NC State
BioResources
Masendra, M., Ashitani, T., Takahashi, K., and Lukmandaru, G. (2018). "Triterpenoids and steroids from the bark of Pinus merkusii (Pinaceae)," BioRes. 13(3), 6160-6170.

Abstract

Serratene triterpenoids are considered chemotaxonomy compounds from the Pinaceae family, within the Pinus genus. However, no studies have been conducted on the constituents of serratene triterpenoids of Pinus merkusii. P. merkusii is the only pine species native to Southeast Asia, including Indonesia. This study aimed to investigate the use of triterpenoids and steroids from the bark of P. merkusii as chemotaxonomic biomarkers. Five lipophilic extractives, including three triterpenoids (3β-methoxyserratt-14-en-21-one (C1), 3α,21β- dimethoxy-D14-serratene (C3), serrate-14-en-3β,21β-diol (C5)), and two steroids (stigmast-4-en-3-one (C2) and β-sitosterol (C4)), were isolated from the n-hexane extract of the bark of P. merkusii using column chromatography and thinlayer chromatography. The structures of the triterpenoids and steroids were characterized on the basis of their spectroscopic data. The discovery of serratene triterpenoids and steroids in P. merkusii bark characterizes this species as being in chemical accordance with other species of the Pinus genus and Pinaceae family.


Download PDF

Full Article

Triterpenoids and Steroids from the Bark of Pinus merkusii (Pinaceae)

Masendra,a Tatsuya Ashitani,b Koetsu Takahashi,b and Ganis Lukmandaru a,*

Serratene triterpenoids are considered chemotaxonomy compounds from the Pinaceae family, within the Pinus genus. However, no studies have been conducted on the constituents of serratene triterpenoids of Pinus merkusiiP. merkusii is the only pine species native to Southeast Asia, including Indonesia. This study aimed to investigate the use of triterpenoids and steroids from the bark of P. merkusii as chemotaxonomic biomarkers. Five lipophilic extractives, including three triterpenoids (3β-methoxyserratt-14-en-21-one (C1), 3α,21β- dimethoxy-14-serratene (C3), serrate-14-en-3β,21β-diol (C5)), and two steroids (stigmast-4-en-3-one (C2) and β-sitosterol (C4)), were isolated from the n-hexane extract of the bark of P. merkusii using column chromatography and thinlayer chromatography. The structures of the triterpenoids and steroids were characterized on the basis of their spectroscopic data. The discovery of serratene triterpenoids and steroids in P. merkusii bark characterizes this species as being in chemical accordance with other species of the Pinus genus and Pinaceae family.

Keywords: Pinus merkusii; Pinaceae; Triterpenoids; Steroids

Contact information: a: Department of Forest Product Technology, Faculty of Forestry, Universitas Gadjah Mada, Jl. Agro No. 1, Bulaksumur, Yogyakarta 55281, Japan; b: Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata 997-855, Japan;

* Corresponding author: glukmandaru@ugm.ac.id

INTRODUCTION

Pinaceae is one of the largest families of conifers, comprised of 11 genera and 225 species. Nearly half of these species (110 species) are considered true pines and belong to the genus Pinus (Farjon 2005). Approximately 80 Pinus species are distributed throughout the northern hemisphere (Li et al. 2012). P. merkusii Jungh & de Vriese is a rather small tree, ranging from 20 m to 30 m in height. This pine is an exception to the general “rule” that pines are restricted to regions with a cool climate. It grows from sea level up to an altitude of 2000 m in Indonesia (Sumatra) and the Philippines (Farjon 1984). Species of P. merkusii belong to the section and subsection Pinus, kingdom of plantae, division of spermatophyte, subdivision of gymnospermae, class of Coniferae, order of Pinales, family of Pinaceae, and genus of Pinus (Gernandt et al. 2005; Siregar 2005; Baharudin and Tasikirawati 2009).

In Indonesia, in addition to being used as material for pulp and paper, P. merkusii has been planted to produce a resin that can be divided into gum and turpentine. The chemical composition of P. merkusii resin has been reported to include mono- and sesquiterpenes (Coppen et al. 1998; Wiyono et al.2006; Hadiyane et al. 2015; Sukarno et al. 2015). The wood from this species has been reported to contain phenolic compounds and diterpenoids (Wijayanto et al. 2015). Terpenoids have been reported as bioactive compounds related to bark function in protecting the tree from exposure to extreme conditions (Gershenzon 1994; Wittstock and Gershenzon 2002; Seki et al. 2012). Triterpenoids of this family have been well reported as biomarkers such as serratene-type triterpenoids (Le Milbeau et al. 2013; Otaka et al. 2016). These serratene-type triterpenoids have also been isolated from other Pinusand Picea species, especially from their bark (Rowe 1964; Rowe et al. 1972; Norin and Winnel 1972; Cheng and Chao 1979; Fang et al. 1991; Fang and Cheng 1992; Yamamoto et al. 2011; Labib et al. 2018). However, there have not been any reports regarding serratene-type triterpenoids through isolation, structure determination, and chemotaxonomy value from the bark of the tropical species of P. merkusii until now.

EXPERIMENTAL

Materials

Samples

Bark samples were taken from a 30-year-old P. merkusii tree at the state-owned enterprise of Perhutani Plantation (Magelang, Central Java, Indonesia) in February 2015. This site is located at 7° 28′ 0″ S, 110° 13′ 0″ E, with an average temperature of 26.2 °C, annual rainfall of 3189 mm, and an elevation of 139 m above sea level. The 1.5-cm-thick bark sample was dried at room temperature, and then finely ground before extraction. A voucher number (PM-3001) was made by the Faculty of Forestry, Universitas Gadjah Mada, Indonesia.

Methods

General equipment

The 13C and 1H nuclear magnetic resonance (NMR) were determined by a JEOL ECZ-400 spectrometer (JEOL, Tokyo, Japan). The NMR spectra were recorded using standard JEOL pulse sequences at 400 MHz and 100 MHz for 1H and 13C, respectively. Chemical shifts were recorded as δ (ppm) values with chloroform-deuterium (Sigma-Aldrich, St. Louis, MO, USA) as the solvent. Coupling constants (J) were recorded in Hz, and multiplicities were abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, br = broad, and m = multiplet. Melting points were not corrected.

Silica gel (60 N, spherical 63 μm to 210 μm; neutral Kanto Chemical Co., Inc., Tokyo, Japan) was used for column chromatography with a glass column (40 cm × 2.5-cm inner diameter). n-hexane-ethyl acetate (EtOAc) in increasing polarity were used as eluents (99:1, 49:1, 9:1, 8:2, 1:1, v/v). Aluminum sheets that were pre-coated with silica gel 60 F254 (Merck, Kenilworth, NJ, USA) were used for thinlayer chromatography (TLC). Spots were visualized using ultraviolet (UV) light irradiation (λ = 254 nm and 360 nm) by spraying with vaniline-sulfuric acid (for color testing), followed by heating at 150 °C for 10 min. The developing solvents used for TLC were n-hexane-EtOAc (8:2, v/v).

Gas chromatography-mass spectrometry (GC-MS) data were collected with a GCMS-QP2010 (Shimadzu, Kyoto, Japan) under the following conditions: DB-1 capillary column (30 mm × 0.25-mm inner diameter and 0.25 μm; GL Sciences, Tokyo, Japan); column temperature from 50 °C (1 min) to 320 °C at 5 °C/min; injection temperature of 250 °C; detection temperature of 320 °C; acquisition mass range of 50 amu to 800 amu using helium as the carrier gas. The components were identified by comparison of the experimental GC-MS data with NIST MS library and literature data (Fang et al. 1991; Barla et al. 2006; Pateh et al. 2009; Halilu et al. 2013; Le Milbeau et al. 2013).

Extraction and isolation

The air-dried P. merkusii bark (1.0 kg) was extracted with n-hexane for two weeks under room temperature and evaporated until fully dry before weighing. The n-hexane extract with a dark yellow color yielded 1.59 g. For separation, 1.0 g of extract was chromatographed into Si gel column chromatography (SiGCC), resulting in 9 fractions (H1-H9) with eluent of n-hexane-EtOAc (99:1, 49:1, 9:1, 8:2, 1:1, v/v). From 9 fractions, H2 and H8 had a high purity, both H2 (14.8 mg) and H8 (71.0 mg) were collected from eluent of n-hexane-EtOAc (8:2, v/v), and H2 and H8 were designated as C1and C5. The fractions of H3 and H4 were re-column chromatographed into SiGCC with H3 eluent of n-hexane-EtOAc (9:1, 1:1, v/v) to obtain H31-H36 fraction, and H4 eluent of n-hexane-EtOAc (8:2, 1:1, 0:1, v/v) to obtain H41-H49 fractions. From H3 separation, high purity fraction of H32 (13.9 mg) was isolated by eluent of n-hexane-EtOAc (9:1, v/v), while from H4 separation, pure fraction of H42 (1.0 mg) and H47 (63.1 mg) were collected from eluent of n-hexane-EtOAc (8:2 and 1:1, v/v, respectively). For further discussion, H32, H42 and H47 were designated as C2, C3 and C4, respectively.

The compound of C1-C5 were then analyzed with1H NMR and 13C NMR. Moreover, due to the paucity of compound C3 (1 mg), only 1H NMR (400 MHz) data that can be analyzed as the 13C NMR (100 MHz) data could not be obtained. The GC-MS chromatogram of n-hexane extract and the scheme of the extraction and isolation is shown in Fig. 1 and Fig. 2.

Fig. 1. Chromatogram of n-hexane extract of P. merkusii bark; 1. Internal standard (heneicosane (ret. time: 32.26 min), 2. β-sitosterol (49. 65 min), 3. Stigmast-4-en-3-one (51.17), 4. 3α,21β-dimethoxy-14-serratene (53.70), 5. 3β-methoxyserratt-14-en-21-one (54.16), 6. Serrate-14-en-3β,21β-diol (55.29).

Fig. 2. Schematic for separation of triterpenoids and steroids from the bark of P. merkusii

3β- Methoxyserratt-14-en-21-one, C31H52O (C1)

Compound C1 (14.8 mg) was isolated as a white crystalline powder from fraction H2. The 1H-NMR (CDCl3) results were as follows: δ 0.93 (3H, s, H-23-Me), 0.73 (3H, s, H-24-Me), 0.77 (3H, s, H-25-Me), 0.80 (3H, s, H-26-Me), 0.90 (3H, s, H-28-Me), 1.02 (3H, s, H-29-Me), 1.06 (3H, s, H-30-Me), 2.60 (1H, dd, = 11.7 and 4.1 Hz, H-3α), 2.73 (1H, dt, = 14.8 and 5.5 Hz, H-20), 3.33 (3H, s, H-3-OMe), and 5.35 (1H, brs, H-15); EI-MS m/z 454 (M+; C31H50O2, 43), 221 (100), 218 (73), and 135 (63); Rf: 0.58; retention time of 54.16 min. The 13C NMR spectral data is provided in Table 1.

Stigmast-4-en-3-one, C29H48O (C2)

Compound C2 (13.9 mg) was obtained as a crystalline powder from fraction H32. The 1H-NMR (CDCl3) results were as follows: δ 1.49, 1.24 (each 1 H, m, H-1), 2.36, 2.25 (each 1 H, m, H-2), 5.70 (1H, br, s, H-4), 2.02, 1.90 (each 1H, m, H-6), 1.18, 1.42 (each 1 H, m, H-7), 1.41 (1H, m, H-8, H-9), 1.56, 1.27 (each 1H, m, H-11), 1.57, 1.31 (each 1 H, m, H-12), 1.02 (1H, m, H-14), 1.62, 1.35 (each 1 H, m, H-15, H-16), 1.13 (1H, m, H-17), 0.69 (3H, s, H-18), 1.16 (3H, s, H-19), 1.65 (1H, s, H-20), 0.84 (3H, d = 6.9 Hz, H-21), 1.27, 1.27 (each 1 H, m, H-22, H-23), 1.48 (1H, m, H-24), 1.83 (1H, m, H-25), 0.80 (3H, d, = 6.9 Hz, H-26), 0.78 (3H, d, = 6.9 Hz, H-27), 1.56, 1.56 (each 1 H, m, H-28), 0.9 (3H, t, = 6.9 Hz, H-29). The EI-MS m/z 412 (M+; C29H48O, 39), 397 (8), 370 (14), 289 (22), 229 (38), and 124 (100); Rf: 0.36; retention time of 51.17 min. The 13C NMR spectral data is provided in Table 1.

3α,21β- Dimethoxy-14-serratene, C32H54O2 (C3)

Compound C3 (1.0 mg) was obtained as a crystalline powder from fraction H42. The 1H-NMR (CDCl3) results were as follows: δ 0.93 (3H, s, H-23-Me), 0.65 (3H, s, H-24-Me), 0.72 (3H, s, H-25-Me), 0.78 (3H, s, H-26-Me), 0.80 (3H, s, H-28-Me), 0.92 (3H, s, H-29-Me), 0.93 (3H, s, H-30-Me), 3.33 (3H, s, 21-OMe), 2.03 (3H, s, H-3-OMe), 2.62 (1H, dd, = 11.9 and 4.1 Hz, H-3-α), 5.28 (1H, brs, H-15). The electron ionized mass spectrometry (EI-MS) m/z 470 (M+; C32H54O2, 54), 455 (35), 438 (18), 423 (18), 234, (52), 221, (81), 189 (100), 149 (20), 135 (72), and 147 (43); Rf: 0.9; retention time of 53.70 min.

β-Sitosterol, C29H50O (C4)

Compound C4 (63.1 mg) was obtained as a crystalline powder from fraction H47. The 1H-NMR (CDCl3) results were as follows: δ 1.13, 1.13 (2H, m, H-1), 1.58, 1.23 (2H, m, H-2), 3.5 (1H, m, H-3), 2.23, 1.97 (2H, m, H-4), 5.33 (1H, m, H-6), 2.15, 1.97 (2H, m, H-7), 1.48 (1H, m, H-8), 1.44 (1H, m, H-9), 1.51, 1.22 (2H, m, H-11), 1.56, 1.47 (2H, m, H-12), 1.41 (1H, m, H-14), 1.63, 1.46 (2H, m, H-15, H-16), 1.47 (1H, m, H-17), 1.04 (3H, m, H-18), 1.05 (3H, m, H-19), 1.64 (1H, m, H-20), 0.98 (3H, d, = 6.9 Hz, H-21), 1.25 (2H, m, H-22, H-23), 1.46 (1H, m, H-24), 1.81 (1H, m, H-25), 0.81 (3H, d, = 4.8 Hz, H-26), 0.89 (3H, d, = 4.8 Hz, H-27), 1.49 (2H, m, H-28), 0.90 (3H, m, H-29). The EI-MS m/z 414 (M+; C29H50O, 91), 396 (43), 81 (85), 55 (100); Rf: 0.34; retention time of 49.65 min. The 13C NMR spectral data is provided in Table 1.

Serrate-14-en-3β,21β –diol, C30H50O2 (C5)

Compound C5 (71.0) was obtained as an amorphous substance from fraction H8. The 1H-NMR (CDCl3) results were as follows: δ 0.94 (3H, s, H-23-Me), 0.74 (3H, s, H-24-Me), 0.77 (3H, s, H-25-Me), 0.81 (3H, s, H-26-Me), 0.91 (3H, s, H-28-Me), 1.02 (3H, s, H-29-Me), 1.06 (3H, s, H-30-Me), 3.17 (1H, dd, J = 11.7 and 4.1 Hz, H-3-β), 3.43 (1H, brs, H-21-β ), and 5.30 (1H, brs, H-15); EI-MS m/z 442 (M+; C30H50O2, 33), 427 (29), 409 (16), 391, 220 (26), and 207 (100); Rf: 0.46; retention time of 55.29. The 13C NMR spectral data is provided in Table 1.

Fig. 3. Triterpenoids and steroids from the bark of P. merkusii

RESULTS AND DISCUSSION

Identification of Triterpenoids and Steroids

The isolation of P. merkusii bark yielded three terpenoids and two steroids. Their chemical structures were established using spectral methods, TLC, GC-MS, NMR, and comparison with literature data. These triterpenoids and steroids were the first to be isolated from P. merkusii. The structure of the C1 to C5 compounds is displayed in Fig. 3. The scheme of extraction and isolation of these compounds is shown in Fig. 2, with the isolated compounds marked with the numbers C1 through C5.

Compound C1, C3, and C5 were identified as 3β-methoxyserratt-14-en-21-one, serrate-14-en-3β,21β-diol, and 3α,21β- dimethoxy-14-serratene (Fang et al. 1991; Le Milebau et al. 2013), respectively, by 1H NMR, 13C NMR, and MS analyses (Fig. 3). Compound C1 (C31H52O) had [M]at m/z 454, and contained a methoxy ether, which demonstrated resonances at δ 3.33 (s) and δ 2.60 (dd, J = 11.7 and 4.1 Hz), attributable to the methoxy group ascribed to the 3β position (Fang et al. 1991). Thus, compound C1 was identified as 3β-methoxyserratt-14-en-21-one. The spectral data of compound C3 (1H NMR and EIMS) was compared with data from the literature (Fang et al. 1991; Le Milebau et al. 2013). This compound had an intake molecule of m/z470, with 1H NMR data showing the appearance of two methoxy ethers at δ 3.33 and δ 2.02 on C-21 and C-3. Therefore, compound C3 was identified as 3α,21β- dimethoxy-14-serratene.

Table 1. 13C NMR Spectral Data of Serratenes (C1 and C5) and Steroids (C2 and C4) (CDCl100 MHz, δ)

Fang et al. 1991a, Barla et al. 2006b, Pateh et al. 2009c, and Halilu et al. 2013d

Compound C5 had [M]+ at m/z 442, and other fragmentation at m/z 207 (100) and 220 (26). The fragmentations at m/z 207 and 220 both resulted from cleavage of the sevenmembered C ring. The 1H, 13C NMR, and EIMS spectral data of compound C5 were similar to that of serrate-14-en-3β,21β-diol (Fang et al. 1991). Compounds C2 and C4 were identified as steroid compounds. The 1H NMR of compound C2 (m/z 412) showed six steroidal methyl groups (Me), signaled at δ 0.69 (3H), δ 0.78 (3H), δ 0.80 (3H), δ 0.84 (3H), δ 0.90 (3H), and δ 1.16 (3H). The other spectral data (13C NMR, and EIMS) was compared with literature (Barla et al. 2006; Halilu et al. 2013), and the compound was confirmed as stigmast-4-en-3-one. The 1H NMR of C4 (m/z 414) also showed six methyl groups (Me), signaled at δ 0.81 (3H), δ 0.89 (3H), δ 0.90 (3H), δ 0.98 (3H), δ 1.04 (3H), and δ 1.05 (3H). The spectral data of C4 was compared to those of previous publications (Pateh et al. 2009; Yamamoto et al. 2011; Halilu et al. 2013), and the compound was identified as β-sitosterol. The structures of C1 through C5 are shown in Fig. 3, and the 13C NMR data is presented in Table 1.

Chemotaxonomic Significance

The characterization of P. merkusii bark resulted in three triterpenoids (C1, C3, and C5) and two steroids (C2 and C4). In previous research, triterpenoids and steroids have been linearly detected in the Pinaceae family (Zullo and Adam 2002; Le Milbeau et al. 2013; Otaka et al. 2016). Compound C1 has been reported in Pinus bark, i.e.P. armandii (Fang et al. 1991; Fang and Cheng 1992), P. luchuensis (Yamamoto et al. 2011), P. contorta (Rowe et al. 1972), P. strobus (Zinkel and Evans 1972), P. radiata (Weston 1973), P. taiwanensis (Cheng and Chao 1979), and P. roxburghii (Labib et al. 2018), while compound C5 has been reported in the bark of P. lambertiana (Rowe 1964). Compound C3 has been detected and isolated in the bark of several pines (Rowe and Bower 1965). The finding of triterpenoids (C1, C3, and C5) in the same section (section PinusP. taiwanensisP. luchuensisP. sylvestris(Farjon 1984)), and in different sections (section StrobusP. lambertianaP. strobusP. armandii; section PinasterP. taedaP. palustrisP. radiata(Farjon 1984)) shows that these compounds have potential as chemotaxonomic biomarkers.

The steroids of P. merkusii were divided into an alcohol and ketone group. The alcohol group, β-sitosterol (C4), is widely distributed in the plant kingdom (Umezawa 2001), while the ketone group, stigmast-4-en-3-one (C2), has been reported in the families of Euphorbiaceae (Barla et al. 2006), Anacardiaceae (Lee et al. 2005), and Chrysobalanaceae (Halilu et al. 2013). In the Pinus genus, compound C4 has been reported in the bark of P. sylvestris (Norin and Winell 1972), P. luchuensis (Yamamoto et al. 2011), P. taiwanensis (Cheng and Chao 1979), and P. radiata (Weston 1973), while compound C2 has been reported in P. sylvestris and P. radiata (Norin and Winell 1972; Weston 1973). Compound C2 is thought to be an artifact formed by autoxidation, related to the age of the tree or a microbial transformation, as was expected in the characterization of the C2 compound from P. sylvestrisbark (Norin and Winell 1972). P. merkusii can therefore be characterized as being chemically in accordance with the other species of the Pinus genus and Pinaceae family.

Ecological Significance

As the outermost component of trees, bark protects the living tissues in the trees from the external environment. Seki et al. (2012) reported that terpenoids in bark can protect the living tissues from chemical deterioration and harm. Terpenoids can also act as a barrier against herbivores, pathogens, allelopathic interactions, nutrient cycling, attraction pollinators, dispersers, and entomophages (Gershenzon 1994; Wittstock and Gershenzon 2002). Therefore, the triterpenoids and steroids in the bark of P. merkusii have important ecological functions.

CONCLUSIONS

  1. P. merkusii bark hexane extract was isolated using column chromatography and three serratene triterpenoids and two steroids were extracted and identified as 3β-methoxyserratt-14-en-21-one (C1), and stigmast-4-en-3-one (C2), 3α,21β-dimethoxy-14-serratene (C3), β-sitosterol (C4), serrate-14-en-3β,21β-diol (C5).
  2. To the researchers’ knowledge, this study is the first report of the isolation of these compounds from the bark of P. merkusii. The compounds C2 and C4 were known to be present in all species, or spread widely in the kingdom Plantae.
  3. Chemotaxonomically, the presence of the isolated compounds, especially the serratene triterpenoids (C1, C3, and C5), in this species demonstrated that these compounds can be considered as biomarkers in the Pinus genus and Pinaceae family.

ACKNOWLEDGEMENTS

This work was supported by BIDIK MISI of the Indonesian Government scholarship and JASSO (Japan Student Services Organization). The authors thank Mr. Sukmono Susanto (Perhutani enterprise) for providing research materials.

REFERENCES CITED

Baharudin and Taskirawati, I. (2009). Non Wood Forest Product, Faculty of Forestry. Universitas Gadjah Mada, Yogyakarta, Indonesia.

Barla, A., Birman, H., Kultur, S., and Oksuz, S. (2006). “Secondary metabolites from Euphorbia and their vasodepressor activity,” Turkish J. Chem.30(3), 325-332.

Cheng, Y. S., and Chao, Y. L. (1979). “The neutral part of the bark of Pinus taiwanensis Hayata,” J. Chinese Chem. Soc. 26(4), 163-167. DOI: 10.1002/jccs.197900029

Coppen, J. J. W., James, D. J., Robinson, J. M., and Subansenee, W. (1998). “Variability in xylem resin composition amongst natural population of Thai and Filipino Pinus merkusii de Vriese,” Flavour and Fragrance Journal 13(1), 33-39. DOI: 10.1002/(SICI)1099-1026(199801/02)13:1<33::AID-FFJ687>3.0.CO;2-U

Fang, J. M., and Cheng, Y. S. (1992). “Chemical constituents of some endemic conifers in Taiwan,” J. Chinese Chem. Soc. 39(6), 647-654. DOI: 10.1002/jccs.199200100

Fang, J. M., Tsai, W. Y., and Cheng, Y. S. (1991). “Serratene triterpene from Pinus armandii bark,” Phytochemistry 30(4), 1333-1336. DOI: 10.1016/S0031-9422(00)95231-2

Farjon, A. (1984). Pines: Drawings and Descriptions of the Genus Pinus, Brill/Backhuys, Leiden, Netherlands.

Farjon, A. (2005). Pines: Drawings and Description of the Genus Pinus, 3rd edition, Brill, Leiden, Netherlands.

Gernandt, D. S., Lopez, G. G., Garefa, O. S., and Liston, A. (2005). “Phylogeny and classification of Pinus,” Taxon 54(1), 29-42. DOI: 10.2307/25065300

Gershenzon, J. (1994). “Metabolic cost of terpenoid accumulation in higher plants,” J. Chem. Ecol. 20(6), 1281-1328. DOI: 10.1007/BF02059810

Hadiyane, A., Sulistyawati, E., Asharina, W.P., and Dungani, R. (2015). “A study on production of resin from Pinus merkusii Jungh. et de Vriese in the Bosscha observatory area, West Java-Indonesia,” Asian J. Plant Sci. 14(2), 89-93. DOI: 10.3923/ajps.2015.89.93

Halilu, M. E., October, N., Balogun, M., Agunu, A., Abubakar, A., and Abubakar, M. S. (2013). “Isolation and characterization of steroids from petroleum ether extract of stem bark of Parinari curatellifolia Planch ex. Benth (Chrysobalanaceae),” J. Nat. Sci. Res. 3(6), 53-61.

Labib, R.M., Srivedavyasasri, R., Youssef, F.S., and Ross, S.A. (2018). “Secondary

metabolites isolated from Pinus roxburghii and interpretation of their cannabinoid and opioid binding properties by virtual screening and in vitro studies,” Saudi Pharm. J. 26, 437-444. DOI: 10.1016/j.jsps.2017.12.017

Le Milbeau, C. L., Lavrieux, M., Jacob, J., Breheret, J. G., and Zocatelli, R. (2013). “Methoxy serratenes in soil under conifers and their potential use as biomarkers of Pinaceae,” Org. Geochem. 55, 45-54. DOI: 10.1016/j.orggeochem.2012.11.008

Lee, T. H, Chiou, J. L., Lee, C. K., and Kuo, Y. H. (2005). “Separation and determination of chemical constituents in the roots of Rhus javanica L. var. Roxburghiana,” J. Chinese Chem. Soc. 52(4), 833-841. DOI: 10.1002/jccs.200500117

Li, B., Shen, Y., Li, C., He, Y., and Zi, W. (2012). “Terpenoids from Pinus densata Mast. and their chemotaxonomic significant,” Biochem. Syst. Ecol. 44, 79-82. DOI: 10.1016/j.bse.2012.04.009

Norin, T., and Winell, B. (1972). “Extractives from the bark of Scots pine, Pinus silvestris L.,” Acta Scandinavica 26, 2297-2304. DOI: 10.3891/acta.chem.scand.26-2297

Otaka, J., Komatsu, M., Miyazaki, Y., Futamura, Y., and Osada, H. (2016). “Two new triterpenoids from the roots of Pinus densiflora,” Biosci. Biotech. Bioch. 81(3), 1-4. DOI: 10.1080/09168451.2016.1263149

Pateh, U. U., Haruna, A. K. Garba, M., Iliya, I., Sule, I. M., Abubakar, M. S., and Ambi, A. A. (2009). “Isolation of stigmasterol, β-sitosterol and 2-hydroxyhexadecanoic acid methyl ester from the rhizomes of Stylochiton lancifolius Pyer and Kotchy (Arecaea),” Niger. J. Pharmaceutical Sci. 8(1), 19-25. DOI: 10.20959/wjpps201712-10694

Rowe, J. W. (1964). “Triterpenes of pine barks: Identity of pinusenediol and serratenediol,” Tetrahedron Letters 5(34), 2347-2353. DOI: 10.1016/S0040-4039(01)89450-8

Rowe, J. W., and Bower, C. L. (1965). “Triterpenes of pine bark: Naturally occurring derivatives of serratendiol,” Tetrahedron Letters 6(32), 2745-2750. DOI: 10.1016/S0040-4039(01)83904-6

Rowe, J. W., Ronald, R. C., and Nagasampi, B. A. (1972). “Terpenoids of lodgepole pine bark,” Phytochemistry 11(1), 365-369. DOI: 10.1016/S0031-9422(00)90015-3

Seki, K., Orihashi, K., Sato, M., Kishino, M., and Saito, N. (2012). “Accumulation of constitutive diterpenoids in the rhytidome and secondary phloem of the branch bark of Larix gmelinii var. japonica,” J. Wood Sci. 58(5), 437-445. DOI: 10.1007/s10086-012-1271-9

Siregar, E. B. M. (2005). Plant Breeding of Pinus merkusii, E-USU Repository, Universitas Sumatra Utara, Medan, Indonesia.

Sukarno, A., Hardiyanto, E.B., Marsoem, S.N., and Na’iem, M. (2015). “Oleoresin production, turpentine yield and components of Pinus merkusii from various Indonesian provenances,” J. Trop. For. Sci. 27(1), 136-141. DOI: –

Umezawa, T. (2001). Chemistry of Extractives in Wood and Cellulosic Chemistry, 2nd Edition, D. N. S. Hon and N. Shiraishi (eds.), Marecel Dekker, New York, USA.

Weston, R. J. (1973). “Neutral extractives from Pinus radiata bark,” Aust. J. Chem. 26(12), 2729-2734. DOI: 10.1071/CH9732729

Wijayanto, A., Dumacay, S., Gerardin-Charbonnier, C., Sari, R. K., Syafii, W., and Gerardin, P. (2015). “Phenolic and lipophilic extractives in Pinus merkusii Jungh. Et de Vries knots and stemwood,” Ind. Crop. Prod. 69, 466-471. DOI: 10.1016/j.indcrop.2015.02.061

Wittstock, U., and Gershenzon, J. (2002). “Constitutive plant toxins and their role in defense against herbivores and pathogens,” Curr. Opin. Plant Biol.5(4), 300-307. DOI: 10.1016/S1369-5266(02)00264-9

Wiyono, B., Tachibana, S., and Tinambunan, D. (2006). “Chemical composition of Indonesian Pinus merkusii turpentine oils, gum oleoresins and rosins from Sumatra and Java,” Pakistan J. Biol. Sci. 9(1), 7-14. DOI: 10.3923/pjbs.2006.7.14

Yamamoto, H., Ookuba, Y., Ikeda, A., Kusano, A., Tanaka, K., and Matsukawa, S. (2011). “Terpenes isolated from the bark and wood of Pinus luchuensis,” Bulletin of the Faculty of Education, Ibaraki University (Natural Sciences) 60, 91-100.

Zinkel, D. F., and Evans, B. B. (1972). “Terpenoids of Pinus strobus cortex tissue,” Phytochemistry 11(11), 3387-3389. DOI: 10.1016/S0031-9422(00)86414-6

Zullo, M. A. T., and Adam, G. (2002). “Brassinosteroid phytohormones structure, bioactivity and applications,” Braz. J. Plant Physiol. 14(3), 143-181. DOI: 10.1590/S1677-04202002000300001

Article submitted: February 1, 2018; Peer review completed: May 13, 2018; Revisions accepted: June 19, 2018; Published: June 22, 2018.

DOI: 6160-6170