Chemical Composition, Antioxidant, and Antibacterial Activity of Wood Vinegar from Litchi chinensis

The antioxidant and antibacterial activities of wood vinegar from Litchi chinensis, and its components have been studied. The chemical compositions of wood vinegar were analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 17 chemical compounds were identified, representing 83.96% of the compositions in the wood vinegar. Three major components, included 2,6-dimethoxyphenol (syringol, 29.54%), 2-methoxyphenol (guaiacol, 12.36%), and 3,5-dimethoxy-4-hydroxytoluene (11.07%), were found in the wood vinegar. Antioxidant activities of the acids were investigated from the aspects of 1,1-Diphyl-2-picrylhydrazyl (DPPH) free radicals scavenging capacity, superoxide anion radical scavenging capacity, and reducing power. The pyroligneous acid exhibited high antioxidant activity which was comparable to the reference standards (vitamin C and butylated hydroxyl toluene) at the same dose with IC50 values of 36.5 ppm calculated by the DPPH radical scavenging assay, 38.38 g Trolox equivalent/100 g DW by the trolox equivalent antioxidant capacity (TEAC) assay, and 67.9 by the reducing power analysis. Antibacterial activity was evaluated using the disc diffusion and microdilution methods against a group of clinically antibiotic resistant isolates. The major components exhibited broad spectrum inhibition against all the bacterial strains with a range of disc inhibition zoon between 15–19 mm. The minimum inhibition concentration and minimum bactericide concentration against the test strains was ranging in 0.95–3.80 μL/100 μL and 1.90–3.80 μL/100 μL, respectively. Most of the antibiotic resistant strains were more susceptible to the wood vinegar than the non-antibiotic resistant strain except the strain of ornithine resistant Staphylococcus aureus. Based on the chemical profile, it was considered that the strongest antioxidant and antibacterial activity of Litchi chinensis wood vinegar was due to its highly phenolic compositions. This study revealed that the Litchi chinensis wood vinegar is valuable to develop as alternative food antioxidant and antibiotics.


Introduction
Wood vinegar, also called pyroligneous acid, is a brown, flavorful liquid produced by distillation of wood in the absence of air condition. When the gas generated from combustion is cooled, it condenses into liquid. The carbonization of many different types of wood can be used to produce various wood
As shown in Table 1, the GC-MS analysis revealed a total of 17 chemical constituents of wood vinegar, most of which were phenol compounds.

Antibacterial Ability
The results for the in vitro antibacterial properties of the wood vinegar are presented in Table 2. According to the disc inhibition assay, all of the test pathogens were sensitive to the wood vinegar with a range of disc inhibition zone (DIZ) between 15 to 19 mm, indicating that the wood vinegar possesses a broad antibacterial spectrum against different pathogens, with a maximum DIZ (19 mm) against the clinical isolate of Staphylococcus aureus, followed by Acinetobacter baumannii, Pseudomonas aeruginosa and Ornithine resistant Staphylococcus aureus with slightly smaller DIZ values (16-17 mm).  Among the phenolic components, catechol is probably produced via the demethylation of guaiacol and 3-methoxy-1,2-benzenediol can be obtained from the demethylation of syringol [15]. The results shown in Table 1 indicate that the major lignin units of the wood vinegar were sinapyl alcohol and coniferyl alcohol.

Antibacterial Ability
The results for the in vitro antibacterial properties of the wood vinegar are presented in Table 2. According to the disc inhibition assay, all of the test pathogens were sensitive to the wood vinegar with a range of disc inhibition zone (DIZ) between 15 to 19 mm, indicating that the wood vinegar possesses a broad antibacterial spectrum against different pathogens, with a maximum DIZ (19 mm) against the clinical isolate of Staphylococcus aureus, followed by Acinetobacter baumannii, Pseudomonas aeruginosa and Ornithine resistant Staphylococcus aureus with slightly smaller DIZ values (16-17 mm). . aeruginosa > E. coli~S. typhi. Their results are consistent to our finding that the Gram-positive strain S. aureus was the most susceptible, while the Gram-negative E. coli showed only light susceptibility to the wood vinegar. Many previous reports suggest that Gram-positive bacteria are generally more susceptible to plant extracts than Gram-negative bacteria because of the absence of an outer membrane of lipoprotein and lipopolysaccharide. An outer membrane of lipoprotein and lipopolysaccharide is selectively permeable and can regulate access of antimicrobials into the underlying cell structures [19]. However, the Gram-positive strain ORSA showed the lowest susceptible to the wood vinegar, and it is presumed that strong biofilm formers (ORSA) might result in stronger drug resistance [20]. Other wood vinegar sources including bamboo, eucalyptus, and rubber also exhibited antimicrobial activity against dermatitis, environment, and plant bacteria, as well as fungi [21]. A concentration of less than 10% is suggested as suitable for use as an antimicrobial agent while preventing leaf burning and some pathogenic fungi on PDA.

Antioxidant Assay
The antioxidant assay results are shown in Table 3. In the DPPH radical scavenging ability assay, the DPPH IC 50 value (the concentration required to scavenge DPPH radical by 50%) of wood vinegar was about 36.53 ppm ( Table 3). The DPPH IC 50 values of butylated hydroxyl toluene (BHT) and vitamin C were about 175 ppm and 7 ppm, respectively. The result indicates that the radical scavenging ability of wood vinegar is higher than the commercial chemical antioxidant (BHT), but lower than the natural antioxidant (vitamin C). The reducing power analysis found that the reducing power increased with the concentration of each sample. The ranking order for reducing power was wood vinegar (67.9 abs/10 −3 ppm) > Vitamin C (7.3 abs/10 −3 ppm) > BHT (2.2 abs/10 −3 ppm). Significantly, the wood vinegar exhibited higher reducing power than the commercial antioxidants (BHT and vitamin C). For the Trolox equivalent antioxidant capacity (TEAC) assay, the wood vinegar (38.38 g Trolox/100 g DW) and Vitamin C (38.47 g Trolox/100 g DW) showed similar TEAC values that were both higher than that of BHT (35.64 g Trolox/100 g DW). The TEAC assay results indicate that wood vinegar possesses antioxidant ability similar to that of vitamin C and higher antioxidant capacity than BHT. The total phenolic and flavonoid contents in the wood vinegar were respectively 37.34 g gallic acid/100 g DW and 4.42 g quercetin/100 g DW (Table 3). These results imply that the wood vinegar contains a high quantity of phenolic compounds and flavonoids, which is consistent with the results obtained from GC-MS analysis. Previous studies have shown that phenolic compounds exhibit strong free radical scavenging capability, reducing power, and antioxidant capability and can, therefore, be used as reductants and antioxidants. Plant phenolic and flavonoid components are widely distributed in the tissues of plants, and also play a pivotal role in highly effective bioactivity. Due to the side effects of chemical synthetic antioxidants (such as BHT and BHA), more attention is now focused on the antioxidant activity of phenolic compounds from plants. Previous reports have verified that the pyroligneous acid exhibits significant antioxidant activity. Ma et al. [22] found that the fruit of S. chinensis exhibits superoxide anion scavenging activity and antioxidant activity. Loo et al. [5,6] demonstrated that pyroligneous acid from the mangrove plant and Rhizophora apiculate exhibit antioxidant and free radical scavenging activities. All these results indicate that pyroligneous acid could potentially be a natural antioxidant.
Many studies have indicated that pyroligneous acid is rich in phenolic compounds [10][11][12], which are pyrolytic products of lignin and hemicellulose, comprising 30%-60% of the total organic compounds in the acid. 2,6-dimethoxyphenol, also known as syringol, has been identified as having a woody/herby flavor and smoky odor [23]. Syringol exhibited antioxidant activities based on its DPPH radical scavenging activity, ABTS radical cation scavenging activity, phosphomolybdenum and ferric reducing antioxidant power [6]. In addition, 3-Methyl-1,2-cyclopentanedione may be regarded as a potent regulator of (ONOO-)-mediated diseases via direct scavenging of the reactivity of and ONOOcan prevent (ONOO-)-induced damage of GSH reductase [24]. Loo et al. reported the isolation of three antioxidative compounds (syringol, catechol, and 3-methoxycatechol) from mangrove wood vinegar [5] which is consistent to our finding in the Lichi wood vinegar. All of these results indicate that the pyroligneous acid from Lichi has potential to be developed as a natural antioxidant.

Materials and Chemicals
The raw wood vinegar was prepared from Litchi chinensis using a traditional Japanese black charcoal kiln and collected by using running water through a shuttle (7-10 in length), which connected with the chimneys, to condense the smoke. Pyroligneous acids were collected from a temperature range of 100-600 • C and prepared by the Bu-Quang charcoal company (Kaohsiung, Taiwan). The raw wood vinegar was stored in the dark at 4 • C for analysis. All chemicals (1,1-diphyl-2-picrylhydrazyl (DPPH), Molecules 2016, 21, 1150 7 of 10 butylated hydroxytoluene (BHT), trichloroacetic acid (TAC), ascorbic acid, Folin Ciocalteu's reagent, potassium ferricyanide, ferric chloride, sodium carbonate, and all analytical chemicals) were purchased from Sigma (Sternheim, Germany). The clinical antibiotic resistant strains (Staphylococcus aureus 985, Acinetobacter baumannii 814, Pseudomonas aeruginosa 717, and ornithine-resistant Staphylococcus aureus 220) used in this research were isolated from blood and phlegm samples provided by Chia-Yi Christian Hospital (Chia-Yi, Taiwan). The standard strain (Escherichia coli ATCC 25257) was purchased from the Taiwan s Food Industry Research and Development Institute Bioresources Collection and Research Center (Hsinchu, Taiwan).

Instrument
Analysis used an Agilent 7890B gas chromatography instrument, combined with an Agilent-5977A mass spectrometer (Agilent Technologies) equipped with electron ionization (EI) and quadrupole analyzer, and an Agilent Chem Station data system. GC separation was performed on a 30 m HP-5ms Ultra Inert capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25 µm (Agilent 19091S-433UI, Agilent Technologies).

Phytochemical Composition Analysis
The components of wood vinegar were subjected to GC-MS analysis on an Agilent system consisting of a model 7890B gas chromatographer and a model 5977A mass selective detector (MSD, electron energy, 70 eV). The carrier gas was helium (99.99%) with a flow rate of 0.8 mL/min. The injector and detector temperatures were respectively set at 250 • C and 250 • C. Spectra were obtained over a scan range of 50 to 550 amu at 2 scans/s. The GC program was set as follows: the initial temperature was 60 • C and held for 10 min, then increased by 2 • C/min to 80 • C and held for 5 min, then raised by 2 • C/min to 110 • C and held 6 min, then raised by 2 • C/min to 120 • C and held for 5 min, and finally raised by 2 • C/min to 180 • C and held at 180 • C for 2 min. The wood vinegar (1.0 µL) was injected automatically while maintaining a solvent delay of 4 min. Interpretation of the mass spectrum was made by comparing the peak distribution against the database of National Institute Standard and Technology (NIST MS 14.0, Gaithersburg, MD, USA). Relative percentages of the chemical compositions were calculated based on the GC peak areas without correction.

Antibacterial Activity Assay
Disc Inhibitory Assay A Petri dish was prepared with a base layer of Muller Hinton (MH) agar (10 mL) and a top layer of 0.75% MH agar (5 mL), then inoculated with 50 µL of each bacterial suspension (10 5 CFU/mL). Paper discs (6 mm in diameter) were impregnated with 30 µL of wood vinegar and placed on the inoculated plates, then incubated at 37 • C for 14 h. The diameters of the inhibition zones (DIZ) were measured [25].

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
A broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) [25]. The wood vinegars (50, 100, 125, 150, and 200 µL) were diluted in 5 mL LB broth. Each strain (10 6 CFU/mL) was added and incubated at 37 • C at 220 rpm for 16 h. Subsequently, 100 µL of the culture mixture was coated on the MH agar medium, and the colony numbers were counted after incubating for 16 hrs at 37 • C. The MIC is defined as the concentration level that can inhibit 90% of bacteria development, with 99% bacterial inhibition set as the MBC.

Determination of Total Phenolic Content
The total phenolic content of wood vinegar was determined using the Folin-Ciocalteu method using gallic acid as a standard [26]. Briefly, 0.1 mL of wood vinegar was added into 0.1 mL of 25% Folin-Ciocalteu reagent and 2 mL of 2% Na 2 CO 3 solution with thorough shaking. After reacting at room temperature for 30 min, the absorbance was measured at 760 nm and the phenolic content was calculated according to the equation that was obtained from the standard gallic acid.

Determination of Flavonoids Content
The total flavonoid content of wood vinegar was determined using the method described as our previous study [26] with slight modifications. Briefly, one mL of the solution containing 1 mg/mL wood vinegar with methanol was added into 0.1 mL of 10% aluminium nitrate, 0.1 mL of 1 M potassium acetate and 3.8 mL of methanol. After reacting at room temperature for 40 min, the absorbance was read spectrophotometrically at 415 nm, using quercetin as a standard. The concentrations of flavonoid compounds were calculated using the equation obtained from the standard quercetin graph.

DPPH Free Radical Scavenging Ability
The DPPH free radical scavenging ability of wood vinegar was evaluated using the method described as our previous study [26] with some modifications. Briefly, one mL of various concentrations of wood vinegar in methanol was added to 0.25 mL of DPPH solution in methanol. The mixture was shaken and allowed to stand for 30 min in room temperature. The absorbance of the resulting solution was then measured at 517 nm with a spectrophotometer. The percentage of DPPH free radical inhibition was calculated with the following equation: DPPH Inhibition (%) = 1 − Abs. of Exp. − Abs. of Comp. Abs. of control × 100% (1)

Reducing Power
The reducing power of wood vinegar was determined using the method described as our previous study [26] with some modifications. Briefly, 150 µL of wood vinegar in methanol was mixed with 150 µL of 200 mM sodium phosphate buffer (pH 6.6) and 150 µL of 1% K 3 Fe(CN) 6 . The mixture was then incubated at 50 • C for 20 min. Subsequently, 150 µL of 10% trichloroacetic acid, 600 µL of double deionized water, and 600 µL of FeCl 3 were added and the mixture was incubated for a further 14 min. The absorbance of each reaction mixture was measured at 700 nm. A higher absorbance indicates a higher reducing power.

Trolox Equivalent Antioxidant Capacity
The Trolox equivalent antioxidant capacity (TEAC) of wood vinegar was determined as our previous study [26] with some modifications. In brief, 20 µL of various concentration of wood vinegar in methanol was mixed with 1 mL of 0.175 mM ABTS solution, and the mixture was shaken and allowed to stand for 10 min at room temperature. The absorbance of the resulting solution was then measured at 734 nm with a spectrophotometer. Percentage of ABTS radical inhibition was calculated with the following equation: 3.3.6. Statistical Analysis Data were expressed as mean and standard deviation (SD). A one way variance was used to analyze data, with p < 0.05 representing a significant difference between means (Duncan's multiple range test).

Conclusions
This is the first study of the chemical composition, and antibacterial and antioxidant activities of wood vinegar from Litchi chinensis. The results reveal that Lichi wood vinegar contains seventeen compounds in various concentrations. More than 70% of the components were identified as phenolic compounds which could be classified into the syringol-type (42.2%), guaiacol-type (16.55%), and the benzenediol-type (12.59%). Antioxidant activity assay results showed significant antioxidant activity comparable to commercial antioxidants. Moreover, the wood vinegar showed significant antibacterial activity against clinical antibiotic-resistant pathogens, which implies they can be developed into useful sterile products for medical, aquaculture, and livestock breeding applications. The results suggest the antioxidant and antibacterial activities of Litchi chinensis wood vinegar are contributed by the three major phenolic compounds, 2,6-dimethoxyphenol (Syringol, 29.54%), 2-methoxyphenol (guaiacol, 12.36%), and 3,5-dimethoxy-4-hydroxytoluene (11.07%). The chemical constituents of the wood vinegar must be identified to maximize utilization.