Pinus brutia extractives analysis for sustainability

. The aim of this study was to determine all the extractives from different samples were collected from heartwood, sapwood, bark and needles and branches of Pinus brutia. A Soxlet device with two solvents (water and ethanol) were used to collect the extracts. Analytical chemical analyses were conducted with Gas C1hromatography-Mass Spectrometry (Agilent 5975C). The greater percentage of the extracts in water and ethanol showed that the greater percentage of them is found in the branches and needles of the trees. Especially the results showed significant amounts of the chemical compounds, such as Borneol, Tetradecane, 6,6-Biquinoline, Butyl citrate, Isopropyl palmitate and Isopropyl myristate, which can be used for sustainability, environmental integration at the aspect of more effective use of natural resources and have many uses in novel – functional food, green chemistry and pharmaceutical industries. Finally, the greater percentage of the extracts in water and ethanol showed that the greater percentage of them is found in the branches and needles of the trees.


INTRODUCTION
The reduction of raw materials leads to the necessity of sustainable development.The primary goal is the more effective use of natural resources and the reduction of forest waste after logging, in the form of wood residues.Extractives are compounds contained in large amount at the material mentioned above.Countries around the world, such as Poland, Canada, are focused on exploiting wood waste in order to produce energy or other materials from the extractives of forest residues [1,2] and production of wooden barrels [3].All forest species contain a large amount of extractives in their wood but also at their branches, bark, needles, cones and roots as well.Extractives are chemical compounds inside cell walls, more rarely in cell cavities, with various synthesis, such as aromatic phenolic compounds, aliphatic compounds, terpenes, terpenoids and others.Since there is no chemical attachment to the wood, extractives can be removed from it with the use of various solvents, for example hot water, methanol, ethanol, dichloromethane and others, without causing transformation of it [4][5][6][7][8][9].Of the genus Pinus several species are native in Greece, such as Pinus brutia, Pinus nigra, Pinus pinea, Pinus leucodermis, Pinus peuce and others [10,11].
The aim of this study was the quantitative and qualitative estimation of the extracts from the species Pinus brutia for further use, at the frame of the most effective exploitation of wood, wood residues and other wood products.

Experimental procedure
The research material originated from the University Forest of Aristotle University of Thessaloniki in Taxiarchis, Chalkidiki (North Greece) (Figure 1.a).From each tree, wood discs were taken from breast height.Each disc was cut and came up a longitudinal strip of wood from pith to bark.The disks were divided into three different protions: bark, sapwood and heartwood (Figure 1.b).All the specimens were cut with the use of a sharp blade into smaller pieces and passed through the Willey mill in order to produce particles smaller than 0,1mm.

Apparatus and separation conditions
A Soxhlet-type device of big size made from glass was used for the extractions.A wood sample from the Willey mill of 2g was placed in the Soxhlet device as well as a glass filter (Figure 2 a, b).The dry weight of the samples and the glass filter was calculated by weighing after drying in an oven at 103 ± 2 °C, before each extraction.The extractions with water lasted six hours, while with ethanol lasted four hours.For each extraction and each solvent a different sample was used.Four complete cycles of solvents were repeated according to the ASTM Standards [17,18].At the end of the extraction the samples were left at room temperature for many hours (more than 24) and then placed in an oven at 103°C for an additional 24 hours.Furthermore, the samples were weighed to determine the dry mass of the wood, after removing the extracts (1).
Qualitative extractives analysis was conducted with an Agilent 5975C GC-MS instrument.The solvents together with the extracts were condensed to about 2 ml final volume, with the use of rotary evaporator instrument (Bȕchi Rotavapor R-215, Bȕchi Heating bath B-491).Τhe rotation speed of the vial ranged between 80 and 120 rpm and the temperature from 15-20 o C (for ethanol and dichloromethane extractions, according to their boiling point).The condensation process was completed using pure nitrogen gas vapor until the appropriate final volume was reached.A glass chromatography column (1 cm internal diameter) was used to clean the samples that could damage the chromatography column.For this reason Florisil (MgO3Si) 2.5g, Al2O3 3.5g and Na2SO4 1.5g were used to absorb moisture (Figure 2.b).The mass spectrometer was equipped with quadrupole non-polar capillary column DB-5ms, 30m length and 0.25mm internal diameter, film thickness 0.25μm, filler 5% phenyl polysiloxane and 95% methyl polysiloxane.The conditions of Gas chromatography Agilent 7890A for the compounds identification and quantification were: flow rate 0.99333 mL/min and pressure 11.656 psi and use of Helium as a carrier gas.The Library used for the identification of the compounds was the one applied in the Gas chromatography and the identification of the compounds based on the largest percentage of appearance and previous knowledge of Pinus extracts, according to literature.Two temperature programs were applied, for better resolution.

RESULTS
The greater percentage of water and ethanol soluble extractives was found in the branches and needles of the trees (Figure 3).More especially chemical analysis showed significant amounts of chemical compounds, such as Borneol, Tetradecane, 6,6-Biquinoline, Butyl citrate Isopropyl palmitate and Isopropyl myristate.The chemical compound 1-bromo-2-nitrobenzene was used as Internal Standard (IS) for the estimation of the quantity sapwood GC-MS analysis revealed the results below [19,20].Table 1 shows that in sapwood there are great amounts of 2-propenoic acid, ethyl oleate, ethyl ester linoleic acid, 2-dodecene (Z), diethoxydimethoxy-silane, borneol and 2,5-bis-(1,1dimethylethyl) Phenol.As it is shown in Table 3, needles contain large quantities (+)-4-careen, butyl citrate, cyclotetradecane, tridecane, diphenyl-Pyrazole, megastigmatrienone and aminosalicylic acid.In all cases, many extractives of scientific and commercial interest were found As shown in figure 4, Borneol and 6,6 Biquinoline are two extracts found in the heartwood and in the highest concentration, alpha-hydroxy-Benzene acetic acid, Butyl citrate and tetrahydro-2-methyl-Thiophene are three extracts found in the needles and in the highest concentration.

Conclusions
From all the above data, it is obvious that the largest number of extractives was found in needles and branches.The results compared with other Pinus species are in agreement with those of other researchers [13,14].Furthermore, many compounds detected in this research were found from previous researchers as well [14,15].In this work an attempt was made to extract the ingredients of the species Pinus brutia using green techniques and solvents that help in the production of green products and help in sustainable development.In summary, all the extracts which were found can have many applications in human life, in the aspect of the optimization of natural resources utilization, taking into consideration the green growth, circular economy and sustainable development, since new uses of natural resources are suggested.
Authors want to acknowledge assistance from colleagues and special work by technical staff.

Figure 1 .
Figure 1.(a).Map of the sample collection area.

Figure 1 .
Figure 1.(b).Pinus brutia discs and samples of bark, needles and branches, from which the research material originated.
The first temperature program was: Initial temperature at 70 o C, for 4 minutes, raising rate 50 o C/min and final temperature up to 280 o C for 10 minutes.The last temperature program was: Initial temperature at 60 o C, for 4 minutes, raising rate 50 o C/min and final temperature up to 240 o C for 5 minutes.Compounds identification was based on mass spectrometry and each peak of diagrams conducted.

Figure 3 .
Figure 3. Extracts amounts from different parts of Pinus Brutia with water.

Figure 4 .
Figure 4. Quantity and type of Pinus brutia extracts.

Table 3 .
Chemical compounds found at Pinus brutia needles specimens (ethanol extracted specimens)

Table 4 .
Chemical compounds found at Pinus brutia bark specimens (ethanol extracted specimens)