Optimization for Green Path in Wood Extractives by Taguchi Analysis Primjena Taguchijeve analize

• Extractive composition of Pinus pinaster (Maritime pine) wood was studied with choline chloride based deep eutectic solvents (DES). Two different eutectic mixtures (ChCl: Et-Gly; ChCl:Urea 1:2 molar ratio), two different extraction methods (hot water bath, ultrasound assisted extraction), and temperature (40 – 60 °C), time (30 – 60 min.) and solid:liquid ratio (1:10 – 1:20 g/mL) parameters were applied. For the optimization of operation conditions, Taguchi analysis was performed. Resin acids formed the chemical composition of Pinus pinaster (Maritime pine) wood. Dehydroabietic acid, abietic acid and isopimaric acid were found to be the major compounds. The impact of parameters on the performance of the system was determined as follows: DES type > solid:liquid ratio > extraction method > temperature > time. So, for the extraction of lipophilic compounds in Maritime pine wood, the optimum conditions were determined as hot water bath extraction at 60 °C with ethylene glycol and 1:10 solid liquid ratio for 60 min.

DES, consisting of two or more compounds, are formed from hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA).DES are negligibly volatile, cheap, non-toxic, low-flammable and thermally stable, often biodegradable, and not requiring purification (Ozturk et al., 2018a).They are classified into four groups: Type I (organic salts + metal salts), Type II (organic salts + metal hydrates), Type III (organic salts + HBD) and Type IV (metal chlorides + HBD).Type III deep eutectic solvents, used in this study, are applied in fractionation of lignocellulosic biomass, biodiesel production, metal processing and extraction of polar molecules and bioactive compounds.They are also used in pharmaceutical and biomedical applications (Ozturk et al., Barbieria et al., 2020).Choline choride (ChCl), a non-toxic and biodegrable compound, is mostly used in the DES mixtures as a HBD.With these features, ChCl based DES solvents are convenient for the pharmaceutical and cosmetic use (Häkkinen, 2020).
Extractives, defined as low molecular compounds in the woody plants, are composed of different chemical substances.These compounds can be classified into two groups -lipophilics and hydrophilics (Sjöström, 1981).For the extraction of lipophilic compounds, non-polar solvents are used, while polar solvents are used for hydrophilics (Vek et al., 2020).Although the amount of extractives is less than 10 % of dry wood, they are used in different areas, e.g lipophilics in pharmacy, food and cosmetic industries.Hydrophilic compounds have antioxidant, antimicrobial, antiviral, cancerogenic and cardio protective effects (Fengel and Wegener, 2003;Benouadah et al., 2018).Because of these features, extractives are becoming increasingly important.The use of non-toxic, biodegradable chemicals has also become popular in the last years.
Pinus pinaster Aiton occurs naturally in Southwest Europe (e.g Spain, Portugal), Western Mediterranean and Northwest Africa as a fast growing species.This species was first planted in 1881 in different regions of Turkey and today it covers a total of 57,378.4ha (Velioglu et al., 2020; Koch, 1972).
It is mainly used in pulp and paper industry both in Turkey and in Europe.It is also used in particleboard and packaging industries.Due to its high resin content, special resin production sites have been created for Pinus pinaster in different countries (e.g Portugal, Spain, France, Italy).The yield of resin is 1457-2500 g/tree with acid-paste method in Turkey (Aydin, 2017).It is reported that the turpentine part of this resin is used as antiseptic, diuretic and anthelmintic.Also, local people use the cone extracts of this species to prevent bronchitis and cough (Kurtca and Tumen, 2020).There are several studies regarding the extractives of Pinus pinaster.In the wood part, simple phenolics, stilbenes, lignans, flavonoids, organic acids, steryl esters and triglycerides are found to be the major extracts in the hot water aliquot (Conde et al., 2014).However, in the bark part, diterpenic compounds that have nutraceutical effects are found to be the main compounds with fatty acids, long-chain alcohols and sterols (Sousa et al., 2018).
In this study, a new generation of choline chloride based on two different deep eutectic solvents was used to determine the extractive composition of Pinus pinaster wood.Different temperatures (40-60 °C), time (30-60 min.),solid:liquid ratio (1:10-1:20 g/mL) and extraction methods (hot water bath-ultrasound assisted extraction) were used.For the optimization of operation conditions, Taguchi method was performed.Although there are studies about the phenolic compositions of wood with deep eutectic solvents, lipophlics are studied for the first time.

Materijal
In this study, Pinus pinaster, obtained from Zonguldak -Turkey, was used as wood material.Bark was removed before chipping the woody parts into small matchstick size.Samples were grounded in a Wiley mill and freeze-dried before the extraction.Choline chloride, urea and ethylene glycol were purchased from Merck in analytical grades and used without further purification.

Deep eutectic solvents (DES) preparation 2.2. Priprema eutektičkih otapala (DES)
Two different DES mixtures were prepared.Molar ratios are listed in Table 1.Mixing procedure was performed at 80 °C until a homogenous clear liquid was obtained.

Metode ekstrakcije
Two different extraction procedures were applied.As a control, sequential extraction with n-hexane and acetone:water (95:5 v/v) was carried out for 6 hours in a soxhlet apparatus.With DES mixtures, hot water extraction (HW) and ultrasound assisted extraction (UAE) were performed.Wood samples and DES were put in a test tube and extracted according to the below conditions.After each extraction method, samples were centrifuged for 15 min.at 4000 rpm. 1 mL of aliquot was taken to a new test tube and extracted (liquid-liquid) with acetone before silylation.For the optimization of extraction, some parameters were tested: type of DES (Table 1), solid:liquid ratio (1:10 -1:20 g/mL), temperature (40 -60 °C) and extraction time (30 -60 min.).Three repetitions were made for each parameter.

FID-GC and GC-MS analysis 2.4. Analiza FID-GC i GC-MS
Shimadzu GCMS-QP2010 GC-MS equipped with TRB-5MS column (30 m × 0.25 mm (0.25 μm thickness)) was used for the identification.Temperature program was 120 °C for 1 min.then raised to 310 °C with a 6 °C/min.waiting for 20 minutes.The injection temperature was 260 °C, split ratio was 1:25, ion source was 200 °C and ionization energy 70eV.Wiley and NIST libraries were used.For quantitative analysis, Shimadzu GC 2010 FID-GC was used.

Dizajn Taguchijeve metode
Taguchi is an effective statistical method to optimize the operation condition settings.With this method, the number of experiments was reduced and so were the costs and time (Kumar et al., 2015;Uslu and Aydin, 2020).The method consists of an orthogonal array, signal-to-noise ratio (S/N or SNR), response table and graph (Main Effect Analysis) (Ozakin and Kaya, 2020).To start with Taguchi, operation parameters, quality characteristics and orthogonal array are selected for designing and doing the experiments.Then, the results are analyzed by using signal-to-noise ratio (S/N).Finally, optimum parameters are obtained with the analysis results (Sun et al., 2013;Liu et al., 2019).

Selection of parameter levels and orthogonal array of Taguchi 2.5.1. Odabir razina parametara i ortogonalni niz Taguchijeve metode
The parameters, affecting the amount of selected compounds (abietic acid, dehydroabietic acid and isopimaric acid, the most abundant compounds), were de-termined as extraction method, extraction temperature, extraction time and solid/liquid ratio.Table 2 illustrates the factors considered and their levels.
According to the number of selected parameters and their levels, the L 32 (2 5 ) orthogonal array of Taguchi was selected, as shown in Table 3.The main feature of the orthogonal indices is that all the factors are included in the experiment with an equal number of trials.

Signal to noise ratio 2.5.2. Omjer signala i šuma
In the Taguchi method, generally, the S/N is adopted as the indicator of quality (Jiang et al., 2020).It is defined as undesired random noise value, desirable signal ratio and shows the quality characteristics of experimental data (Kurt et. al., 2009;Gunay et al., 2011;Gunay and Yucel, 2013).Depending on the particular characteristics of the design problem, different S/N ratios may be applicable, including "lower is better", "nominal is best", or "higher is better" (Chen et al., 2007;Kurt and Can, 2021).In this study, for the calculation of S/N ratio "higher-is-better" performance character was preferred as shown in Eq. 1.
(1) n is the number of observations of the experiment and y i is the observed data at the i th experiment (Taguchi et al., 2005).

Grey relations analysis 2.5.3. Siva relacijska analiza
In order to verify the Taguchi results and specify the best experiment conditions, Grey relation analysis (GRA) was done.GRA is a part of a system theory, improved for solving complex relationships between alternatives and variables (Yang and Chen, 2006).In case of many criteria and alternatives, this method is  Step 1. Form the decision matrix and uniform the data in order to prohibit unit variations.It is actually necessary because variations between data can be different.Reproduce a value to form the array between 0 to 1 from original value.Three different equation "higher is better", "lower is better" and "nominal is best" are used according to the problem in the normalization process.In this study, "higher-is-better" performance character was preferred as shown in Eq. 2. (2) Where x i * (k) specifies the sequence after data preprocessing, is the measured results, is the minimum value , and is the maximum value of , i is the number of experiments, and k represents the measurement values.
Step 2. Calculation of grey relational coefficient: ( Where, is the deviation sequence of the reference sequence and comparability sequence, is the minimum value in the sequence, is the maximum value in the sequence.is defined as identification coefficient and the range is between 0 to 1. Generally, the value of is taken as 0.5. Step 3. Calculation of grey relational grade is defined as final step.It is calculated according to Eq. 4 averaging the sum of the grey relational coefficients (4) Where γ i changes in the range of 0 to 1, and n is the number of experiments.The higher grey relational grade signifies more ideal results.

Kemijski sastav
The amount of lipophilic compounds after the sequential extraction with n-hexane and acetone are given in Table 4.As seen, it is mainly formed from resin acids and the main compounds are abietic, dehydroabietic and levopimaric acids.In addition, oxidized resin acids (6 %) were found in the n-hexane extract.
Two types of resin acids (Abietane-type and pimarane-type) were found in the DES extracts of P. pinaster wood.Compared to organic solvent (eg.acetone), no fatty acids were found in the DES extracts.Also, hydroxy resin acids were not seen.In between all DES types and all experimental conditions, dehydroabietic acid (18-37 %) and abietic acid (14-28 %) were found to be the major compounds (Table 5 and  Table 6).Isopimaric acid, a pimarane type, was determined as 7.3-15 % in both DES.However, the highest amount was obtained with ChCl:Et-Gly in HW at 60 °C from 1 g of P. pinaster wood.With the organic solvent (n-hexane), the amount of isopimaric acid was 7.7 %.
PSMME (pinosylvin monomethyl ether) a typical stilbene for Pinus species was not found in ultrason bath extraction with ChCl:Ur (Table 6), whereas in hot water extraction (HW) the amount of PSMME was 0.4-2.4%.ChCl:Ur is more viscous and it is difficult to handle during silylation.The effects of experimental conditions, e.g.time and temperature, on the main compounds like dehydroabietic, abietic and isopimaric acids are shown in Figure 1 and 2. As seen, the amount of the main compounds are higher with ChCl:Et-Gly than with ChCl:Ur.At the optimum conditions discussed in Taguchi analysis (solid:liquid ratio of 1:10, extraction temperature: 60 °C, extraction time: 60 min, extraction method HW), dehydroabietic acid was found to be (31.1 ± 1.5) % with ChCl:Et-Gly and (28±3) % with ChCl:Ur.The second important compound, abietic acid, was (16.2±1.1)% and (20±0.5)% in ChCl:Et-Gly and ChCl:Ur, respectively.
Temperature is another factor affecting the efficiency of extraction.Increasing the temperature from 40 to 60 °C, changed the amount of dehydroabietic acid from (28.7±0.1)% to (31.1±1.5)%.For isopimaric acid, this amount is (10.8±0.6)% to (13.1±1.9)% respectively.Increasing the temperature, decreased the solvent vis-cosity and mass transfer limitations, but increased the diffusivity.Similar results were obtained by Ozturk et al. (2018b) for polyphenolics from orange peel.
Solid:liquid ratio is an important parameter for the operation.The increase of the amount of liquid increases positive interaction between the solid and liquid states.Two different ratios were applied in this study (1:10 and 1:20 g/mL).Changing the liquid ratio from 10 mL to 20 mL, decreased the amount of dehydroabietic acid (31.1±1.5 -28±0.4%).Similar decline was observed for isopimaric acid (13.1±1.9 -9.5±0.3 %).However, for abietic acid, increasing the liquid ratio positively affects the amount (16.2±1.1 -18.7±1.0 %).
As seen in Figure 1, at the optimum conditions, the amount of dehydroabietic was found to be (31.1±1.5)% with hot water bath and (28±3.2) % with UAE.To the contrary, the amount of abietic acid was found low with HW.UAE is a simple method that requires less time and solvent.In the recent studies, UAE

Taguchijeva analiza
S/N ratios related to control parameters obtained from Taguchi analysis are given in Table 7.The highest S/N of each parameter indicates the optimal level of that parameter.For example, as seen in Table 7, the 23.11 S/N ratio value in the method factor is defined as level 1 and shows that HW is the method with the best results.Likewise, with the 23.24 S/N ratio value, Et-Gly is the best DES solvent.Overall, among all parameters, the most effective factor was DES solvent type.
As seen in Figure 3. the greatest S/N ratio ensures the best levels of experimental parameters.The optimal factors for this study were determined as A: Hot water bath extraction; B: 60 min.;C: 60 °C; D: Ethylene glycol; E: 1:10 solid:liquid ratio.

Grey relations analysis 3.3. Siva relacijska analiza
Table 8 shows grey relational grade values obtained by using Eqs.2, 3 and 4. As seen from the table, the optimum parameters were observed in the experiment number 13, which has the highest grey grade value (0.6588).These parameters belong to the combination of experiments, as obtained in the Taguchi analysis.A: Hot water bath extraction; B: 60 min.;C: 60 °C; D: Ethylene glycol; E:1:10 solid:liquid ratio (g/mL).

CONCLUSIONS 4. ZAKLJUČAK
Extraction of lipophilic compounds, e.g fatty and resin acids which have antimicrobial and antifungal activities, was investigated with deep eutectic solvents.Choline chloride based on two different eutectic mixture urea (1:2) and ethylene glycol (1:2) was used.Ten different compounds, mainly resin acids, were identified in the DES mixtures with GC-MS.Dehydroabietic acid, abietic acid, isopimaric acid and palustric acid were found to be the major compounds.L 32 orthogonal array from Taguchi was applied for optimization.Extraction method, extraction time, extraction temperature, solid:liquid ratio and DES type were the main parameters.
The sequence of individual parameters in this study is ranked as follows: DES type > solid:liquid ratio > extraction method > temperature > time.Ethylene glycol was found to be more effective compared to urea to extract the lipophilic compounds from Pinus pinaster wood. 1 g of wood meal and 10 mL of DES mixture (solid:liquid ratio) were found to be sufficient.Hot water extraction at 60 °C for 60 min.are the optimum factors for this study.Compared to traditional wood extraction with soxhlet apparatuses, DES application only 1 g of wood meal and 60 min.
Further, with different DES mixtures and wood species, more effective extraction methods can be developed in the concept of green chemistry and wood extractives.