Laccase-Mediated Enhancement of the Antioxidant Activity of Propolis and Poplar Bud Exudates.

The treatment of propolis and poplar bud exudates with laccase from Trametes versicolor and 2,2,6,6-tetramethyl-1-piperidinyloxy free radical increased the antioxidant activity, as evaluated by the 2,2'-diphenyl picrylhydrazyl (DPPH)- and t-butyl-OOH-induced DNA breakage comet assay analyses. The effect was highest for shorter reaction times. Propolis showed the highest antioxidant activity in the DPPH test, whereas poplar bud exudates were more active in reducing the t-butyl-OOH-induced lesions in the Chinese hamster ovary cell line. Even if the concentration of polyphenols decreased during the oxidation, the formation of low-molecular-weight phenols phloroglucinol 4 (1,3,5-trihydroxy benzene), hydroquinone 5 (1,4-dihydroxy benzene), and catechol 6 (1,2-dihydroxy benzene), characterized by the radical-scavenging activity, can account for the observed increase in the antioxidant activity.

Introduction

Propolis is a dark-colored resinous substance collected by Apis mellifera from various tree buds for the construction and adaptation of nests.[1−3] The composition of propolis is chemically related to that of bud exudates from which it derives, reflecting the specificity of the local ecological conditions.[4,5] Propolis and poplar bud exudates show antitumoral,[6] antibacterial,[7] anti-inflammatory,[8] antiviral,[9] and antioxidant properties.[10−14] Phenyl propanoid acids and flavonoids (polyphenols) are mainly responsible for the antioxidant activity.[15,16] The position and stereoelectronic properties of the substituents on the aromatic rings of polyphenols[17,18] as well as the presence of catechol and pyrogallol pharmacophores tune the antioxidant effect.[19−21]

The main transformations of polyphenols during food processing are catalyzed by phenol oxidases, such as laccases.[22,23] Laccases (EC 1.10.3.2, para-diphenol, dioxygen oxidoreductase) are multicopper enzymes that catalyze the oxidation of four molecules of substrate through the reduction of dioxygen (O2) to water.[24] They are tolerant to non-natural media (organic solvents and ionic liquids) and resistant to different types of inhibitors.[25] Examples of the applications of nonaqueous enzymology in the oxidation of polyphenols with laccases have already been reported.[26,27] The reactivity of laccases (E0 = 0.5–0.8 V vs normal hydrogen electrode)[28] toward high-redox-potential substrates can be increased in the presence of redox mediators in the so-called laccase-mediator systems (LMSs).[29] Redox mediators, such as 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO; E0 = 0.75 V),[30] diffuse far away from the active site of the enzyme in the bulk of the solution, increasing the possible biotechnological applications.[31] TEMPO undergoes a one-electron transfer mechanism to form the oxoammonium ion intermediate.[32] TEMPO-based LMSs were shown to be the best catalytic systems in the oxidation of different recalcitrant compounds.[33] LMSs oxidize polyphenols by radical cross-coupling reactions,[34] oxidative ring-opening reactions,[35] and aromatic ring hydroxylations.[36,37] The oxidation of catechins to theaflavins by laccases is recognized as a model for the chemical transformation of green tea into black tea.[38] Moreover, laccases and vanillin (i.e., a natural redox mediator) act as sensoring systems to analyze poplar hydrolysates.[39] Preliminary data reported that the oxidation of different propolis (geographical locations and floral origin: Livada, Carei, Cristian, Fagaras, and Gura) with laccase from Sclerotinia sclerotiorum without redox mediators generally leads to the pro-oxidant activity in a complex laccase–hemoglobin system. In this latter case, the pro-oxidant activity was not correlated with the redox potential.[40] We describe here that the oxidation of propolis and poplar bud exudates with laccase from Trametes versicolor in the presence of TEMPO increased the antioxidant activity for a shorter reaction time, as evaluated by the 2,2′-diphenyl picrylhydrazyl (DPPH)[41] test and by the t-butyl-OOH-induced DNA breakage in the alkaline comet assay.[42] The antioxidant activity was compared with the oxidation pathway of polyphenols because they are recognized to be the main factors responsible for the antioxidant activity of propolis and poplar bud exudates.[43,44]

Results and Discussion

Commercially available black bud poplar exudate (Populus nigra) and propolis (from China) were extracted with MeOH (80% v/v water) to afford PN-A and PROP samples, respectively. The extraction of the black bud poplar exudate was also performed with EtOH (80% v/v water) to afford PN-B. The samples were preliminarily characterized by gas chromatography–mass spectrometry (GC–MS) after treatment with N,O-bis-trimethylsilyl trifluoroacetamide (NBSA) in the presence of oleic acid as internal standard. The GC profiles of PROP, PN-A, and PN-B samples are in Supporting Information SI #1 (Figures SI1A–C). To identify the chemical structure of polyphenols, two strategies were followed. First, the spectra of identifiable peaks were compared with commercially available mass spectrum libraries, such as NIST (Fison, Manchester, U.K.). In contrast, the GC–MS analyses were repeated using commercially available standard compounds (method of addition). Tables –3 report the most abundant polyphenols detected in the samples [mass-to-charge ratio (m/z) values and the abundance of peaks of identified polyphenols are in Table SI2A; mass-to-charge ratio fragmentation spectra of identified polyphenols are in SI #3]. We identified a large panel of compounds, including (a) phenol derivatives: 2-hydroxybenzyl alcohol, salicylic acid, and vanillin; (b) phenyl propanoid derivatives: cinnamyl alcohol, cinnamic acid, 4-hydroxy cinnamic acid, 4-hydroxy cinnamic acid methyl ester, 3,4-dimethoxy cinnamic acid, ferulic acid, caffeic acid, and caffeic acid phenethyl ester; (c) flavanons: pinostrobin, naringenin, and sakuranetin; (d) diidro flavonols: pinobanksin and 3-O-acetyl pinobanksin; (e) flavons: chrysin, tectochrysin, and genkwanin; and (f) flavanols: galangin, kaempferol, isorhamnetin, and quercetin (only in the case of PN-A and PN-B samples). The structures of the identified polyphenols are presented in SI #4.

Table 1

Composition of the Most Abundant Polyphenols in the PN-A Sample before and after Treatment with Laccase from T. versicolor and TEMPOa

		starting material	t = 1 h	t = 7 h	t = 15 h
type	compound	yield (mg/g)b
phenols	hydroquinone	n.d.	8.83	6.35	3.75
	catechol	n.d.	0.41	0.19	0.05
	phloroglucinol	n.d.	0.18	0.07	0.03
	2-hydroxybenzyl alcohol	0.01	0.01	0.01	0.005
	salicylic acid	0.94	0.60	0.54	0.35
	vanillin	0.16	0.14	0.03	0.01
phenyl propanoids	cinnamyl alcohol	0.51	0.25	0.29	0.11
	cinnamic acid	9.40	4.33	4.95	2.11
	4-hydroxy cinnamic acid	1.53	1.51	1.47	1.22
	4-hydroxy cinnamic acid MEc	2.87	0.93	0.87	0.32
	3,4-dimethoxy cinnamic acid	4.63	6.66	2.46	1.11
	ferulic acid	7.12	1.38	1.36	1.28
	caffeic acid	9.20	9.83	1.01	0.81
	caffeic acid PEd	10.24	10.67	10.24	8.10
flavanones	pinostrobin	8.43	4.99	4.71	4.65
	naringenin	16.63	7.93	7.52	3.32
	sakuranetin	5.23	4.81	4.75	4.13
dihydro flavonols	pinobanksin	17.85	11.81	9.53	8.44
	3-O-acetyl pinobanksin	9.4	6.27	5.10	2.50
flavones	chrysin	16.03	13.94	13.23	13.81
	tectochrysin	4.35	5.84	5.67	3.71
flavonols and flavonol derivatives	galangin	2.55	1.74	0.39	0.09
	kaempferol	2.50	1.13	0.27	0.07
	isorhamnetin	3.12	2.17	2.14	2.0
	quercetin	0.63	0.50	0.40	0.12
	compound 1e	n.d.	n.d.	1.18	n.d.
	compound 2	n.d.	n.d.	0.16	n.d.
	compound 3	n.d.	n.d.	1.02	n.d.

The oxidations were performed by treating the appropriate sample (0.1 g) with laccase (10 U) and TEMPO (6 mM) in 0.1 M sodium acetate buffer (3.0 mL, pH 5–5.5) at 40 °C, in the presence of a low amount of CH2Cl2 (5.0% v/v).

The yield was reported as milligrams of compound per gram of starting material.

ME: methyl ester.

PE: Phenethyl ester.

Compound 1: 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone; compound 2: 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone; compound 3: galangin dimer.

Table 3

Composition of the Most Abundant Polyphenols in the PROP Sample before and after Treatment with Laccase from T. versicolor and TEMPOa

		starting material	t = 1 h	t = 7 h	t = 15 h
type	compound	yield (mg/g)b
phenols	hydroquinone	n.d.	8.83	5.35	2.15
	catechol	n.d.	0.37	0.12	0.03
	phloroglucinol	n.d.	0.14	0.07	0.01
phenyl propanoids	cinnamyl alchol	4.56	5.17	2.55	2.98
	cinnamic acid	2.37	2.96	1.87	1.80
	4-hydrocinnamic acid	0.40	0.20	0.13	0.14
	3,4-dimethoxy cinnamic acid	1.91	2.30	1.17	1.47
	4-hydroxy cinnamic acid MEc	1.12	1.62	1.67	1.58
	caffeic acid	1.34	1.14	0.98	0.31
	caffeic acid PEd	18.23	10.85	7.29	7.18
	ferulic acid	9.25	2.59	2.34	2.13
flavanones	pinostrobin	4.30	3.26	2.95	2.30
	naringenin	0.25	0.13	0.11	0.08
	sakuranetin	0.17	0.14	0.08	0.10
dihydro flavonols	pinobanksin	3.32	2.68	2.70	1.82
	3-O-acetyl pinobanksin	0.03	0.02	0.01	n.d.
flavones	chrysin	35.57	33.25	29.36	17.96
	tectochrysin	2.58	3.42	3.11	2.12
	genkwanin	1.48	1.34	1.27	1.11
flavonols and flavonol derivatives	galangin	13.23	10.84	7.12	6.68
	kaempferol	0.19	0.09	0.08	0.04
	isorhamnetin	3.33	2.47	0.46	traces
	compound 1e	n.d.	n.d.	0.05	n.d.
	compound 3	n.d.	n.d.	5.32	n.d.

The oxidations were performed by treating the appropriate sample (0.1 g) with laccase (10 U) and TEMPO (6 mM) in 0.1 M sodium acetate buffer (3.0 mL, pH 5–5.5) at 40 °C, in the presence of a low amount of CH2Cl2 (5.0% v/v).

The yield was reported as milligrams of compound per gram of starting material.

ME: methyl ester.

PE: phenethyl ester.

Compound 1: 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone; compound 3: galangin dimer.

The oxidation of PROP, PN-A, and PN-B samples was performed with laccase from T. versicolor,[45] in the presence of TEMPO.[46−51] Laccase from T. versicolor showed the optimal pH value of 5.5 and the following kinetic parameters: Km = 1.6 mM, Vmax = 1381 × 10–3 ΔAbs (min μgenzyme)−1, Vmax/Km = 8752 (average errors in the kinetic parameters were ±2–4% for Km and ±1–3% for Vmax). Briefly, the appropriate sample (0.1 g) was treated with laccase (10 U) and TEMPO (6 mM) in 0.1 M sodium acetate buffer (3.0 mL, pH 5–5.5) at 40 °C, in the presence of a low amount of CH2Cl2 (5.0% v/v).[31] Nonaqueous enzymology shows numerous advantages with respect to traditional conditions, such as increased solubility of substrates, improved stability and selectivity, and limited side reactions.[52] The reaction mixtures were vigorously stirred at room temperature, quenched after 1, 7, and 15 h, and analyzed by GC–MS. The GC profiles at 1 h are reported in Supporting Information SI #1 (Figures SI #1D–SI #1F). The reaction mixtures quenched at 7 h were also purified by high-performance liquid chromatography (HPLC, as selected samples) to isolate possible degradation products. Tables –3 report the composition of polyphenols in the reaction mixtures (columns 4–6). Figure depicts the pattern of different types of polyphenols at different reaction times.

Figure 1

Variation of the concentration of polyphenols (mg/g) with the reaction time (h) in the oxidation of PROP, PN-A, and PN-B samples with T. versicolor and TEMPO.

Regardless of the experimental conditions, polyphenols decreased continuously during the oxidation process, reaching ca. 50% of the initial concentration at 15 h. The PN-A sample showed a higher depletion of polyphenols than the PN-B sample (Figure ) probably due to the presence of an appreciable amount of vanillin, which is a natural redox mediator for laccase.[53] The general order of the reactivity of flavonoids observed in all samples was: flavonols > flavanones, dihydro flavonols > flavones (very low reactivity). Phenyl propanoid derivatives were all efficiently oxidized by laccase (Tables –3, Figure ).

The high reactivity of flavonols was in accordance with the data previously reported for the oxidation of purified flavonoids with laccase from T. versicolor. This latter study highlighted the important role played by the hydroxy group at C3 of the chromene ring in the first step of the reaction. The mechanism requires the initial H abstraction from C3-OH followed by the formation of a carbocation at C-2 (by dismutation reaction between two radicals), nucleophilic addition, and rearrangement of the C-ring (see Figure for the nomenclature of the aromatic rings in flavonols).[54] The occurrence of this oxidative pathway was confirmed by the detection of 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone 1, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone 2, and the galangin dimer 3 in the reaction mixtures quenched at 7 h after HPLC purification (at a selected reaction time) (Scheme ).[54] Compounds 1 and 2 were produced by the addition of water to the respective C2 carbocation intermediates, followed by the C-ring rearrangement, whereas compound 3 was derived from the addition of a second molecule of galangin on the first formed C2 galangin carbocation.[54−58] Under similar experimental conditions, flavones were found to be stable, thereby highlighting the crucial role of the C3-OH group in the oxidation of flavonoids with laccase.[59]

Figure 2

Mean tail moment (TM) values obtained at different recovery times for both control and t-butyl-OOH-treated samples with and without pretreatment with PROP. Student’s t-test, § t-butyl-OOH vs control, p < 0.01; * Propolis 1, 7, and 15 h vs t-butyl-OOH, p < 0.01.

Scheme 1

Oxidation Products Detected during the Treatment of PROP, PN-A, and PN-B Samples with T. versicolor and TEMPO

Simple low-molecular-weight phenols phloroglucinol 4, hydroquinone 5, and catechol 6 were also detected in appreciable amounts by GC–MS analyses (Tables –3, Figure , and Scheme ). Phloroglucinol (1,3,5-trihydroxy benzene) is usually produced during the degradation of the A-ring of kaempferol and naringenin by oxidative metabolism.[60,61] Moreover, 2,4,6-trihydroxy benzoic acid, which is a chemical precursor of phloroglucinol by decarboxylation, was obtained by the treatment of quercetin with the peroxyl-radical-generating agent, azobisisobutyronitrile.[55] Hydroquinone (1,4-dihydroxy benzene) and catechol (1,2-dihydroxy benzene) have been observed during the oxidation of flavonoids and other complex phenols.[62] The concentration of compounds 4–6 was highest at 7 h of the oxidation (Figure ).

The antioxidant activities of the PROP, PN-A, and PN-B samples before and after the treatment with laccase were initially evaluated by the DPPH radical-scavenging analysis.[63] Briefly, the appropriate sample was dissolved in EtOH (0.01–100 mg/mL) and added to a freshly prepared DPPH solution (6 × 10–5 M in EtOH). The decrease in absorbance (475 nm) was determined at different times until the reaction reached a plateau. The kinetics of the process was analyzed for each concentration tested, and the DPPH activity remaining at the steady state was estimated. This value was used to calculate IC50 (i.e., the concentration of substrate in milligrams per milliliter that causes 50% loss of the DPPH activity).[64] PROP showed an IC50 value (110 μg/mL) of the same order of magnitude as Brazilian, Korean, and Australian red propolis (Table , entry 1).[65,66] The IC50 values of the PN-A and PN-B samples were higher than those of PROP (300 and 290 μg/mL, respectively) and similar to those of Populus tremula and P. nigra (337 and 485 μg/mL, respectively).[67] Irrespective of the nature of the sample, the DPPH antioxidant activity increased during the first hour of the treatment, reaching the lowest IC50 value (35 μg/mL) in the case of PROP (Table , entry 2). For a longer reaction time, a progressive increase in the IC50 values was observed (Table , entries 3 and 4).

Table 4

IC50 Values of PROP, PN-A, and PN-B Samples before and after Treatment with Laccase from T. versicolor and TEMPO

		IC50a,b
entry	reaction time (h)	PN-A	PN-B	PROP
1	0	300 ± 0.13	290 ± 0.16	110 ± 0.12
2	1	50 ± 0.09	53 ± 0.07	35 ± 0.07
3	7	314 ± 0.18	251 ± 0.27	99 ± 0.10
4	15	456 ± 0.21	339 ± 0.12	294 ± 0.24

IC50 is defined as the drug concentration (μg/mL) causing 50% inhibition of the desired activity.

Each experiment was performed in triplicate.

To have a more realistic scenario of the complex interactions within the cell in biological systems, the PROP and the more DPPH-active PN-A samples were selected for the evaluation of the antioxidant activity in Chinese hamster ovary (CHO) cell line. Mammalian cells are endowed with several levels of defense mechanisms against oxidative damage, the first of which is based on the quenching of reactive oxygen species by antioxidants, such as ferritin and ceruloplasmin.[68] The antioxidant activity was assessed by the ability to reduce the extent of DNA breakage [single-strand breaks (SSBs)] induced by tert-butyl-hydroperoxide (t-butyl-OOH), using a slightly modified version of the alkaline comet assay, which is a rapid and sensitive procedure to quantify DNA lesions in mammalian cells.[69,70] The frequency of SSBs of DNA and “alkali-labile sites” was measured immediately after the treatment and at different recovery times in CHO, with and without pretreatment with the samples. This technique detects both SSBs and the so-called alkali-labile sites, which are observed as SSBs after denaturation under alkaline condition (both types of lesions will be referred to as SSBs because real SSBs and alkali-labile sites in this assay cannot be distinguished). The results obtained in control cells and in cells pretreated with the samples after treatment with t-butyl-OOH are reported in Figures and 3. Pretreatment with the samples alone did not increase the TM values (i.e., the product of the tail length and the fraction of total DNA in the tail), suggesting the absence of pro-oxidant activity in the samples after the laccase treatment, whereas the presence of t-butyl-OOH significantly enhanced the frequencies of SSBs, as shown by the increase of TM. The kinetics of the repair of the oxidative DNA damage was assessed at 0, 0.5, and 1 h after treatment with t-butyl-OOH (with or without pretreatment with the selected samples). We observed a statistically significant reduction in the mean TM in the presence of PROP with respect to t-butyl-OOH, except for the sample treated at 15 h. In particular, the most effective compound in reducing the t-butyl-OOH-induced DNA damage was PROP after 7 h of treatment with laccase (Figure ).

Figure 3

Mean TM values obtained at different recovery times for both control and t-butyl-OOH-treated samples with and without pretreatment with PN-A. Student’s t-test, § t-butyl-OOH vs control, p < 0.01; * PN-A 1, 7, and 15 h vs t-butyl-OOH, p < 0.01.

In a similar way, we observed a statistically significant reduction in the mean TM in the presence of PN-A treated with laccase for short time compared with t-butyl-OOH alone. In this latter case, the most effective compound in reducing the t-butyl-OOH-induced DNA damage was the PN-A sample after 1 h of treatment (Figure ).

Conclusions

In conclusion, polyphenols decreased during the treatment of propolis and poplar bud exudates with laccase from T. versicolor and TEMPO. Phenyl propanoid acids and flavonoids (with the only exception of recalcitrant flavones) were efficiently oxidized. Low-molecular-weight phenols hydroquinone, catechol, and phloroglucinol were produced as degradation products during the first period of the oxidation, in addition to some flavonol derivatives modified on the C-ring.[71] The antioxidant activities of propolis and poplar bud exudates did not display a reactivity-related similarity to the trend of polyphenol derivatives. The antioxidant activity increased immediately (from 0 to 7 h) and then decreased rapidly for higher reaction times. In the mammalian cells (comet assay), t-butyl-OOH markedly induced primary damage, whereas pretreatments with propolis and poplar bud exudates did not have effects on the DNA integrity, indicating the absence of pro-oxidant effects. The comet assay data clearly indicated that the samples treated with laccase and TEMPO were able to enhance significantly the extent and the rate of DNA repair of t-butyl-OOH-induced lesions. A marked protection against the oxidative DNA breakage was observed for PN-A (treatment at 1 h) and PROP (treatment at 7 h). On the basis of these data, the enhancement of the antioxidant properties of propolis and poplar bud exudates after treatment with laccase and TEMPO could be reasonably attributed to the formation of hydroquinone, catechol, and phloroglucinol because of their ability to scavenge radical species,[72−75] even if a general modulatory effect on the endogenous antioxidant systems of the cell cannot be completely ruled out.[76] Noteworthy, the PROP sample showed the highest antioxidant activity in the DPPH test, whereas PN-A was most effective in the comet assay, confirming a different responsiveness of the mammalian cells with respect to the DPPH analysis.

Experimental Section

Laccase from T. versicolor (Sigma-Aldrich) is a monomer, organized in three sequentially arranged domains with a molecular mass of approximately 62–70 kDa, having dimensions of about 6.5 nm × 5.5 nm × 4.5 nm and isoelectric point of 4,2.[77,78] TEMPO, tert-butyl-hydroperoxide (t-butyl-OOH) [75-91-2], and organic solvents were purchased from Sigma-Aldrich. Commercially available black bud poplar exudates (P. nigra) and propolis were obtained from Shaanxi Teng Yun Biotechnology Co. Ltd (Lianhu District, Xian, China). All spectrophotometric measurements were made with a Varian Cary 50 UV–vis spectrophotometer equipped with a single-cell Peltier thermostated cell holder. Spectrophotometric data were analyzed with Cary UV software. All experiments were carried out in triplicate. GC–MS analysis was performed using a combined LC–GC–MS instrument by Varian. The following are the parameters used for the analysis: SLB-5ms capillary GC column; film thickness, 30 m × 0.25 mm × 0.25 mm (Supelco-28471-U); flow velocity of the carrier (helium), 1.0 mL min–1; column gradient temperature, 140 °C (2 min) to 300 °C (5 min) at the rate of 5.0 °C min–1; and injection temperature, 250 °C. The analysis was performed using an electron impact ion source (70 eV) and interface (transfer-line temperature) of 250 °C. The MS Data Review software was applied for the remote control of the instrument. The HPLC analyses were performed by Agilent 1260 Infinity II DAD Detector equipped with a Capcell Pak C18 column. The NMR spectra were recorded on a Bruker 400 MHz spectrophotometer.

Preparation of Propolis and Poplar Samples

Raw propolis and poplar (10 g) were manually ground, pretreated with petroleum ether to remove resins (600 mL, 6 h at 45 °C), and extracted in Soxhlet with MeOH (80% v/v water) or EtOH (80% v/v water) at reflux temperature for 48 h. The obtained macerate was removed by filtration, and the solid portion was discarded. The recovered extract was distilled under reduced pressure (650 mmHg, 80 rpm) to yield PROP and PN-A/PN-B samples. For the GC–MS analysis, the appropriate sample (10 mg) was treated with 300 μL of NBSA (stabilized with 1% trimethylchlorosilane) in 100 μL of pyridine, in the presence of oleic acid as internal standard at 90 °C for 2 h.

Treatment of Propolis and Poplar Samples with Laccase from T. versicolor and TEMPO

The activity of laccase from T. versicolor was determined spectrophotometrically using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as a substrate. Briefly, ABTS (300 μL, 5 mM) and sodium acetate buffer (0.1 M, pH 5, 2.6 mL) were incubated under vigorous stirring at 40 °C for 10 min. Then, an appropriate amount of laccase in the sodium acetate buffer (100 μL) was added to the mixture and the initial rate was immediately measured with an increase in the optical density at 436 nm. One activity unit (U) is defined as the amount of enzyme that oxidized 1.0 μL of ABTS per minute. The kinetic parameters (Km, Vmax, Vmax/Km) were determined by measuring the enzyme activity at different concentrations of ABTS (0.1–5 mM) and plotting the data in a double-reciprocal Lineweaver–Burk plot. The reaction was carried out by the same procedure as for the activity assay, using laccase (0.1 μg) and measuring the absorbance at 436 nm, as described above.[79] The appropriate sample (1.0 g) and TEMPO (6 mM) were treated with laccase (100 U) in 0.1 M sodium acetate buffer (10 mL, pH 5–5.5) in the presence of a low amount of organic solvent CH2Cl2 (5.0% v/v). The amount of TEMPO was selected on the basis of data recently reported on the use of the LMS for the oxidation of recalcitrant aldehydes.[31] At defined reaction times (1, 7, and 15 h), the reaction was extracted with ethyl acetate (EtOAc) (30 mL × 2). The organic layers were treated with a saturated solution of NaCl, dried over anhydrous Na2SO4, and then filtered and concentrated under vacuum to yield the crude to be analyzed, as previously reported for parent substrates.

In the case of the reactions quenched at 7 h, the crude was purified by HPLC using a procedure previously reported for the isolation of flavonol derivatives, with some modifications.[80] The operating conditions were as follows: MeCN/H2O (30%) containing 0.1% trifluoroacetic acid; flow rate, 0.8 mL/min; and UV detection at 254 nm. The retention times of compounds 1–3 were identified by co-injection with standard compounds.[54] NMR data of compounds 1–3 were consistent with those reported previously.[80]

2-(4-Hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone 1: m/z 446 (detected as dimethyl silyl ether derivative); HRMS [EI]−: m/z calcd for C15H7O6 283.0243 [M – H]−, found 283.0246. 1H NMR [dimethylsulfoxide (DMSO)-d6, 400 MHz]: 5.86 (m, 1H, H-6), 5.92 (m, 1H, H-8), 6.80 (d, J = 6.7 Hz, 2H, H-3′ and H-5′), 7.57 (d, J = 6.7 Hz, 2H, H-2′ and H-6′), 9.39 (s, 1H, OH), 9.96 (s, 1H, OH), 10.77 (s, 1H, OH). 13C NMR (DMSO-d6, 125 MHz): 90.41 (CH), 95.84 (CH), 103.76 (C), 105.51 (C), 115.80 (CH), 125.28 (CH), 127.39 (C), 142.93 (C), 161.27 (CH), 166.31 (C), 173.85 (C), 190.47 (CO), 191.83 (CO).

2-(3,4-Dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone 2: HRMS [EI]−: m/z calcd for C15H6O7 298.0114 [M – H]−, found 298.0118; 1H NMR (DMSO-d6, 400 MHz): 5.87 (m, 1H, H-6), 5.93 (m, 1H, H-8), 6.77 (m, 1H, H-5′), 7.50–7.55 (m, 2H, H-2′ and H-6′), 9.37 (s, 1H, OH), 9.97 (s, 1H, OH), 10.72 (s, 1H, OH), 10.80 (s, 1H, OH). 13C NMR (DMSO-d6, 125 MHz): 90.33 (CH), 96.54 (CH), 100.46 (C), 104.57 (C), 114.89 (CH), 117.33 (C), 123.82 (CH), 124.99 (C), 144.73 (C), 151.36 (C), 158.53 (CH), 168.46 (C), 171.86 (C), 189.87 (CO), 190.24 (CO).

Galangin dimer 3: HRMS [EI]−: m/z calcd for C30H14O10 534.0587 [M – H]−, found 534.0582; 1H NMR (DMSO-d6, 400 MHz): 5.79 (m, 1H, H-6″), 5.86 (m, 1H, H-6), 5.90–5.93 (m, 2H, H-8-H″), 6.74–6.81 (m, 4H, H-3′, H-5′, H-3‴, and H-5‴), 7.23–7.32 (m, 2H, H-3′ and H-3‴), 7.38 (m, 2H, H-2′ and H-6′), 7.49 (m, 2H, H-2‴ and H-6‴), 8.32 (s, 1H, OH), 8.99 (s, 1H, OH), 9.84 (s, 1H, OH), 10.61 (s, 1H, OH). 13C NMR (DMSO-d6, 125 MHz): 93.01 (CH), 94.19 (CH), 95.43 (CH), 97.84 (CH), 102.66 (C), 104.50 (C), 125.81 (CH), 126.28 (CH), 126.67 (CH), 127.89 (CH), 128.21 (CH), 129.45 (CH), 130.19 (C), 130.79 (C), 133.53 (C), 136.48 (C), 157.88 (C), 158.80 (C), 161.08 (C), 162.27 (C), 163.91 (C), 166.31 (C), 166.74 (C), 178.45 (CO), 190.72 (CO), 196.53 (CO).

Antioxidant Activities of PROP, PN-A, and PN-B Samples as Evaluated by the DPPH Method

The quantitative assessment of the antioxidant activity of propolis and poplar samples was performed with a slight modification of the procedures reported in the literature.[81] EtOH was used as blank. The inhibition of free radical DPPH by the samples was monitored by measuring the decrease in the absorbance of solutions with different concentrations. In particular, the sample at an initial concentration of 1.0 mg mL–1 was diluted with ethanol until it achieved final concentrations of 25.0, 15.0, 10.0, 5.0, and 2.5 μg mL–1. Then, 1.0 mL of 0.3 mM DPPH in ethanol was added to 2.5 mL of the appropriate solution and they were left to react in dark at room temperature (26 °C) over 30 min. The absorbance readings were then taken with a spectrophotometer at 518 nm. To make the results more reliable, the measurements were performed on five different concentrations of the same sample and, for each concentration, three readings were taken. The antioxidant activity was expressed as the IC50 value. The results were expressed as the obtained values plus standard deviation (SD) of three independent experiments for each extract analyzed. The results were statistically processed through the GraphPad Prism 5.1 software.[82]

Antioxidant Activities of PROP, PN-A, and PN-B Samples as Evaluated by the Comet Assay

CHO cell line was cultured in Ham’s F10 medium with 10% fetal calf serum, 1% glutamine (Lonza), and 1% streptomycin and penicillin (Lonza) at 37 °C in a humified atmosphere of 95% air and 5% CO2. Moreover, to eliminate variations due to culture conditions, control and treated cultures were maintained concurrently and the same batches of the culture medium and preparation methods of chemicals were used for each experiment. t-Butyl-OOH (tert-butyl peroxide) was diluted with distilled water at the appropriate treatment concentration and used freshly made. In particular, a dose of 500 μM was used. The samples were dissolved in DMSO at a concentration of 500 μg/mL. For the experiments, the stock solutions were diluted with DMSO at the appropriate treatment concentration of 100 μg/mL and used freshly made.

Cell Treatments with t-Butyl-OOH and Selected Compounds

Briefly, the cell lines were seeded at a density of 5 × 105 cells/mL in Ham’s F10 complete medium and pretreated with the selected compounds (100 μg/mL) for 3 h at 37 °C. The cells were exposed to t-butyl-OOH for 1 h, washed in phosphate-buffered saline (PBS) to remove the oxidative agent, and suspended again in Ham’s F10 complete medium. The treated cells were used for the comet assay (t = 0, 1/2, and 1 h). All of the doses used here were chosen on the basis of pilot experiments.

Single-Cell Gel Electrophoresis (SCGE) Analysis (Comet Assay)

The standard alkaline (pH > 13) SCGE, or comet assay, analysis was performed as described earlier under visible fluorescent light.[83] After the treatment, the cells were collected and processed for the assay. In short, 20 μL of the cell suspension (5 × 105 cells) was mixed with 80 μL of 0.75% low-melting-point agarose in PBS at 37 °C and immediately pipetted onto a frosted glass microscope slide precoated with a layer of 1% normal-melting-point agarose, similarly prepared in PBS. Two slides for each experimental point were then incubated in a lysis solution (2.5 M NaCl, 10 mM Tris–HCl, 100 mM EDTA, pH 10, with 1% Triton and 10% DMSO freshly added) for 1 day at 4 °C. After lysis, the slides were placed on a horizontal electrophoresis unit containing fresh electrophoresis buffer (1 mM EDTA, 300 mM NaOH, pH 13) and incubated for 15 min to allow unwinding of DNA. Electrophoresis was then conducted for 20 min at 25 V, 300 mA, and 4 °C. Subsequently, the slides were gently washed three times in a neutralization solution (0.4 M Tris–HCl, pH 7.5) for 5 min and fixed in fresh 100% methanol for 3 min. The slides were stained with 50 μL of ethidium bromide (20 μg/mL) and covered with a coverslip. The stained nucleoids were examined at 400× magnification with an automatic image analyzer (Comet Assay III; Perceptive Instruments, U.K.) connected to a fluorescence microscope (Axioskop 2; Zeiss). To evaluate the amount of DNA damage, computer-generated TM values and percentages of DNA damage were used. For each experimental point, 100 cells were scored from two slides, for a total of 200 cells. The significance of the induction of mean TM in the effect of the t-butyl-OOH treatment versus that in control samples and the effects of the oxidative agent under different experimental conditions (i.e., with and without propolis or PN-A) were analyzed by Student’s t-test for paired samples (p < 0.01). The results are represented as the mean ± standard error of repeated experiments.

Table 2

Composition of the Most Abundant Polyphenols in the PN-B Sample before and after Treatment with Laccase from T. versicolor and TEMPOa

		starting material	t = 1 h	t = 7 h	t = 15 h
type	compound	yield (mg/g)b
phenols	hydroquinone	n.d.	8.04	7.47	4.51
	catechol	n.d.	0.89	0.32	0.15
	phloroglucinol	n.d.	0.23	0.09	0.13
phenyl propanoids	cinnamyl alcohol	0.14	0.03	0.02	0.01
	cinnamic acid	0.12	0.38	0.12	0.04
	3,4-dimethoxy cinnamic acid	0.13	0.23	0.07	0.02
	ferulic acid	0.25	0.04	0.03	0.03
	caffeic acid	1.17	1.26	1.03	0.54
	caffeic acid PEc	8.75	1.72	0.57	0.46
flavanons	pinostrobin	23.22	10.75	8.73	8.06
	sakuranetin	2.30	1.69	1.64	0.84
dihydro flavonols	pinobanksin	33.06	18.27	13.39	11.85
	3-O-acetyl pinobanksin	5.75	2.06	2.01	0.76
flavons	chrysin	3.77	2.97	2.51	2.12
	tectochrysin	27.67	30.74	30.59	26.65
flavonols and flavonol derivatives	galangin	42.11	34.25	23.31	19.27
	kaempferol	6.97	5.72	4.47	2.68
	quercetin	1.81	1.12	0.71	0.18
	compound 1d	n.d.	n.d.	1.80	n.d.
	compound 2	n.d.	n.d.	0.75	n.d.
	compound 3	n.d.	n.d.	8.02	n.d.

The oxidations were performed by treating the appropriate sample (0.1 g) with laccase (10 U) and TEMPO (6 mM) in 0.1 M sodium acetate buffer (3.0 mL, pH 5–5.5) at 40 °C, in the presence of a low amount of CH2Cl2 (5.0% v/v).

The yield was reported as milligrams of compound per gram of starting material.

PE: phenethyl ester.

Compound 1: 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone; compound 2: 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone; compound 3: galangin dimer.