Antiviral Activity of Phenolic Derivatives in Pyroligneous Acid from

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Antiviral Activity of Phenolic Derivatives in Pyroligneous Acid from Hardwood, Softwood, and Bamboo Ruibo Li, Ryo Narita, Hiroshi Nishimura, Shinsuke Marumoto, Seiji P. Yamamoto, Ryota Ouda, Mitsuyoshi Yatagai, Takashi Fujita, and Takashi Watanabe ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01265 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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ACS Sustainable Chemistry & Engineering

Antiviral Activity of Phenolic Derivatives in Pyroligneous Acid from Hardwood, Softwood, and Bamboo

Ruibo Li§1, Ryo Narita§#1,2, Hiroshi Nishimura1, Shinsuke Marumoto1,3, Seiji P. Yamamoto‡1,2, Ryota Ouda1,2, Mitsuyoshi Yatagai4, Takashi Fujita2, Takashi Watanabe1* 1. Research Institute for Sustainable Humanosphere, Uji campus, Kyoto University, Gokasyo, Uji, Kyoto, 611-0011, Japan 2. Institute for Frontier Life and Medical Sciences, Kyoto University, Shogoin, Kawahara-Cho, Sakyo-Ku, Kyoto, 606-8507, Japan 3. Joint Research Center, Kinki University, 3-4-1 Kowakae, Higashi Osaka, Osaka, 577-8502, Japan 4. Emeritus Professor of Tokyo University, 7-3-1 Hongo, Bunkyo-Ku, Tokyo, 113-8656, Japan *

Corresponding author. *Tel: +81-774-38-3640. Fax: +81-774-38-3681. E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Pyroligneous acids (PA) from hardwood, softwood and bamboo significantly disinfected encephalomyocarditis virus (EMCV). Twenty-five kind of phenolic derivatives in the PAs were identified and quantified. The total amount of phenolic compounds in bamboo PA is higher than those in the PAs from softwood and hardwood. Phenol, 2-methoxyphenol, 2-methoxy-4-methylphenol, and 2-methoxy-4-ethylphenol are the most abundant compounds in the PAs examined. The activities of all the phenolic compounds against the encephalomyocarditis virus were assessed. The number of phenolic hydroxyl groups significantly affects the antiviral activity, and catechol and its derivatives exhibit higher viral inhibition effects than other phenolic derivatives. In addition, substituents affect the antiviral activity of the compounds. Phenolic compounds with a methyl group show higher activities than with a methoxyl group (e.g., 2-methylphenol > 2-methoxyphenol). Moreover, the relative position of functional groups also plays a key role in the viral inhibition activity (e.g., 2,6-dimethoxyphenol > 3,4-dimethoxyphenol). Thus, PAs contain phenol derivatives with considerable structural diversity and viral inhibition activities, providing a new strategy for virus-inactivation treatment through the optimization of PA-derived phenol structures.

KEYWORDS: bio-oil, viral inactivation, picornavirus, phenolic compound, lignin biomass, encephalomyocarditis virus

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INTRODUCTION Pyroligneous acids (PAs), also called wood vinegar, pyroligneous liquor, pyrolysis bio-oil, or liquid smoke, are the crude condensate of smoke produced through carbonization,1, 2 and consist of a pyrolyzate of cellulose, hemicelluloses, and lignin. Wood and bamboo PAs are complex mixtures of water, alcohols, organic acids, esters, aldehydes, ketones, phenolics, and nitrogen compounds.3-5 Acetic acid is the primary component of wood and bamboo PAs.6 Wood and bamboo PAs are used for sterilization, food additives, smoke flavoring and antimicrobial agents. Furthermore, it has been shown that bamboo PA exerts a promotional effect on the germination and radicle growth of some types of seeds, e.g., lettuce.7, 8 Interestingly, the chemical composition of PAs depends on the original wood species. For example, the chemical composition of moso bamboo (Phyllostachys pubescens) PA is different from that of madake bamboo (Phyllostachys bambusoides) despite their close phylogenetic relationship.7 In addition, preparation of crude PA critically affects its efficacy. Regarding its effect on the regulation of germination and growth, it is known that bamboo PA collected at temperatures of up to 250°C promotes radicle and hypocotyl growth, whereas bamboo PA collected at temperatures from 250°C to 400°C inhibits their growth.8, 9 Previous studies have shown that PA has the potential to modulate immune responses.10, 11 For example; cresol derived from Moso bamboo PA inhibits inflammasome activation through reactive oxygen species production and inactivation of the protein kinase C-α/δ.10 Furthermore, wood PA derived from sawtooth oak inhibits the phosphorylation of the signal transducer STAT3, and thus shows anti-inflammatory activity.11 The antioxidant activity of PAs has also attracted a great deal of attention.12-14 Sharma et al.12 investigated the antioxidant activity of PA extracted from birch wood under slow pyrolysis conditions and found that the phenolic fractions obtained 3



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from PA show comparable antioxidant activity to those of the commercial antioxidant butylated hydroxytoluene (BHT). A series of phenols in the phenolic fractions with molecular weights of 302, 316, and 344 corresponding to a group of dimer were suggested to be responsible for the observed antioxidant activities. In addition to their use as insecticidal and antimicrobial agents,15-17 it has been reported that PAs directly inactivate viruses such as the tobacco mosaic virus18 and the porcine reproductive and respiratory syndrome virus.19 Previously, we have demonstrated that the phenols in bamboo PAs exhibit antiviral activity against the encephalomyocarditis virus (EMCV), which belongs to the Picornaviridae family.20 We also proposed the synergistic effect of phenol and acetic acid against the EMCV. However, despite these studies on the antiviral activity of wood and bamboo PAs, the compounds responsible for the activities are not well understood. Phenolic compounds, one of the main components of PAs, are generated from lignin degradation during pyrolysis,21-23 and different wood species have different lignin structures. Thus, the phenolic compounds in PAs vary depending on the wood species.4, 24 In this study, we aimed to identify the antiviral phenolic compounds in PAs from softwood, hardwood, and bamboo, and elucidate the relationship between their structures and their antiviral activities.

MATERIALS AND METHODS Wood and Bamboo PAs. Six kinds of wood and bamboo PAs (A–F) from hardwood, moso bamboo, and softwood were used for this study. PA distillates (Ad, Bd, and Dd) were obtained by distilling the original PAs (e.g Ad was distilled from A). All the PAs were produced according to the guidelines set out by the Japan Mokusaku-eki Association. Briefly, during the wood and 4


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bamboo charcoal making process, the smoke from the funnel of the furnace were collected at temperature from 80 °C to 150°C monitored by a thermocouple at the exit of the smoke funnel, then cooling down.10 The obtained liquid was kept at room temperature for 3 months. After removing the upper and the bottom layer, the middle layer was PA. The pH values of the wood and bamboo PAs were measured with a pH meter (Horiba D-51) at room temperature. Detailed information on these PAs is given in Table 1. For gas chromatography-mass spectrometry (GC-MS) analysis, wood and bamboo PAs were diluted with an equal amount of methanol and filtered using 0.45 µm syringe filters (Star Lab Sci, Japan). A sample volume of 1.0 µL was injected into the GC-MS apparatus. For quantification of acetic acid, PAs were diluted 10 times with methanol. Chemicals.

Phenol,

2-methylphenol,

2-methoxyphenol,

2,4-dimethylphenol,

2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, BHT, 4-allyl-2,6-dimethoxyphenol, and ethyl acetate (EtOAc) were purchased from Wako Pure Chemical Industry Co., Ltd. (Osaka, Japan). 3-Methylphenol, 2,5-dimethylphenol, 4-ethylphenol, and 1,2,3-trimethoxybenzene were obtained from Sigma-Aldrich Corp. (Tokyo, Japan). 2,6-Dimethylphenol, 2-ethylphenol, 3-ethylphenol, 2,3-dimethylphenol, 3,4-dimethylphenol, 1,2-benzenediol, 4-methoxyphenol, 3-methoxyphenol, 2,6-dimethoxyphenol,

3-methyl-1,2-benzenediol,

2,3,5-trimethylphenol,

2-methoxy-4-propylphenol,

4-methylphenol,

1,2,4-trimethoxybenzene,

3,4-dimethoxyphenol, 4-hydroxyacetophenone, and ethanone-1-(4-hydroxy-3-methoxyphenyl) were bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All of the chemicals used in this research were analytical grade or special grade (i.e., their purities were higher than 99.5%). GC-MS. Wood and bamboo PAs were analyzed by GC-MS using a Shimadzu GCMS-QP 2010 plus (Shimadzu, Co., Ltd., Kyoto, Japan) fitted with an AOC-20i auto sampler and a DB-5MS 5



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column (25 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific). The GC oven temperature program was as follows: 50°C held for 3 min, raised to 130°C at a rate of 3.2°C/min, and then increased at 12°C/min to 280°C. For quantification of acetic acid in wood and bamboo PAs, the column oven temperature was set as follows: 50°C held for 4.0 min, raised to 130°C at a rate of 3.0°C/min, and then increased by 40°C/min to 280°C. The other GC-MS parameters employed were an injection and ion source temperature of 280°C, helium carrier gas of 1.5 mL/min, an injection volume of 1.0 µL, a split ratio of 1:13, ion source energy of 70 eV in EI mode, and a mass range of m/z 40–600. Phenolic compounds were identified by comparing their retention times and mass spectra with authentic standards. Quantifications were performed using BHT as an internal standard. Six different concentrations (0.005, 0.01, 0.05, 0.1, 0.5, and 1.0 mg/mL) were measured for calibration curves, and all the curves showed good linearity (R2 higher than 0.996). The concentrations (expressed as weight percentage) of acetic acid in wood and bamboo PAs were determined (quantification ion m/z of 60.0) using a calibration curve obtained from standard solutions at 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, and 3.0 mg/mL with a high correlation (R2 = 0.997). All of the quantifications were performed in selected ion monitoring mode. Cell Culture. L929 cells (Murine fibroblast cell line purchased from ATCC) were maintained in minimum essential medium Eagle (MEM, Nacalai Tesque) supplemented with 5% fetal bovine serum (BioWest), 100 U/mL penicillin (Nacalai Tesque), and 100 µg/mL streptomycin (Nacalai Tesque). The cells were incubated at 37°C with 5.0% CO2. Virus Inactivation Assay. EMCV was propagated in Vero cells and its titer was determined by standard plaque assay on L929 cells as described previously.25 For the inactivation assay, 10 µL of medium (MEM) containing 1 × 105 pfu EMCV was incubated with 10 µL PA, or phenolic 6


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compound (10.0%, w/w) on ice for 1 h. Methanol (for phenolic compounds) or H2O (for wood and bamboo PAs) was used as solvent control. 80 µL of culture medium was added to the mixture, and 10 µL of the mixture was added to 1 mL of culture medium containing 2.5 × 105 L929 cells in a 12-well plate. After 6 h of incubation, the cells were harvested and subjected to quantitative real-time polymerase chain reaction (PCR). The IC50 value of a compound was defined as the concentration at which it inactivated the relative EMCV RNA levels of the treated cells by 50%. Quantitative Real-Time PCR. Total RNA content of EMCV-infected cells was extracted with Sepazol (Nacalai Tesque). A high-capacity cDNA reverse transcription kit (Applied Biosystems) was used for cDNA synthesis. Viral RNA levels were monitored with the StepOnePlus real-time PCR system (Applied Biosystems) and Fast SYBR Green Master Mix (Applied Biosystems), using

the

following

EMCV-specific

primers:

forward,

5’-TTA-TAG-TGC-CGG-ACC

-TGG-CA-3’ and reverse, 5’-CCC-AAG-CTC-CCA-GTG-TTG-TC-3’. The RNA copy number of EMCV

was

normalized

to

that

of

internal

β-Actin:

forward,

5’-GAC-ATG-GAG-AAG-ATC-TGG-CAC-CAC-A-3’ and reverse, 5’-ATC-TCC-TGC-TCG -AAG-TCT-AGA-GCA-A-3’. Amido Black Staining. The cells were washed in phosphate buffer saline (PBS, pH 7.4, Nacalai Tesque) and fixed with methanol. Then, 0.5% (w/v) Amido Black solution (Nacalai Tesque) was added to stain the cell. After 20 min of incubation at room temperature, the Amido Black solution was removed and the dye was eluted with 0.1 mol/L NaOH. Absorption was measured at 630 nm. The 50% cytotoxic concentration against L929 cell (CC50) of a compound was defined as the concentration at which it reduced the absorbance at 630 nm by 50%. Inhibition of phenolic compounds. Inhibition of phenolic compound was measured at 5.0% (w/w), and calculated with the formula: 7



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Inhibition (%) =

1 - X × 100% 1

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(1)

X is relative EMCV RNA copies, calculated with the formula: X =

EMCV RNA Copies (virus incubated with phenolic compound) EMCV RNA Copies (virus incubated with blank solvent)

(2)

Statistical analyses. The results are shown as the mean ± SD of at least three independent replicates. Student t-test was used to determine statistical analyses. P < 0.05 was considered significant.

RESULTS AND DISCUSSION Antiviral activities of wood and bamboo PAs. Antiviral activities of PAs from softwood, hardwood, and bamboo were evaluated according to the method previously reported.20 For the inactivation assay, 10 µL of medium containing EMCV was incubated with 10 µL PA for 1 h at room temperature. Cell was infected by the virus pretreatment with PA. Virus RNA copies were quantified by PCR. As shown in Figure 1, all of the PAs inhibited EMCV completely, except D and its distillate Dd from Japanese red pine. Phenolic Compounds in Wood and Bamboo PAs. Phenolic compounds, one of the major components of PAs, have attracted a great deal of interest because of their potential applications as antioxidants, transportation fuel additives, and precursors for chemical products such as pesticides, dyes, and pharmaceutical products.26-28 Phenolic compounds in PAs from hardwood, bamboo, and softwood were determined by GC-MS as presented in Table 2. In total, 25 different phenolic compounds were identified. The concentrations of these compounds in the different PAs are given in Table 3. The components of the PAs vary with the raw materials used in pyrolysis. The total amount of phenolic compounds from bamboo PA (C) is the highest, followed by that from ubame oak (A). The concentrations of phenolic compounds in distillates PAs (Ad, Bd, and 8


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Dd) are higher than that in the original PAs (A, B, and D). Phenol, 2-methoxyphenol, 2-methoxy-4-methylphenol, and 4-ethyl-2-methoxyphenol are the most abundant phenolic components in all of the PAs investigated. The phenol and 2,6-dimethoxyphenol contents in bamboo PA are higher than those in the PAs from softwood and hardwood. More amount of 2,6-dimethoxyphenol was found in the PA from hardwood (A). The concentrations of 1,2-benzenediol in the softwood PAs E and F are much higher than those in the hardwood PAs A and B. Phenolic compounds result from the thermal degradation of lignin,21-23 which has a heterogeneous phenolic structure comprising three main building blocks, i.e., guaiacyl (G), syringyl (S), and hydroxyphenyl (H) units.29-31 Hardwood and softwood lignins have different structures. Softwood lignin is mainly composed of guaiacyl units and contains far fewer p-hydroxyl units, while hardwood lignin contains mainly syringyl and guaiacyl units in addition to some p-hydroxyl units.29-31 This accounts for the higher amount of 2,6-dimethoxyphenol (syringol) in PAs from hardwood than that from softwood. Softwood lignin comprises of G nucleus with a small amount of H unit. The presence of 2,6-dimethoxyphenol, a phenol having S nucleus in the PAs from softwood may generate from radical reactions of 2-methoxyphenol at high temperature (Scheme S1 in supporting information) as reported.32-34 Actually 2,6-dimethoxyphenol has been identified in pyrolysis products of softwood.35-37 1,2-Benzenediol (catechol) is not an original component in most naturally occurring lignins. However, catechol and its derivatives are found as the main components in PAs from softwood owing to the demethylation of 2-methoxyphenol (guaiacol).38-39 The abundance of 1,2-benzenediol in PAs E and F is explained by the higher content of guaiacyl structures in softwood than that in hardwood. Evaluation of Antiviral Activities and Cytotoxicities of Phenolic Compounds. As shown in 9



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Table 3, a wide range of phenolic compounds is present in wood and bamboo PAs. However, the antiviral activities of these phenol derivatives have not been well studied. Consequently, in this study, all the phenolic compounds identified in wood and bamboo PAs were evaluated for their inhibitory effects on EMCV. The structures of these compounds are shown in Figure 2 and their antiviral activities are presented in Figure 3. Compounds 1–7, 9–10, 15, 17–23, and 27–29 inhibit EMCV by more than 80%, whereas compounds 11, 12, 25, and 30 do not inactivate EMCV to any degree (Figure 3). To evaluate the antiviral potency of these compounds, the half maximal concentrations for cytotoxicity (CC50) and for EMCV inhibition (IC50) were assessed (Table 4). 3-Methyl-1,2-benzenediol and 3-methoxy-1,2-benzenediol are the most potent compounds, followed by 1,2-benzenediol. Moreover, compounds 1–3, 5, 7–8, 15, 17–21, and 23 also exhibit potent antiviral effects against EMCV with IC50 values lower than 10.0 mg/mL. The cell cytotoxicity of these phenolic compounds was also assessed. The CC50 values for most of the compounds tested are higher than 1.0 mg/mL, except for those of compounds 3, 5–7, 17–18, 22–23, and 28–29, which are around 0.5 mg/mL. As shown in Table 4, some of the IC50 values are higher than the corresponding CC50 values. However, the virus was first incubated with the phenolic compound and then used to infect cells after 1,000 times of dilution. Therefore, the final concentration of the phenolic compound mixed with the cells was 1,000 times lower than that incubated with the virus. For instance, the IC50 of phenol is 3.0 mg/mL, but that of the final concentration mixed with the cells was 0.003 mg/mL, which is much lower than CC50. Structure-Activity Relationship. From these results, the following primary structure-activity relationship features can be asserted: The number of free hydroxyl groups on the benzene ring plays a key role in strong antiviral activity (10 cf. 11; 27–29 cf. 1), as previously reported for cinnamic acid derivatives.40 Alkyl substituents (methyl or ethyl) provide no enhancement of the 10


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antiviral activity compared to phenol (2–7 cf. 1). 2-Methoxyphenol (guaiacol), one of the main phenolic compounds in PAs, only moderately suppresses EMCV. A methyl or ethyl substituent at the 4 (12–13) or 5 (16) position of 2-methoxyphenol leads to a decrease in the antiviral activity. Interestingly, the higher activity of 2-methoxyophenol with a 4-propyl- (14) or 4-allyl- (15) group than that with a methyl- (11) or ethyl- (12) group implies that a longer carbon side chain enhances viral inactivation. In addition, phenolic compounds with unsaturated carbon side chains inactivate the virus more efficiently (14 cf. 15). A comparison of methoxyl and methyl substituents shows that methyl derivatives have higher activity than methoxyl analogs (2–4 cf. 8–10; 20 cf. 21; 23 cf. 24). However, increasing the number of methyl groups does not result in increased viral inhibition (2–4 cf. 17–20). The relative positions of the substituents also clearly affect the antiviral activity. For example, 2,6-dimethoxyphenol exhibits a lower IC50 than 3,4-dimethoxyphenol. Furthermore, the carboxylic acid group appears to promote the antiviral activity (24 cf. 26).41 Moreover, electron-withdrawing substituents decrease the antiviral activity significantly (33, 34 cf. 1), although it is not clear how the inductive effects are related to the activity. Thus, we conclude that the antiviral activities of phenolic compounds depend on the number, structure, and position of substituents (Table S1). The antiviral activities of phenolic compounds from natural sources have attracted increasing research interest in recent years.42-45 For example, Yang et al.43 found that some of the phenolic compounds isolated from Arundina gramnifolia exhibit anti-HIV and anti-tobacco mosaic virus activities; phenolic glycosides that inhibit chikungunya and dengue virus replication have been isolated from Flacourtia ramontchi by Litaudon et al.44 and McKee et al.45 have reported the HIV-1 RNase inhibition effects of phenolic glycosides from the evergreen species Eugenia hyemalis. 11



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Several studies have been conducted on the isolation of natural antiviral compounds from plants, microorganisms, and animals. In contrast to these studies, we have demonstrated that a large number of antiviral phenolic compounds can be produced by simple pyrolysis of renewable lignocellulosic biomass such as waste agricultural residues and unutilized wood resources. We found that the structures and positions of the substituents attached to the aromatic ring significantly affect antiviral activity.

CONCLUSION In this study, antiviral activities of PAs from hardwood, softwood and bamboo against EMCV were evaluated. All the PAs except for Japanese red pine wood exhibited strong antiviral activity. Twenty-five kind of phenolic derivatives in the PAs were identified and quantified. An assessment of the antiviral activity of all the phenolic compounds against EMCV demonstrated that they are strongly dependent on the functional groups and their relative positions attached to the aromatic ring. More potent antiviral activities were determined for catechol and it derivatives. The antiviral activities of phenolic compounds with methyl groups are higher than those of compounds with methoxyl groups. These results present a new strategy to optimize the production of antiviral agents from PAs by monitoring the concentration of each phenol derivative from different plant origins. Understanding the structure-activity relationship of phenol derivatives will lead to the synthesis of more effective agents for virus-inactivation treatments. The abundance and renewability of waste biomass makes the exploitation of PAs as a source of antiviral phenolic compounds an extremely attractive and environmentally friendly strategy for the treatment of viral conditions.

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ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publication website at: Table S1 shows the structure-activity relationship of phenolic compounds. Scheme S1 shows the possible pathway for the formations of 2,6-dimethoxyphenol from 2-methoxyphenol at high temperature.

AUTHOR INFORMATION Corresponding Author Takashi Watanabe *

Tel: +81-774-38-3640. Fax: +81-774-38-3681.

E-mail: [email protected] Author Contributions §

These authors contributed equally to this work

Present Address #

Center for Structural Biology, Department of Molecular Biology and Genetics

Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark ‡

Department of Microbiology, Tennoji Center, Osaka Institute of Public Health

Tojo-Cho 8-34, Tennoji-ku, Osaka, Japan 543-0026 Note The authors declare no competing financial assistance

AKNOWLEDGEMENTS 13



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This work was supported by Frontier Research Grant from the Research Institute for Sustainable Humanosphere, Kyoto University. The authors also acknowledge Dr. Koji Onomoto from Division of Molecule Immunology, Medical Mycology Research Center, Chiba University for his contribution to establishing the experimental basis on antiviral assay of PAs.

REFERENCES (1)

Mohan, D.; Pittman, C. U.; Steele, P. H. Pyrolysis of wood/biomass for bio-oil: A critical review. Energy Fuels 2006, 20 (3), 848-889, DOI: 10.1021/ef0502397.

(2)

Mukarakate, C.; Evans, R. J.; Deutch, S.; Evans, T.; Starace, A. K.; Dam, J.; Watson, M. J.; Magrini, K. Reforming biomass derived pyrolysis bio-oil aqueous phase to fuels. Energy Fuels 2017, 31 (2), 1600-1607, DOI: 10.1021/acs.energyfuels.6b02463.

(3)

Fagernäs, L.; Kuoppala, E.; Tiilikkala, K.; Oasmaa, A. Chemical composition of birch wood slow pyrolysis products. Energy Fuels 2012, 26 (2), 1275-1283, DOI: 10.1021/ef2018836.

(4)

Zhou, S.; Xue, Y.; Sharma, A.; Bai, X. Lignin valorization through thermochemical conversion: Comparison of hardwood, softwood and herbaceous lignin. ACS Sustainable Chem. Eng. 2016, 4 (12), 6608-6617, DOI: 10.1021/acssuschemeng.6b01488.

(5)

Fagernäs, L.; Kuoppala, E.; Arpiainen, V. Composition, utilization and economic assessment of torrefaction condensates. Energy Fuels 2015, 29 (5), 3134-3142, DOI: 10.1021/acsenergyfuels5b00004.

(6)

Zhang, J.; Choi, Y. S.; Shanks, B. H. Tailoring the composition of bio-oil by vapor-phase removal

of

organic

acids.

ChemSusChem

2015,

10.1002/cssa.201500884. 14


8

(24),

4256-4265,

DOI:

Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7)

Mu, J.; Uehara, T.; Furuno, T. Effect of bamboo vinegar on regulation of germination and radicle growth of seed plants. J Wood Sci. 2003, 49 (3), 262-270, DOI: 10.1007/s10086-002-0472-z.

(8)

Mu, J.; Uehara, T.; Furuno, T. Effect of bamboo vinegar on regulation of germination and radicle growth of seed plants II: composition of moso bamboo vinegar at different collection temperature and its effects. J Wood Sci. 2004, 50 (5), 470-476, DOI: 10.1007/s10086-003-0586-y.

(9)

Wu, Q.; Zhang, S.; Hou, B.; Zheng, H.; Deng, W.; Liu, D.; Tang, W. Study on the preparation of wood vinegar from biomass residues by carbonization process. Bioresour Technol. 2015, 179, 98-103, DOI: 10.1016/j.biortech.2014.12.026.

(10)

Ho, C.-L.; Lin, C.-Y.; Ka, S.-M.; Chen, A.; Tasi, Y.-L.; Liu, M.-L.; Chiu, Y.-C.; Hua, K.-F. Bamboo vinegar decreases inflammatory mediator expression and NLRP3 inflammasome activation by inhibiting reactive oxygen species generation and protein kinase C-alpha/delta

activation.

PLoS

One.

2013,

8

(10),

e75738,

DOI:

10.10731/journal.pone.0075738. (11)

Lee, C. S.; Yi, E. H.; Kim, H.-R.; Huh, S.-R.; Sung, S.-H.; Chung, M.-H.; Ye, S.-K. Anti-dermatitis effects of oak wood vinegar on the DNCB-induced contact hypersensitivity via STAT3 suppression. J. Ethnopharmacol. 2011, 135 (3), 747-753, DOI: 10.1016/j.jep.2011.04.009.

(12)

Chandrasekaran, S. R.; Murali, D.; Marley, K. A.; Larson, R.A.; Doll, K. M.; Moser, B. R.; Scott, J.; Sharma, B. K. Antioxidants from slow pyrolysis bio-il of birch wood: Application for biodiesel and biobased lubricants. ACS Sustainable Chem. Eng. 2016, 4 (3), 1414-1421, DOI: 10.1021/acssuschemeng.5b.01302. 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(13)

Page 16 of 30

Loo, A. Y.; Jain, K.; Darah, I. Antioxidant activity of compounds isolated from the pyroligneous acid, Rhizophora apiculata. Food Chem 2008, 107 (3), 1151-1160, DOI: 10.1016/j.foodchem.2007.09.044.

(14)

Ma, C.; Song, K.; Yu, J.; Yang, L.; Zhao, C.; Wang, W.; Zu, G.; Zu, Y. Pyrolysis process and antioxidant activity of pyroligneous acid from Rosmarinus officinalis leaves. J. Anal. Appl. Pyrolysis 2013, 104, 38-47, DOI: 10.1016/j.jaap.2013.09.011.

(15)

Suqi, L.; Caceres, L.; Schieck, K.; Booker, C. J.; McGarver, B. M.; Yeung, K. K.-C.; Pariente, S.; Briens, C.; Berruti, F.; Scott, I .M. Insecticidal activity of bio-oil from pyrolysis of straw from Brassica spp. J. Agric. Food Chem. 2014, 62 (16), 3610-3618, DOI: 10.1021/jf500048t.

(16)

Booker, C. J.; Bedmutha, R.; Vogel, T.; Gloor, A.; Xu, R.; Ferrante, L.; Yeung, K. K.-C.; Scott, I .M.; Conn, K. L.; Berruti, F.; Briens, C. Experimental investigations into the insecticidal, fungicidal, and bactericidal properties of pyrolysis bio-oil from tobacco leaves using a fluidized bed pilot plant. Ind. Eng. Chem. Res. 2010, 49 (20), 10074-10079, DOI: 10.1021/ie100329z.

(17)

Hossain, M. M.; Scott, I. M.; McGarvey, B .D.; Conn, K.; Ferrante, L.; Berruti, F.; Briens, C. Insecticidal and anti-microbial activity of bio-oil derived from fast pyrolysis of lignin, cellulose,

and

hemicellulose.

J.

Pest.

Sci.

2015,

88

(1),

171-179,

DOI:

10.1007/s10340-014-0568-4. (18)

Miyamoto, Y.; Takeuchi, T.; Taniguchi, K. Inactivation of tobacco mosaic virus by "Mokusaku-eki". NihonShokubutsu Byorigaku Kaihou. 1965, 27, 261.

(19)

Lin, F-H.; Chueh, L-L.; Lee, Y-H.; Ho, I-S. The effect of motmorillonite and bamboo vinegar on porcine reproductive and respiratory syndrome virus. Curr. Nanosci. 2011, 7 16


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6), 839-844, DOI: 10.2174/157341311798220646. (20)

Marumoto, S.; Yamamoto, S.; Nishimura, H.; Onomoto, K.; Yatagai, M.; Yazaki, K.; Fujita, T.; Watanabe, T. Identification of a germicidal compound against picornavirus in bamboo pyroligneous acid. J. Agric. Food. Chem. 2012, 60 (36), 9106-9111, DOI: 10.1021/jf3021317.

(21)

Patwardhan, P. R.; Brown, R. C.; Shanks, B. H. Understanding the fast pyrolysis of lignin. ChemSusChem 2011, 4 (11), 1629-1636, DOI: 10.1002/cssc.201100133.

(22)

Zhou, S.; Garcia-Perez, M.; Pecha, B.; Kersten, S. R. A.; McDonald, A. G.; Westerhof, R. J. M. Effect of the fast pyrolysis temperature on the promary and secondary products of lignin. Energy Fuels 2013, 27 (10), 5867-5877, DOI: 10.1021/ef4001677.

(23)

Kozliak, E. I.; Kubátová, A.; Artemyeva, A. A.; Nagel, E.; Zhang, C.; Rajappagowda, R. B.; Smirnova, A. L. Thermal liquefaction of lignin to aromatics: Efficiency, selectivity, and product analysis. ACS Sustainable Chem. Eng. 2016, 4 (10), 5106-5122, DOI: 10.1021/acssuschemeng.6b01046.

(24)

Negahdar, L.; Gonzalez-Quiroga, A.; Otyuskaya, D.; Toraman, H. E.; Liu, L.; Jastrzebski, J. T. B. H.; Geem, K. M. V.; Marin, G. B.; Thybaut, J. W.; Weckhuysen, B. M. Characterization and comparison of fast pyrolysis bio-oils from pinewood, rapeseed cake, and wheat straw using

13

C NMR and comprehensive GC × GC. ACS Sustainable Chem.

Eng. 2016, 4 (9), 4974-4985, DOI: 10.1021/acssuschemeng.6b01329. (25)

Yoneyama, M.; Kikuchi, M.; Shinobu, N.; Imaizumi, T.; Miyagishi, M.; Taira, K.; Akira, S.; Fujita, T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 2004, 5(7), 730-737, DOI: 10.1038.ni1087. 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(26)

Elliott, D. C.; Wang, H. Hydrocarbon liquid production via catalytic hydroprocessing of phenolic oils fractionated from fast pyrolysis of red oak and corn stover. ACS Sustainable Chem. Eng. 2015, 3 (5), 892-902, DOI: 10.1021/acssuschemeng.5b00015.

(27)

Lopes, A. M. C.; Brenner, M.; Falé, P.; Roseiro, L. B.; Bogel-Lukasik, R.; Extraction and purification of phenolic compounds from lignocellulosic biomass assisted by ionic liquid, polymeric resins, and supercritical CO2. ACS Sustainable Chem. Eng. 2016, 4 (6), 3357-3367, DOI: 10.1021/acssuschemeng.6b00429.

(28)

Toledano, A.; Serrano, L.; Balu, A. M.; Luque, R.; Pineda, A.; Labidi, J. Fractionation of organosolv lignin from olive tree clipping and its valorization to simple phenolic compounds. ChemSusChem 2013, 6 (3), 529-536, DOI: 10.1002/cssc.201200755.

(29)

Zakzeski, J.; Bruijinincx, P. C. A.; Jongerius, A. L.; Weckhusen, B. M. The catalytic valorization of lignin for the production of renewable chemicals. Chem. Rev. 2010, 110 (6), 3552-3599, DOI: 20.1021/cr900354u.

(30)

Liu, W-J.; Jiang, H.; Yu, H-Q. Thermochemical conversion of lignin to functional materials: a review and future directions. Green Chem. 2015, 17 (11), 4888-4907, DOI: 10.1039/C5GC01054C.

(31)

Upton, B. M.; Kasko, A. M. Strategies for the conversion of lignin to high-value polymeric materials: Review and prospective. Chem. Rev. 2016, 116 (4), 2275-2306, DOI: 10.1021/acschemrev.5b00345.

(32)

Ben, H.; Ragauskas, A. J. NMR characterization of pyrolysis oils from Kraft lignin. Energy Fuels 2011, 25 (5), 2322-2332, DOI: 10.1021/ef2001162.

(33)

Britt, P. F.; Buchanan III, A. C.; Cooney, M. J.; Martineau, D. R. Flash vacuum pyrolysis of methoxy-substituted lignin model compounds. J. Org. Chem. 2000, 65 (5), 1376-1389, 18


Page 18 of 30

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DOI: 10.1021/jo991479k. (34)

Britt, P. F.; Kidder, M. K.; Buchanan III, A. C. Oxygen substituent effects in the pyrolysis of phenethyl phenyl ethers. Energy Fuels 2007, 21

(6), 3102-3108,

DOI:

10.1021/ef700354y. (35)

Hu, J.; Shen, D.; Wu, S.; Zhang, H.; Xiao, R. Composition analysis of organosolv lignin and its catalytic solvolysis in supercritical alcohol. Energy Fuels 2014, 28 (7), 4260-4266, DOI: 10.1021/ef500068h.

(36)

Asmadi, M.; Kawamoto, H.; Saka, S. Gas- and solid/liquid-phase reactions during pyrolysis of softwood and hardwood lignins. J. Anal. Appl. Pyrolysis. 2011, 92 (2), 417-425, DOI: 10.1016/j.jaap.2011.08.003.

(37)

Mun, S. P.; Ku, C. S. Pyrolysis GC-MS analysis of tars formed during the aging of wood and bamboo vinegars. J Wood Sci 2010, 56 (1), 47-52, DOI: 10.1007/s10086-009-1054-0.

(38)

DeSisto, W. J.; Hill II, N.; Beis, S. H.; Mukkamala, S.; Joseph, J.; Baker, C.; Ong, T-H.; Stemmler, E. A.; Wheeler, M. C.; Frederick, B. G.; Heiningen, A. Fast pyrolysis of pine sawdust in a fluidized-bed reactor. Energy Fuels 2010, 24 (4), 2642-2651, DOI: 10.1021/ef901120h.

(39)

Klein, M. T; Virk, P. S. Modeling of lignin thermolysis. Energy Fuels 2008, 22 (4), 2175-2182, DOI: 10.1021/ef800285f.

(40)

Chiang, L.C.; Chiang, W.; Chang, M. Y.; Ng, L.T.; Lin, C. C. Antiviral activity of plantago major extracts and related compounds in vitro. Antiviral Res 2002, 55 (1), 53-62, DOI: 10.1016/S0166-3542(02)00007-4.

(41)

Das, P.; Deng, X.; Zhang, L.; Roth, M. G.; Fontoura, B. M. A.; Phillips, M. A.; Brabander, J. K. D. SAR-based optimization of a 4-quinoline carboxylic acid analogue with potent 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

antiviral activty. ACS Med. Chem. Lett. 2013, 4 (6), 517-521, DOI: 10.1021/ml300464h. (42)

Chávez, J. H.; Leal, P. C.; Yunes, R .A.; Nunes, R. J.; Baradi, C. R M.; Pinto, A. R.; Simões, C. M. O.; Zanetti, C. R. Evaluation of antiviral activity pf phenolic compounds and derivatives against rabies virus. Vet. Microbiol. 2006, 116 (1), 53-59, DOI: 10.1016/j.vetmic.2006.03.019.

(43)

Hu, Q.-F.; Zhou, B.; Huang, J.-M.; Gao, X.-M.; Shu, L.-D.; Yang, G.-Y.; Che, C.-T. Antiviral phenolic compounds from Arundina gramnifolia. J. Nat. Prod. 2013, 76 (2), 292-296, DOI: 10.1021/np300727f.

(44)

Bourjot, M.; Leyssen, P.; Eydoux, C.; Guillemot, J.-C.; Canard, B.; Rasoanaivo, P.; Guéritte, F.; Litaudon, M. Flacourtosides A-F, phenolic glucosides isolated from Flacourtia ramontchi. J. Nat. Prod. 2012, 75 (4), 752-758, DOI: 10.1021/np300059n.

(45)

Bokesch, H. R.; Wamiru, A.; Grice, S. F. J. L.; Beutler, J. A.; McKee, T. C.; McMahon, J. B. HIV-ribonuclease H inhibitory phenolic glycosides from Eugenia hyemalis. J. Nat. Prod. 2008, 71 (10), 1634-1636, DOI: 10.1021/np8002518.

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Figure Captions Figure 1. Antiviral activities of wood and bamboo PAs. Figure 2. Phenolic compounds identified in wood and bamboo PAs. Figure 3. Antiviral activities of phenolic compounds identified in PAs.

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Table 1. Source and Physicochemical Properties of Wood and Bamboo PAs. no classification species academic name source acetic acid (%a) A hardwood ubame oak quercus Wakayama, 12.4 phillyraeoides Japan Ad hardwood ubame oak quercus Wakayama, 9.03 phillyraeoides Japan B hardwood mongolian quercus crispula Iwate, Japan 2.96 oak Bd hardwood mongolian quercus crispula Iwate, Japan 2.96 oak C bamboo mosochiku phyllostachys Yamanashi, 4.75 pubescens Japan D softwood Japanese red pinus densiflora Akita, Japan 0.75 pine Dd softwood Japanese red pinus densiflora Akita, Japan 0.65 pine E softwood Japanese chamaecyparis Gifu, Japan 1.59 cypress obtusa F softwood Japanese larix kaempferi Hokkaido, 1.15 larch Japan a Expressed as weight percentage. b Measured at room temperature.

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pHb 2.09 2.17 2.59 2.51 3.39 3.61 2.70 2.33 3.22

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Table 2. Phenolic Compounds in Wood and Bamboo PAs (A–F). compounds retention time chemical molecular (min) formula weight phenol 9.33 C6H6O 94.1 2-methylphenol 12.44 C7H8O 108.1 3- or 4-methylphenol 13.50 C7H8O 108.1 2-methoxyphenol 13.86 C7H8O2 124.1 2,6-dimethylphenol 14.79 C8H10O 122.2 2-ethylphenol 16.13 C8H10O 122.2 2,4-dimethylphenol 16.53 C8H10O 122.2 2,5-dimethylphenol 16.74 C8H10O 122.2 122.2 3- or 4-ethylphenol 17.48 C8H10O 2,3-dimethylphenol 17.90 C8H10O 122.2 2-methoxy-4-methylphenol 18.41 C8H10O2 138.2 122.2 3,4-dimethylphenol 18.65 C8H10O 1,2-benzenediol 18.82 C6H6O2 110.1 4-methoxyphenol 19.77 C7H8O2 124.1 3-methyl-1,2-benzenediol 21.20 C7H8O2 124.1 3-methoxy-1,2-benzenediol 21.37 C7H8O3 140.1 2,3,5-trimethylphenol 21.99 C9H12O4 184.2 4-ethyl-2-methoxyphenol 22.18 C9H12O2 152.2 1,2,3-trimethoxybenzene 23.69 C9H12O3 168.2 2,6-dimethoxyphenol 25.05 C8H10O3 154.2 2-methoxy-4-propylphenol 25.84 C10H14O2 166.2 154.2 3,4-dimethoxyphenol 28.52 C8H10O3 4-hydroxyacetophenone 29.04 C8H10O2 136.2 ethanone-1-(4-hydroxy29.95 C9H10O3 166.2 3-methoxyphenyl) 4-allyl-2,6-dimethoxyphenol 32.02 C11H14O3 194.2

23


quantification ion (m/z) 94.0 108.1 108.1 109.0 107.1 107.1 107.1 107.1 107.1 107.1 138.1 107.0 110.0 109.0 124.0 140.1 121.1 137.1 168.0 154.0 137.1 154.0 121.0 151.0 194.0

24


17.37 ± 4.19

4-methoxyphenol

1,2-benzenediol

3,4-dimethylphenol

125.27 ± 2.77 3.95 ± 0.13 60.32 ± 8.97

2-methoxy-4-methyl phenol

11.41 ± 0.35

212.13 ± 76.22 81.98 ± 16.02 86.73 ± 17.97 1276.58 ± 144.40 17.80 ± 0.58 5.87 ± 1.19 19.55 ± 1.60 34.07 ± 4.77 13.02 ± 1.61 1.31 ± 0.50 223.25 ± 32.43 6.63 ± 0.44 3.49 ± 0.13

254.94 ± 6.60 52.63 ± 4.48 69.92 ± 4.56 961.48 ± 35.45 17.03 ± 0.61 4.57 ± 1.56 11.97 ± 0.42 17.45 ± 0.61 11.11 ± 2.47 ND

Ad

A

2,3-dimethylphenol

3- or 4-ethylphenol

2,5-dimethylphenol

2,4-dimethylphenol

2-ethylphenol

2,6-dimethyl phenol

2-methoxyphenol

3- or 4-methyl phenol

2-methylphenol

phenol

compound

13.05 ± 0.61

178.16 ± 21.31 42.84 ± 2.92 65.76 ± 6.15 190.49 ± 19.62 16.17 ± 0.08 2.55 ± 0.20 5.80 ± 0.20 21.27 ± 1.65 10.75 ± 0.38 0.50 ± 0.14 57.05 ± 5.64 4.21 ± 0.22 69.69 ± 15.60

B

12.19 ± 0.73

320.94 ± 8.58 56.75 ± 1.35 106.07 ± 2.78 200.39 ± 8.89 15.66 ± 0.02 2.53 ± 0.06 4.53 ± 1.68 21.19 ± 1.23 13.19 ± 0.06 1.51 ± 0.03 55.03 ± 2.47 6.33 ± 0.94 9.14 ± 0.16

Bd

21.97 ± 1.06

932.12 ± 88.30 119.47 ± 7.88 238.65 ± 17.58 444.06 ± 34.61 19.33 ± 0.30 9.77 ± 0.74 19.39 ± 0.10 31.72 ± 0.96 224.69 ± 18.65 3.09 ± 0.23 105.07 ± 7.99 7.10 ± 0.06 60.32 ± 8.97

C

10.96 ± 0.17

192.65 ± 8.96 44.60 ± 1.30 86.68 ± 4.14 175.77 ± 6.84 16.43 ± 0.02 2.27 ± 0.06 9.85 ± 2.14 24.45 ± 1.46 19.65 ± 0.51 0.66 ± 0.09 122.18 ± 5.10 4.57 ± 0.21 54.93 ± 0.16

D

10.24 ± 0.14

473.73 ± 16.62 92.97 ± 2.08 188.93 ± 8.10 381.93 ± 12.59 17.12 ± 0.05 4.43 ± 0.08 19.54 ± 1.45 29.96 ± 9.06 30.01 ± 10.74 1.75 ± 0.10 217.14 ± 6.60 10.24 ± 1.07 5.04 ± 0.79

Dd

12.51 ± 0.20

236.80 ± 23.99 52.81 ± 3.53 94.36 ± 7.45 397.41 ± 38.80 16.72 ± 0.09 2.65 ± 0.15 14.16 ± 0.22 28.75 ± 1.38 11.27 ± 0.48 0.23 ± 0.08 245.50 ± 22.02 4.65 ± 0.13 247.71 ± 26.41

E

12.26 ± 1.53

220.01 ± 14.77 63.21± 2.94 133.17 ± 9.56 310.08 ± 15.71 17.79 ± 0.11 3.38 ± 0.12 25.04 ± 5.05 22.12 ± 0.05 14.60 ± 1.81 2.75 ± 0.24 279.78 ± 13.49 8.96 ± 0.39 593.95 ± 111.93

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 3. Concentrations of Phenolic Compounds in Wood and Bamboo PAs (A–F)a, b.

ACS Sustainable Chemistry & Engineering Page 24 of 30

25


96.14 ± 5.26 30.61 ± 5.47 16.00 ± 1.33 0.28 ± 0.08 2314.9 ± 106.2 6.55 ± 1.90 0.61 ± 0.16 2915.2 ± 362.7

ND

1.21 ± 0.09 44.79 ± 1.49 4.55 ± 0.53 160.09 ± 0.82 22.41 ± 3.59 587.21 ± 51.02 3.88 ± 0.14 93.59 ± 1.27

1.39 ± 0.13 52.57 ± 8.94 1.85 ± 0.46 159.35 ± 0.42 21.60 ± 5.98 321.00 ± 10.81 NDc

Ad

93.73 ± 1.06 29.00 ± 0.76 9.09 ± 1.89 0.37 ± 0.06 1160.0 ± 89.8 6.79 ± 1.31 0.57 ± 0.01 1557.5 ± 130.4

ND

ND

ND

158.19 ± 0.32 13.07 ± 0.30 552.70 ± 99.08

158.35 ± 0.32 11.51 ± 0.33 131.57 ± 9.81 ND

ND

ND

0.78 ± 0.37

Bd

ND

1.29 ± 0.11 46.81 ± 0.74

B

93.72 ± 0.65 28.20 ± 0.01 13.99 ± 1.17 0.28 ± 0.06 3322.6 ± 212.8

ND

17.26 ± 1.02 123.67 ± 1.78 1.85 ± 0.46 160.01 ± 0.81 14.12 ± 0.37 632.76 ± 19.06

C

93.15 ± 0.56 29.05 ± 0.30 17.01 ± 2.23 0.30 ± 0.03 1140.7 ± 42.6

ND

1.53 ± 0.23 44.21 ± 0.16 0.96 ± 0.25 158.37 ± 0.21 8.94 ± 0.02 21.53 ± 7.37

D

93.12 ± 0.17 27.72 ± 0.03 6.21 ± 0.96 0.25 ± 0.13 1870.8 ± 103.0

ND

2.71 ± 0.33 42.80 ± 0.11 2.68 ± 0.03 191.16 ± 28.63 9.17 ± 0.09 11.95 ± 3.07

Dd

93.97 ± 0.89 27.85 ± 0.06 40.26 ± 2.26 0.43 ± 0.11 1773.6 ± 135.2

ND

26.08 ± 3.84

ND

158.81 ± 0.62

ND

13.80 ± 1.28 46.78 ± 1.24

E

27.89 ± 0.10 34.21 ± 7.22 0.68 ± 0.06 2047.8 ± 189.4

ND

ND

160.07 ± 0.10 8.93 ± 0.01 46.80 ± 0.44

ND

14.89 ± 2.11 47.25 ± 1.67

F

Concentrations of phenolic compounds are in mg/L. b All the concentrations are average values of triplicate determinations, and are expressed as means ± standard deviations. c ND means the concentration is lower than the quantification limit.

a

Total concentrations of phenolic compounds

4-allyl-2,6-dimethoxyphenol

ethanone-1-(4-hydry3-methoxyphenyl)

4-hydroxy acetophenone

3,4-dimethoxyphenol

2-methoxy-4-propylphenol

2,6-dimethoxyphenol

1,2,3-trimethoxybenzene

4-ethyl-2-methoxyphenol

2,3,5-trimethoxyphenol

3-methoxy-1,2-benzenediol

3-methyl-1,2-benzenediol

compound

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 3. Continued

Page 25 of 30 ACS Sustainable Chemistry & Engineering


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Table 4. Evaluation of Anti-EMCV Activities of Phenolic Compounds in PAs (A–F).a no compound IC50 CC50 (mg/mL) inhibitionb (mg/mL) (%) 1 phenol 3.00 ± 0.13 >1.0 >99.9 2 2-methylphenol 3.80 ± 1.51 >1.0 >99.9 3 3-methylphenol 2.97 ± 1.20 0.42 ± 0.18 >99.9 4 4-methylphenol 12.8 ± 1.48 >1.0 98.8 5 2-ethylphenol 5.75 ± 0.72 0.39 ± 0.04 >99.9 6 3-ethylphenol 7.04 ± 5.19 0.43 ± 0.09 >99.9 7 4-ethylphenol 5.12 ± 0.08 0.50 ± 0.05 99.9 8 2-methoxyphenol 17.1 ± 10.9 >1.0 78.3 9 3-methoxyphenol 15.5 ± 1.66 >1.03 99.2 10 4-methoxyphenol 31.5 ± 27.0 >1.0 93.2 11 4-methoxybenzenylalcohol >51.7 >1.0 1.93 12 4-methyl-2-methoxyphenol >51.3 >1.0 3.70 13 4-ethyl-2-methoxyphenol >50.1 >1.0 63.4 14 4-propyl-2-methoxyphenol 14.1 ± 2.92 >1.0 80.6 15 4-allyl-2-methoxyphenol 7.63 ± 0.35 >1.0 98.3 16 5-methyl-2-methoxyphenol >51.2 >1.0 54.7 17 2,3-dimethylphenol 4.26 ± 2.46 0.44 ± 0.10 99.8 18 2,4-dimethylphenol 9.26 ± 0.51 0.50 ± 0.07 99.1 19 2,5-dimethylphenol 9.38 ± 6.10 >1.0 98.7 20 2,6-dimethylphenol 4.06 ± 1.19 >1.0 99.2 21 2,6-dimethoxyphenol 15.6 ± 0.79 >1.0 98.0 22 4-allyl-2.6-dimethoxyphenol 7.40 ± 1.44 0.58 ± 0.04 99.4 23 3,4-dimethylphenol 6.61 ± 1.69 0.51 ± 0.03 98.4 24 3,4-dimethoxyphenol >50.8 >1.0 38.2 25 3,4-dimethoxybenzylalcohol >51.4 >1.0 4.65 26 3,4-dimethoxyphenylacetic acid >50.7 >1.0 79.3 27 1,2-benzenediol 0.67 ± 0.18 >1.0 >99.9 28 3-methyl-1,2-benzenediol 99.9 29 3-methoxy-1,2-benzenediol 99.9 30 1,2,3-trimethoxybenzene >50.4 >1.0 5.22 31 1,2,4-trimethoxybenzene >51.8 >1.0 22.8 32 2,3,5-trimethoxyphenol >51.4 >1.0 79.2 33 ethanone-1-(4-hydroxy>50.1 >1.0 59.9 3-methoxyphenyl) 34 4’-hydroxyacetophenone >51.1 >1.0 68.0 a The concentrations of phenolic compounds mixed with cells are 1/1000 of the concentrations mixed with virus. More details were shown in experimental part. b Inhibition of phenolic compound was measured at the concentration of 5.0% (w/w). The details were given in the experimental part. 26


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Figure 1. Antiviral activities of wood and bamboo PAs. Ultrapure water was used as solvent control. * indicate a significant difference at the respective level of P < 0.05 compared to the solvent control (H2O) by Student’s t-test.

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Figure 2. Phenolic compound identified in wood and bamboo PAs. No. 9, 11, 15, 16, 25, 26, and 31 were not contained in wood and bamboo PAs.

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Figure 3. Antiviral activities of phenolic compounds at concentrations of 5.0% (w/w). * indicate a significant difference at the respective level of P < 0.05 compared to solvent control (MeOH) by Student’s t-test.

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Graphical Abstract

This research supplies a strategy to obtain huge amount of antiviral compounds from woody biomass resources just by pyrolysis.

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Antiviral Activity of Phenolic Derivatives in Pyroligneous Acid from

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