Lipophilic Extractives of the Barks from Podocarpus imbricartus, chemical components in the inner dan outer barks (Fengel & Wegener 1989; Seki et al. Agathis loranthifolia, Araucaria cunninghamii, and Araucaria papuana 2012). To provide more information concerning the conifers in Indonesia, the objectives in this work were to explore the bark lipophilic components of Podocarpus imbricartus, in Indonesia Agathis loranthifolia, Araucaria cunninghamii, and Araucaria papuana by gas

chromatography-mass spectrometry (GC-MS). The differences between inner and outer Dipta Dwi Pratiwi*, Masendra, Rizki Arisandi, & Ganis Lukmandaru bark regions were also discussed. It is expected that the obtained results create a

Department of Forest Products Technology, Faculty of Forestry, Universitas Gadjah Mada background for their possible application in medicines, cosmetics, and as bioactive *Email: [email protected] agents in the pharmaceutical industry.

ABSTRACT Materials and Methods The chemical composition of the lipophilic extractive in toluene extracts from Materials Pododcarpus imbricartus, Agathis loranthifolia, Aracauria cunninghamii, and Aracauria papuana has been investigated by gas chromatography-mass spectrometry (GC/MS). Different groups of The research materials used were A. cunninghamii (19 years), P. imbricartus (19 compounds, such as fatty acids, aliphatic hydrocarbons, and other components were identified. years), A. loranthifolia (37 years), and A. papuana (37 years) obtained from the site of The contents of toluene extract were higher in outer bark than those of inner bark. In general, Plant Biotechnology and Breeding Research Center (BBPBPT), Situbondo, East Java. The the major group of the lipophilic components in both the inner and outer bark was found to be bark specimens were collected from two individuals for each species and were taken at varied and dependent on species and radial position. Various fatty acids and aliphatics 1.0-1.5 m height. From each species, the barks were divided into two parts in radial hydrocarbons were characterized. The predominating lipophilic compounds identified in inner direction, i.e. the inner and outer bark based on visual inspection. Then, each part was and outer bark were hexadicanoic acid, tetradecanoic acid, and octadecenoic acid, 1,2 benzene ground to size 100-mesh-bark meals. dicarboxyclic, and n-undecane. Extraction Keywords: aliphatic hydrocarbons, bark, conifer, fatty acids, gas chromatography The bark powder (2 g oven-dry-weight) was then extracted with toluene through a soxhlet apparatus for 6 hours. The dissolved extract from the extraction process was Introduction concentrated by evaporating the solvent in the vacuo and the extractive content was Coniferous species are distributed in a limited number in Indonesia although calculated. their timbers exhibit good properties (Kosasih & Rochayat 2000). Besides Pinus GC-MS analysis merkusii, there are several conifer genus that the woods have been utilized and give potential economic values such as damar (Agathis sp), jamuju (Podocarpus sp), and Gas Chromatography–Mass Spectrometry (GC–MS) analysis was performed for all cypress (Araucaria sp). Unfortunately, the basic properties of their stem remain toluene extracts both by direct injection and trimethylsilylated (TMS derivatization). For unknown, particularly the bark parts. To increase their utilization, basic information of derivatization, 40 µl sample (1 mg/ml) were evaporated then mixed with trimethylsylil- their barks should be explored. hydroxyl (TMS-IH) and bis (trimethylsylil)-trifluroacetamide (BSA) each 20 µl The bark is the second most important tissues in the tree after the woods (Masendra et al. 2018a). The analysis was performed according to the GC-MS (Sjostrom 1993). The chemical composition of bark varies among different tree species equipments by Shimadzu QP 2010: RTX - column type is 5 ms, Restek Corp (30 m and also depends on the morphological parts (Sakai 2001). Based on their extractives, length). Helium was used as a carrier gas. The injector and detector temperatures were 0 0 barks have more enormous components of lipophilic and phenolic groups compared to both maintained at 250 C, operation temperature at 50-300 C. The column 0 0 the woods. Lipophilics, in particular, showed various bioactivities for medicinal temperature was programmed at 70-120 C, with 4 C increase per min which was 0 0 purposes (Hichri et al. 2003; Chen et al. 2013). In general, the lipophilic components maintained for 1 min. Then it was programmed at 120-300 C, with 6 C increase per min consist of fatty acids, resinous acids, waxes, alcohols, terpenes, sterols, sterol esters, and held on for 15 min. The compounds of each extract were identified by using NIST aliphatic hydrocarbons, and glycerides (Sjostrom 1993). data base library computer software. The compound quantification was calculated by One study has addressed the lipophilics in the barks of pines grown Indonesia the relative peak area. (Masendra et al. 2018a). Previous studies have shown the variation between the 82 83

— 78 — chemical components in the inner dan outer barks (Fengel & Wegener 1989; Seki et al. 2012). To provide more information concerning the conifers in Indonesia, the objectives in this work were to explore the bark lipophilic components of Podocarpus imbricartus, Agathis loranthifolia, Araucaria cunninghamii, and Araucaria papuana by gas chromatography-mass spectrometry (GC-MS). The differences between inner and outer bark regions were also discussed. It is expected that the obtained results create a background for their possible application in medicines, cosmetics, and as bioactive agents in the pharmaceutical industry.

Materials and Methods Materials The research materials used were A. cunninghamii (19 years), P. imbricartus (19 years), A. loranthifolia (37 years), and A. papuana (37 years) obtained from the site of Plant Biotechnology and Breeding Research Center (BBPBPT), Situbondo, East Java. The bark specimens were collected from two individuals for each species and were taken at 1.0-1.5 m height. From each species, the barks were divided into two parts in radial direction, i.e. the inner and outer bark based on visual inspection. Then, each part was ground to size 100-mesh-bark meals. Extraction The bark powder (2 g oven-dry-weight) was then extracted with toluene through a soxhlet apparatus for 6 hours. The dissolved extract from the extraction process was concentrated by evaporating the solvent in the vacuo and the extractive content was calculated. GC-MS analysis Gas Chromatography–Mass Spectrometry (GC–MS) analysis was performed for all toluene extracts both by direct injection and trimethylsilylated (TMS derivatization). For derivatization, 40 µl sample (1 mg/ml) were evaporated then mixed with trimethylsylil- hydroxyl (TMS-IH) and bis (trimethylsylil)-trifluroacetamide (BSA) each 20 µl (Masendra et al. 2018a). The analysis was performed according to the GC-MS equipments by Shimadzu QP 2010: RTX - column type is 5 ms, Restek Corp (30 m length). Helium was used as a carrier gas. The injector and detector temperatures were both maintained at 2500C, operation temperature at 50-3000 C. The column temperature was programmed at 70-1200C, with 40C increase per min which was maintained for 1 min. Then it was programmed at 120-3000C, with 60 C increase per min and held on for 15 min. The compounds of each extract were identified by using NIST data base library computer software. The compound quantification was calculated by the relative peak area.

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— 79 — Results and Discussion Extractive content The determination of extractive content by toluene extraction was presented in Table 1. Generally, the outer bark has higher values compared to the inner bark. The highest amount of inner bark was measured from P. imbricartus (1.7%) whereas the highest amount of outer bark was also from P. imbricartus (2.0%). The trends of lipophilic extracts were in agreement with the previous works (Domingues et al. 2011; Masendra et al. 2018a).

Table 1. The results of toluene extraction (percentage of oven-dry wood) Bark sample Extractive content (%) Inner bark Outer bark P. imbricartus 1.7 2.0 A. loranthifolia 0.9 1.3 A. cunninghamii 0.8 1.0 A. papuana 1.1 1.4

Lipophilic composition Components from the bark extracts were detected by GC-MS. The lipophilic constituents in the bark samples can be grouped into fatty acids, aliphatic hydrocarbons, and other components. The latter was composed of esters, phenolics, alcohols, and terpenes. The fatty acids were detected by derivatization method whereas the others by direct injection. Lipophilic constituents from the four species are listed in Table 2. In the chromatograms of outer barks, on the basis of aliphatic hydrocarbons, five main peaks i.e. n-decane, benzene-1,2,4 trimethyl, benzyl undecanoate, and p-cresol have been detected in P. imbricartus whereas four major peaks i.e. 2,3,3 trymethyloctane, 1,3,5-cyloheptarine, benzyl ester, and benzenedicarboxylic acid were found in A. loranthifolia. Further, A. cunninghamii contained four major peaks i.e n- decane, 1,2,4 trimethylbenzene, tetradecenyl acetate, and henicosyl formate as A. papuana detected two main peaks i.e. tetradecenyl acetate, dan 9,19- cycloergosterol. In the inner bark, the aliphatic hydrocarbons were detected from five minutes as the high molecular components were detected after twenty minutes. P. imbricartus showed the main peaks for themethyl-1 undecane, 1,2- dipropylcylopentane, n-decane, pyridine, benzene, 1,2,4-trimethyl. A. loranthifolia detected the main peaks i.e. n-decane, pseudocumane, n-dodocenylsuccinic, and dodecanoic acid. A. cunninghamii consisted three major peaks, i.e. 8-methyl undecane, hemelitol, and n-dodocane as for as A. papuana detected three main peaks i.e. undecane, n-decane, and napthalane.

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— 80 — Table 2. Lipophilic constituents of the outer and inner bark in four coniferous species –MS (percentage of dry extract, average of two measurements)

Constituentsidentified by GCRetentio and GC P.imbricartu A. A. A. papuana Similari n time s loranthifolia cunninghamii ty index (min) IB OB IB OB IB OB IB OB (%) Fatty acids 5.34 14.7 19.8 8.66 2.50 21.95 59.2 24.5 6 8 8 Methacrylic acid 4.9 ------12.84 - 96 Butanedioic acid 5.2 - - - - 2.5 - - - 86 Acetic acid 6.1 2.34 - 84 Pentanedioic acid 7.1 ------1.57 - 84 Propenoic acid 12.4 - - 2.92 - - 2.18 - - 91 Palmitic acid 24.6 - 1.16 4.43 - - - - 5.9 94 Undecanoic acid 32.7 - - 7.76 - - - - - 88 Carboxylic acid 32.4 - - - 8.66 - - - - 97 Dodecanoic acid 35.3 - - 2.36 - - - - - 87 Hexadecanoic acid 22.92 5.34 6.92 2.39 - - 10.97 24.35 - 95 Trichloracetic acid 24.01 - 3.10 - - - 2.11 - - 92 Octadecenoic acid 25.81 - 3.52 - - - 4.6 - 8.68 95 Tetradecanoic acid 24.55 - 3.10 ------83 Aliphatic 36.1 17.4 18.1 hydrocarbons 6.51 12.2 16.3 7 5 21.45 8 8.48 n-decane 4.6 1.74 6.27 7.52 - 5.41 5.41 7.56 - 96 Heptane, 5-ethyl-2- - - 4.98 methyl- 1.76 - - 3.74 - - 96 1,2,4- - - - - 4.64 trimethylbenzene 5.41 - - - 95 n-undecane 6.3 - 1.24 4.37 9.01 3.31 5.41 - - 96 1-tricosene 36.56 - 3.41 2.44 - 97 n-dodecane 43.31 - 1.82 2.77 6.3 1.5 - 4.83 - 94 1,2 benzene 16.93 2.87 dicarboxyclic 3.01 1.64 20.86 1.82 3.48 3.35 8.48 96 23.8 28.5 31.4 Other components 6.5 7 3.18 5 5.52 22.03 4.04 5 2-Propenoic acid, 2- methyl-, 2- 4.88 hydroxyethyl ester 4.53 - 3.18 13.89 - 13.89 - - 92 Benzyl undecanoate 32.71 - - - - 4.73 - - - 88 Benzyl stearate 35.31 - - - - 0.79 - - - 88 Hemellitol 5.1 - - - - - 2.84 - - 95 1,2- - - Benzenedicarboxylic 32.35 97 acid, mono(2- ethylhexyl) ester - 8.66 - - - - Biphenol 27.22 - - - 6 - - - - 92 Tetracyclo[3.3.1.0(2,8). ------5.4 0.74 0(4,6)]-non-2-ene - 92

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— 81 — Continues table 2 100% Phenol, 4,4'(1- - - - 5.3 - -

22.63 22.63 80% methylethylidene - 90 Others component Tetrasiloxane, ------4.04 31.45 60% 4.04 Alipathic hydrogen decamethyl 94 40% p-Cymene 6.2 1.23 1.24 ------86 Fatty acids 9,19-cycloergosterol 43.788 - - - - - 2.09 - 3.3 84 (%) extractive 20%

24------6.7 lipophilic of Composition 0% 44.781 methylenecycloartan 87 IB OB IB OB IB OB IB OB Remarks : IB = inner bark OB = outer bark – : not detected P. imbricartus A. loranthifolia A. cunninghamii A. papuana

Variations in species Figure 2. Composition of lipophilic extractives (% of the dry weight of toluene extracts) Fatty acids were more detected in species A. papuana, whereas they were less in four coniferous species detected in A. cunninghamii. The aliphatic hydrocarbons were the most abundant type of alkanes in this research. The aliphatic hydrocarbons were found mostly in A. cunninghamii (outer bark) as they were not detected at all in the outer bark of A. Aliphatic hydrocarbons were dominating alkanes in this experiment i.e. benzene papuana. As for the other components, the outer bark of A. loranthifolia gave the highest dicarboxyclic 1.2, n-undecane, n-dodocane, and n-decane. In addition, in a lesser amount, concentration while the inner bark of A. papuana showed the lowest values. the study also detected some bioactive compounds such phenols i.e. benzyl benzoate, The lipophilic contents were affected by the radial direction (Figure 1). It showed and triterpenoid i.e. 9,19-cycloergosterol or monoterpene i.e. p-cymene. Previously, that the values in the outer bark were higher than in the inner bark, except for fatty acid triterpenoids and resin acids were the most abundant group in certain pines (Masendra components in A. papuana and A. loranthifolia. As for A. loranthifolia, the other et al. 2018a; 2018b). Those compounds, however, were less detected in this experiment. component group dominated in the outer bark while aliphatic hydrocarbons were most More than twenty different fatty acids have been identified in a variety of softwood abundant in the inner bark. A. cunninghamii showed lower concentrations of fatty acids (Conner et al. 1980, Foster et al. 2010). This indicates that each species of conifer has to in the inner bark whereas A. papuana gave higher amounts of fatty acids in the inner dominate specific components of fatty acids depending on the type of tissues, or tree bark than the outer bark. Composition lipophilic by weight of extracts is shown in Figure age. The most abundant fatty acids in the research were the high saturated fatty acids i.e. 2 as it showed various tendencies among the species. The highest content of lipophilics hexadecanoic acid (C16), tetradecanoic acid (C14) whereas the unsaturated fatty acids were measured for aliphatic hydrocarbons (91.23%) in A. cunninghamii (outer bark) were octadecenoic acid (C18). Those findings are in line with Bikonvens et al. (2013) and other component groups (61 %) in A. loranthifolia (outer bark). which showed the same results in Alnus incana. Unsaturated fatty acids would be beneficial for human health as they have an important role in the prevention or reducing the risk of cancer, cardiovascular diseases (Chen et al. 2013). 70 Fatty acids 60

Alipathic hydrogen 50 %) 40 Conclusions 30 20 From the different radial parts, the outer bark samples contained more lipophilic 10 extractives than the inner bark. The outer and inner bark consisted of fatty acid, 0 aliphatic hydrocarbon, and other component groups. Hexadecanoic acid was the most

Lipophilic content IB OB IB OB IB OB IB OB dominant fatty acids whereas n-decane, n-undecane, n-dodocane, and 1.2-benzene P. imbricartus A. loranthifolia A. cunninghamii A. papuana dicarboxyclic were the most abundant aliphatic hydrocarbons. The other component groups consisted of esters, phenolics, alcohols, and terpenes. Further, the use of Figure 1. Lipophilic contents (% of dry weight of toluene extracts) with error bar as standard components as well as comparation of fragmentation patterns in mass- standard deviation. 86 87

— 82 — 100%

80% 60% Others component Alipathic hydrogen 40% Fatty acids extractive (%) extractive 20%

Composition of lipophilic lipophilic of Composition 0% IB OB IB OB IB OB IB OB P. imbricartus A. loranthifolia A. cunninghamii A. papuana

Figure 2. Composition of lipophilic extractives (% of the dry weight of toluene extracts) in four coniferous species

Aliphatic hydrocarbons were dominating alkanes in this experiment i.e. benzene dicarboxyclic 1.2, n-undecane, n-dodocane, and n-decane. In addition, in a lesser amount, the study also detected some bioactive compounds such phenols i.e. benzyl benzoate, and triterpenoid i.e. 9,19-cycloergosterol or monoterpene i.e. p-cymene. Previously, triterpenoids and resin acids were the most abundant group in certain pines (Masendra et al. 2018a; 2018b). Those compounds, however, were less detected in this experiment. More than twenty different fatty acids have been identified in a variety of softwood (Conner et al. 1980, Foster et al. 2010). This indicates that each species of conifer has to dominate specific components of fatty acids depending on the type of tissues, or tree age. The most abundant fatty acids in the research were the high saturated fatty acids i.e. hexadecanoic acid (C16), tetradecanoic acid (C14) whereas the unsaturated fatty acids were octadecenoic acid (C18). Those findings are in line with Bikonvens et al. (2013) which showed the same results in Alnus incana. Unsaturated fatty acids would be beneficial for human health as they have an important role in the prevention or reducing the risk of cancer, cardiovascular diseases (Chen et al. 2013).

Conclusions From the different radial parts, the outer bark samples contained more lipophilic extractives than the inner bark. The outer and inner bark consisted of fatty acid, aliphatic hydrocarbon, and other component groups. Hexadecanoic acid was the most dominant fatty acids whereas n-decane, n-undecane, n-dodocane, and 1.2-benzene dicarboxyclic were the most abundant aliphatic hydrocarbons. The other component groups consisted of esters, phenolics, alcohols, and terpenes. Further, the use of standard components as well as comparation of fragmentation patterns in mass-

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— 83 — spectrophometer should be conducted to confirm the actual components in the future Seki K, Orihashi K, Sato M. 2012. Accumulation of constitutive diterpenoids in the works. rhytidome and secondary phloem of the branch bark of Larix gmelinii var. japonica. Journal of Wood Science 58:437–445 Acknowledgements Sj str m E. 1993. Wood chemistry: Fundamentals and application. 2nd Edition. Gadjah We thank the Dr. Mudji Susanto (Centre for Forest Biotechnology and Tree Mada University Press. Yogyakarta. Improvement Research) for providing research materials. ̈ ̈

References Bikonvens O, Roze L, Pranovich A, Reunanen M, Telysheva G. 2013. Chemical composition of liphophilic extractives from Alnus incana. BioResources 8: 350- 357. Chen B, McClements DJ, Decker EA. 2013. Design of foods with bioactive lipids for improved health. The Annual Review of Food Science and Technology 4 (1): 35–56. Conner RN. 1980. Foraging habitats of woodpeckers in southwestern Virginia. Journal Field Ornithology 51 : 119-127. Domingues RMA, Sousa GDA, Silva CM, Freire CSR, Silvestre AJD, Pascoal-Neto C. 2011. High value triterpenic compounds from the outer barks of several Eucalyptus species cultivated in Brazil and in Portugal. Industrial Crops and Products 33:158-164. Foster B, Martin TM, Pauly M. 2010. Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part II. Journal of Visualized Experiment 37:1837. Fengel D, Wegener G. 1989. Wood: Chemistry, ultrastructure, reactions. Walter de Gruyter, Hichri F, Jannet HB, Cheriaa J, Jegham S, Mighri Z., 2003. Antibacterial activities of a few prepared derivatives of oleanolic acid and of other natural triterpenic compounds. Comptes Rendus Chimie 6(4): 473–483. Kosasih AS, Rochayat N. 2000. Pengaruh pemberian hormon terhadap keberhasilan perbanyakan jamuju (Podocarpus imbricarta). Buletin Penelitian Hutan 619 : 1-11. Masendra, Ashitani T, Takahashi K, Lukmandaru G. 2018a. Liphophilic extractives of the inner and outer barks from six different pinus species grown in Indonesia. Journal of Forestry Research 29(5):1329-1336 https://doi.org/10.1007/ s11676-017-0545-x. Masendra, Ashitani T, Takahashi K, Lukmandaru G. 2018b. Triterpenoids and steroids from the Bark of Pinus merkusii (Pinaceae). BioResources 13(3):6160-6170. Sakai K. 2001. Chemistry of bark. In : Hon DNS, Shiraishi N (ed). Wood and Cellulosic Chemistry. New York: Marcel Dekker.

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