Unlike floral honeys, which derive from the nectar of flowering plants, honeydew honey is obtained by secretions of the living parts of plants or excretions onto them produced by sap-sucking insects. Honeydew honeys differ in chemical composition from nectar honeys [1
] as well in the volatile composition and/or antioxidant activity. Screening of natural organic compounds that characterize these honey types is of particular interest because palynological analysis cannot be carried out [2
-β-Methyl-γ-octalactone, a characteristic volatile compound of oak wood, is proposed as a chemical marker for the plant origin of oak honeydew honeys [2
]. Other compounds such as aminoacetophenone and propylanisole can be considered characteristic of holm-oak honeydew honeys. 1-(2-Furanyl)-ethanone, butane-2,3-diol, 3-hydroxy-butan-2-one and 1-hydroxy-propan-2-one were suggested compounds for discrimination among nectar and honeydew honeys [1
Antioxidant capacity of different honeys varies by floral source [3
] as well by processing and storage conditions [4
]. Components that were identified and/or quantified as honey antioxidants included phenolic compounds, ascorbic acid, the enzymes glucose oxidase, catalase, peroxidase and others. Additional research on single phenolic and other compounds in honey indicate that the antioxidant capacity is due to combination of a wide range of honey active compounds beyond phenolics [6
]. Many different methods are appropriate for assessing the antioxidant activity (FRAP assay (ferric reducing antioxidant power), DPPH (1,1-diphenyl-2-picrylhydrazyl) method, ORAC (oxygen radical absorbance capacity), TEAC (Trolox equivalent antioxidant activity) and others) and in most cases it is necessary to use several tests to obtain good reliability [7
]. Little information is available on the potential antioxidant activity of the honey ultrasonic solvent extracts. In our previous research [8
] we reported the scavenging ability of the series of concentrations of the Amorpha fruticosa
honey ultrasonic solvent extracts and the corresponding honey samples that was tested by a DPPH assay. Approximately 25 times lower concentration ranges (up to 2 g/L) of the extracts exhibited significantly higher free radical scavenging potential with respect to the honey samples.
Ten. belongs to the oak species. The growth process of oak fruits is extremely rich in cycles of 5-8 years. At the point of natural reduction of overproduction of fruits, the sweet sap from fruits appears. Fruit sap starts to flow over the cuticle of the fruit often with a foamy appearance. Therefore, the corresponding honeydew can also be produced without the mediation of plant sucking insects. Oak honeydew volatiles from Spain were previously identified by a microscale SDE apparatus after dichloromethane extraction [2
] and trans
-β-methyl-γ-octalactone, a characteristic volatile compound of oak wood, was proposed as a chemical marker. This compound is well known in winemaking, because it is responsible for the oak aroma of barrel-aged wines [9
The scope of this research is to obtain new information of oak honeydew volatile organic composition by combined use of headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). In addition, the obtained USE extracts were tested by DPPH and FRAP assay for the first time in order to unlock their antiradical and antioxidant potential. DPPH and FRAP assay of the USE extracts were compared to the activity of the honeydew samples to obtain data of potential extra value of the solvent extracts, not just for organic analyses.
3.1. Honey Samples
Two oak honeydew samples that were collected in different years were investigated: sample I (2005) and sample II (2009). The samples were obtained from professional beekeepers and no mechanical treatment or heat was used. The combs were placed in the area of wild growing Quercus frainetto
Ten. Melissopalynological analysis was performed by the methods recommended by the International Commission for Bee Botany [12
]. Microscopical examination was carried out on a Hund h 500 (Wetzlar, Germany) light microscope attached to a digital camera (Motic m 1000) and coupled to an image analysis system (Motic Images Plus software) for morphometry of pollen grains. Water content was determined by refractometry, measuring the refractive index, using a standard model Abeé refractometer at 20 ºC. Water content (%) was obtained from the Chataway table [13
]. Electrical conductivity was measured in a solution of 20 g honeydew honey in low conductivity water at 20 ºC using conductometer (Hanna HI 8733). All the samples were stored in hermetically closed glass bottles at 4 ºC until the volatiles isolation.
3.4. Gas Chromatography and Mass Spectrometry (GC, GC/MS)
Gas chromatography analyses were performed on an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A equipped with flame ionization detector, mass selective detector, model 5975C and capillary column HP-5MS ((5%-phenyl)-methylpolysiloxane Agilent J & W GC column, 30 m, 0.25 mm i.d., coating thickness 0.25 μm). Chromatographic conditions were as follows: helium was carrier gas at 1 mL·min−1, injector temperature was 250 ºC, and FID detector temperature was 300 ºC. HP-5MS column temperature was programmed at 70 ºC isothermal for 2 min, and then increased to 200 ºC at a rate of 3 ºC·min−1 and held isothermal for 18 min. The injected volume was 1 μL and the split ratio was 1:50. MS conditions were: ionization voltage 70 eV; ion source temperature 230 ºC; mass scan range: 30–300 mass units. The analyses were carried out in duplicate.
3.5. Data Analysis and Data Evaluation
The individual peaks were identified by comparison of their retention indices (relative to C9
alkanes for HP-5MS) to those of authentic samples and literature [14
], as well as by comparing their mass spectra with the Wiley 275 MS library (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral database. The percentage composition of the samples was computed from the GC peak areas using the normalization method (without correction factors). The component percentages (Table 1
and Table 2
) were calculated as mean values from duplicate GC and GC-MS analyses.
3.6. Antiradical Activity (DPPH Assay)
The antiradical capacity was determined by the 2,2,diphenyl-1-picrylhydrazyl (DPPH) assay [15
]. Oak honeydew samples were diluted first in ultra pure water (1:10, w/v) and then in methanol with different concentrations (g/L) shown in Figure 2
. Ultrasonic solvent extracts were carefully evaporated to dryness under nitrogen and dissolved in methanol with different g/L concentrations showed in Figure 2
. Spectrophotometric readings were carried out with a UV-Vis Perkin-Elmer Lambda EZ 201 spectrophotometer at 517 nm. DPPH assay was carried out in triplicate for each sample.
The percent of inhibition (I%) of the DPPH radical by the samples was calculated in the following way: I% = [(AC(0) -AA(t))/AC(0)] × 100, where AC(0) is the absorbance of the control at t = 0min and AA(t) is the absorbance of the samples at t = 60 min. Pure methanol was used to zero the spectrofotometer. The absorbance of DPPH radical without the sample, i.e. the control, was determined.
Quantitative analysis was done using the external standard method (Trolox). A calibration curve in the range of 0.05-1.0 mmol/L was used for Trolox and data were expressed as Trolox equivalent antioxidant capacity (TEAC, mmol/kg). Each sample (50 μL of previously prepared concentration 2 g/L) was dissolved in 2 mL of DPPH 0.04 mmol/L in methanol. The mixtures was shaken and left for 60 min at room temperature in the dark. The absorbance was measured against a control made of 50 µL of methanol and 2 mL of DPPH (the bank was read at t = 0 min and at t = 60 min).
3.7. Total Antioxidant Activity (FRAP Assay)
The ferric reducing-antioxidant assay (FRAP) is based on the reduction at low pH of ferric 2,4,6-tris(2-pyridyl)-1,3,5-triazine [Fe(III)-TPTZ] to the ferrous complex followed by spectrophotometric analysis [15
]. The reagent was prepared by mixing 10mM TPTZ with 20mM ferric chloride in acetate buffer (pH 3.6). Quantitative analysis was done using the external standard method (ferrous sulfate, 0.1–2 mmol), correlating the absorbance (λ = 593 nm, UV-Vis Perkin-Elmer Lambda EZ 201) with the concentration. The results were expressed as millimoles per kilogram of Fe2+