with ethyl acetate (2 × 1.8 L). The petroleum ether and ethyl acetate solutions were evaporated under hypobaric conditions to produce the petroleum ether-extracted residues (43.85 g) and ethyl acetate-extracted (58.29 g). Preliminary experimental results demonstrated that the petroleum ether extract had potent antibacterial activity against Xoo, Xoc, Xac and Xcm. Petroleum ether extract was subjected to passing through a column of MCI gel (methanol-H2O: 70-100%) to obtain five components (Fr. A-Fr. E). Fr. C and Fr. D were separated on a column of Sephadex LH-20 column and eluted with methanol to obtain two fractions (Fr. CD1 and Fr. CD2). Afterward, Fr. CD1 was separated on a silica gel column by an elution with petroleum ether: ethyl acetate (15:1) to obtain compound 1 (30.0 mg). Fr. CD2 was applied to a silica gel column (petroleum ether/ethyl acetate =3:1) to yield compound 2 (14.3 mg). The characteristics of both compounds were identified as follows:

Experimental Materials and Reagents
Leaves and stems of P. austrosinense were randomly collected in Xinglong Tropical Botanical Garden, Wanning City, Hainan Province, China (18 • 44 N, 110 • 11 E). The obtained plants were identified by the Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences. Eight phytopathogenic bacterial strains used for in vitro antibacterial screening included Xoo (bacterial leaf blight of rice), X. oryzae pv. oryzicola (Xoc, bacterial leaf streak of rice), Xac (citrus bacterial canker), X. campestris pv. mangiferaeindicae (Xcm, bacterial black spot of mango), X. fragariae (Xf, bacterial angular leaf spot of strawberry), X. campestris pv. campestris (Xcc, black rot of cabbage), Pectobacterium carotovorum subsp. brasiliense (Pcb, bacterial soft rot of potato) and P. carotovorum subsp. carotovorum (Pcc, bacterial soft rot of Chinese cabbage). All of the strains were incubated in 20% glycerol and preserved at −80 • C for further use. Strains were cultured on Luria-Bertani (LB) agar plates (containing 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 16 g of agar and 1 L of distilled water) or in LB broth without agar at 28 • C in the dark. All the bacteria were obtained from the Environment and Plant Protection Institute of the Chinese Academy of Tropical Agricultural Science (Haikou, China).

Extraction and Isolation
The leaves and stems (2 kg) of P. austrosinense were air-dried and powdered, and then macerated in 10 L methanol for 7 days under room temperature. After filtration, the filtrate was evaporated under hypobaric condition to yield the crude extracts. The methanol extracts (320 g) were re-suspended in water (1.8 L) and partitioned with petroleum ether (3 × 1.8 L), followed by extraction with ethyl acetate (2 × 1.

In Vitro Antibacterial Bioassay
The antibacterial activities of 4-allylbenzene-1,2-diol against Xoc, Xac, Xcm, and Xoo were measured according to the method of Zhao et al., 2019 [28], with some modifications. Compounds 1 and 2 were dissolved in sterile distilled water with 1% acetone and diluted to a final concentration of 1000 µmol/L. Sterile distilled water with 1% acetone was used as the negative control, and Kasugamycin (Shanghai Macklin Biochemical Technology Co. Ltd., Shanghai, China) was used as the positive control. Approximately 10 µL of bacteria suspensions cultured on the phase of logarithmic growth was added to 190 µL LB containing tested compounds. The cultures were inoculated at 28 • C at 180 rpm for 12-18 h. The OD 600 value was recorded to evaluate the bactericidal activity. Each treatment was repeated three times.
4-Allylbenzene-1,2-diol determination of the bactericidal spectrum was set up identically to that of the antibacterial activity assay in vitro. Antibacterial activities of 4allylbenzene-1,2-diol on 8 phytopathogenic bacteria, including Xoo, were evaluated using turbidity assays. The 4-allylbenzene-1,2-diol was tested at final concentrations of 250 µmol/L and 500 µmol/L respectively. The OD value was measured after incubation to evaluate the antibacterial activity.

Determination of the Minimum Inhibitory Concentration (MIC)
The activities of 4-allylbenzene-1,2-diol against Xoo, Xoc, Xac, Xcm, Xf, Xcc, Pcc, and Pcb were examined by referring to the twofold dilution method, by which the minimum inhibitory concentrations were obtained [29]. The bacterial suspension (OD 600 = 0.6) was added to the drug-contained medium to get the concentrations of 2670, 1335, 667.5, 333.75, 166.88, 83.44, 41.72, and 20.86 µmol/L, respectively. Kasugamycin at 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 µmol/L were used as positive controls, while 1% acetone was used as a negative control. Finally, the 96-well plate was incubated for 12 h at 28 • C in the incubator, and the lowest concentration was recorded as MIC when the blank control group became turbid. All measurements were repeated three times.

Growth Curve Assay
The effect of 4-allylbenzene-1,2-diol on the growth curve of Xoo was determined according to a previous method [30] with some modifications. 4-Allylbenzene-1,2-diol was dissolved in sterilized distilled water with 1% acetone and added to the culture medium to obtain final concentrations of 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC, and MIC. Sterilized distilled water with 1% acetone was used as a blank control. The bacterial suspension (OD 600 = 0.6) was inoculated into LB medium-contained agents. Cell densities were monitored by measuring the optical density at 600 nm every 12 h during the cultivation of 84 h. All measurements were conducted in triplicate and means were considered.

In Vivo Antibacterial Activity against Xoo
The protective and curative activities of 4-allylbenzene-1,2-diol against rice bacterial leaf blight in potted plants were evaluated under greenhouse conditions. The experimental procedures followed the reference [31] with slight modifications. Rice seeds of 'Xiangliangyou 900' were germinated in the greenhouse and grown for 5 weeks. 4-Allylbenzene-1,2-diol in acetone was diluted with sterile distilled water (containing 0.1% Tween-20) to final concentrations of 2 MIC (667.5 µmol/L) and 4 MIC (1335 µmol/L). Sterile distilled water with 0.1% Tween-20 and 1% acetone served as blank controls, while kasugamycin (2% aqueous solution, 2000 µmol/L) was used as a positive control. For the protective activity experiment, the tips of rice leaves were cut using sterile scissors, and the Xoo in the logarithmic growth phase was inoculated at 24 h after evenly spraying 4-allylbenzene-1,2-diol solutions. In the curative activity experiment, the solutions were sprayed on the leaves at 24 h after inoculation. Additionally, the assay was repeated three times, with seven plants for each treatment. Inoculated rice plants were placed in a greenhouse at 70-80% relative humidity and 28 ± 2 • C. The disease index of the inoculated leaves was evaluated and photographed for 15 days. The degree of disease was graded as follows: level 0, no onset; level 1, lesions accounted for less than 1-5% of the leaf area; level 3, lesions accounted for 6-15% of the leaf area; level 5, lesions accounted for 16-25% of the leaf area; level 7, lesions accounted for 26-50% of the leaf area; level 9, lesions accounted for more than 50% of the leaf area. The disease index and control effect were calculated based on the following formula; see Equations (1)

Scanning Electronic Microscope (SEM)
Sample preparation for scanning electron microscopy was carried out according to the method provided by Liu et al., 2021 [32] with slight modifications. The bacterial sus pensions (OD 600 = 0.6) were washed three times with 0.1 mol/L phosphate buffer (pH 7.2) and resuspended. Thereafter, 4-allylbenzene-1,2-diol was added to the bacterial suspension to make the final concentrations reaching to MIC, 2 MIC and 4 MIC, respectively, and then the mixture was shaken at 180 rpm for 5 h at 28 • C. The cells were obtained after centrifugation at 5000 rpm for 5 min, and then washed three times with 0.1 mol/L PBS (pH 7.2). Subsequently, the bacterial cells were fixed to dehydrate with 2.5% glutaraldehyde at 4 • C for 12 h, and then washed 3 times with 0.1 mol/L PBS (pH 7.2), dehydrated with 30%, 50%, 70%, 80%, 90% and 100% ethanol solution for 15 min in sequence, and freezedried for 12 h. Finally, the samples were flattened and sprayed with gold. A SEM (Quorum Technologies, SC7620, East Sussex, UK) was used to observe the morphological change of the bacterial membrane.

Membrane Permeability
The permeability of the bacterial membrane was expressed in the relative electric conductivity that was measured using the method of Ernst et al., 2000 [33] with minor modifications. Bacteria were cultured at 28 • C until the logarithmic growth phase, followed by centrifugation at 2000 rpm for 20 min. The supernatant was discarded, and the cells were re-suspended with sterile water. The concentration of bacterial suspensions was approximately 10 8 CFU/mL. Different concentrations of 4-allylbenzene-1,2-diol were respectively added to the bacterial suspension and incubated at 37 • C for 24 h. Conductivity was measured at 0, 2, 4, 6, 8, 10 and 24 h after additions of 4-allylbenzene-1,2-diol and recorded as L 1 . The conductivity of the mixture in boiling water for 10 min was recorded as L 2 . The cell membrane permeability was calculated using the following formula 3: Membrane permeability (%) = L 1 L 2 × 100% (3)

Bacterial Motility Assay
Bacterial motility was measured using a swimming assay according to the method of Di et al., 2008 [34]. Cultures of Xoo (OD 600 = 0.6) were prepared. The LB solid medium containing 0.3% agar powder was heated in a microwave oven and boiled until completely dissolved. After cooling down to 40 • C, the reagent containing 4-allylbenzene-1,2-diol was added to the culture medium, with the final concentrations being 1/4 MIC, 1/2 MIC, MIC and 2 MIC, respectively. The overnight cultured bacterial suspension containing 4-allylbenzene-1,2-diol was drop-inoculated to the center of semisolid medium plates and incubated for 48 h at 28 • C. The bacterial solution without containing 4-allylbenzene-1,2diol served as a blank group. The motility of bacterial cells was evaluated by measuring the diameter of the longest bacterial circles, and the measurement was repeated three times.

Assay of Biofilm Formation
The biofilm formation assay was performed based on the crystal violet staining method, as described by Du et al., 2018 [35] with slight modifications. The overnight cultured bacterial suspension (OD 600 = 0.6) was inoculated into the LB medium containing 4-allylbenzene-1,2-diol whose final concentration was 1/4 MIC, 1/2 MIC, MIC and 2 MIC, respectively. For promoting the growth of biofilms, the mixed cultures in glass tubes were incubated at 28 • C for 5 d. After that, the culture medium was poured out and gently washed three times with distilled water. The cultures in each glass tube were stained with 2.5 mL of crystal violet (0.1%) for 30 min. After staining, the glass tubes were washed three times with 0.1 mol/L PBS (pH 7.2) to remove excess stains. Finally, the crystal violet-stained cells were solubilized with glacial 3 mL of glacial acetic acid. The absorbance of the biofilm was measured at 590 nm. Three replicates were performed.

Extracellular Polysaccharide (EPS) Production
EPS production was determined according to previous reports [22,36]. The bacterial cells were shakenly (180 rpm) cultured in LB media containing different concentrations (1/4 MIC, 1/2 MIC, MIC and 2 MIC) of 4-allylbenzene-1,2-diol for 72 h at 28 • C. Afterward, the cultures were centrifuged at 3000 rpm for 20 min, and the supernatants were collected. Finally, the supernatants were mixed with three-fold volumes of absolute ethanol and incubated overnight to precipitate EPS. The obtained EPS was pelleted via centrifugation and desiccation. The assay was performed three times.

Statistical Analyses
Data were presented as means ± standard deviations, and the data were subjected to variance analysis using the SPSS software (version 20.0, IBM Corp., Armonk, NY, USA). Significant differences between means were analyzed using Duncan's Multiple Range Test at 0.05 levels. All assays for evaluating the activity of 4-allylbenzene-1,2-diol were performed with three replicates. The graphs were generated using Sigma Plot (version 12.5, Systat Software Inc., San Jose, CA, USA).
4-Allylbenzene-1,2-diol is a simple phenolic compound that has been reported to have a variety of biological functions such as antifungal, anticancer and antioxidant properties [37]. Ali et al., 2010 [38] reported that 4-allylbenzene-1,2-diol is the most active compound extracted from Piper betle L. and has antibacterial properties, particularly for treating topical infections. According to a report by Sharma et al., 2009 [39], 4-allylbenzene-1,2-diol may be a promising compound that is developed as an antibacterial agent to treat oral diseases. In addition, 4-allylbenzene-1,2-diol was clinically confirmed to increase the sensitivity of bacteria to antibiotics due to its property of damage to the membrane in bacteria [40]. These findings indicate that 4-allylbenzene-1,2-diol is a promising functional compound. However, the bactericidal activity of 4-allylbenzene-1,2-diol against plant pathogens has not been reported. In our study, in vitro activity results showed that 4-allylbenzene-1,2-diol had good bactericidal activities against eight plant pathogenic bacteria, especially for Xoo and Xoc, with MIC values of 333.75 µmol/L, which were lower than that of kasugamycin. Kasugamycin has systemic activity and has been widely used to control disease in rice [41]. Thus, we used kasugamycin as the positive control to evaluated in vivo controlled efficacy of 4-allylbenzene-1,2-diol against Xoo on rice. 4-Allylbenzene-1,2diol was found to have excellent protective activity, but its curative activity was relatively poor, which indicates that 4-allylbenzene-1,2-diol had a poor systemic activity or permeability in rice leaves, and its bactericidal activity might be induced by directly contacting with the pathogen. Furthermore, we explored the mechanism of bactericidal action 4allylbenzene-1,2-diol in vitro. The results of SEM showed that 4-allylbenzene-1,2-diol caused the concaves and perforations in bacteria, which indicated that 4-allylbenzene-1,2diol might disrupt cell membrane integrity. Moreover, 4-allylbenzene-1,2-diol promoted the increase in the relative conductivity of Xoo in a dose-effect manner, which further confirmed that this active compound could trigger damage to the cell membrane, resulting in the leakage of cellular contents. These findings indicated that the bactericidal mechanism of 4-allylbenzene-1,2-diol against plant pathogenic bacteria could be related to its damage to the cell membrane, which was consistent with the results obtained by Singh et al., 2021 [40].
Further results showed that 4-allylbenzene-1,2-diol limited the movement of Xoo in addition to inhibiting the growth of pathogenic bacteria. The swimming mobility of plant pathogenic bacteria is considered to be directly correlated with pathogenicity, and is also able to promote the formation of biofilm while helping the interaction between the bacterium and host, thus serving to enhance the infectious ability of the bacteria [42]. In this study, the swimming mobility of Xoo decreased by more than 50% when applied 4-allylbenzene-1,2-diol at the minimum concentration (1/4 MIC). Interestingly, 4-allylbenzene-1,2-diol showed a weaker inhibition on the growth of Xoo under the same conditions (1/4 MIC, 48 h), which indicated that the motility of Xoo could be more sensitive to 4-allylbenzene-1,2-diol. Tans-Kersten et al., 2001 [43] found that the loss of motility could significantly reduce the pathogenicity of Ralstonia solanacearum-caused bacterial wilt disease in tomato plants. In this study, the decreased infectious ability of Xoo due to the treatment of 4-allylbenzene-1,2-diol could be associated with the inhibition of swimming mobility. The formation of bacterial biofilms can enhance the tolerance of bacteria to bactericides, which increases the difficulty of preventing and controlling the infection of bacteria [44,45]. Exocellular polysaccharides, as one of the main constituents of biofilm, are closely related to the pathogenicity of bacteria of plant pathogens [46][47][48]. Chen et al. 2016 [49] reported that the natural product resveratrol inhibited the formation of biofilms of Ralstonia solanacearum, which contributed to the improvement of antibacterial ability. In our study, 4-allylbenzene-1,2-diol inhibited the biofilm and reduced the exopolysaccharide production of Xoo. Based on the above results, we speculated that the antibacterial effect of 4-allylbenzene-1,2-diol against Xoo might be associated with reduced pathogenicity by inhibiting polysaccharide synthesis and secretion, bacterial swimming mobility, and biofilm formation.

Conclusions
In this study, 4-allylbenzene-1,2-diol, an active compound was separated from the endemic plant P. austrosinense in China, which exhibited strong antibacterial activity against plant pathogenic bacteria with a broad spectrum. The antibacterial mechanism of 4-allylbenzene-1,2-diol might involve the loss of cell membrane integrity and reduced pathogenicity in plant pathogens. The results suggested that 4-allylbenzene-1,2-diol and the medicinal plant P. austrosinense could be potential sources of developing novel bactericides. Further studies will aim to elucidate the antibacterial molecular mechanisms as well as investigate their controlling efficiency in field conditions. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28083572/s1, Figure S1: Picture of Piper austrosinense; Figure S2: The chemical structures of kasugamycin.