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β-Cyclodextrins as sustained-release carriers for natural wood preservatives
Industrial Crops & Products 130 (2019) 42–48
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β-Cyclodextrins as sustained-release carriers for natural wood preservatives ⁎
Lili Cai, Dragica Jeremic, Hyungsuk Lim , Yunsang Kim
⁎
T
Department of Sustainable Bioproducts, Mississippi State University, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Wood protection Natural wood preservatives Allyl isothiocyanate β-cyclodextrins Fungi-resistant Encapsulation
Natural preservatives for wood protection have gained increasing attention due to their intrinsic antimicrobial properties, renewability, and lower impact on the environment. We report a study on the use of β-cyclodextrin (βCD) derivatives as sustained-release carriers of allyl isothiocyanate (AITC), as a model natural preservative compound. The formation of AITC inclusion complex in βCDs is qualitatively confirmed by FT-IR and the maximum inclusion yield is estimated to be 39%. Impregnation of wood with the water-borne βCD-AITC complexes allows penetration and even distribution of the preservative in the lumen and possibly in the cell walls. The efficacy of the βCD-AITC complexes as wood preservatives of southern yellow pine is examined by the AWPA E10-16 standard. Compared with the water-treated and AITC-treated wood, βCD-AITC-treated wood exhibits decrease in mass loss from 45% to 25% and no visible cell wall damage after exposure to brown and white rot fungi. The results indicate that βCD suppresses the premature leaching of otherwise volatile AITC and suggests a novel approach of application of volatile or water-immiscible natural preservatives for wood protection.
1. Introduction Despite numerous favorable characteristics of wood as a construction material, its susceptibility to biological decay under humid conditions requires appropriate protection methods to extend its service life. Approaches to protecting wood from various decay microorganisms can be categorized into passive and active methods. Passive protection renders wood less susceptible to decay mechanisms, and it involves wood surface coating with varnish (Chen et al., 2009; Gobakken and Westin, 2008), bulking or coating cell walls with metallic compounds (Guo et al., 2018; Usmani et al., 2018), chemical modification to block the hydroxyl groups (Rowell et al., 2009), physical modification by heat (Kamdem et al., 2002) and mechanical densification (Schwarze and Spycher, 2005). Active protection relies on chemical compounds that inhibit the growth or kill wood-decay organisms (Stirling and Temiz, 2014), and typically include oil-borne or waterborne preservatives (Groenier and Lebow, 2006). Although many of currently used fungicides and insecticides are highly efficient wood protectants, a lot of research emphasis is put on use of natural biocides that can provide long term protection and have a lower impact on the environment. Among studied bio-based compounds, extractives (Tascioglu et al., 2013), tannins (Thevenon et al., 2008), chitosan (ElGamal et al., 2016), and essential oils (Chittenden and Singh, 2011;
Saha Tchinda et al., 2018; Xie et al., 2017) showed good antimicrobial properties, but their use was restricted due to their long-term leachability or volatility. One of the approaches examined to overcome this problem was a study of grafting extractives into wood through the action of an enzyme, namely, laccase, but its further application is hindered by the deactivation of the enzyme and limited development of enzymatic grafting (Fernández-Costas et al., 2017). Thus, developing effective wood preservatives based on the use of natural compounds still poses a challenge. Allyl isothiocyanate (AITC), also known as mustard oil, can be obtained from cruciferous vegetables, such as wasabi, broccoli and cabbage. These plants are rich in allyl glucosinolate (sinigrin), which can be hydrolyzed to AITC by the enzyme myrosinase released from the plant tissues when they are disrupted. The yield of AITC can reach to as far as 90% by proper cellulolytic pretreatment (Szakacs-Dobozi et al., 1988). The isothiocyanate functional group (N]C]S) is known to confer insecticidal, antibacterial and fungicidal properties (Lin et al., 2000). This functional group affects the metabolic functions of microorganisms by inhibiting the oxygen uptake in yeasts and fungi (Park et al., 2012). It was also reported that AITC could serve as an inhibitor for dehydrogenases and other sulfhydryl enzymes in fungi (Kalnins, 1982). In the wood preservation field, AITC was shown to form a polymer within wood in the presence of pyridine (Kalnins, 1982).
Abbreviations: MβCDs, methylated β-cyclodextrin; HPβCDs, hydroxypropylated βCDs; AITC, allyl isothiocyanate ⁎ Corresponding author. E-mail addresses: [email protected] (H. Lim), [email protected] (Y. Kim). https://doi.org/10.1016/j.indcrop.2018.12.061 Received 17 September 2018; Received in revised form 23 November 2018; Accepted 19 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2.2. Preparation of βCD-AITC complexes
Gaseous AITC treatment was proven to be effective in retarding the fungal growth and as a remedy treatment for wood already infested by fungi (Tsunoda, 2000). However, the further use of AITC in wood preservation has been limited by toxicity of pyridine and the long-term volatility of AITC. Moreover, strong pungent odor and poor solubility of AITC in water have hampered its widespread use. Cyclodextrin, a donut-shaped oligosaccharide derived from starch, is one of the commonly used host materials for encapsulating guest substances with limited water solubility, particularly in food and pharmaceutical industries. Much work has been done on the inclusion of AITC in different types of cyclodextrins and their derivatives to suppress its volatility and mask the unpleasant odor. The studies examined inclusion of AITC into α-cyclodextrin, β-cyclodextrin (βCD), methylated βCDs (MβCDs) and hydroxypropylated βCDs (HPβCDs) (Zhang et al., 2007; Neoh et al., 2012). It has been suggested that the inclusion process was mainly driven by the Van der Waals forces and the decomposition of AITC in the aqueous solution was reduced by CDs (Ohta et al., 2000). The retention of AITC in MβCD-AITC was reported to be as high as 80% at 120 °C exposures for 30 min (Neoh et al., 2012). The controlled release of AITC from βCD-AITC can effectively suppress fungal and bacterial growth on food packaging materials (Aytac et al., 2014; Piercey et al., 2012). In summary, when encapsulated in βCDs, the improved stability of AITC and its preserved antifungal and antibacterial properties suggest the potential application of βCD-AITC complexes for wood preservation. Our objective is to investigate the utility of βCD as a sustained-release carrier of AITC that serves as a natural wood preservative. In this proof-of-principle study, AITC was encapsulated within the cavity of βCDs and the βCD-AITC complexes were tested as natural wood preservatives. Two βCD derivatives, MβCD and HPβCD, were selected because of their enhanced water solubility compared to pristine βCD. The formation of βCD-AITC complexes was confirmed by Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and the maximum inclusion yield of AITC in MβCD and HPβCD was estimated by ultraviolet–visible (UV–vis) spectroscopy. The βCD-AITC complexes were applied to southern yellow pine wood cubes via impregnation in the aqueous complex solution. SEM/EDX (scanning electron microscope / energy-dispersive X-ray) mapping of the crosssections of treated wood show the homogeneous dispersion of the βCDAITC complex in the samples. Fungal resistance of the treated wood was evaluated by soil block decay test. The decreased mass loss and preserved microstructure of βCD-AITC-treated wood specimens subjected to the decay test indicate that encapsulation of AITC in βCDs significantly suppressed leaching of AITC and provided release of AITC from the complex even upon treatment in aqueous solution. This study suggests the potential of AITC encaged in the submicron-size βCD as a carrier for an environmental friendly wood preservative.
MβCD-AITC complex was prepared using a co-precipitation method with minor modifications (Li et al., 2007). Briefly, aqueous solutions of different concentrations of MβCD (1, 3, 10, 20, 50, 60 and 65 w/v (%)) were prepared by using distilled water at room temperature. AITC in ethanol (1:1, v/v) was slowly added to the solution with continuous stirring. The molar ratio of AITC to MβCD was kept constant in the ratio of 3:1. A vial containing the mixture was sealed and kept under stirring for 6 h. Then, resulting slurry was equilibrated at 4 °C for 24 h and the free AITC on the surface was pipetted out. The slurry was subjected to liquid nitrogen before freeze-drying for 36 h. The dry complex in a form of powder was stored in an airtight glass desiccator at room temperature before further analysis. The HPβCD-AITC complex was prepared by the same procedure except that the concentration of HPβCD solution used was 10–70 w/v (%) at an interval of 20 w/v (%). 2.3. Characterization of βCD-AITC complexes To confirm the formation of MβCD-AITC and HPβCD-AITC complexes, spectra of obtained powders were analyzed by ATR-FTIR (PerkinElmer Spectrum Two equipped with a universal ATR element, Perkin Elmer Ltd, Bucks, UK). Three replicates of ATR-FTIR spectra in the spectral range of 4000 to 400 cm−1 at the resolution of 4 cm−1 were obtained for every sample. The spectra were baseline-corrected and normalized to 1050 cm−1 (CeOe(H)) peak using Spectrum® Quant software (PerkinElmer, Waltham, MA, USA). The inclusion yield of AITC in βCDs was estimated using a UV–vis method (Zhang et al., 2007), which consists of 1) estimation of the amount of encapsulated AITC in βCDs and 2) taking the ratio between encapsulated and theoretical maximum of encapsulated AITC in βCDs. In a typical example, 10 mg of βCD-AITC complex dissolved in 200 μl DI water was mixed with 10 ml hexane in a test tube, and ultrasonicated in 2 cycles of 10 min of ultrasonication to free encapsulated AITC. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was analyzed by UV–vis (Cary 100 Bio UV–vis double-beam spectrophotometer). Every sample was extracted in triplicate and all measurements were carried out at room temperature. The amount of encapsulated AITC in βCDs was determined by using the calibration curve in Figure S1, which displays linear relationship between the concentration of AITC (CAITC) and the absorbance intensity in UV–vis at 249 nm in the range of 0.1–2 mM with R2 = 0.999. Thus, the mass ratio of encapsulated AITC in the βCD-AITC complex was calculated as follow:
AITCencapsulated (g / g ) =
CAITC × MAITC × V m
(1)
where, CAITC − Concentration of AITC from βCD-AITC complexes extracted by hexane (mol/L) MAITC − molecular weight of AITC (g/mol) V − total volume of the supernatant (L) m − amount of βCD-AITC used for inclusion yield estimation (g) Theoretical maximum of AITC encapsulation in βCD is known to be 1 mol of AITC in 1 mol of βCD (Zhang et al., 2007). Thus, the inclusion yield of AITC in βCD can be determined using the equation below:
2. Materials and methods 2.1. Materials Southern yellow pine (Pinus spp.) sapwood samples were cut to 14 mm × 14 mm × 14 mm (L × T × R, end matched) according to AWPA E10 standard (American Wood Protection Association, 2016). Methyl-β-cyclodextrin (MβCD, ≥ 98%, ACROS Organics, Mw 1303.3 g/ mol), hydroxypropyl-β-cyclodextrin (HPβCD, 97%, ACROS Organics, Mw 1180.05 g/mol), allyl isothiocyanate (AITC, ≥94.5%, Sigma-Aldrich), ethanol (99.5%, ACROS Organics), and toluene (99.8%, SigmaAldrich) were used as received without further purification. The degree of substitution (DS, the average number of substituents on a cyclodextrin molecule) of MβCD is between 1.7 and 1.9, which corresponding to 0.24 to 0.27 substituents per glucose units. DS of HPβCD is between 2 and 6, which corresponding to 0.28 to 0.86 substituents per glucose units. These substituents in HPβCD are located mainly at O (2) of the anhydroglucopyranose units.
Inclusion yeild (%) =
AITCencapsulated (g / g ) AITCtheoretical (g / g )
× 100
(2)
where AITCtheoretical is the maximum mass ratio of encapsulated AITC in the βCD-AITC complex, which is based on the mole ratio of AITC to MβCD-AITC or HPβCD-AITC. 2.4. Soxhlet extraction of extractives from southern yellow pine The wood cubes were treated with ethanol/toluene solution for 48 h 43
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by drying in a laminar flow cabinet. Six replicates from each treatment were exposed to a brown rot fungus (Gloeophyllum trabeum) for 8 weeks, and another six to a white rot fungus (Trametes versicolor) for 16 weeks. Both weathered and un-weathered samples, were used for soil block test. Resistance to fungi was evaluated in terms of mass loss, which was calculated as follows:
to remove extractives (ASTM D1107, 2013) and reduce their interference during chemical analysis. The amount of removed extractives was 2.45% ± 0.2% and the density of the resultant wood cubes was 0.62 ± 0.02 g/cm3, respectively. 2.5. Preservatives treatment by impregnation of wood in βCD-AITC solutions in water
Mass loss (%) = All wood cubes were submerged in a treating solution and kept under vacuum of 25 in. of Hg for 3 h. The samples were then left in the solution under atmospheric pressure for 6 h. Aqueous solutions of 50 w/ v (%) MβCD- and HPβCD-AITC were chosen as preservative treatment based on the maximum encapsulation yield of AITC in βCD-AITC. Negative control samples were treated in the same manner with distilled water, while positive controls were treated with 2% AITC in 10% ethanol.
(Vtrt . −Vuntrt . ) × 100 Vuntrt .
× 100
(6)
2.9. SEM-EDX analysis of wood before and after exposure to fungi Wood samples used for SEM imaging were microtomed cross-sections of 200 μm thickness, sputter-coated with platinum (30 nm thick). SEM images and elemental mapping of wood before exposure to fungi were taken at 15 kV (EVO50, Zeiss coupled with EDX, Bruker 133 eV). Elemental mapping was collected at a working distance of 10 mm and the acquisition time of 300 s. SEM images of wood after fungi exposure were recorded by SEM (JSM-6500 F, JEOL USA. Inc) at 5 kV.
The amount of the impregnated chemicals retained in wood samples was measured in terms of mass and volume. The oven-dry mass of the samples before and after the treatment was measured to calculate the mass gain. It should be noted that the mass gain in wood includes the mixture of βCD and βCD-AITC complexes. The treated wood cubes were first air-dried at room temperature for 24 h, and subsequently ovendried at 40 °C for another 24 h. Volumetric changes or bulking due to the incorporation of the chemicals into the cell walls were measured by buoyancy force in mercury. The mass gain and bulking are calculated as follows:
Bulking (%) =
mtrt .
where mtrt. and mtrt.&expo. are oven-dried mass of samples before and after exposure to fungi.
2.6. Mass gain and bulking
(mtrt . −muntrt . ) Mass gain (%) = × 100 muntrt .
(mtrt . −mtrt . & expo . )
2.10. Statistical analysis One-way analysis of variance (ANOVA) was performed on all data using a commercial software, SAS (9.4, SAS Institute Inc., Cary, NC). The results were interpreted at 95% confidence interval. 3. Results and discussion 3.1. Formation of βCD-AITC complexes
(3)
The inclusion of AITC in MβCD and HPβCDs is qualitatively investigated by ATR-FTIR. The neat AITC is characterized by the appearance of N]C]S out-of-phase stretching band at 2082 cm−1 and at 2165 cm−1, as shown in Fig. 1a and 1b at different spectra ranges. In MβCD- and HPβCD-AITC complexes, peaks in the vicinity of the neat AITC peaks indicated the presence of AITC in these compounds, with peak maxima shifting from 2165 and 2082 cm-1 to 2161 and 2105 cm−1, respectively, due to the different bond environment. Overall, the relative intensity of the peaks from isothiocyanate in HPβCD-AITC complex is lower than that in the MβCD-AITC complex in the spectra normalized to 1050 cm−1 (CeOeH) peak, which may suggest the lower level of AITC inclusion in HPβCD. This can be ascribed to the varying structure of βCD derivatives (Buchanan et al., 2007). The inclusion yield of AITC was quantitatively determined by the UV–vis absorbance. The inclusion yield of AITC reached a maximum of 39% in aqueous MβCD concentration of 50 w/v (%) (Figure S2b). This is in accordance with the study showing AITC inclusion yield plateau at the MβCD concentration around 50% (Landy et al., 2000). Thus, 50 w/v (%) concentration of MβCD was chosen for further wood treatment. However, the inclusion yield of AITC in HPβCD was not significantly affected by concentration of HPβCD, as confirmed by ANOVA statistical analysis, and was estimated to be around 14% (Figure S3). The lower inclusion yield of AITC in HPβCD as compared to MβCD estimated by UV–vis is in accordance with the ATR-FTIR results shown in Fig. 1b.
(4)
where muntrt. and mtrt. are oven-dried mass of samples before and after treatment, respectively. Vuntrt. and Vtrt. are oven-dried volume of samples before and after treatment, respectively. 2.7. AITC amount in wood The amount of AITC in wood (mass gain by AITC) is calculated according to the inclusion yield and mass gain data using the following equation:
Mass gain by AITC (%) = mass gain× inclusion yield × theoretical maximum of encapsulated AITC (5) 2.8. Fungal resistance test (soil block test) Prior to the exposure of the samples to fungi, and according to AWPA E10-16 standard, the samples were weathered per the procedure for oil-type preservatives in order to compare the long-term performance of βCD-AITC preservatives with AITC-treated controls. Six replicates of wood blocks for each treatment method were immersed in 400 ml of distilled water for 2 h under room temperature and then dried in a forced-air oven for 334 h at 50 °C. The blocks after weathering test were further used in the soil block test. The effect of βCD-AITC complexes on the fungal resistance of southern yellow pine was evaluated according to AWPA E10-16 standard with minor modifications (American Wood Protection Association, 2016). The modifications include numbers of the replicates and sterilization of the samples. The surface of samples was sterilized by dipping wood cubes into 70% ethanol solution in water for 10 s, followed
3.2. βCD-AITC complexes in wood The effectiveness of a wood preservative treatment can be evaluated by the amount of preservatives retained (retention/mass gain), preservative location (penetration not only into cell lumens, but also into cell walls), and the preservative distribution across the sample width (Koch, 1972). The bulking and mass gain of wood specimens after each treatment and drying is shown in Table 1. There was no volume change 44
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Fig. 1. a) Infrared spectra of (A) neat AITC, (B) MβCD, (C) HPβCD, (D) MβCD-AITC complex, and (E) HPβCD-AITC complex. Spectra shown in (B) to (D) are normalized to 1050 cm−1 (CeOeH) peak. b) The same spectra in the range of 2500–1700 cm−1.
To further confirm that the bulking and mass gain results were not simply the result of presence of the βCDs, and that AITC in fact remained in wood upon the treatment, the distribution of the preservatives in wood, SEM-EDX mapping was conducted on cross-sections of the treated wood. No sulfur was detected in the water-treated wood energy spectrum (Figure S4d), so the elemental distribution of S was not presented. As illustrated in Fig. 2c, sulfur arising from MβCD-AITC is distributed evenly across the sample. The distribution of the preservative across the cell walls is difficult to examine at the magnification level of the analysis, limited by the instrument capabilities. Although sulfur was visible in the sample treated with HPβCD-AITC (Fig. 2e), its overall intensity was lower, as in agreement with UV–vis results, and the distribution less uniform when compared to MβCDAITC-treated samples.
Table 1 Bulking and mass gain of treated southern yellow pine. Treatment
Bulking (% ( x¯ ± SD)*
Mass gain (%) ( x¯ ± SD)
Estimated amount of AITC in wood (%) ( x¯ ± SD)
Control AITC MβCD-AITC HPβCD-AITC
0 (A)** 0 (A) 0.5 ± 0.1 (B) 0.7 ± 0.1 (B)
0 (A) 0 (A) 52.1 ± 4.1 (B) 61.2 ± 9.4 (C)
0 (A) 0 (A) 1.42 ± 0.11 (B) 0.60 ± 0.08 (C)
* x¯ ± SD: Mean and standard deviation. ** Letters after x¯ ± SD represent statistical grouping (p < 0.05) in bulking and mass gain based on one-way analysis of variance (ANOVA) at 95% confidence interval.
or mass gain after neat AITC treatment, indicating its evaporation after 24 h of drying at the 40 °C. The low bulking and high mass gain of βCDAITC modified wood may be attributed to the loading of the preservatives primarily on the surfaces of wood lumens and marginal cell walls penetration into the cell walls.
3.3. Decay resistance of βCD-AITC complexes against brown-rot (G.t.) and white-rot (T.v.) fungi Long-term efficacy of the βCD-AITC complexes treatment on wood against brown-rot and white-rot fungi was evaluated following AWPA
Fig. 2. SEM images of (a) water-treated wood, (b) MβCD-AITC treated wood, (c) the corresponding EDX mapping of sulfur (S), (d) HPβCD-AITC treated wood, and (e) the corresponding EDX mapping of sulfur. 45
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Fig. 3. Photographs of southern yellow pine wood blocks with different treatments after exposure to 8-week G.t. and 16-week T.v..
of MβCD-AITC in wood samples. Mass loss of AITC-treated wood was not significantly different from the mass loss of the water-treated, and it was significantly higher than that of βCD-AITC-treated samples, indicating βCD suppressed the volatility of AITC even after subjecting samples to weathering. The stability of the complexes was also evident by no significant difference in mass loss of weathered and un-weathered MβCD- and HPβCD-AITC-treated wood. In the case of the white rot fungus, T.v., βCD-AITC treatment shows no effectiveness when mass losses are compared. This could be a result of both higher resistance of T.v. to AITC, or to the longer exposure of samples to this fungus because of its lower attacking speed on softwood (Rowell, 1995). Despite high mass loss, treated wood exposed to T.v. still retained its visual appearance. The macroscopic preserved structure of βCD-AITC treated wood decayed samples was examined on the microscopic level (Fig. 5). In the case of water-treated wood, fungal hyphae readily grew and penetrated through pits (Fig. 5c), which resulted in the severely collapsed cell wall structure. The severe damages in cell wall structures can be further seen in the high magnification SEM images in Fig. 5d and h. Conversely, no substantial fungal hyphae presence was observed in the cell wall of βCD-AITC treated wood (Fig. 5e and i), and those cell wall structures retained their overall shape, although it was modified (Fig. 5a and b). These results corroborate the fungi degradation on wood cell wall was suppressed by the presence of βCD-AITC complexes.
Fig. 4. Mass loss of water-treated and preservative-treated wood with and without weathering pretreatment after exposure to G. trabeum (8-week) and T. versicolor (16-week).
E10-16 standard. It was first noticed that in case G.t., mycelia on the feeder strips changed the color in presence of the βCD-AITC treated wood blocks, as shown in Figure S5. The mycelia of T.v. did not change the color, but its growth on the feeder strips was also suppressed in the presence of βCD-AITC treated wood blocks. The decayed wood blocks subjected to different treatments are shown in Fig. 3. The extensive decay of water-treated and neat AITC-treated wood is obvious. The significant decay of AITC-treated wood is probably due to high volatility of AITC (Figure S5), as indicated by mass gain upon treatment. In contrast, the complex-treated specimens were visually unchanged, even in the case of samples subjected to accelerated weathering before exposure to fungi. Mass loss of water-treated and preservative-treated wood samples after fungi exposure is displayed in Fig. 4. For G.t., βCD-AITC-treated wood shows a significantly lower mass loss compared to water-treated and neat AITC-treated wood. Specifically, mass loss of the water-treated samples by G.t. (45% ± 6%) was reduced to 25% ± 1% and 28% ± 3% after MβCD- and HPβCD-AITC treatments, respectively. As expected from the higher inclusion yield of AITC in MβCD than HPβCD (39% vs 14%), MβCD-AITC exhibited overall lower, although not significantly different decay mass loss, despite the lower preservative mass retention
4. Conclusions In this study, we show that βCDs can not only enhance solubility and decrease volatility of AITC (mustard oil), but also could potentially be used as carriers of antimicrobial compounds that otherwise would not be considered for wood protection. The βCD-AITC complexes were applied to wood in aqueous solutions and showed uniform distribution across wood samples, although their penetration into wood cell walls seemed to be limited to the layers bordering cell lumens. Combined results from mass loss, SEM, and EDX before and after decay test show that the βCD-AITC complexes suppressed the attack of brown and white rot fungi on wood, and helped maintain the original appearance of wood. Although the mass losses of wood were still significantly higher than the mass losses of commercially available preservatives, the use of the βCD-AITC complexes still improves the applications of AITC for remedial treatments, for example, in which AITCs volatility renders 46
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Fig. 5. SEM images taken from the cross sections of: (a and b) the water-treated wood before decay, (c and d) water-treated wood after 8-week G.t. exposure, (e and f) MβCD-AITC treated wood after 8-week of G.t. exposure, (g and h) water-treated wood after 16-week T.v. exposure, and (i and j) MβCD-AITC treated wood after 16week T.v. exposure.
References
them inapplicable (Lutomski, 1975). Future investigations are planned to examine approaches for increasing the inclusion yield of AITC in βCD, with the aim of improving wood protection performance of the AITC. The complexation of other natural compounds such as cinnamaldehyde, eugenol, and thymol with βCDs as carriers will be also pursued to develop natural wood preservatives.
American Wood Protection Association, 2016. Laboratory Method for Evaluating the Decay Resistance of Wood-based Materials Against Pure Basidiomycete Cultures: soil/block Test. pp. E10–E16. Aytac, Z., Dogan, S.Y., Tekinay, T., Uyar, T., 2014. Release and antibacterial activity of allyl isothiocyanate/β-cyclodextrin complex encapsulated in electrospun nanofibers. Colloids Surf. B Biointerfaces 120, 125–131. Buchanan, C.M., Buchanan, N.L., Edgar, K.J., Ramsey, M.G., 2007. Solubilty and dissolution studies of antifungal drug:hydroxybutenyl-$β$-cyclodextrin complexes. Cellulose 14, 35–47. https://doi.org/10.1007/s10570-006-9076-x. Chen, F., Yang, X., Wu, Q., 2009. Antifungal capability of TiO2 coated film on moist wood. Build. Environ. 44, 1088–1093. https://doi.org/10.1016/J.BUILDENV.2008. 07.018. Chittenden, C., Singh, T., 2011. Antifungal activity of essential oils against wood degrading fungi and their applications as wood preservatives. Int. Wood Prod. J. 2, 44–48. https://doi.org/10.1179/2042645311Y.0000000004. El-Gamal, R., Nikolaivits, E., Zervakis, G.I., Abdel-Maksoud, G., Topakas, E., Christakopoulos, P., 2016. The use of chitosan in protecting wooden artifacts from damage by mold fungi. Electron. J. Biotechnol. 24, 70–78. https://doi.org/10.1016/j. ejbt.2016.10.006. Fernández-Costas, C., Palanti, S., Charpentier, J.P., Sanromán, M.Á., Moldes, D., 2017. A sustainable treatment for wood preservation: enzymatic grafting of wood extractives. ACS Sustain. Chem. Eng. 5, 7557–7567. https://doi.org/10.1021/acssuschemeng. 7b00714. Gobakken, L.R., Westin, M., 2008. Surface mould growth on five modified wood substrates coated with three different coating systems when exposed outdoors. Int. Biodeterior. Biodegrad. 62, 397–402. https://doi.org/10.1016/J.IBIOD.2008.03.004. Groenier, J.S., Lebow, S., 2006. Preservative-treated Wood and Alternative Products in the Forest Service. MT US Dept. Agric. For. Serv. Technol. Dev. Program, Missoula 2006 iv, 44 pages. Guo, Huizhang, Bachtiar, Erik Valentine, Ribera, Javier, Heeb, Markus, Francis, W.M.R.Schwarze, Burgert, Ingo, 2018. Non-biocidal preservation of wood against brown-rot fungi with TiO2/Ce Xerogel. Green Chem. 19. https://doi.org/10.1039/ c7gc00195a. Kalnins, M.A., 1982. Chemical modification of wood for improved decay resistance. Wood Sci. 15, 81–89. Kamdem, D.P., Pizzi, A., Jermannaud, A., 2002. Durability of heat-treated wood. Holz als Roh- und Werkst. 60, 1–6. https://doi.org/10.1007/s00107-001-0261-1. Koch, P., 1972. Utilization of the Southern Pines-Volume 1, Agricultural Handbook SFESAH-420. USDA-Forest Service, Southern Forest Experiment Station, Asheville, NC, pp. 1–734. Landy, D., Fourmentin, S., Salome, M., Surpateanu, G., 2000. Analytical improvement in measuring formation constants of inclusion complexes between β-cyclodextrin and
Author contributions Lili Cai designed the experiments, acquired and analyzed data, and drafted the manuscript. Dr. Jeremic proposed the study and obtained the funds, assisted with the experiments, and reviewed the manuscript. Dr. Lim managed and supervised the work, and reviewed the manuscript. Dr. Kim gave suggestions on optimizing the preservatives preparation process, analyzed data and critically revised the manuscript. All authors have given approval to the final version of the manuscript. Acknowledgments This project was supported by USDA National Institute of Food and Agriculture Competitive Grant No. 2016-67022-25125. Any opinions, findings, conclusions, or recommendations expressed in the publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. The authors gratefully acknowledge Dr. Hui Wan and Dr. Jeff Morrell as co-PI for giving valuable suggestions on this project. The authors would also like to thank Dr. Frank Owens at Mississippi State University, Dr. Brian K. Via and Dr. Charles Essien at Auburn University for providing technical assistance in preparing wood sections by microtome. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.12.061. 47
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natural durability of five Cameroonian wood species. Ind. Crops Prod. 123, 183–191. https://doi.org/10.1016/j.indcrop.2018.06.078. Schwarze, F.W.M.R., Spycher, Melanie, 2005. Resistance of thermo-hygro-mechanically densified wood to colonisation and degradation by brown-rot fungi. Holzforschung. https://doi.org/10.1515/HF.2005.059. Stirling, R., Temiz, A., 2014. Fungicides and Insecticides Used in Wood Preservation. Deterior. Prot. Sustain. Biomater. pp. 185–201. Szakacs-Dobozi, M., Halasz, A., Kozma-Kovacs, E., Szakacs, G., 1988. Enhancement of mustard oil yield by cellulolytic pretreatment. Appl. Microbiol. Biotechnol. 29 (1), 39–43. https://doi.org/10.1007/BF00258348. Tascioglu, C., Yalcin, M., Sen, S., Akcay, C., 2013. Antifungal properties of some plant extracts used as wood preservatives. Int. Biodeterior. Biodegrad. 85, 23–28. https:// doi.org/10.1016/j.ibiod.2013.06.004. Thevenon, M.-F., Tondi, G., Pizzi, A., 2008. High performance tannin resin-boron wood preservatives for outdoor end-uses. Eur. J. Wood Wood Prod. 67, 89. https://doi.org/ 10.1007/s00107-008-0290-0. Tsunoda, K., 2000. Gaseous treatment with allyl isothiocyanate to control established microbial infestation on wood. J. Korean Wood Sci. Technol. 46, 154–158. https:// doi.org/10.1007/BF00777363. Usmani, S., Stephan, I., Huebert, T., Kemnitz, E., 2018. Nano metal fluorides for wood protection against fungi. ACS Appl. Nano Mater. 2018, 1–6. https://doi.org/10.1021/ acsanm.8b00144. Xie, Y., Wang, Z., Huang, Q., Zhang, D., 2017. Antifungal activity of several essential oils and major components against wood-rot fungi. Ind. Crops Prod. 108, 278–285. https://doi.org/10.1016/j.indcrop.2017.06.041. Zhang, Q.F., Jiang, Z.T., Li, R., 2007. Complexation of allyl isothiocyanate with β-cyclodextrin and its derivatives and molecular microcapsule of allyl isothiocyanate in βcyclodextrin. Eur. Food Res. Technol. 225, 407–413. https://doi.org/10.1007/ s00217-006-0431-9.
phenolic compounds. J. Incl. Phenom. Macrocycl. Chem. 38, 187–198. https://doi. org/10.1023/a:1008156110999. Li, X., Jin, Z., Wang, J., 2007. Complexation of allyl isothiocyanate by α- and β-cyclodextrin and its controlled release characteristics. Food Chem. 103, 461–466. https:// doi.org/10.1016/j.foodchem.2006.08.017. Lin, C.-M., PRESTON III, J.F., Wei, C.-I., 2000. Antibacterial mechanism of allyl isothiocyanate. J. Food Prot. 63, 727–734. Lutomski, K., 1975. Resistance of Beech Wood Modified With Styrene, Methyl Methacrylate and Diisocyanate Against the Action of Fungi. Mater. und Org. Neoh, T.L., Yamamoto, C., Ikefuji, S., Furuta, T., Yoshii, H., 2012. Heat stability of allyl isothiocyanate and phenyl isothiocyanate complexed with randomly methylated βcyclodextrin. Food Chem. 131, 1123–1131. https://doi.org/10.1016/j.foodchem. 2011.09.077. Ohta, Y., Takatani, K., Kawakishi, S., 2000. Kinetic and thermodynamic analyses of the cyclodextrin-allyl isothiocyanate inclusion complex in an aqueous solution. Biosci. Biotechnol. Biochem. 64, 190–193. Park, S.-Y., Barton, M., Pendleton, P., 2012. Controlled release of allyl isothiocyanate for bacteria growth management. Food Control 23, 478–484. https://doi.org/10.1016/J. FOODCONT.2011.08.017. Piercey, M.J., Mazzanti, G., Budge, S.M., Delaquis, P.J., Paulson, A.T., Hansen, L.T., 2012. Antimicrobial activity of cyclodextrin entrapped allyl isothiocyanate in a model system and packaged fresh-cut onions. Food Microbiol. 30, 213–218. Rowell, R.M., 1995. Chemical modification of wood for improved adhesion in composites. Proceedings of Wood Adhesives 7296, 55–60. Rowell, R.M., Ibach, R.E., McSweeny, J., Nilsson, T., 2009. Understanding decay resistance, dimensional stability and strength changes in heat-treated and acetylated wood. Wood Mater. Sci. Eng. 4, 14–22. https://doi.org/10.1080/ 17480270903261339. Saha Tchinda, J.B., Ndikontar, M.K., Fouda Belinga, A.D., Mounguengui, S., Njankouo, J.M., Durmaçay, S., Gerardin, P., 2018. Inhibition of fungi with wood extractives and
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
β-Cyclodextrins as sustained-release carriers for natural wood preservatives ⁎
Lili Cai, Dragica Jeremic, Hyungsuk Lim , Yunsang Kim
⁎
T
Department of Sustainable Bioproducts, Mississippi State University, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Wood protection Natural wood preservatives Allyl isothiocyanate β-cyclodextrins Fungi-resistant Encapsulation
Natural preservatives for wood protection have gained increasing attention due to their intrinsic antimicrobial properties, renewability, and lower impact on the environment. We report a study on the use of β-cyclodextrin (βCD) derivatives as sustained-release carriers of allyl isothiocyanate (AITC), as a model natural preservative compound. The formation of AITC inclusion complex in βCDs is qualitatively confirmed by FT-IR and the maximum inclusion yield is estimated to be 39%. Impregnation of wood with the water-borne βCD-AITC complexes allows penetration and even distribution of the preservative in the lumen and possibly in the cell walls. The efficacy of the βCD-AITC complexes as wood preservatives of southern yellow pine is examined by the AWPA E10-16 standard. Compared with the water-treated and AITC-treated wood, βCD-AITC-treated wood exhibits decrease in mass loss from 45% to 25% and no visible cell wall damage after exposure to brown and white rot fungi. The results indicate that βCD suppresses the premature leaching of otherwise volatile AITC and suggests a novel approach of application of volatile or water-immiscible natural preservatives for wood protection.
1. Introduction Despite numerous favorable characteristics of wood as a construction material, its susceptibility to biological decay under humid conditions requires appropriate protection methods to extend its service life. Approaches to protecting wood from various decay microorganisms can be categorized into passive and active methods. Passive protection renders wood less susceptible to decay mechanisms, and it involves wood surface coating with varnish (Chen et al., 2009; Gobakken and Westin, 2008), bulking or coating cell walls with metallic compounds (Guo et al., 2018; Usmani et al., 2018), chemical modification to block the hydroxyl groups (Rowell et al., 2009), physical modification by heat (Kamdem et al., 2002) and mechanical densification (Schwarze and Spycher, 2005). Active protection relies on chemical compounds that inhibit the growth or kill wood-decay organisms (Stirling and Temiz, 2014), and typically include oil-borne or waterborne preservatives (Groenier and Lebow, 2006). Although many of currently used fungicides and insecticides are highly efficient wood protectants, a lot of research emphasis is put on use of natural biocides that can provide long term protection and have a lower impact on the environment. Among studied bio-based compounds, extractives (Tascioglu et al., 2013), tannins (Thevenon et al., 2008), chitosan (ElGamal et al., 2016), and essential oils (Chittenden and Singh, 2011;
Saha Tchinda et al., 2018; Xie et al., 2017) showed good antimicrobial properties, but their use was restricted due to their long-term leachability or volatility. One of the approaches examined to overcome this problem was a study of grafting extractives into wood through the action of an enzyme, namely, laccase, but its further application is hindered by the deactivation of the enzyme and limited development of enzymatic grafting (Fernández-Costas et al., 2017). Thus, developing effective wood preservatives based on the use of natural compounds still poses a challenge. Allyl isothiocyanate (AITC), also known as mustard oil, can be obtained from cruciferous vegetables, such as wasabi, broccoli and cabbage. These plants are rich in allyl glucosinolate (sinigrin), which can be hydrolyzed to AITC by the enzyme myrosinase released from the plant tissues when they are disrupted. The yield of AITC can reach to as far as 90% by proper cellulolytic pretreatment (Szakacs-Dobozi et al., 1988). The isothiocyanate functional group (N]C]S) is known to confer insecticidal, antibacterial and fungicidal properties (Lin et al., 2000). This functional group affects the metabolic functions of microorganisms by inhibiting the oxygen uptake in yeasts and fungi (Park et al., 2012). It was also reported that AITC could serve as an inhibitor for dehydrogenases and other sulfhydryl enzymes in fungi (Kalnins, 1982). In the wood preservation field, AITC was shown to form a polymer within wood in the presence of pyridine (Kalnins, 1982).
Abbreviations: MβCDs, methylated β-cyclodextrin; HPβCDs, hydroxypropylated βCDs; AITC, allyl isothiocyanate ⁎ Corresponding author. E-mail addresses: [email protected] (H. Lim), [email protected] (Y. Kim). https://doi.org/10.1016/j.indcrop.2018.12.061 Received 17 September 2018; Received in revised form 23 November 2018; Accepted 19 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2.2. Preparation of βCD-AITC complexes
Gaseous AITC treatment was proven to be effective in retarding the fungal growth and as a remedy treatment for wood already infested by fungi (Tsunoda, 2000). However, the further use of AITC in wood preservation has been limited by toxicity of pyridine and the long-term volatility of AITC. Moreover, strong pungent odor and poor solubility of AITC in water have hampered its widespread use. Cyclodextrin, a donut-shaped oligosaccharide derived from starch, is one of the commonly used host materials for encapsulating guest substances with limited water solubility, particularly in food and pharmaceutical industries. Much work has been done on the inclusion of AITC in different types of cyclodextrins and their derivatives to suppress its volatility and mask the unpleasant odor. The studies examined inclusion of AITC into α-cyclodextrin, β-cyclodextrin (βCD), methylated βCDs (MβCDs) and hydroxypropylated βCDs (HPβCDs) (Zhang et al., 2007; Neoh et al., 2012). It has been suggested that the inclusion process was mainly driven by the Van der Waals forces and the decomposition of AITC in the aqueous solution was reduced by CDs (Ohta et al., 2000). The retention of AITC in MβCD-AITC was reported to be as high as 80% at 120 °C exposures for 30 min (Neoh et al., 2012). The controlled release of AITC from βCD-AITC can effectively suppress fungal and bacterial growth on food packaging materials (Aytac et al., 2014; Piercey et al., 2012). In summary, when encapsulated in βCDs, the improved stability of AITC and its preserved antifungal and antibacterial properties suggest the potential application of βCD-AITC complexes for wood preservation. Our objective is to investigate the utility of βCD as a sustained-release carrier of AITC that serves as a natural wood preservative. In this proof-of-principle study, AITC was encapsulated within the cavity of βCDs and the βCD-AITC complexes were tested as natural wood preservatives. Two βCD derivatives, MβCD and HPβCD, were selected because of their enhanced water solubility compared to pristine βCD. The formation of βCD-AITC complexes was confirmed by Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and the maximum inclusion yield of AITC in MβCD and HPβCD was estimated by ultraviolet–visible (UV–vis) spectroscopy. The βCD-AITC complexes were applied to southern yellow pine wood cubes via impregnation in the aqueous complex solution. SEM/EDX (scanning electron microscope / energy-dispersive X-ray) mapping of the crosssections of treated wood show the homogeneous dispersion of the βCDAITC complex in the samples. Fungal resistance of the treated wood was evaluated by soil block decay test. The decreased mass loss and preserved microstructure of βCD-AITC-treated wood specimens subjected to the decay test indicate that encapsulation of AITC in βCDs significantly suppressed leaching of AITC and provided release of AITC from the complex even upon treatment in aqueous solution. This study suggests the potential of AITC encaged in the submicron-size βCD as a carrier for an environmental friendly wood preservative.
MβCD-AITC complex was prepared using a co-precipitation method with minor modifications (Li et al., 2007). Briefly, aqueous solutions of different concentrations of MβCD (1, 3, 10, 20, 50, 60 and 65 w/v (%)) were prepared by using distilled water at room temperature. AITC in ethanol (1:1, v/v) was slowly added to the solution with continuous stirring. The molar ratio of AITC to MβCD was kept constant in the ratio of 3:1. A vial containing the mixture was sealed and kept under stirring for 6 h. Then, resulting slurry was equilibrated at 4 °C for 24 h and the free AITC on the surface was pipetted out. The slurry was subjected to liquid nitrogen before freeze-drying for 36 h. The dry complex in a form of powder was stored in an airtight glass desiccator at room temperature before further analysis. The HPβCD-AITC complex was prepared by the same procedure except that the concentration of HPβCD solution used was 10–70 w/v (%) at an interval of 20 w/v (%). 2.3. Characterization of βCD-AITC complexes To confirm the formation of MβCD-AITC and HPβCD-AITC complexes, spectra of obtained powders were analyzed by ATR-FTIR (PerkinElmer Spectrum Two equipped with a universal ATR element, Perkin Elmer Ltd, Bucks, UK). Three replicates of ATR-FTIR spectra in the spectral range of 4000 to 400 cm−1 at the resolution of 4 cm−1 were obtained for every sample. The spectra were baseline-corrected and normalized to 1050 cm−1 (CeOe(H)) peak using Spectrum® Quant software (PerkinElmer, Waltham, MA, USA). The inclusion yield of AITC in βCDs was estimated using a UV–vis method (Zhang et al., 2007), which consists of 1) estimation of the amount of encapsulated AITC in βCDs and 2) taking the ratio between encapsulated and theoretical maximum of encapsulated AITC in βCDs. In a typical example, 10 mg of βCD-AITC complex dissolved in 200 μl DI water was mixed with 10 ml hexane in a test tube, and ultrasonicated in 2 cycles of 10 min of ultrasonication to free encapsulated AITC. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was analyzed by UV–vis (Cary 100 Bio UV–vis double-beam spectrophotometer). Every sample was extracted in triplicate and all measurements were carried out at room temperature. The amount of encapsulated AITC in βCDs was determined by using the calibration curve in Figure S1, which displays linear relationship between the concentration of AITC (CAITC) and the absorbance intensity in UV–vis at 249 nm in the range of 0.1–2 mM with R2 = 0.999. Thus, the mass ratio of encapsulated AITC in the βCD-AITC complex was calculated as follow:
AITCencapsulated (g / g ) =
CAITC × MAITC × V m
(1)
where, CAITC − Concentration of AITC from βCD-AITC complexes extracted by hexane (mol/L) MAITC − molecular weight of AITC (g/mol) V − total volume of the supernatant (L) m − amount of βCD-AITC used for inclusion yield estimation (g) Theoretical maximum of AITC encapsulation in βCD is known to be 1 mol of AITC in 1 mol of βCD (Zhang et al., 2007). Thus, the inclusion yield of AITC in βCD can be determined using the equation below:
2. Materials and methods 2.1. Materials Southern yellow pine (Pinus spp.) sapwood samples were cut to 14 mm × 14 mm × 14 mm (L × T × R, end matched) according to AWPA E10 standard (American Wood Protection Association, 2016). Methyl-β-cyclodextrin (MβCD, ≥ 98%, ACROS Organics, Mw 1303.3 g/ mol), hydroxypropyl-β-cyclodextrin (HPβCD, 97%, ACROS Organics, Mw 1180.05 g/mol), allyl isothiocyanate (AITC, ≥94.5%, Sigma-Aldrich), ethanol (99.5%, ACROS Organics), and toluene (99.8%, SigmaAldrich) were used as received without further purification. The degree of substitution (DS, the average number of substituents on a cyclodextrin molecule) of MβCD is between 1.7 and 1.9, which corresponding to 0.24 to 0.27 substituents per glucose units. DS of HPβCD is between 2 and 6, which corresponding to 0.28 to 0.86 substituents per glucose units. These substituents in HPβCD are located mainly at O (2) of the anhydroglucopyranose units.
Inclusion yeild (%) =
AITCencapsulated (g / g ) AITCtheoretical (g / g )
× 100
(2)
where AITCtheoretical is the maximum mass ratio of encapsulated AITC in the βCD-AITC complex, which is based on the mole ratio of AITC to MβCD-AITC or HPβCD-AITC. 2.4. Soxhlet extraction of extractives from southern yellow pine The wood cubes were treated with ethanol/toluene solution for 48 h 43
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by drying in a laminar flow cabinet. Six replicates from each treatment were exposed to a brown rot fungus (Gloeophyllum trabeum) for 8 weeks, and another six to a white rot fungus (Trametes versicolor) for 16 weeks. Both weathered and un-weathered samples, were used for soil block test. Resistance to fungi was evaluated in terms of mass loss, which was calculated as follows:
to remove extractives (ASTM D1107, 2013) and reduce their interference during chemical analysis. The amount of removed extractives was 2.45% ± 0.2% and the density of the resultant wood cubes was 0.62 ± 0.02 g/cm3, respectively. 2.5. Preservatives treatment by impregnation of wood in βCD-AITC solutions in water
Mass loss (%) = All wood cubes were submerged in a treating solution and kept under vacuum of 25 in. of Hg for 3 h. The samples were then left in the solution under atmospheric pressure for 6 h. Aqueous solutions of 50 w/ v (%) MβCD- and HPβCD-AITC were chosen as preservative treatment based on the maximum encapsulation yield of AITC in βCD-AITC. Negative control samples were treated in the same manner with distilled water, while positive controls were treated with 2% AITC in 10% ethanol.
(Vtrt . −Vuntrt . ) × 100 Vuntrt .
× 100
(6)
2.9. SEM-EDX analysis of wood before and after exposure to fungi Wood samples used for SEM imaging were microtomed cross-sections of 200 μm thickness, sputter-coated with platinum (30 nm thick). SEM images and elemental mapping of wood before exposure to fungi were taken at 15 kV (EVO50, Zeiss coupled with EDX, Bruker 133 eV). Elemental mapping was collected at a working distance of 10 mm and the acquisition time of 300 s. SEM images of wood after fungi exposure were recorded by SEM (JSM-6500 F, JEOL USA. Inc) at 5 kV.
The amount of the impregnated chemicals retained in wood samples was measured in terms of mass and volume. The oven-dry mass of the samples before and after the treatment was measured to calculate the mass gain. It should be noted that the mass gain in wood includes the mixture of βCD and βCD-AITC complexes. The treated wood cubes were first air-dried at room temperature for 24 h, and subsequently ovendried at 40 °C for another 24 h. Volumetric changes or bulking due to the incorporation of the chemicals into the cell walls were measured by buoyancy force in mercury. The mass gain and bulking are calculated as follows:
Bulking (%) =
mtrt .
where mtrt. and mtrt.&expo. are oven-dried mass of samples before and after exposure to fungi.
2.6. Mass gain and bulking
(mtrt . −muntrt . ) Mass gain (%) = × 100 muntrt .
(mtrt . −mtrt . & expo . )
2.10. Statistical analysis One-way analysis of variance (ANOVA) was performed on all data using a commercial software, SAS (9.4, SAS Institute Inc., Cary, NC). The results were interpreted at 95% confidence interval. 3. Results and discussion 3.1. Formation of βCD-AITC complexes
(3)
The inclusion of AITC in MβCD and HPβCDs is qualitatively investigated by ATR-FTIR. The neat AITC is characterized by the appearance of N]C]S out-of-phase stretching band at 2082 cm−1 and at 2165 cm−1, as shown in Fig. 1a and 1b at different spectra ranges. In MβCD- and HPβCD-AITC complexes, peaks in the vicinity of the neat AITC peaks indicated the presence of AITC in these compounds, with peak maxima shifting from 2165 and 2082 cm-1 to 2161 and 2105 cm−1, respectively, due to the different bond environment. Overall, the relative intensity of the peaks from isothiocyanate in HPβCD-AITC complex is lower than that in the MβCD-AITC complex in the spectra normalized to 1050 cm−1 (CeOeH) peak, which may suggest the lower level of AITC inclusion in HPβCD. This can be ascribed to the varying structure of βCD derivatives (Buchanan et al., 2007). The inclusion yield of AITC was quantitatively determined by the UV–vis absorbance. The inclusion yield of AITC reached a maximum of 39% in aqueous MβCD concentration of 50 w/v (%) (Figure S2b). This is in accordance with the study showing AITC inclusion yield plateau at the MβCD concentration around 50% (Landy et al., 2000). Thus, 50 w/v (%) concentration of MβCD was chosen for further wood treatment. However, the inclusion yield of AITC in HPβCD was not significantly affected by concentration of HPβCD, as confirmed by ANOVA statistical analysis, and was estimated to be around 14% (Figure S3). The lower inclusion yield of AITC in HPβCD as compared to MβCD estimated by UV–vis is in accordance with the ATR-FTIR results shown in Fig. 1b.
(4)
where muntrt. and mtrt. are oven-dried mass of samples before and after treatment, respectively. Vuntrt. and Vtrt. are oven-dried volume of samples before and after treatment, respectively. 2.7. AITC amount in wood The amount of AITC in wood (mass gain by AITC) is calculated according to the inclusion yield and mass gain data using the following equation:
Mass gain by AITC (%) = mass gain× inclusion yield × theoretical maximum of encapsulated AITC (5) 2.8. Fungal resistance test (soil block test) Prior to the exposure of the samples to fungi, and according to AWPA E10-16 standard, the samples were weathered per the procedure for oil-type preservatives in order to compare the long-term performance of βCD-AITC preservatives with AITC-treated controls. Six replicates of wood blocks for each treatment method were immersed in 400 ml of distilled water for 2 h under room temperature and then dried in a forced-air oven for 334 h at 50 °C. The blocks after weathering test were further used in the soil block test. The effect of βCD-AITC complexes on the fungal resistance of southern yellow pine was evaluated according to AWPA E10-16 standard with minor modifications (American Wood Protection Association, 2016). The modifications include numbers of the replicates and sterilization of the samples. The surface of samples was sterilized by dipping wood cubes into 70% ethanol solution in water for 10 s, followed
3.2. βCD-AITC complexes in wood The effectiveness of a wood preservative treatment can be evaluated by the amount of preservatives retained (retention/mass gain), preservative location (penetration not only into cell lumens, but also into cell walls), and the preservative distribution across the sample width (Koch, 1972). The bulking and mass gain of wood specimens after each treatment and drying is shown in Table 1. There was no volume change 44
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Fig. 1. a) Infrared spectra of (A) neat AITC, (B) MβCD, (C) HPβCD, (D) MβCD-AITC complex, and (E) HPβCD-AITC complex. Spectra shown in (B) to (D) are normalized to 1050 cm−1 (CeOeH) peak. b) The same spectra in the range of 2500–1700 cm−1.
To further confirm that the bulking and mass gain results were not simply the result of presence of the βCDs, and that AITC in fact remained in wood upon the treatment, the distribution of the preservatives in wood, SEM-EDX mapping was conducted on cross-sections of the treated wood. No sulfur was detected in the water-treated wood energy spectrum (Figure S4d), so the elemental distribution of S was not presented. As illustrated in Fig. 2c, sulfur arising from MβCD-AITC is distributed evenly across the sample. The distribution of the preservative across the cell walls is difficult to examine at the magnification level of the analysis, limited by the instrument capabilities. Although sulfur was visible in the sample treated with HPβCD-AITC (Fig. 2e), its overall intensity was lower, as in agreement with UV–vis results, and the distribution less uniform when compared to MβCDAITC-treated samples.
Table 1 Bulking and mass gain of treated southern yellow pine. Treatment
Bulking (% ( x¯ ± SD)*
Mass gain (%) ( x¯ ± SD)
Estimated amount of AITC in wood (%) ( x¯ ± SD)
Control AITC MβCD-AITC HPβCD-AITC
0 (A)** 0 (A) 0.5 ± 0.1 (B) 0.7 ± 0.1 (B)
0 (A) 0 (A) 52.1 ± 4.1 (B) 61.2 ± 9.4 (C)
0 (A) 0 (A) 1.42 ± 0.11 (B) 0.60 ± 0.08 (C)
* x¯ ± SD: Mean and standard deviation. ** Letters after x¯ ± SD represent statistical grouping (p < 0.05) in bulking and mass gain based on one-way analysis of variance (ANOVA) at 95% confidence interval.
or mass gain after neat AITC treatment, indicating its evaporation after 24 h of drying at the 40 °C. The low bulking and high mass gain of βCDAITC modified wood may be attributed to the loading of the preservatives primarily on the surfaces of wood lumens and marginal cell walls penetration into the cell walls.
3.3. Decay resistance of βCD-AITC complexes against brown-rot (G.t.) and white-rot (T.v.) fungi Long-term efficacy of the βCD-AITC complexes treatment on wood against brown-rot and white-rot fungi was evaluated following AWPA
Fig. 2. SEM images of (a) water-treated wood, (b) MβCD-AITC treated wood, (c) the corresponding EDX mapping of sulfur (S), (d) HPβCD-AITC treated wood, and (e) the corresponding EDX mapping of sulfur. 45
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Fig. 3. Photographs of southern yellow pine wood blocks with different treatments after exposure to 8-week G.t. and 16-week T.v..
of MβCD-AITC in wood samples. Mass loss of AITC-treated wood was not significantly different from the mass loss of the water-treated, and it was significantly higher than that of βCD-AITC-treated samples, indicating βCD suppressed the volatility of AITC even after subjecting samples to weathering. The stability of the complexes was also evident by no significant difference in mass loss of weathered and un-weathered MβCD- and HPβCD-AITC-treated wood. In the case of the white rot fungus, T.v., βCD-AITC treatment shows no effectiveness when mass losses are compared. This could be a result of both higher resistance of T.v. to AITC, or to the longer exposure of samples to this fungus because of its lower attacking speed on softwood (Rowell, 1995). Despite high mass loss, treated wood exposed to T.v. still retained its visual appearance. The macroscopic preserved structure of βCD-AITC treated wood decayed samples was examined on the microscopic level (Fig. 5). In the case of water-treated wood, fungal hyphae readily grew and penetrated through pits (Fig. 5c), which resulted in the severely collapsed cell wall structure. The severe damages in cell wall structures can be further seen in the high magnification SEM images in Fig. 5d and h. Conversely, no substantial fungal hyphae presence was observed in the cell wall of βCD-AITC treated wood (Fig. 5e and i), and those cell wall structures retained their overall shape, although it was modified (Fig. 5a and b). These results corroborate the fungi degradation on wood cell wall was suppressed by the presence of βCD-AITC complexes.
Fig. 4. Mass loss of water-treated and preservative-treated wood with and without weathering pretreatment after exposure to G. trabeum (8-week) and T. versicolor (16-week).
E10-16 standard. It was first noticed that in case G.t., mycelia on the feeder strips changed the color in presence of the βCD-AITC treated wood blocks, as shown in Figure S5. The mycelia of T.v. did not change the color, but its growth on the feeder strips was also suppressed in the presence of βCD-AITC treated wood blocks. The decayed wood blocks subjected to different treatments are shown in Fig. 3. The extensive decay of water-treated and neat AITC-treated wood is obvious. The significant decay of AITC-treated wood is probably due to high volatility of AITC (Figure S5), as indicated by mass gain upon treatment. In contrast, the complex-treated specimens were visually unchanged, even in the case of samples subjected to accelerated weathering before exposure to fungi. Mass loss of water-treated and preservative-treated wood samples after fungi exposure is displayed in Fig. 4. For G.t., βCD-AITC-treated wood shows a significantly lower mass loss compared to water-treated and neat AITC-treated wood. Specifically, mass loss of the water-treated samples by G.t. (45% ± 6%) was reduced to 25% ± 1% and 28% ± 3% after MβCD- and HPβCD-AITC treatments, respectively. As expected from the higher inclusion yield of AITC in MβCD than HPβCD (39% vs 14%), MβCD-AITC exhibited overall lower, although not significantly different decay mass loss, despite the lower preservative mass retention
4. Conclusions In this study, we show that βCDs can not only enhance solubility and decrease volatility of AITC (mustard oil), but also could potentially be used as carriers of antimicrobial compounds that otherwise would not be considered for wood protection. The βCD-AITC complexes were applied to wood in aqueous solutions and showed uniform distribution across wood samples, although their penetration into wood cell walls seemed to be limited to the layers bordering cell lumens. Combined results from mass loss, SEM, and EDX before and after decay test show that the βCD-AITC complexes suppressed the attack of brown and white rot fungi on wood, and helped maintain the original appearance of wood. Although the mass losses of wood were still significantly higher than the mass losses of commercially available preservatives, the use of the βCD-AITC complexes still improves the applications of AITC for remedial treatments, for example, in which AITCs volatility renders 46
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Fig. 5. SEM images taken from the cross sections of: (a and b) the water-treated wood before decay, (c and d) water-treated wood after 8-week G.t. exposure, (e and f) MβCD-AITC treated wood after 8-week of G.t. exposure, (g and h) water-treated wood after 16-week T.v. exposure, and (i and j) MβCD-AITC treated wood after 16week T.v. exposure.
References
them inapplicable (Lutomski, 1975). Future investigations are planned to examine approaches for increasing the inclusion yield of AITC in βCD, with the aim of improving wood protection performance of the AITC. The complexation of other natural compounds such as cinnamaldehyde, eugenol, and thymol with βCDs as carriers will be also pursued to develop natural wood preservatives.
American Wood Protection Association, 2016. Laboratory Method for Evaluating the Decay Resistance of Wood-based Materials Against Pure Basidiomycete Cultures: soil/block Test. pp. E10–E16. Aytac, Z., Dogan, S.Y., Tekinay, T., Uyar, T., 2014. Release and antibacterial activity of allyl isothiocyanate/β-cyclodextrin complex encapsulated in electrospun nanofibers. Colloids Surf. B Biointerfaces 120, 125–131. Buchanan, C.M., Buchanan, N.L., Edgar, K.J., Ramsey, M.G., 2007. Solubilty and dissolution studies of antifungal drug:hydroxybutenyl-$β$-cyclodextrin complexes. Cellulose 14, 35–47. https://doi.org/10.1007/s10570-006-9076-x. Chen, F., Yang, X., Wu, Q., 2009. Antifungal capability of TiO2 coated film on moist wood. Build. Environ. 44, 1088–1093. https://doi.org/10.1016/J.BUILDENV.2008. 07.018. Chittenden, C., Singh, T., 2011. Antifungal activity of essential oils against wood degrading fungi and their applications as wood preservatives. Int. Wood Prod. J. 2, 44–48. https://doi.org/10.1179/2042645311Y.0000000004. El-Gamal, R., Nikolaivits, E., Zervakis, G.I., Abdel-Maksoud, G., Topakas, E., Christakopoulos, P., 2016. The use of chitosan in protecting wooden artifacts from damage by mold fungi. Electron. J. Biotechnol. 24, 70–78. https://doi.org/10.1016/j. ejbt.2016.10.006. Fernández-Costas, C., Palanti, S., Charpentier, J.P., Sanromán, M.Á., Moldes, D., 2017. A sustainable treatment for wood preservation: enzymatic grafting of wood extractives. ACS Sustain. Chem. Eng. 5, 7557–7567. https://doi.org/10.1021/acssuschemeng. 7b00714. Gobakken, L.R., Westin, M., 2008. Surface mould growth on five modified wood substrates coated with three different coating systems when exposed outdoors. Int. Biodeterior. Biodegrad. 62, 397–402. https://doi.org/10.1016/J.IBIOD.2008.03.004. Groenier, J.S., Lebow, S., 2006. Preservative-treated Wood and Alternative Products in the Forest Service. MT US Dept. Agric. For. Serv. Technol. Dev. Program, Missoula 2006 iv, 44 pages. Guo, Huizhang, Bachtiar, Erik Valentine, Ribera, Javier, Heeb, Markus, Francis, W.M.R.Schwarze, Burgert, Ingo, 2018. Non-biocidal preservation of wood against brown-rot fungi with TiO2/Ce Xerogel. Green Chem. 19. https://doi.org/10.1039/ c7gc00195a. Kalnins, M.A., 1982. Chemical modification of wood for improved decay resistance. Wood Sci. 15, 81–89. Kamdem, D.P., Pizzi, A., Jermannaud, A., 2002. Durability of heat-treated wood. Holz als Roh- und Werkst. 60, 1–6. https://doi.org/10.1007/s00107-001-0261-1. Koch, P., 1972. Utilization of the Southern Pines-Volume 1, Agricultural Handbook SFESAH-420. USDA-Forest Service, Southern Forest Experiment Station, Asheville, NC, pp. 1–734. Landy, D., Fourmentin, S., Salome, M., Surpateanu, G., 2000. Analytical improvement in measuring formation constants of inclusion complexes between β-cyclodextrin and
Author contributions Lili Cai designed the experiments, acquired and analyzed data, and drafted the manuscript. Dr. Jeremic proposed the study and obtained the funds, assisted with the experiments, and reviewed the manuscript. Dr. Lim managed and supervised the work, and reviewed the manuscript. Dr. Kim gave suggestions on optimizing the preservatives preparation process, analyzed data and critically revised the manuscript. All authors have given approval to the final version of the manuscript. Acknowledgments This project was supported by USDA National Institute of Food and Agriculture Competitive Grant No. 2016-67022-25125. Any opinions, findings, conclusions, or recommendations expressed in the publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. The authors gratefully acknowledge Dr. Hui Wan and Dr. Jeff Morrell as co-PI for giving valuable suggestions on this project. The authors would also like to thank Dr. Frank Owens at Mississippi State University, Dr. Brian K. Via and Dr. Charles Essien at Auburn University for providing technical assistance in preparing wood sections by microtome. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.12.061. 47
Industrial Crops & Products 130 (2019) 42–48
L. Cai et al.
natural durability of five Cameroonian wood species. Ind. Crops Prod. 123, 183–191. https://doi.org/10.1016/j.indcrop.2018.06.078. Schwarze, F.W.M.R., Spycher, Melanie, 2005. Resistance of thermo-hygro-mechanically densified wood to colonisation and degradation by brown-rot fungi. Holzforschung. https://doi.org/10.1515/HF.2005.059. Stirling, R., Temiz, A., 2014. Fungicides and Insecticides Used in Wood Preservation. Deterior. Prot. Sustain. Biomater. pp. 185–201. Szakacs-Dobozi, M., Halasz, A., Kozma-Kovacs, E., Szakacs, G., 1988. Enhancement of mustard oil yield by cellulolytic pretreatment. Appl. Microbiol. Biotechnol. 29 (1), 39–43. https://doi.org/10.1007/BF00258348. Tascioglu, C., Yalcin, M., Sen, S., Akcay, C., 2013. Antifungal properties of some plant extracts used as wood preservatives. Int. Biodeterior. Biodegrad. 85, 23–28. https:// doi.org/10.1016/j.ibiod.2013.06.004. Thevenon, M.-F., Tondi, G., Pizzi, A., 2008. High performance tannin resin-boron wood preservatives for outdoor end-uses. Eur. J. Wood Wood Prod. 67, 89. https://doi.org/ 10.1007/s00107-008-0290-0. Tsunoda, K., 2000. Gaseous treatment with allyl isothiocyanate to control established microbial infestation on wood. J. Korean Wood Sci. Technol. 46, 154–158. https:// doi.org/10.1007/BF00777363. Usmani, S., Stephan, I., Huebert, T., Kemnitz, E., 2018. Nano metal fluorides for wood protection against fungi. ACS Appl. Nano Mater. 2018, 1–6. https://doi.org/10.1021/ acsanm.8b00144. Xie, Y., Wang, Z., Huang, Q., Zhang, D., 2017. Antifungal activity of several essential oils and major components against wood-rot fungi. Ind. Crops Prod. 108, 278–285. https://doi.org/10.1016/j.indcrop.2017.06.041. Zhang, Q.F., Jiang, Z.T., Li, R., 2007. Complexation of allyl isothiocyanate with β-cyclodextrin and its derivatives and molecular microcapsule of allyl isothiocyanate in βcyclodextrin. Eur. Food Res. Technol. 225, 407–413. https://doi.org/10.1007/ s00217-006-0431-9.
phenolic compounds. J. Incl. Phenom. Macrocycl. Chem. 38, 187–198. https://doi. org/10.1023/a:1008156110999. Li, X., Jin, Z., Wang, J., 2007. Complexation of allyl isothiocyanate by α- and β-cyclodextrin and its controlled release characteristics. Food Chem. 103, 461–466. https:// doi.org/10.1016/j.foodchem.2006.08.017. Lin, C.-M., PRESTON III, J.F., Wei, C.-I., 2000. Antibacterial mechanism of allyl isothiocyanate. J. Food Prot. 63, 727–734. Lutomski, K., 1975. Resistance of Beech Wood Modified With Styrene, Methyl Methacrylate and Diisocyanate Against the Action of Fungi. Mater. und Org. Neoh, T.L., Yamamoto, C., Ikefuji, S., Furuta, T., Yoshii, H., 2012. Heat stability of allyl isothiocyanate and phenyl isothiocyanate complexed with randomly methylated βcyclodextrin. Food Chem. 131, 1123–1131. https://doi.org/10.1016/j.foodchem. 2011.09.077. Ohta, Y., Takatani, K., Kawakishi, S., 2000. Kinetic and thermodynamic analyses of the cyclodextrin-allyl isothiocyanate inclusion complex in an aqueous solution. Biosci. Biotechnol. Biochem. 64, 190–193. Park, S.-Y., Barton, M., Pendleton, P., 2012. Controlled release of allyl isothiocyanate for bacteria growth management. Food Control 23, 478–484. https://doi.org/10.1016/J. FOODCONT.2011.08.017. Piercey, M.J., Mazzanti, G., Budge, S.M., Delaquis, P.J., Paulson, A.T., Hansen, L.T., 2012. Antimicrobial activity of cyclodextrin entrapped allyl isothiocyanate in a model system and packaged fresh-cut onions. Food Microbiol. 30, 213–218. Rowell, R.M., 1995. Chemical modification of wood for improved adhesion in composites. Proceedings of Wood Adhesives 7296, 55–60. Rowell, R.M., Ibach, R.E., McSweeny, J., Nilsson, T., 2009. Understanding decay resistance, dimensional stability and strength changes in heat-treated and acetylated wood. Wood Mater. Sci. Eng. 4, 14–22. https://doi.org/10.1080/ 17480270903261339. Saha Tchinda, J.B., Ndikontar, M.K., Fouda Belinga, A.D., Mounguengui, S., Njankouo, J.M., Durmaçay, S., Gerardin, P., 2018. Inhibition of fungi with wood extractives and
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