Influence of Lyophilisation and Oven-Drying on Extraction Yield of Oregonin from European Black Alder (Alnus glutinosa (L.) Gaertn.) Bark

Oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(β-D-xylopyranosyloxy)-heptan-3-one) is the fi rst discovered and reported naturally occurring diarylheptanoid glycoside. It exhibits high biological activity, but it is also the compound of interest because of its ability to form reddish-orange and reddish-brown colours. In this research, European black alder (Alnus glutinosa (L.) Gaertn.) bark was separately subjected to lyophilisation and oven-drying before oregonin extraction with two different solvents. According to the results, methanol has proven to be more suitable as solvent compared to deionised water for oregonin extraction by maceration from black alder bark matrix regardless of the dehydration method. Accordingly, in the case of methanol, much higher yields of oregonin were obtained after lyophilisation, than after oven-drying. Furthermore, extraction by deionised water produced slightly higher yield of oregonin after oven-drying than after lyophilisation, as opposed to methanol. However, in much lower oregonin concentrations. Among other things, oregonin propensity to degradation at elevated temperature could probably be applied for improving and facilitating alder wood hydrothermal processing by minimizing uneven discolouration.

In the previous papers of other researchers, oregonin has been isolated and reported from the bark of several alder species (Alnus spp.) (Table 1).Its biological activity was reported together with its anti-oxidant, anti-infl ammatory, anti-microbial, anti-atopic dermatitis, and anti-cancer properties (Kuo et al., 2008;Choi et al., 2010;Tung et al., 2010;Sati et al., 2011;Telysheva et al., 2011).Therefore, black alder bark could be used as a resource of bioactive compounds, and not only as fuel in wood processing companies as currently used.Besides exhibiting high biological activity, oregonin is also the compound of interest because of its ability to form reddish-orange and reddish-brown colours, which particularly affects alder wood hydrothermal processing.
Among other factors, heat can have negative infl uence on bioactive compounds during their acquisition and chemical analysis.On the other hand, however, heat could be used for inactivation of chemical compounds (if prone to thermal degradation) that impede hydrothermal wood processing.As for the lyophilisation process, there is an unwarranted and unexamined assumption that it properly and optimally preserves the plant constituents, but these assumptions may be erroneous in some cases (Abascal et al., 2005).In this research, the infl uence of heat and dehydration procedure on oregonin extraction yield from black alder bark was investigated and reported.Bark was used instead of wood because it contains larger quantities of oregonin than wood (Klarić, 2015).The stated research results will help understanding the infl uence of lyophilisation on oregonin extraction yield as compared to oven-drying.Furthermore, methanol and deionised water were compared as solvents for the oregonin extraction.

Kemikalije
High purity (≥95 %) analytical standard of oregonin was obtained from Sigma-Aldrich (Germany).For the extraction process, methanol (MeOH) of HPLC grade was supplied by J. T. Baker (USA), while deionized water (dH 2 O) (ASTM Type II) was prepared on TKA/Thermo Scientifi c MicroMed Pure system (Thermo Fisher Scientifi c, USA).Formic acid of analytical grade (Orka Lab, Croatia) and deionised water (ASTM Type I, (MiliQ)) prepared on Millipore Simplicity Purifi cation system (Millipore Corporation, USA) were used for mobile phase in high performance liquid chromatography (HPLC) analysis.Bark (inner & outer bark included) was collected from fi ve-centimetre thick cross-section segment (disk) sawn at the height of 1.5 meters (from the ground) from black alder tree (30 cm breast height diameter).The tree was sampled during July 2014 in thirty-year old forest department "98" section "b" within the management unit "Đurđevačke nizinske šume" owned and governed by the Croatian state forest enterprise Hrvatske šume Ltd.The bark sample was milled and homogenized according to the previously described procedure (Klarić et al., 2016).A certain amount of frozen homogenized raw bark was oven-dried at 103 ± 2 °C till constant mass has been reached.Another amount of frozen homogenized raw bark was lyophilised (Christ alpha 1-2 LD, Germany) by the main drying phase (-55 °C, 0.021 bar, 24 hours) and fi nal drying phase (-50 °C, 0.040 bar, 4 hours).

Extraction procedure 2.3. Ekstrakcija
Extraction by maceration was conducted on magnetic stirrer (IKA C-MAG HS 7, Germany) for 24 hours (mot 1.5) at 20 ± 1 °C.Two grams of oven-dried and lyophilized bark were separately extracted in 250 mL of MeOH and dH 2 O, in triplicates with regard to solvent type and dehydration procedure.The obtained extracts were fi ltered through grade 388 quantitative ashless fi lter paper (Munktell, Sweden), and stored in amber glass jars and kept in refrigerator till further analysis.Before HPLC analysis, extracts were additionally fi ltered through syringe nylon fi lters (0.22 μm) in amber glass vials with rubber/FEP sept.The amount of extracted oregonin from bark was determined by HPLC-DAD method.

Liquid chromatography 2.4. Tekućinska kromatografi ja
The liquid chromatography analysis was conducted using a Varian ProStar 500 (USA) HPLC system consisting of a ProStar 330 diode array detector (DAD), ProStar 410 autosampler, ProStar 230 tertiary pump system and column compartment.Instrument control, data acquisition and evaluation were done with Star Chromatography Work station v5.5 (Varian ProStar 360).Separation was performed on Nucleosil-C18 column 150 × 4.6 mm, particle size 5 μm (Supelco Analytical, USA).The analysis was performed using 1.4 % for reproducibility.Extracts of oregonin from bark were analysed and expressed as mg of oregonin per g of dry bark mass.The descriptive results of the research are shown in Table 2.
As it is evident from the presented results (Table 2), statistically signifi cant difference for oregonin yields was established between dehydration methods for MeOH (F(1, 8.220 = 17297.271,p < 0.001), and for dH 2 O (F(1, 15.191) = 10136.376,p < 0.001).A statistically signifi cant difference was also observed in oregonin yields between MeOH and dH 2 O after lyophilisation (F(1, 8.033) = 22078.953,p < 0.001), and after oven-drying (F(1, 11.646) = 4657.159,p < 0.001), as determined by Welch's ANOVA.With methanol, much higher yields of oregonin from black alder bark were obtained after lyophilisation than after oven-drying at 103 ± 2 °C.However, in case of dH 2 O, this trend is reversed.This reversed trend in the case of dH 2 O was unexpected, but regardless of dehydration method applied, much higher oregonin yield was obtained with MeOH than with dH 2 O as a solvent.During the dehydration process, changes that take place within wood are dependent on time-temperature-moisture-oxygen interrelationship (Navi and Sandberg, 2012).Consequently, in specifi c cases, the degradation of compounds present in wood and/or formation of newly formed compounds is possible, due to a wide range of chemical reactions, such as oxidation, hydrolysis, deacetylation, depolymerisation, etc. (Hon and Minemura, 2001;Fengel and Wegener, 2003;Hill, 2006;Navi and Sandberg, 2012;Németh et al., 2013).Although it is generally considered that the lyophilisation is a preferable dehydration method, which retains higher levels of phenols i.e. bioactive compounds in the sample, it may not always be the case (Abascal et al., 2005;Dai and Mumper, 2010;Jiang et al., 2016).As regards VOCs (volatile organic compounds), they are probably lost to a greater extent during lyophilisation, as opposed to other dehydration methods at lower temperatures during which no vacuum is applied, or if fresh plant material is used (Abascal et al., 2005).In addition to low temperatures, another important advantage of lyophilisation during the acquisition of natural bioactive compounds is that the sample oxidation is prevented during dehydration process due to the lack of oxygen, considering bark and wood great internal structural voids and hygroscopicity of structural components of the matrix.On the other hand, if an elevated temperature near 100 °C is applied, catechol oxidases will most likely be inactivated, i.e. subjected to denaturation causing the loss of activity.This inactivation of the enzymes can then facilitate the successful implementation of wood hydrothermal processing.As regards methanol, it is often a solvent of choice for ex- Further research regarding oregonin will be focused on its presence, concentration and infl uence on black alder wood discolouration during hydrothermal processing.

ZAKLJUČAK
It is more preferable to use MeOH as a solvent for oregonin extraction by maceration from black alder bark than dH 2 O, regardless of the applied dehydration method.According to the obtained results, the highest yields of oregonin were achieved by conducting extraction with MeOH after lyophilisation, while significantly lower yields were achieved after oven-drying at 103 ± 2 °C.On the basis of the above mentioned fi ndings, it could be concluded that, among others, oregonin is presumably prone to degradation at elevated temperature (103 ± 2 °C).This fi nding could probably be implemented in hydrothermal wood processing to minimize the uneven discolouration of wood by conducting pre-steaming or similar high temperature procedures immediately after sawing and before kiln drying.If oregonin is the compound of interest because of its high biological activity, lyophilisation should be a preferred method of dehydration, accompanied by a suitable extraction solvent.
In the case of dH 2 O, yields of oregonin obtained after lyophilisation and oven-drying were reversed compared to MeOH as can be seen in Figure 3 and Figure 4.This reversed trend in the case of dH 2 O is an interesting and unexpected phenomenon that should be further researched.

3
RESULTS AND DISCUSSION 3. REZULTATI I RASPRAVA In this research, the effect of lyophilisation and oven-drying on maceration extraction yield of oregonin with MeOH and dH 2 O was investigated.The amount of extracted oregonin was determined by HPLC-DAD analysis.To confi rm the presence of the target compound in bark samples, retention time of peak obtained by analysing extracts of bark were compared with retention time of standard compound.Further, confi rmation of peak identity was provided by comparison of UV spectra obtained by DAD.Strong agreement of retention time (t R = 16.50 min) and UV spectra (λ max = 280 nm) from oregonin standard and peak from bark extracts were obtained, thus confi rming the identity of target analyte.The chromatogram of oregonin is shown in Figure 2. The quantifi cation of oregonin was carried out on the basis of the calibration curve (y = 5.8621 • x -2.322).Coeffi cient of determination (R 2 ) was 0.9993 confi rming high degree of correlation and good linearity of the method.Acceptable RSD values were obtained, 0.4 % for repeatability and 2.2 Bark acquisition and preparation 2.2.Dobivanje i priprema kore

Table 1
Alder species (Alnus spp.) in which oregonin presence in bark was reported Tablica 1. Vrste johe (Alnus spp.) u kojima je utvrđeno postojanje oregonina u kori .1 % formic acid in MeOH as eluent A and 0.1 % formic acid in MiliQ water as eluent B in gradient elution mode.The elution started with 90 % of eluent B for 25 min, following 25 min gradient to 0 % of B and then back to initial conditions within 5 min.Flow rate was 0.5 mL•min -1 and injection volume was 10 μL.The separation was monitored at absorbance wavelength of 280 nm.Stock standard solution of oregonin was prepared by dissolving accurate quantity of the standard in MeOH and stored in the dark at 4 °C.The working standard solutions of different concentrations were prepared by appropriate dilution of the stock solution.