*2.9. Cd2*+ *Adsorption*

A 1.5 mM stock solution of the heavy metal was prepared by dissolving 10 mg of cadmium carbonate in 39 mL of 0.1 M HCl. Prior to the adsorption experiments a 1.5 mg/mL suspension of each wood sample in 0.01 M phosphate bu ffer (pH 7.0) was obtained by homogenization in a Tenbroeck glass to glass homogenizer for 4 min. 0.7 mL of the wood suspensions and 0.1 mL of the metal solution were added to 10 mL of 0.01 M phosphate bu ffer at pH 7.0. After 2 h, the mixtures were filtered through a 0.45 μm nylon membrane, acidified by addition of 69% nitric acid (1:100 *v*/*v*), properly diluted with 1% nitric acid, and analyzed by ICP-MS [34]. A calibration curve was built with cadmium solutions at five di fferent concentrations. For each binding experiment a blank experiment was planned in which the metal ion was added in the phosphate bu ffer and incubated for 2 h without addition of the wood sample. Experiments were run in triplicate.

#### *2.10. Evaluation of the Solubility of Woods and Tannins in the Assay Media*

Wood or tannin samples (3 mg) were added to methanol (20 mL), 0.3 M acetate bu ffer (pH 3.6) (20 mL), or water (15 mL), and taken under magnetic stirring. After 10 or 30 min the

supernatants obtained after centrifugation (8247× *g*, room temperature, 15 min) were analyzed by UV-Vis spectrophotometry.

#### *2.11. Determination of the Amount of Tannins in the Wood Samples*

Wood or tannin samples (10 mg) were stirred in 1 mL of a 1:1 *v*/*v* acetone/water mixture containing 1% acetic acid [35]. After 60 min the supernatants obtained after centrifugation (3534× *g*, room temperature, 20 min) were analyzed by UV-Vis spectrophotometry after 1:500 *v*/*v* dilution in methanol. The amount of tannins in each wood sample was determined by comparison of the absorbance at 269 nm (chestnut-derived samples) or 280 nm (quebracho-derived samples) with that measured for chestnut or quebracho tannins, respectively.

#### *2.12. Measurement of Total Phenolic Content (TPC)*

Wood or tannin samples (10 mg) were stirred in DMSO (1 mL) for 1 h. After centrifugation (3534× *g*, room temperature, 20 min) 1–50 μL of the supernatant were added to 1.4 mL of water followed by 0.3 mL of a 75 g/<sup>L</sup> Na2CO3 solution and 0.1 mL of Folin & Ciocalteu's reagent. After 30 min incubation at 40 ◦C, absorbance at 765 nm was measured [36]. Gallic acid was used as reference compound. Experiments were run in triplicate.

#### **3. Results and Discussion**

#### *3.1. Antioxidant Properties of Exhausted Woods*

In a first series of experiments the antioxidant properties of chestnut wood fiber and exhausted chestnut and quebracho woods were investigated with respect to the corresponding fresh woods and tannins by widely used assays, i.e. DPPH, FRAP, superoxide scavenging, and ORAC assays following the "QUENCHER" method which allows one to measure the e fficiency of electron transfer processes from a solid antioxidant [27–31].

#### 3.1.1. DPPH and FRAP Assays

Table 1 reports the EC50 value, which is the dose of the material at which a 50% DPPH reduction is observed, determined for tannins and wood samples in the DPPH assay. For comparison data for the reference antioxidant Trolox are also reported.


**Table 1.** Antioxidant properties of tannins and wood samples.<sup>1</sup>

1 Reported are the mean ± SD values of at least three experiments. 2 EC50 is the dose of the material at which a 50% 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction is observed.

Among the waste materials, chestnut wood fiber displayed the most promising DPPH-reducing ability, with an EC50 value of 0.054 mg/mL that compares well with that of Trolox (3.6-fold higher). Also quebracho and particularly chestnut exhausted wood exhibited quite low EC50 values, much lower than those reported for other agro-food wastes, such as spent co ffee grounds (EC50 = 5.00 mg/mL) [4]. As expected, higher antioxidant activities were exhibited by fresh wood samples, still containing tannins, and tannins themselves, characterized by EC50 values approaching that of Trolox.

The marked di fferences observed in the antioxidant activity may be interpreted considering the solubility of the materials in the assay medium. Spectra shown in Figure 3a clearly indicate release of UV absorbing species in the case of pure tannins, chestnut wood fiber, and fresh woods, whereas exhausted woods do not give rise to appreciable absorbance.

The results of the FRAP assay (Table 1) looked less encouraging than those obtained in the DPPH assay, with all the waste materials exhibiting an iron(III)-reducing activity far lower than Trolox (Trolox equivalents <<1). Only chestnut tannins showed a satisfactory antioxidant power in this assay, whereas the fresh wood samples as well as quebracho tannins performed much less. Here again the antioxidant activity parallels fairly well the solubility in the medium used for the FRAP assay (Figure 3b).

To obtain information about the compounds responsible for the antioxidant properties observed, the amounts of tannins and the TPC were determined for each wood sample (Table 2). As shown in Figure 4, a good linear correlation was found between Trolox equivalents determined in the FRAP assay and the tannin content (for both the hydrolyzable and non-hydrolyzable class), but not TPC (*R*<sup>2</sup> = 0.59), pointing to residual tannins as the main determinants of the iron-reducing properties. On the contrary, DPPH reducing ability was not apparently related to either TPC ( *R*<sup>2</sup> = 0.37) or tannin content ( *R*<sup>2</sup> = 0.33 and 0.50 for chestnut and quebracho-derived samples, respectively), suggesting that several factors (e.g., relative solubility in the assay medium) might be involved in the di fferent activities observed.

The exhausted woods and the chestnut wood fiber that have been subjected to the hydrolytic treatment according to the protocol developed in previous studies [4,25,26] were then evaluated for their antioxidant activity by the DPPH and FRAP assays. Data shown in Figure 5a indicate for both exhausted quebracho and chestnut woods a decrease of EC50 values in the range of 25%–30% compared to the values obtained for the untreated materials. By contrast no significant increase of the activity was observed in the FRAP assay following the hydrolytic treatment, apart for chestnut wood fiber, which exhibited a two-fold increase in Trolox eqs (Figure 5b).

**Figure 3.** UV-Vis spectra of wood and tannin samples (0.15 mg/mL) in methanol (**a**) or 0.3 M acetate bu ffer (pH = 3.6) (**b**).


**Table 2.** Tannin content and total phenolic content (TPC) of wood samples.

1 Reported are the mean values of at least three experiments (SD ≤ 5%). 2 Reported are the mean values ± SD of at least three experiments.

**Figure 4.** Correlation between tannin content and Fe3<sup>+</sup>-reducing activity of tannin and wood samples. (**a**) Chestnut-derived samples; (**b**) Quebracho-derived samples.

**Figure 5.** Antioxidant properties of exhausted wood samples before and after hydrolytic activation. (**a**) DPPH assay; (**b**) ferric reducing/antioxidant power (FRAP) assay. Reported are the mean ± SD values of at least three experiments.

#### 3.1.2. Superoxide Scavenging Assay

A very high efficiency, compared to other agri-food waste products such as spent coffee grounds [4], was observed in the superoxide scavenging assay (Figure 6), with tannins and fresh woods being always the most active samples, although in the case of chestnut-derived materials both the exhausted woods exhibited an activity comparable to that of the native sample. Only a modest correlation (*R*<sup>2</sup> = 0.90 and 0.82 for chestnut and quebracho-derived samples, respectively) was found between the percentage of superoxide scavenging and the tannin content, whereas no correlation was found with TCP. Moreover, no significant improvement in the scavenging ability was detected in exhausted woods subjected to the hydrolytic treatment.

## 3.1.3. ORAC Assay

The relative fluorescence intensities determined in the ORAC assay are reported in Table 3. In this case major differences were apparent between tannins/fresh woods and exhausted samples, with the first ones almost totally inhibiting fluorescein oxidation and the second ones being completely inactive, with the only exception of chestnut wood fiber, which showed an activity comparable to that of fresh chestnut wood. Notably, in this case a good linear correlation (*R*<sup>2</sup> = 0.95) was found between the antioxidant activity of the wood samples and TPC; on the contrary no significant correlation was found with the amount of residual tannins. No effect of the hydrolytic treatment was apparent either in this assay.

**Figure 6.** Superoxide scavenging activity of tannins and wood samples. Reported are the mean ± SD values of at least three experiments.



1 Reported are the mean values of at least three experiments (SD ≤ 5%). 2 Mixture containing only fluorescein and APPH, without any antioxidant.

The results of the antioxidant assays further add to the potential of wood fiber as a reinforcement in polymers [37,38] or as an ecological-friendly medium for horticultural practice to increase the antioxidant activity of fruit and vegetables [39].

#### *3.2. Pollutant Adsorption Properties of Exhausted Woods*

In a further series of experiments the adsorption capacity of the exhausted woods toward various environmental pollutants was evaluated. These included MB, as a model organic dye, NOx, that is nitric oxide (NO) and nitrogen dioxide (NO2) which are reactive nitrogen species commonly present in cigarette smoke and exhaust gases able to induce oxidative and nitrosative stress in humans, and cadmium ions (Cd<sup>2</sup>+), as a model of toxic heavy metals.

#### 3.2.1. MB Adsorption Assay

Table 4 reports the percentages of MB adsorption by the wood samples.


**Table 4.** Pollutant adsorption properties of wood samples.<sup>1</sup>

1 Reported are the mean ± SD values of at least three experiments. 2 Determined with methylene blue (MB) at 5 mg/L. 3 Determined with 0.6 mL of gas.

Both exhausted chestnut and quebracho woods proved to be very efficient at a dose of 0.2 mg/mL, and their adsorption properties were comparable to those exhibited by the fresh wood samples. Indeed, UV-Vis spectrophotometric analysis (Figure 7) indicated a lower solubility in the assay medium (water) of chestnut wood fiber and exhausted woods compared to the fresh samples, a feature that further adds to the potential of these materials for removal of organic compounds from waste waters.

**Figure 7.** UV-Vis spectra of wood samples (0.2 mg/mL) in water.

A 20%–30% dye removal was still observed when wood samples were added to a more concentrated MB solution (25 mg/mL) (Figure 8). For comparison, under the same conditions a 70% MB removal was obtained with activated carbon, taken as a reference material. In contrast to what observed in the antioxidant assays, the MB adsorption ability of the exhausted woods did not change appreciably following to acid treatment.

**Figure 8.** MB adsorption by chestnut wood fiber and exhausted woods before and after hydrolytic activation (MB starting concentration 25 mg/L). Reported are the mean ± SD values of at least three experiments. For comparison data relative to activated carbon are also reported.

#### 3.2.2. NOx Scavenging Assay

All the samples examined led to >70% scavenging when 0.2 mL of NOx gases were passed through 5 mg of wood (data not shown). The percentages of NOx scavenging determined in the experiments run with 0.6 mL of gas are reported in Table 4, from which the superior ability of quebracho woods stands out compared to chestnut woods. These data would sugges<sup>t</sup> a higher trapping efficiency of condensed tannins with respect to hydrolyzable tannins that could likely be ascribed to the reactivity of the resorcinol moieties of profisetinidins toward electrophiles like nitric oxides [40]. As observed above for MB adsorption, also in this case fresh woods were only slightly more active than exhausted samples. Notably, further to acid treatment a lower efficiency was observed for most of the tested woods (not shown), pointing to a role of the hydrolyzable cellulosic matrix in NOx scavenging.

#### 3.2.3. Cd2+ Removal Assay

In a last series of experiments the ability of wood samples to remove heavy metals from aqueous solutions was investigated using Cd2+ as model ions. The assay was performed at pH 7.0, based on previous observations showing that natural phenolic polymers do not exhibit significant chelating properties at acidic pH [34]. In this case fresh woods were not evaluated because of the release of significant amounts of material under the testing conditions, likely due to solubilization of residual tannins and/or other low molecular weight components.

Based on the data reported in Figure 9, chestnut wood fiber was again the most active among the waste materials, followed by exhausted quebracho wood. An increase in Cd2+ removal was observed following hydrolytic activation for all the woods examined.

**Figure 9.** Cd2+ removal by exhausted woods before and after hydrolytic activation. Reported are the mean ± SD values of at least three experiments.
