Lipophilic Extractives of ( macrophylla King) several studies from mahogany tree parts i.e fruits, seeds, leaves, and with regard Barks to their bioactivities and cell wall components have been published (Dewanjee et al. 2009; Sayyad et al. 2016; Corales et al. 2016; Sun et al. 2018). Rizki Arisandi, Masendra, Brandon AV Purba, Fatimah Zulaikha Wati, Fatra Valahatul Ihda, Fuad To our knowledge, however, only few investigations have been done related to Sumantri, & Ganis Lukmandaru* mahogany barks (Falah et al. 2008; Rosdiana et al. 2017). In the present study, the lipophilic extractives from n-hexane extract, and its neutral-acidic fractionations of inner Department of Forest Products Technology, Faculty of Forestry, Universitas Gadjah Mada, Jl. Agro No.1, and outer bark of mahogany were analyzed by gas chromatography-mass Bulaksumur, Yogyakarta 55281, *Email: [email protected] spectrophotometry (GC-MS).

Materials and Method ABSTRACT Bark collection and extraction The aim of this study was to investigate the lipophilic extractives in the bark of mahogany (Swietenia macrophylla). The bark was separated into inner and outer bark region. The bark of S. macrophylla was collected from SRIKANDIRATU, a furniture The bark was ground to form powder and then was extracted by n-hexane at 90 oC for 6 hours. manufacturer in Jepara, Central Java. The mahogany bark (± 20 year-old) was separated Further, n-hexane soluble extract was fractionated to obtain neutral-acidic fraction. The into inner and outer parts. The bark sample was ground to produce powder (40-60 amounts of n-hexane extract of inner and outer bark were 0.63 and 0.54 % , respectively (based on oven dried ). The extracts of n-hexane extract, neutral, and acidic fractions were mesh) before extraction. The inner and outer of S. macrophylla bark each (500 g) was identified by gas chromatography-mass spectrometry (GC-MS) to investigate the lipophilic refluxed for 6 h with n-hexane solvent. The solution then was evaporated and the crude constituents. The lipophilic constituents composed of hydrocarbons, aliphatic alcohols, fatty was weighed as the result. acids, terpenoids and sterols. After fractionation, sterols and terpenoids, was the most abundant in neutraf fractions whereas fatty acids were the major compounds in acidic fraction, particulary Neutral-acid fractionation in the outer bark. The high levels of terpenoids and sterols in the barks should be followed by compound isolation and toxicity test in medicinal purposes point of view. The n-hexane extract was dissolved in dichloromethane (50 ml) and was entered into a separator funnel. To obtain a fraction of neutral and acidic fractions, 50 ml of 10 % Keywords: bark extractives, bioactive compounds, fatty acids, neutral fraction, sterols Na2CO3 was added. After separation, the layer of dichloromethane solvent was washed by aquades and the sodium sulfate was added to bind the water. For 24 hours, the solution was filtered to obtain a neutral fraction. Further, the fraction was evaporated by Introduction rotary evaporator then the amounts of neutral fraction were dried and calculated. Swietenia macrophylla King or mahogany is one of the popular timbers for Separately, the aqueous layer was acidified until pH 3 with hydrochloric acid and wooden industries in Indonesia. This species was distributed in more than 40 countries dissolved by dichloromethane (50 ml) into separator funnel. After that, the fraction of including in Brazil, Bolivia, Mexico, Guatemala, Peru, and from Central and South dichloromethane layer was treated similarly to neutral fraction to collect the acidic America (Brown et al. 2003; Blundel & Gullison 2003; Andre et al. 2008; Goh & Kadir fraction (Lukmandaru 2012). 2011). In Indonesia, especially in Jepara (Central Java), many furniture home-industries GC-MS analyses choose the mahogany wood to produce the chair, the frame of the bed, and other stuffs. Consequently, the utilization of mahogany wood as above mentioned is uncommercial or Each sample was prepared for direct injection for n-hexane extract and neutral they discards the bark as residues. fraction. Derivatization was carried out by trimethylsilylated method (TMS The bark is interesting and available byproducts from the forest plantation that derivatization) to analyze the acidic fraction. The amount of 40 µl sample (1 mg/ml) was can be used as biomass for biorefineries (Lima et al. 2018). Tree barks have a rich evaporated then was mixed with trimethylsyli-hydroxyl (TMS-IH) and bis chemical composition, for example in lipophilics and polyphenolics, as well as they (trimethylsylil)-trifluroacetamide (BSA) each 20 µl. GC-mass spectrometry (GC-MS) data contain a high amount of inorganic material that allows a wide range of potential were collected with a GCMS-QP 2010 (Shimadzu, Japan) under the following conditions: applications and valorization over the usual burning for energy (Pereire et al. 2003; RTX - column type is 5 ms, Restek Corp (30 m length); column temperature from 500 C Miranda et al. 2012; Baptista et al. 2013). Additionally, phytoconstituents such as (1 min) to 3200 C at 50 C/min; injection temperature of 2500 C; detection temperature of flavonoids, alkaloids, tannins, and triterpenoids are bioactive compounds for many 320 0 C; acquisition mass range from of 50-800 amu using helium as the carries gas. medicinal , particularly from triterpenoids (Swati & Rica 2011). In this context, the 96 97

— 92 — several studies from mahogany tree parts i.e fruits, seeds, leaves, and woods with regard to their bioactivities and cell wall components have been published (Dewanjee et al. 2009; Sayyad et al. 2016; Corales et al. 2016; Sun et al. 2018). To our knowledge, however, only few investigations have been done related to mahogany barks (Falah et al. 2008; Rosdiana et al. 2017). In the present study, the lipophilic extractives from n-hexane extract, and its neutral-acidic fractionations of inner and outer bark of mahogany were analyzed by gas chromatography-mass spectrophotometry (GC-MS).

Materials and Method Bark collection and extraction The bark of S. macrophylla was collected from SRIKANDIRATU, a furniture manufacturer in Jepara, Central Java. The mahogany bark (± 20 year-old) was separated into inner and outer parts. The bark sample was ground to produce powder (40-60 mesh) before extraction. The inner and outer of S. macrophylla bark each (500 g) was refluxed for 6 h with n-hexane solvent. The solution then was evaporated and the crude was weighed as the result. Neutral-acid fractionation The n-hexane extract was dissolved in dichloromethane (50 ml) and was entered into a separator funnel. To obtain a fraction of neutral and acidic fractions, 50 ml of 10 %

Na2CO3 was added. After separation, the layer of dichloromethane solvent was washed by aquades and the sodium sulfate was added to bind the water. For 24 hours, the solution was filtered to obtain a neutral fraction. Further, the fraction was evaporated by rotary evaporator then the amounts of neutral fraction were dried and calculated. Separately, the aqueous layer was acidified until pH 3 with hydrochloric acid and dissolved by dichloromethane (50 ml) into separator funnel. After that, the fraction of dichloromethane layer was treated similarly to neutral fraction to collect the acidic fraction (Lukmandaru 2012). GC-MS analyses Each sample was prepared for direct injection for n-hexane extract and neutral fraction. Derivatization was carried out by trimethylsilylated method (TMS derivatization) to analyze the acidic fraction. The amount of 40 µl sample (1 mg/ml) was evaporated then was mixed with trimethylsyli-hydroxyl (TMS-IH) and bis (trimethylsylil)-trifluroacetamide (BSA) each 20 µl. GC-mass spectrometry (GC-MS) data were collected with a GCMS-QP 2010 (Shimadzu, Japan) under the following conditions: RTX - column type is 5 ms, Restek Corp (30 m length); column temperature from 500 C (1 min) to 3200 C at 50 C/min; injection temperature of 2500 C; detection temperature of 320 0 C; acquisition mass range from of 50-800 amu using helium as the carries gas.

97

— 93 — Components were identified using comparison of the experimental GC-MS data with the Further, hydrocarbons, fatty alcohols or aliphatic alcohols were observed of inner and NIST MS library and calculated based on percentage of peak area. outer bark of E. globulus (Gutierrez et al. 1999; Freire et al. 2002).

Aliphatic alcohols Results and Discussion Fatty acids Hydrocarbons Sterols and Terpenoids Extractive content and fractionation The bark was extracted by n-hexane solvent. From the physical observation of the colour extract, n-hexane solvent was yellow-brown and oily-like appearance as apolar solvents will dissolve the oil compounds, waxes, fats, and terpenes (Fengel & Wegener 1989; Sjöström 1993). The amounts of n-hexane extract in the inner bark (0.63 %) was higher compared to outer bark (0.54 %) part based on dried extract. On the acidic fraction other hand, neutral fraction was the larger fraction in the inner bark (42.7 %) than outer bark part (3.5 %) based on dried n- hexane extract. The reverse pattern was found in the acidic fraction (Table 1).

neutral fraction Table 1. Yield of n-hexane extract of mahagony % based on dried wood % based on dried extract Radial Yield Neutral fraction Acidic fraction n- hexane extract 0 10 20 30 40 50 Inner bark 0.63 42.7 28.0 Outer bark 0.54 3.5 67.6 Retention time (minutes) (a) Inner bark The similar trend was observed in P. merkusii which dicholomethane extract content in the inner bark was larger than in the outer bark part (Masendra et al. 2018a) Hydrocarbons Sterols and Terpenoids Fatty acids but not for other species such as E. globulus, E. urograndis, E. grandis, E. maidenii, and Aliphatic alcohols Norway (Anas et al. 1983; Domingues et al. 2011; Krogell et al. 2012; Freire et al. 2002). The acetone and dichloromethane soluble extracts of Falcataria moluccana, S. macrophylla, Acacia mangium, and grandis bark ranged from 0.74 to 8.99 % based on dried wood (Baptista et al. 2013; Rosdiana et al. 2017). Generally, neutral acidic fraction fraction contains unsaponified substances in alkaline, glycerols, and acids (fatty and resin), whereas free acidic fraction composed of fatty acids and resin acids (Baeza & Freer 2010). Gas chromatography-mass spectrometry neutral fraction The GC-MS analysis detected lipophilic constituents, i.e. hydrocarbons, aliphatic alcohols, fatty acids, sterols and terpenoids (Fig. 1a-b and Table 2). The lipophilic constituents of n-hexane extract and its neutral-acidic fractionation can be grouped into n-hexane extract four categories i.e hydrocarbons (ret. time 19.3-38.7), aliphatic alcohols (18.4-39.9), 0 10 20 30 40 50 fatty acids (ret. time 15.1-38.2 min), sterols and terpenoids (ret. time 38.4-45 min) (Fig. 1a-b). Previously, the fatty acids and terpenoids were detected in seed and bark of Retention time (minutes) (b) Outer bark mahogany (Chan et al. 1976; Falah et al. 2008; Eid et al. 2013; Rosdiana et al. 2017). Fig. 1a-b. Cromatogram of n-hexane extract, and neutral-acidic fractionation from mahogany barks 98 99

— 94 — Further, hydrocarbons, fatty alcohols or aliphatic alcohols were observed of inner and outer bark of E. globulus (Gutierrez et al. 1999; Freire et al. 2002).

Aliphatic alcohols Fatty acids Hydrocarbons Sterols and Terpenoids

acidic fraction

neutral fraction

n- hexane extract 0 10 20 30 40 50

Retention time (minutes) (a) Inner bark

Hydrocarbons Sterols and Terpenoids

Fatty acids Aliphatic alcohols

acidic fraction

neutral fraction

0 10 20 30 40 50 n-hexane extract

Retention time (minutes) (b) Outer bark Fig. 1a-b. Cromatogram of n-hexane extract, and neutral-acidic fractionation from mahogany barks 99

— 95 — Table 2. Lipophilic constituents from mahogany inner and outer bark detected by GC- Continues Table 2 MS (% dry extract) 38.8 1-Triacontanol - - - 2.83 - - 94 Similarity 39.9 1-Pentacontanol - - - 0.67 - - 88 Extract Neutral Acidic index (%) Fatty acids 22.7 10.0 6.9 9.8 19.8 15.5 Ret.Time Groups Inner Outer Inner Outer Inner Outer 15.1 Pentanoic acid 1.11 - - - 0.49 - 86 Hydrocarbons 4.35 11.97 14.47 15.21 3.96 0.31 16.1 Dodecanoic acid - - - 0.43 0.27 4.28 94 19.3 1-Dodecanone - - 0.71 - - - 85 21.1 Tetradecanoic acid - 1.15 0.57 1.25 0.62 - 87 20.1 Tetradecane 0.73 - - 0.12 0.34 - 75 22.9 Palmitic acid 2.82 3.37 1.55 1.5 5.04 6.36 94 20.5 1-Octadecene - 0.97 1.6 3.22 0.51 - 96 23.5 Pentadecanoic acid 0.32 0.75 - 3.15 - 1.29 91 20.6 Eicosane 0.97 1.57 1.53 0.76 0.51 0.31 93 23.8 Docosanoic acid - 1.52 - 1.28 - - 83 21.5 2-Pentadecanone - - - - 0.36 - 91 24.9 Tetracosanoic acid - - - - 0.21 - 76 24.0 10-Heneicosene 0.53 - 3.23 - 0.62 - 92 25.8 Oleic acid 8.32 - 1.39 1.97 6.69 0.66 88 24.1 Docosane 0.53 - 1.64 - - - 92 26.1 Octadecanoic acid - - - 0.19 - - 87 25.7 1-Hexadecyne - - - 0.55 - - 82 26.4 Linoleic acid 8.16 1.76 - - 5.95 - 85 25.7 Tetracosane - 0.96 0.78 - - - 86 27.2 Butanoic acid 0.28 - - - - 1.12 79 26.3 Nonadecane 0.79 - - 0.08 - - 84 35.5 Decanedioic acid - 1.47 3.38 - 0.31 0.31 89 26.9 1-Tetradecene - - - 0.11 - - 74 38.2 Propenoic acid 1.67 - - - 0.22 1.49 67 27.0 9-Eicosene 0.3 - - 0.07 - - 73 Sterols and 36.7 16.7 17.4 32.0 11.9 4.5 27.1 9-Octadecenamide - - - 0.31 - - 65 terpenoids 28.7 Heptadecane - 1.51 0.58 0.2 0.27 - 93 Ergost-25-ene- 38.4 - - 0.71 - - - 71 29.2 Heptacosane - - - 0.38 - - 72 3,5,6,12-tetrol 30.1 9-Hexacosene - 2.06 1.82 - - - 94 39.2 Stigmast-5-en-3-ol - - 1.29 - 0.42 - 78 30.1 Heneicosane - - - 0.59 - - 91 39.5 Cholestane-3,5-diol - - - 0.28 - - 83 30.1 Pentadecane - - 0.69 1.79 - - 91 41.0 Campesterol 5.28 3.13 1.72 3.92 3.61 0.94 91 31.6 Hexacosane - 2.27 - 0.55 - - 83 41.5 Stigmasterol 4.88 - 0.96 - - - 85 .gamma.- 32.8 Tridecane - - 0.62 0.7 - - 74 41.9 - - - 0.36 - - 82 Ergostenol 32.8 Hexadecane - 0.94 - 0.38 0.33 - 83 42.0 Lanosterol - - 1.47 0.27 - - 68 34.1 Cyclooctacosane - - - 2.13 - - 95 42.4 .beta.-Sitosterol 24.31 12.4 10.44 20.55 7.85 3.51 87 35.2 1-Tricosene - 0.74 - 2.43 - - 94 42.6 Stigmastanol - - - 0.67 - - 77 35.7 Squalene - - 0.54 - 0.79 - 87 9,19-Cycloergost- 43.1 1.43 - 0.8 1.75 - - 82 36.5 Nonane - - - 0.36 - - 79 24(28)-en-3-ol Stigmast-4-en-3- 37.6 Octadecane 0.5 0.95 0.73 0.26 0.23 - 74 43.2 0.83 1.15 - - - - 76 one 38.7 Tritetracontane - - - 0.22 - - 84 43.4 Stigmast-7-en-3-ol - - - 0.43 - - 81 Aliphatic alcohols 1.0 1.9 6.3 6.2 0.0 0.0 9,19- 45.0 - - - 3.72 - - 89 18.4 1-Hexadecanol 0.95 0.84 3.03 - - - 96 Cyclolanostan-3-ol 21.5 1-Dodecanol - - - 0.14 - - 82 Remarks : IB: inner bark; OB: outer bark; (-): not detected 22.1 1-Pentadecanol - - - 1.1 - - 96 22.1 1-Tetradecanol - - 0.95 - - - 94 In this study, chemical composition of lipid was detected in two conditions, 23.5 1,12-Dodecanediol - - 1.1 - - - 94 before and after neutral-acidic fractionation. The similar constituents of before and after 26.8 1-Octanol - - - 0.49 - - 88 fractionation in the neutral fraction were from hydrocarbons in the inner bark (i.e. 29.7 1-Heptacosanol - - - 0.49 - - 84 eicosane, 10-heneicosane, docosane, and octadecane), aliphatic alcohols (i.e. 1- 31.4 1-Octadecanol - - - 0.47 - - 84 hexadecanol), fatty acids (palmitic acid and oleic acid), sterols and terpenoids (i.e 32.7 1-Nonadecanol - 1.07 1.25 - - - 92 campes -sitosterol and 9,19-cycloergost-24(28)-en-3-ol). In the outer bark, the hydrocarbons were 1-octadecane, heptadecane, hexacosane, hexadecane, 1-tricosane, terol, β 100 101

— 96 — Continues Table 2 38.8 1-Triacontanol - - - 2.83 - - 94 39.9 1-Pentacontanol - - - 0.67 - - 88 Fatty acids 22.7 10.0 6.9 9.8 19.8 15.5

15.1 Pentanoic acid 1.11 - - - 0.49 - 86 16.1 Dodecanoic acid - - - 0.43 0.27 4.28 94 21.1 Tetradecanoic acid - 1.15 0.57 1.25 0.62 - 87 22.9 Palmitic acid 2.82 3.37 1.55 1.5 5.04 6.36 94 23.5 Pentadecanoic acid 0.32 0.75 - 3.15 - 1.29 91 23.8 Docosanoic acid - 1.52 - 1.28 - - 83 24.9 Tetracosanoic acid - - - - 0.21 - 76 25.8 Oleic acid 8.32 - 1.39 1.97 6.69 0.66 88 26.1 Octadecanoic acid - - - 0.19 - - 87 26.4 Linoleic acid 8.16 1.76 - - 5.95 - 85 27.2 Butanoic acid 0.28 - - - - 1.12 79 35.5 Decanedioic acid - 1.47 3.38 - 0.31 0.31 89 38.2 Propenoic acid 1.67 - - - 0.22 1.49 67 Sterols and 36.7 16.7 17.4 32.0 11.9 4.5 terpenoids Ergost-25-ene- 38.4 - - 0.71 - - - 71 3,5,6,12-tetrol 39.2 Stigmast-5-en-3-ol - - 1.29 - 0.42 - 78 39.5 Cholestane-3,5-diol - - - 0.28 - - 83 41.0 Campesterol 5.28 3.13 1.72 3.92 3.61 0.94 91 41.5 Stigmasterol 4.88 - 0.96 - - - 85 .gamma.- 41.9 - - - 0.36 - - 82 Ergostenol 42.0 Lanosterol - - 1.47 0.27 - - 68 42.4 .beta.-Sitosterol 24.31 12.4 10.44 20.55 7.85 3.51 87 42.6 Stigmastanol - - - 0.67 - - 77 9,19-Cycloergost- 43.1 1.43 - 0.8 1.75 - - 82 24(28)-en-3-ol Stigmast-4-en-3- 43.2 0.83 1.15 - - - - 76 one 43.4 Stigmast-7-en-3-ol - - - 0.43 - - 81 9,19- 45.0 - - - 3.72 - - 89 Cyclolanostan-3-ol Remarks : IB: inner bark; OB: outer bark; (-): not detected

In this study, chemical composition of lipid was detected in two conditions, before and after neutral-acidic fractionation. The similar constituents of before and after fractionation in the neutral fraction were from hydrocarbons in the inner bark (i.e. eicosane, 10-heneicosane, docosane, and octadecane), aliphatic alcohols (i.e. 1- hexadecanol), fatty acids (palmitic acid and oleic acid), sterols and terpenoids (i.e campes -sitosterol and 9,19-cycloergost-24(28)-en-3-ol). In the outer bark, the hydrocarbons were 1-octadecane, heptadecane, hexacosane, hexadecane, 1-tricosane, terol, β 101

— 97 — and octadecane), fatty acids consisted of tetradecanoic acid, palmitic acid, pentadecanoic compound of hydrocarbons such as tetradecane (12.68 min), 1-octadecane (20.54 min), acid, and docosanoic acid whereas sterols and terpenoids were campesterol and b- pentadecane (27.28 min), 1-tricosane (30.11), eicosane (30.17 min), and heptadecane sitosterol. On the other hand, the different result in the inner bark as well as in the outer (31.53 min) were also observed. In the E. globulus wood, hydrocarbons such as bark parts were from hydrocarbons The similar pattern was also observed for fatty nonacosane, hentriacontane, tritiacontane, and stigmasta-3,5-diene were detected acids, sterols, and terpenoids. (Gutierrez et al. 1999). For acidic fraction, the similar constituents of hydrocarbons were detected In treatment before and after neutral-acidic fractionation, some differences were (tetradecane, eicosane, heptadecane, hexadecane, and octadecane, 10-heneicosane, and found. In the neutral fraction, hydrocarbons and aliphatic alcohols increased in the inner octadecane), as well as for fatty acids (pentanoic acid, palmitiac acid, oleic acid, linoleic bark, whereas sterols and terpenoids increased in the outer bark (Fig. 2a-b). In addition, acid, propenoic acid) and for sterols and terpenoids (campasterol, b-sitosterol). fatty acids content were lower than that of before neutral fractionation. Further, in the However, different components were also observed in the inner bark (1-octadecane, 2- acidic fraction, fatty acids concentration were larger than hydrocarbons, sterols and pentadecanone, heptadecane, hexadecane and squalene), fatty acids (dodecanoic acid, terpenoids. On the other hand, there is no aliphatic alcohols were found in the acidic tetradecanoic acid, tetracosanoic acid, decanedioic acid), sterols and terpenoids fraction. (stigmast-5-en-3-ol). Further, in the outer bark, similar components of hydrocarbon were only eicosane, fatty acids were palmitic acids, pentadecanoic acid, and decanedioic acid, as well as sterols and terpenoids, were campesterol an -sitosterol. The different 70 60 Hyrdrocarbons components of fatty acids were also detected (dodecanoic acid, oleic acid, butanoic acid, 50 d β and propenoic acid). 40 Aliphatic alcohols Gutierrez et al. (1999) reported that the neutral fraction of contained 30 E. globulus (%) 20 sterols (cholesterol, campesterols, ergostanol, sitosterol, fucosterol) and fatty alcohols 10 Fatty acids (n-tetradecanol, n-pentadecanol, n-hexadecanol, n-heptadecanol, n-octadecanol, n- Proportion of 0 lipophilic content lipophilic based based on peak area Inner Outer Inner Outer Inner Outer nonadecanol, n-eicosanol, n-heicosanol, n-docosanol, n-tricosanol, n-tetracosanol, n- Sterols and pentacosanol, n-hexacosanol). For acidic fraction, it contained several fatty acids i.e. n- Extract Neutral Acid terpenoids tetradecanoic acid, n-pentadecanoic acid, 9-hexadecanoic acid, palmitic acid, n- heptadecanoic acid, 9,12-octadecanoic acid, 9-octadecenoic acid, n-octadecanoic acid, n- (a) eicosanoic acid. Fatty acids were also a main family of inner and outer bark of mahogany. Fatty 100% 90% acids accounted for 25-35 % based on dried extract as the dominant component being 80% 70% Hydrocarbons oleic acid, followed by palmitic acid and linoleic acid. Palmitic acid (22.9 min), linoleic 60% acid (26.45 min), and oleic acid (26.51 min) were found also in seed and bark of 50% 40% Aliphatic alcohols mahogany (Chan et al. 1976; Eid et al. 2012; Rosdiana et al. 2017). On the other hand, 30% the groups of sterols, -sitosterol was the most abundant compound. Another area (%) 20% 10% Fatty acids compound, campesterol was the second major components of sterols. In the previous 0% content based on peak

β Proportion of lipophilic -sitosterol was the main components of the sterols group in all Inner Outer Inner Outer Inner Outer Sterols and wood, both in free and esterified forms (Freire et al. 2006). That compound was also Extract Neutral Acid terpenoids studies,found as β the most abundant in the inner and outer bark from E. globulus, 3 other eucalypts (E. grandis, E. urograndis, and E. maidenii) and six different (P. elliotii, P. oocarpa, P. caribeae, P. merkusii, P. montezumae, and P. insularis) (Freire et al. 2002; (b) Dominguez et al. 2011; Masendra et al. 2018a, 2018b). Terpenoid and sterol Fig. 2a-b Major families of compounds identified in inner and outer bark of mahogany components such as squalene (35.80 min), campesterol (41.06 min), and stigmasterol and their acidic-neutral fractions (41.53 min) were also mentioned in neutral fraction and dichloromethane extract of E. globulus wood and outer bark (Gutierrez et al. 1999; Freire et al. 2002). Further, minor

102 103

— 98 — compound of hydrocarbons such as tetradecane (12.68 min), 1-octadecane (20.54 min), pentadecane (27.28 min), 1-tricosane (30.11), eicosane (30.17 min), and heptadecane (31.53 min) were also observed. In the E. globulus wood, hydrocarbons such as nonacosane, hentriacontane, tritiacontane, and stigmasta-3,5-diene were detected (Gutierrez et al. 1999). In treatment before and after neutral-acidic fractionation, some differences were found. In the neutral fraction, hydrocarbons and aliphatic alcohols increased in the inner bark, whereas sterols and terpenoids increased in the outer bark (Fig. 2a-b). In addition, fatty acids content were lower than that of before neutral fractionation. Further, in the acidic fraction, fatty acids concentration were larger than hydrocarbons, sterols and terpenoids. On the other hand, there is no aliphatic alcohols were found in the acidic fraction.

70 60 Hyrdrocarbons 50 40 Aliphatic alcohols 30

(%) 20 10 Fatty acids Proportion of Proportion of 0 lipophilic content lipophilic based based on peak area Inner Outer Inner Outer Inner Outer Sterols and Extract Neutral Acid terpenoids

(a)

100% 90% 80% 70% Hydrocarbons 60% 50% 40% Aliphatic alcohols 30% area (%) 20% 10% Fatty acids 0% content based on peak Proportion of lipophilic Proportion of lipophilic Inner Outer Inner Outer Inner Outer Sterols and Extract Neutral Acid terpenoids

(b) Fig. 2a-b Major families of compounds identified in inner and outer bark of mahogany and their acidic-neutral fractions

103

— 99 — The most predominant compounds in the lipid of inner and outer bark of Brown NS, Jennings S, Clements T. The ecology, silviculture and biogeography of mahogany were sterols, terpenoids, and fatty acids. Sterols and terpenoids were the mahagony (Swietenia macrophylla): a critical review of the evidence. Persp major class (41-57% based on dried extract), and followed by fatty acids (25-35%), Eco, Evol System. 6: 37-49. whereas aliphatic alcohols (1-5%) were the minor class. Hydrocarbon levels in the inner Blundell AG, Gullison RE. 2003. Poor regulatory capacity limits the ability of science to bark (7%) were lower than in the outer bark part (29%). As reported by Gutierrez et al. influence the management of mahogany. For. Pol. Econ. 5: 395-405. (1999) and Freire et al. (2002) that sterols were the major class in the inner and outer Baeza J, Freer J. 2001. Chemical characterization of wood and its components. In DN-S bark of E. globulus, whereas fatty alcohols were the minor group. Hon, Shiraishi N, editor. Wood and Cellulosic Chemistry. New York: Marcel Dekker. The similar result was also found that fatty acid was the major class in the outer Baptista I, Miranda I, Quilhó T, Gominho J, Pereira H. 2013. Characterisation and bark of P. caribeae, P. insularis, and P. montezuma whereas steroids and triterpenoids fractioning of Tectona grandis bark in view of its valorisation as a biorefinery were the dominant groups identified in the inner bark of P. elliotii, P. insularis, and P. raw-material. Ind Crop Prod. 50:166-175. merkusii (Masendra et al. 2018a). On the other hand, fatty acids and sterols were the Dewanjee S, Maiti A, Das, AK, Mandal SC, Dey SP. 2009. Swietenine: A potential oral most abundant group in the outer bark compared to inner bark part in E. globulus, E. hypoglycemic from Swietenia macrophylla seed. Fitoterapia 80:249-251. grandis and E. urograndis (Freire et al. 2002; Domingues et al. 2011), and Betula species Domingues RMA, Sausa GDA, Silva CM, Freire CSR, Silvestre AJD, Neto CP. 2011. High (Pinto et al. 2009; Ferreira et al. 2013). value triterpenic compound from the outer barks of several Eucalyptus species It should be noted that sterols and terpenoids was the most abundant of the cultivated in Brazil and in Portugal. Ind Crop Prod. 33:158-164. lipids in this study. Triterpenoids are rich source of bioactive compounds (Swati & Rica Freire CSR, Silvestre AJD, Neto .P, Cavaleiro JAS. 2002. Lipophilic extractives of the inner 2011). Mahogany contained limonoids, swietenioides, metyl angolensate and diacetyl and outer barks of Eucalyptus globulus. Holzforschung 56:372-379. swietenioides (Mootoo et al. 1999; Goh et al. 2010; Goh & Kadir 2011). Its seeds Fengel D, Wegener G. 1989. Wood: chemistry ultrastructure, reactions. Walter de exhibited anti-hypertensive, anti-diabetic, and anti-inflamatory agents (Dewanjee et al. Gruyter, Berlin. 2009; King et al. 2013). Thus, the constituents of sterol and terpenoid in this study Freire CSR, Silvestre AJD, Neto CP, Cavaleiro JAS. 2002. Lipophilic extractives of the inner should be isolated to confirm the chemical structures as well as its toxicity in and outer barks of Eucalyptus globulus. Holzforschung 56:372-379. subsequent works. Ferreira R, Garcia H, Sausa AF, Freira CSR, Silvestre AJD, Rebelo LPN, Pereira CS. 2013.

Isolation of suberin from outer bark and cork using ionic liquids: A new Conclusions source of macromonomers. Ind Crop Prod. 44: 520-527. Four groups of lipid i.e hydrocarbons, aliphatic alcohols, sterols and terpenoids of Freire CSR, Pinto PCR, Santiaogo AS, Silvestre AJD, Evtuguin, D.M, Neto CP. 2006. inner and outer bark of mahogany bark have been studied. Fatty acids and sterols were Comparative study of lipophilic extractives of and corresponding ECF bleached craft pulp. BioResources 1(1):3-17. the major groups, whereas a minor group was found in the hydrocarbons and aliphatic alcohols. After fractionation, the contents of sterols, terpenoids, hydrocarbons and Gutierrez A, del Rio JC, Gonzalez-Vila, Martin F. 1999. Chemical composition of lipophilic aliphatic alcohols increased. On the other hand, the contents of fatty acids increased in extractives from Eucalyptus globulus Labill. wood. Holzforschung 53(5):481–486. the acidic fraction. Due to the high concentration of sterols and terpenoids, it is thought Goh BH, Kadir A. 2011. In vitro cytotoxic potential of Swietenia macrophylla King seeds that the barks of mahogany have potential to be developed for medicinal substances. againts human carcinoma cell lines. J. Med. Plant Res. 5: 1395-1404. Krogell J, Holmbom B, Pranovich A, Hemming J, Willfor S. 2012. Extraction and chemical characterization of Norway spruce inner and outer bark. Nord Pulp and Paper J. Acknowledgement 27 (1): 6-17. The authors thank to SRIKANDIRATU a furniture industry in Jepara, for providing Lima L, Miranda I, Knapic S, Quilho T, Pereira H. 2018. Chemical and anatomical characterization, and antioxidant properties of barks from 11 Eucalyptus species. sample of mahogany wood. Eur. J. Wood Prod. 76(2):783-792. References Lukmandaru G. 2012. Komposisi ekstraktif pada kayu mangium (Acacia mangium). J. Anas E, Ekman R, and Holmbom J. 1983. Composition of nonpolar extractives in bark of Ilmu Tek Kayu Trop. 10(2): 150-158. norway spruch and scots . J Wood Chem Techno. 3 (3): 119-130.

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— 100 — Brown NS, Jennings S, Clements T. The ecology, silviculture and biogeography of mahagony (Swietenia macrophylla): a critical review of the evidence. Persp Plant Eco, Evol System. 6: 37-49. Blundell AG, Gullison RE. 2003. Poor regulatory capacity limits the ability of science to influence the management of mahogany. For. Pol. Econ. 5: 395-405. Baeza J, Freer J. 2001. Chemical characterization of wood and its components. In DN-S Hon, Shiraishi N, editor. Wood and Cellulosic Chemistry. New York: Marcel Dekker. Baptista I, Miranda I, Quilhó T, Gominho J, Pereira H. 2013. Characterisation and fractioning of Tectona grandis bark in view of its valorisation as a biorefinery raw-material. Ind Crop Prod. 50:166-175. Dewanjee S, Maiti A, Das, AK, Mandal SC, Dey SP. 2009. Swietenine: A potential oral hypoglycemic from Swietenia macrophylla seed. Fitoterapia 80:249-251. Domingues RMA, Sausa GDA, Silva CM, Freire CSR, Silvestre AJD, Neto CP. 2011. High value triterpenic compound from the outer barks of several Eucalyptus species cultivated in Brazil and in Portugal. Ind Crop Prod. 33:158-164. Freire CSR, Silvestre AJD, Neto .P, Cavaleiro JAS. 2002. Lipophilic extractives of the inner and outer barks of Eucalyptus globulus. Holzforschung 56:372-379. Fengel D, Wegener G. 1989. Wood: chemistry ultrastructure, reactions. Walter de Gruyter, Berlin. Freire CSR, Silvestre AJD, Neto CP, Cavaleiro JAS. 2002. Lipophilic extractives of the inner and outer barks of Eucalyptus globulus. Holzforschung 56:372-379. Ferreira R, Garcia H, Sausa AF, Freira CSR, Silvestre AJD, Rebelo LPN, Pereira CS. 2013. Isolation of suberin from birch outer bark and cork using ionic liquids: A new source of macromonomers. Ind Crop Prod. 44: 520-527. Freire CSR, Pinto PCR, Santiaogo AS, Silvestre AJD, Evtuguin, D.M, Neto CP. 2006. Comparative study of lipophilic extractives of hardwood and corresponding ECF bleached craft pulp. BioResources 1(1):3-17. Gutierrez A, del Rio JC, Gonzalez-Vila, Martin F. 1999. Chemical composition of lipophilic extractives from Eucalyptus globulus Labill. wood. Holzforschung 53(5):481–486. Goh BH, Kadir A. 2011. In vitro cytotoxic potential of Swietenia macrophylla King seeds againts human carcinoma cell lines. J. Med. Plant Res. 5: 1395-1404. Krogell J, Holmbom B, Pranovich A, Hemming J, Willfor S. 2012. Extraction and chemical characterization of Norway spruce inner and outer bark. Nord Pulp and Paper J. 27 (1): 6-17. Lima L, Miranda I, Knapic S, Quilho T, Pereira H. 2018. Chemical and anatomical characterization, and antioxidant properties of barks from 11 Eucalyptus species. Eur. J. Wood Prod. 76(2):783-792. Lukmandaru G. 2012. Komposisi ekstraktif pada kayu mangium (Acacia mangium). J. Ilmu Tek Kayu Trop. 10(2): 150-158.

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— 101 — Miranda I, Gominho J, Pereira H. 2012. Incorporation of bark and tops in Eucalyptus The Effect of Wood Species on The Growth of Two Kinds of Edible wood pulping. BioResources :4350–4361. globulus 7 Mushroom Species Masendra, Ashitani T, Takahashi K, Lukmandaru G. 2018a. Lipophilic extractives of the inner and outer barks from six different Pinus species grown in Indonesia. J. Forestry Research 29(5):1329-1336 https://doi.org/10.1007/s11676-017-0545-x Denny Irawati*, Dahayu Rananindha, Yustika Ami Meirista, & J. Pramana Gentur Sutapa

Masendra, Ashitani T, Takahashi K, Lukmandaru G. 2018b. Triterpenoids and steroids Fakultas Kehutanan, Universitas Gadjah Mada, Yogyakarta from the bark of Pinus merkusii (Pinaceae). BioResources 13(3):6160-6170. *Email: [email protected] Pereira H, Graça J, Rodrigues JC. 2003. Wood chemistry in relation to quality. In: Barnett JR, Jeronimidis G (eds) Wood quality and its biological basis. CRC Press, Blackwell Publishing, Oxford, pp 53–83. ABSTRACT Pinto PCRO, Sausa AF, Silvestre AJD, Neto CP, Gandini A, Eckermen C, Holmbom B. 2009. So far, many species of edible mushrooms have been cultivated by the Indonesian farmer Querqus suber and Betula pendula outer barks as renewable sources of using Sengon as their medium. However, there has been no research which uses other oleochemicals: A comparative study. Ind Crop Prod. 29: 126-132. wood species for the cultivation of those edible mushrooms. In Indonesia, there are several woody trees belonging to the Leguminoceae family which are the same as Sengon that the Rosdiana NA, Dumarcay S, Gerardin C, Chapuis H, Medina FJS, Sari RK, Syafii W, Gelhay E, potentiality is still unknown as the mushroom growth media. This study was carried out by Raharivelomanana P, Mohammed R, and Gerardin P. 2017. Characterization of growing ear mushrooms ( ) and Shiitake mushrooms ( bark extractives of different industrial Indonesian wood species for potential Auricularia auricula-judae Lentinula ) on media made from Gamal, Lamtoro, and Johar sawdusts. Fungal mycelia growth was valorization. Ind Crop Prod. 108: 121-127. edodes measured by measuring the length of mycelia which appeared on the surface of the media every Sayyad M, Tiang N, Kumari Y, Goh BH, Jaiswal Y, Rosli R, Williams L, Sahikh MF. 2017. 2 days. An interaction between the wood species and the mushroom species on the growth of Acute toxicity profiling of the ethyl acetate fraction of Swietenia macrophylla mycelia was found in this researh result. Lamtoro and Gamal wood are species that good for the seeds and in-vitro neuroprotection studies. Saud Pharmac J. 25:195-205. growth of ear mushroom mycelia with an average growth rate of 2.56 and 2.16 mm/day, while Sjöström, E. 1993. Wood Chemistry. Fundamentals and Applications, 2nd edition, Gamal and Johar wood are best for Shiitake mushroom mycelia growth, with an average growth Academic Press, San Diego, USA. rate of 2.25 and 2.04 mm/day. The results of statistical analysis showed that there was no Sun YP, Zhu LL, Liu JS, Yu Y, Zhou ZY, Wang G, Wang GK. 2018. Limonoids and significant relationship between the chemical components of wood and mycelia growth rate of triterpenoid from fruit of Switenia macrophylla. Fitoterapia. 125: 141-146. each mushroom species. Keywords : edible mushrooms, gamal, johar, lamtoro, mycelia growth Introduction Currently, the edible mushroom, such as ear mushrooms (Auricularia auricula- judae), oyster mushroom (Pleorotus ostreatus), and Shiitake mushrooms (Lentinula edodes), cultivation in Indonesia has begun to develop quite high, especially in the wood- mushrooms species and there are many mushroom cultivation industries ranging from small to large classes that produce mushrooms. This happened because of the increasing need for mushroom consumption among the community. The average Indonesian mushroom consumption is 0.25 kg/capita/year (Ministry of Agriculture 2017). So far these mushroom species as above has been cultivated by the community by using Sengon wood as a medium. Actually, Sengon is not the only wood which can be used as a medium. Quimio in Chang and Quimio (1982) stated that ear mushrooms can grow well on media made from Lamtoro (Leucaena leucocephala Lam de Wit) wood compared to media made from other woods. Therefore, there are only a few research that use other species of wood for the cultivation of edible mushrooms, especially in Indonesia. 107 105

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