13 Experimental
Samples: 8 trees were sampled from selected varieties (Nr. 21, 23, 26, 52, 59 and H1) during the June of 2013. 30 leaves were taken from each tree and were stored in dry ice until the extraction process.
Chemicals: Water for the extraction and chromatographic separation was produced with double distillation using conventional distillation equipment. Acetonitrile (LC-MS grade) was obtained from VWR-International (Budapest, Hungary). 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (trolox), 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid), potassium persulfate, potassium hydrogen phosphate, potassium dihydrogen phosphate and formic acid (98%) were obtained from Sigma–Aldrich (Budapest, Hungary).
Sample preparation: Leaves were treated to inactivate their polyphenol-oxidising enzymes for 2 min. with 750 W microwave energy in a household microwave oven. Leaves were ground and extracted (0.15 g leaf material with 15 ml 4:1 MeOH:H2
O by stirring for 24 h
in the dark). Extracts were fi ltered through a 0.45 µm cellulose acetate fi lter, and two fold dilutions of the extracts (using clean extraction solvent) were taken to HPLC-MS/MS analysis.
Chromatography conditions: HPLC measurements were carried out using a Shimadzu LC-20 type liquid chromatograph coupled with a Shimadzu SPD-M20A type diode array detector (PDA) (Shimadzu Corporation, Kyoto, Japan) and an AB Sciex 3200 QTrap triple quadrupole/linear ion trap LC/MS/MS detector (AB Sciex, Framingham, USA). A Phenomenex Kinetex C18, 150 mm × 4.6 mm, 2.6 µm core–shell column was used for the separation at 40ºC. The mobile phase consisted of A (H2 (CH3
O + 0.1% HCOOH) and B
CN + 0.1% HCOOH). A gradient elution was run with a 1.2 mL/min fl ow-rate using the following time gradient: 10% B (0–1 min), 12% B (8 min), 18% B (10 min), 22% B (13 min), 28% B (19 min), 98% B (23 min), 98% B (23–32 min), 10% B (33 min), 10% B (33–40 min). 4 µl of the diluted extracts were injected. PDA detection was carried out in the wavelength range between 250–380 nm. Because of the relatively high fl ow rate of the mobile phase, fl ow-splitting was applied using a split valve, which allowed 0.6 mL/min fl ow to enter the MS ion source. Negative electrospray ionisation mode was used for the MS detector with the following ion source settings: ion spray voltage: −4500V, curtain gas (N2) pressure: 30 psi, spray gas (N2) pressure: 40 psi, drying gas (N2) pressure: 30 psi, ion source temperature: 500ºC. Respective MRM transitions and other optimised compound- dependent settings of the MS were used for the relative quantifi cation of the compounds. Relative quantifi cation involved the determination of peak areas respecting the limit of quantifi cation (LOQ) for each compound. Measurements and evaluations were run in triplicate from each sample. Chromatographic data were acquired and evaluated using the Analyst 1.6.1 software.
Antioxidant capacity: the ABTS assay was run as described by Stratil et al. [9] at 734 nm, using the ABTS•+
Correlation analysis: investigations and evaluation was done using the Statistica 12 software (StatSoft Inc, Tulsa, USA)
Results and Discussion
Primary results The typical chromatograms of the separation of a beech leaf extract are depicted in Figure 3.
radical ion and trolox standard for 10 min of reaction time. ABTS
antioxidant power was given in mg equivalents of trolox/g dry leaf units (mg TE/g dw.). Measurements and evaluations were run in triplicate from each sample.
Figure 4. Calculation of the signal-to-noise (S/N) ratio and lowest limit of quantifi cation (LOQ) for a given MRM channel.
The peak areas indicated in Table 1 are average values of 8 individual trees of one variety. Table 1 also includes average antioxidant capacity values (ABTS) of the leaf extracts and average trunk diameters of the sample trees according to their variety. The EQ value represents the Ellenberg’s Climate quotient, which indicates if a variety originated from a site where it had adapted to dry/humid and hot/cold climate by calculating the EQ value by a simple equation in which the mean temperature of the warmest month (in ºC) is divided by the annual precipitation (in mm) and multiplied by 1000 [10].
EQ= T ⎛
EQ= T⎟ ⋅1000 ⎛
⎜ ⎝
⎜ P
annual July
⎜ ⎝
⎞ ⎜ P
⎟ ⎠
annual July
The amounts of individual compounds were assessed by their respective MRM peak areas instead of determining absolute concentrations. This type of evaluation was chosen because no standard compounds were available apart from (+)-catechin and (−)-epicatechin, and because some of the identifi ed compounds showed coelution and inadequate peak resolution in the PDA chromatogram so quantifi cation was not feasible. The MRM measurement mode of a triple quadrupole mass spectrometer provides a selective and reproducible method for quantifi cation even if chromatographic separation could not be achieved with adequate peak resolution. By the evaluation of MRM peak areas, the composition of the extracts was compared without the need to know absolute concentrations. The characteristic MRM transitions were determined by the infusion of the extracts into the MS detector and performing MRM optimisation for each compound at its characteristic [M-H]- m/z value (Q1). Precise structural data of the 44 quantifi ed compounds had been determined in earlier studies of the authors. The lowest limits of quantifi cation (LOQ) were evaluated using the formula presented in Figure 4. Only those peaks were considered for the quantitative evaluation which had a peak area larger than the corresponding LOQ value.
⎞
⎟ ⎠
⎟ ⋅1000
According to Table 1 there are apparent differences between varieties respecting trunk diameter, polyphenolic composition and antioxidant capacities. As a general tendency it was observed that varieties with the poorest growth parameters (Gråsten, Torup) had the highest ABTS levels and in these varieties the concentrations of some of the compounds were also the highest (Caffeic acid-O-hexoside, Unknown 2; Quercetin-O-hexoside 1 and 2; Quercetin-O-pentoside; Kaempferol-O-pentoside) or surprisingly the lowest (Unknown 1, 3 and 6; Procyanidin B dimer 5 and 6; Procyanidin C trimer 6). From these results the following questions arise: which compounds are the most powerful antioxidants? Which compounds can take part most effi ciently as antioxidants in the defence reactions of the leaves? Can certain compounds act as markers of climatic adaptation and vitality, and is there a direct and statistically provable relationship between polyphenol levels and trunk diameter? To answer these questions, a comprehensive and systematic correlation analysis was performed to reveal all relationships, including the average values of the different varieties, between concentrations of individual compounds and ABTS levels, average trunk diameters as well as EQ values.
Statistical Evaluation of Data
Figure 3. The typical PDA (250–380 nm) and MRM chromatograms of a beech leaf extract. In the MRM chromatogram, different colours indicate individual MRM channels.
The results of the correlation analysis are summarised in Table 2. In the case of n=6 data and p<0.05 level, the limit of signifi cance of a correlation was |R| ≥ 0.812. It was presupposed that a signifi cant positive correlation between ABTS antioxidant capacity levels and concentration of a given compound indicates that this compound has a strong infl uence on the antioxidant properties of the leaf extracts, hence it is an ‘effi cient’ antioxidant compound.
Table 1. Average trunk diameters, ABTS antioxidant capacity and Elle climate quotient of the investigated varieties (upper part of the table). average peak areas for each compound according to variety. The colu the MRM transition used for the quantitative assessment of the individ HPLC-MS/MS.
Table 1. Average trunk diameters, ABTS antioxidant capac climate quotient of the investigated varieties (upper part of average peak areas for each compound according to varie the MRM transition used for the quantitative assessment o HPLC-MS/MS.
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