J Appl Phycol (2014) 26:1189–1198 DOI 10.1007/s10811-013-0161-x
Structural diversity and geographical distribution of halogenated secondary metabolites in red algae, Laurencia nangii Masuda (Rhodomelaceae, Ceramiales), in the coastal waters of North Borneo Island Charles S. Vairappan & Intan Irna Zanil & Takashi Kamada
Received: 24 May 2013 / Revised and accepted: 17 September 2013 / Published online: 5 October 2013 # Springer Science+Business Media Dordrecht 2013
Abstract The red algae genus Laurencia (Rhodomelaceae, Ceramiales) is known as a prolific producer of halogenated secondary metabolites with a high level of species diversity and geographical distribution. In North Borneo Island, Malaysia, there are four main Laurencia species: Laurencia snackeyi , Laurencia majuscula , Laurencia similis and L . nangii. Although the chemistry of Laurencia is well studied, the diversity of compounds in L. nangii has not been thoroughly investigated. Therefore, we studied the chemical constituents of seven populations of L. nangii from Tunku Abdul Rahman Marine Park (two populations), Dinawan Island (one population), Tun Mustapha Marine Park (two populations) and Tun Sakaran Marine Park (two populations). Halogenated compounds were isolated and the structures determined via spectroscopic methods. A total of 20 metabolites belonging to the classes of sesquiterpenes, acetylenes, bromoallenes, diterpenes and triterpenes were identified. Populations from Tunku Abdul Rahman Marine Park and Dinawan Island contained non-chamigrane-type sesquiterpenes, acetylenes and diterpenes. Populations from Tun Mustapha Marine Park contained chamigrane-type sesquiterpenes, acetylenes and diterpenes. However, the chemical compositions of populations from Tun Sakaran Marine Park were found to differ significantly, containing chamigrane-type and non-chamigrane-type sesquiterpenes, bromoallenes and triterpenes. This investigation has revealed the presence of interesting chemotaxonomical markers in populations of L . nangii and the existence of chemical races in this species. C. S. Vairappan (*) : I. I. Zanil : T. Kamada Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia e-mail:
[email protected]
Keywords Red algae . Laurencia nangii . North Borneo Island . Halogenated metabolites . Chemotaxonomical markers . Chemical race
Introduction Members of the genus Laurencia are well known as prolific producers of halogenated secondary metabolites with diverse chemical skeletons such as sesquiterpenes, diterpenes, triterpenes and C-15 acetogenins (Kamada and Vairappan 2012). To date, more than 500 halogenated secondary metabolites have been isolated from more than 60 species of Laurencia. Laurencia is a relatively large genus with almost 135 species, which is distributed widely in tropical, subtropical and temperate waters (Masuda et al. 1996). New species within this genus are being discovered and have been shown to produce further new and structurally interesting halogenated secondary metabolites. Laurencia nangii Masuda is a relatively new species that was described by Masuda (1997) based on specimens collected from Vietnam. It is readily identified by its fresh and soft green colour and the fact that it grows on dead rocks, ropes and on other seaweeds. It has also been reported to have multiple (one to four) “corps en cerise” in each superficial cortical cell and trichoblast cell; thus, it can be distinctly differentiated from other similar species such as Laurencia intricata J. V. Lamouroux and Laurencia mariannensis Yamada, which contain multiple “corps en cerise” in their cortical cells and single “corps en cerise” in trichoblast cells (Masuda et al. 2002). The presence of “corps en cerise” also indicates that it could biosynthesize halogenated secondary metabolites, as suggested by Young et al. (1980).
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Chemical constituents in L . nangii have not been well studied, and there are only a few reports on populations from Malaysia. In the North Borneo Island of Malaysia, this species is widely distributed along the coastal waters of Sabah, commonly found growing epiphytically on various algae or on dead corals in the lower intertidal to the upper subtidal zone. Over the years, populations of this species have been reported to produce halogenated metabolites belonging to the C15 acetylenes, diterpenes and C-15 bromoallenes class of compounds (Masuda et al. 2002; Vairappan and Tan 2009; Kamada and Vairappan 2012). Hence, it is apparent that the diversity of halogenated metabolites isolated from this genus is very limited. In this paper, we report the chemical diversity of L . nangii from seven different locations in four geographical areas (Tunku Abdul Rahman Marine Park, Dinawan Island, Tun Mustapha Marine Park and Tun Sakaran Marine Park) of North Borneo Island in Sabah, Malaysia. This study was conducted in an effort to determine the structural diversity of halogenated metabolites among the populations of L. nangii and to shed light on the possible occurrence of chemical races in this population.
Materials and methods Sampling locations Specimens of Laurencia nangii were collected by snorkeling at a depth of 1–3 m at seven locations in North Borneo Island: Sulug Island (05°95′952″ N, 115°99′562″ E) and Manukan Island (05°58′100″ N, 116°00′00″ E) of Tunku Abdul Rahman Marine Park (TARMP); Dinawan Island (DINA; 05°84′330″ N, 115°99′082″ E); Carrington Island (07°13′ 321″ N, 117°25′002″ E) and Banggi Island (07°21′711″ N, 117°15′223″ E) from Tun Mustapha Marine Park (TMMP); and Selakan Island (04°57′509″ N, 118°70′352″ E) and Lohok Butun (Bum-Bum Island; 04°04′933″ N, 118°59′833″ E) from Tun Sakaran Marine Park (TSMP; Fig. 1). Samples were identified on site based on the external morphology and the number of “corps en cerise” in the superficial cortical cells and trichoblast cells as examined under a light microscope. Fresh specimens were brought back to the laboratory in cool boxes at 4 °C. The samples were then washed in several changes of double distilled water and air-dried under shaded conditions for 3 days. Voucher specimens were deposited in BORNEENSIS, Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah (BORH2010014). Dr. Tsuyoshi Abe, an expert from the Hokkaido University Museum, Sapporo, Japan, did the exact species identification of these populations.
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Extraction and isolation of halogenated secondary metabolites Partially dried samples (∼100 g) of each population were weighed and soaked separately in methanol (100 %, v/v) for 5 days. The methanol extract was then evaporated in vacuo and the dried extract was partitioned between EtOAc and H2O. The EtOAc layer was then further treated with anhydrous sodium sulphate to remove moisture and concentrated in vacuo to yield a dark green crude extract. Chemical profiling of crude extracts for each sample was carried out by subjecting the extracts to SiO2 F254 thin-layer chromatography developed with toluene (100 %) and hexane/EtOAc (3:1). The EtOAc extract was chromatographed on a Si gel (Kieselgel 60 F254, Merck, Germany) column and eluted with a stepwise gradient of hexane and EtOAc (9:1, 8:2, 7:3 and 1:1 hexane/ EtOAc and 100% EtOAc) to yield five fractions. The fractions were then subjected to repetitive SiO2 F254 preparative thinlayer chromatography to yield compounds 1–20, as reported in this paper. The percentage yield of the isolated compounds was calculated based on the weight of crude extracts. Structure elucidation of halogenated secondary metabolites The structure of compounds 1–20 was determined based on the 1H-NMR (600 MHz), 13C-NMR (150 MHz) and 2D NMR (1H-1H COSY, HSQC, HMBC, NOESY) spectroscopy data that were measured using JEOL ECA 600. High-resolution mass spectroscopy data were acquired using LC-MS-IT-TOF (Shimadzu, Japan).
Results Seven ethyl acetate (EtOAc) extracts were obtained from the seven populations of L . nangii in North Borneo: 0.95 % (Sulug), 0.96 % (Manukan), 0.63 % (Dinawan), 0.53 % (Carrington), 0.56 % (Banggi), 0.63 % (Selakan) and 0.60 % (Lohok Butun). The extracts were subjected to repetitive chromatographic separation and profiling techniques to yield a total of 20 halogenated secondary metabolites. Independent spectroscopic analysis of the 1D and 2D NMR data coupled with comparison with published data revealed all the 20 compounds as known Laurencia-derived halogenated secondary metabolites. Detailed analysis revealed non-chamigrane-type (1–4) and chamigrane-type (5–7) sesquiterpenes (Fig. 2). In addition, another seven compounds were isolated and identified as C-15 acetogenins belonging to C-15 acetylene-type acetogenin (8 –11 ) and C-15 bromoallene-type acetogenin (12 –14 ; Fig. 3). The remaining six compounds were identified as diterpenes (15–19) and triterpene (20; Fig. 4). Spectroscopic data for compounds 1–20 are given below.
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Fig 1 North Borneo Island (Sabah, Malaysia) map illustrating the sites where L. nangii populations were collected
α-snyderol (1): C15H25OBr, colourless oil, [α]D28 +10.4° (CHCl3; 0.5), 1H-NMR (CDCl3, 600 MHz) δ: 0.91 (3H, s, H312), 1.12 (3H, s, H3-13), 1.30 (3H, s, H3-15), 1.32 (1H, m, H5), 1.53 (1H, m, H-4), 1.67 (3H, s, H3-14), 1.70 (1H, m, H-5), 1.76 (1H, m, H-4), 1.78 (1H, m, H-6), 2.53 (2H, m, H-9), 4.17 (1H, dd, J =10.3, 6.2 Hz, H-10), 5.08 (1H, d, J =11.0 Hz, H1), 5.20 (1H, br s, H-8), 5.23 (1H, d, J =17.2 Hz, H-1), 5.91 (1H, dd, J = 17.2, 11.0 Hz, H-2); 13 C-NMR (CDCl 3 , 150 MHz) δ: 112.8 (t, C-1), 145.4 (d, C-2), 74.2 (s, C-3), 44.9 (t, C-4), 24.1 (t, C-5), 51.0 (d, C-6), 137.5 (s, C-7), 121.4 (d, C-8), 36.0 (t, C-9), 65.9 (d, C-10), 39.8 (s, C-11), 16.3 (q, C-12), 29.1 (q, C-13), 22.8 (q, C-14), 28.6 (q, C-15). (6R ,10S ,11S )-6-(3-hydroxy-3-methylpent-2-enyl)-7,10, 11-trimethylcyclohex-7-enol (2 ): C15H26O2, colourless oil, [α]D28 +10.4° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 0.93 (3H, d, J =6.9 Hz, H3-13), 1.06 (3H, s, H3-12), 1.28 (3H, s, H3-15), 1.33 (1H, m, H-5), 1.59 (1H, m, H-9), 1.68 (3H, s, H3-14), 1.71 (1H, m, H-5), 1.74 (1H, m, H-4), 1.76 (1H, m, H4), 1.79 (1H, m, H-6), 1.85 (1H, m, H-10), 2.11 (1H, m, H-9), 5.05 (1H, dd, J =11.0, 1.4 Hz, H-1), 5.16 (1H, br s, H-8), 5.21 (1H, dd, J = 17.2, 1.4 Hz, H-1), 5.88 (1H, dd, J = 17.2, 11.0 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) δ: 112.5 (t, C-1), 145.7 (d, C-2), 74.4 (s, C-3), 43.6 (t, C-4), 25.4 (t, C-5), 53.4 (d, C-6), 138.4 (s, C-7), 120.1 (d, C-8), 34.2 (t, C-9), 33.5 (d, C-10), 75.6 (s, C-11), 22.7 (q, C-12), 15.1 (q, C-13), 23.6 (q, C-14), 29.2 (q, C-15). (6R ,10R ,11S )-6-(3-hydroxy-3-methylpent-2-enyl)-7,10, 11-trimethylcyclohex-7-enol (3 ): C15H26O2, colourless oil, [α]D28 +31.1° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 0.91 (3H, d, J =6.2 Hz, H3-13), 1.20 (3H, s, H3-12), 1.28 (3H, s, H3-15), 1.35 (1H, m, H-5), 1.49 (1H, m, H-5), 1.62 (1H, m, H-4), 1.63 (1H, m, H-4), 1.68 (3H, s, H3-14), 1.72 (1H, m, H-
10), 1.74 (1H, m, H-9), 1.79 (1H, t, J =4.8 Hz, H-6), 2.06 (1H, m, H-9), 5.07 (1H, dd, J =11.0, 1.4 Hz, H-1), 5.20 (1H, dd, J = 17.2, 1.2 Hz, H-1), 5.38 (1H, br s, H-8), 5.88 (1H, dd, J =17.2, 1.4 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) δ: 112.7 (t, C-1), 145.4 (d, C-2), 74.0 (s, C-3), 42.9 (t, C-4), 26.8 (t, C-5), 53.5 (d, C-6), 136.4 (s, C-7), 121.8 (d, C-8), 32.0 (t, C-9), 32.8 (d, C-10), 74.3 (s, C-11), 24.4 (q, C-12), 15.3 (q, C-13), 23.9 (q, C-14), 28.5 (q, C-15). Dactyloxene A (4): C15H24O, colourless oil, [α]D28 −5.9° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 0.96 (3H, d, J = 6.0 Hz, H3-13), 1.01 (3H, s, H3-12), 1.20 (3H, s, H3-15), 1.44 (1H, m, H-4), 1.49 (1H, m, H-5), 1.61 (1H, m, H-9), 1.65 (3H, s, H3-14), 1.67 (1H, m, H-6), 1.80 (1H, m, H-5), 2.03 (1H, m, H-4), 2.06 (1H, m, H-9), 2.20 (1H, m, H-10), 4.92 (2H, dd, J = 17.8, 11.0 Hz, H-1), 5.17 (1H, br s, H-8), 6.06 (1H, ddd, J = 17.8, 11.0, 1.4 Hz, H-2); 13C-NMR (CDCl3, 150 MHz) δ: 111.4 (t, C-1), 145.9 (d, C-2), 73.9 (s, C-3), 33.9 (t, C-4), 24.1 (t, C-5), 49.0 (d, C-6), 137.3 (s, C-7), 120.3 (d, C-8), 34.9 (t, C-9), 30.0 (d, C-10), 77.6 (s, C-11), 23.1 (q, C-12), 16.0 (q, C-13), 22.5 (q, C-14), 33.2 (q, C-15). 2,10-Dibromo-3-chloro-α-chamigrene (5 ): C15H23Br2Cl, colourless oil, [α] D 28 −24.5° (CHCl 3 ; 0.3), 1 H-NMR (CDCl3, 600 MHz) δ: 0.94 (3H, s, H3-13), 1.22 (3H, s, H312), 1.63 (1H, m, H-5), 1.71 (3H, s, H3-15), 1.99 (3H, s, H314), 2.02 (1H, m, H-5), 2.24 (2H, m, H-1), 2.31 (2H, m, H-4), 2.55 (1H, m, H-9), 2.66 (1H, m, H-9), 4.52 (1H, dd, J =11.0, 6.9 Hz, H-10), 4.92 (1H, dd, J =11.0, 6.9 Hz, H-2), 5.23 (1H, br s, H-8); 13C-NMR (CDCl3, 150 MHz) δ: 40.2 (t, C-1), 63.8 (d, C-2), 71.9 (s, C-3), 41.1 (t, C-4), 32.3 (t, C-5), 48.4 (s, C-6), 140.4 (s, C-7), 123.6 (d, C-8), 37.0 (t, C-9), 61.6 (d, C-10), 43.5 (s, C-11), 25.4 (q, C-12), 17.9 (q, C-13), 26.7 (q, C-14), 24.8 (q, C-15).
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Fig 2 Structural diversity of C15 sesquiterpenes isolated from L. nangii collected from the coastal waters of North Borneo Island
Deoxyprepacifenol (6 ): C15H21OBr2Cl, colourless oil, [α]D28 +54.3° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 1.18 (3H, s, H3-13), 1.21 (3H, s, H3-12), 1.34 (1H, m, H-5), 1.63 (3H, s, H3-14), 1.70 (3H, s, H3-15), 1.77 (1H, m, H-5), 2.16 (1H, t, J =13.8 Hz, H-1), 2.21 (1H, m, H-4), 2.30 (1H, m, H-4), 2.42 (1H, m, H-1), 2.94 (1H, d, J =2.8 Hz, H-8), 4.68 (1H, dd, J =13.1, 4.1 Hz, H-2), 6.24 (1H, d, J =2.8 Hz, H-9); 13 C-NMR (CDCl3, 150 MHz) δ: 39.6 (t, C-1), 63.8 (d, C-2), 71.9 (s, C-3), 40.2 (t, C-4), 26.5 (t, C-5), 49.8 (s, C-6), 58.9 (s, C-7), 57.0 (d, C-8), 124.8 (d, C-9), 144.4 (s, C-10), 46.5 (s, C-11), 25.0 (q, C-12), 25.4 (q, C-13), 24.2 (q, C-14), 24.5 (q, C-15). Cycloelatanene B (7 ): C16H24O2BrCl, colourless oil, [α]D28 −62.0° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 1.09 (3H, s, H3-13), 1.18 (3H, s, H3-12), 1.28 (3H, s, H3-14), 1.58 (1H, m, H-1), 1.60 (3H, s, H3-15), 1.67 (1H, br d, J = 12.4 Hz, H-5), 1.71 (1H, m, H-1), 1.95 (1H, dd, J =13.1, 5.5 Hz, H-2), 2.22 (1H, m, H-5), 2.58 (1H, ddd, J = 13.8, 13.8, 6.9 Hz, H-2), 3.54 (3H, s, H3-16), 4.17 (1H, br s, H-8), 4.30 (1H, d, J =6.9 Hz, H-4), 5.98 (1H, d, J =1.4 Hz, H-9); 13C-NMR (CDCl3, 150 MHz) δ: 31.2 (t, C-1), 38.9 (t, C-2), 73.4 (s, C-3), 85.2 (d, C-4), 33.3 (t, C-5), 50.8 (s, C-6), 87.5 (s, C-7), 81.0 (d, C-8), 131.7 (d, C-9), 135.9 (s, C-10), 45.9 (s, C-11), 23.4 (q, C-12), 29.3 (q, C-13), 23.4 (q, C-14), 27.6 (q, C-15), 58.9 (q, C-16). Z -pinnatifidenyne (8): C15H20OBrCl, yellow oil, [α]D28 −31.7° (CHCl3; 2.0); 1H-NMR (CDCl3, 600 MHz) δ: 1.08 (3H, t, J =6.8 Hz, H3-15), 1.80 (1H, m, H-14), 2.07 (1H, m, Fig 3 Structural diversity of C15 acetogenins (acetylenes and bromoallenes, 8–14) isolated from L. nangii collected from the coastal waters of North Borneo Island
H-14), 2.38 (1H, dd, J =7.8, 5.5 Hz, H-11), 2.52 (1H, m, H-5), 2.52 (1H, m, H-8), 2.61 (1H, ddd, J =16.9, 10.1, 5.5 Hz, H-11), 2.81 (1H, ddd, J =13.7, 8.3, 7.8 Hz, H-5), 2.96 (1H, dd, J = 11.5, 11.0 Hz, H-8), 3.13 (1H, d, J = 2.1 Hz, H-1), 3.48 (1H, d, J =10.1 Hz, H-12), 3.88 (1H, m, H-6), 3.96 (1H, m, H-7), 3.99 (1H, m, H-13), 5.57 (1H, d, J =11.0 Hz, H-3), 5.69 (1H, dd, J =9.1, 6.9 Hz, H-9), 5.91 (1H, ddd, J =16.9, 9.1, 7.8 Hz, H10), 6.02 (1H, dd, J =11.0, 8.3 Hz, H-4); 13C-NMR (CDCl3, 150 MHz) δ: 83.2 (d, C-1), 80.7 (s, C-2), 111.7 (d, C-3), 141.5 (d, C-4), 35.7 (t, C-5), 80.5 (d, C-6), 65.5 (d, C-7), 35.2 (t, C-8), 129.5 (d, C-9), 131.6 (d, C-10), 30.9 (t, C-11), 84.2 (d, C-12), 61.7 (d, C-13), 27.8 (t, C-14), 13.5 (q, C-15). (3Z )-laurenyne (9 ): C 15H 19OCl, colourless needles, [α]D28 +30.4° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 1.69 (3H, d, J =6.2 Hz, H3-15), 2.16 (1H, ddd, J =14.2, 8.6, 1.5 Hz, H-11β), 2.48 (1H, m, H-11α), 2.53 (1H, m, H-8β), 2.54 (1H, m, H-5β), 2.72 (1H, m, H-5α), 2.97 (1H, dddd, J = 12.7, 11.5, 10.0, 1.2 Hz, H-8α), 3.10 (1H, d, J =2.1 Hz, H-1), 3.76 (1H, dd, J =8.8, 5.9 Hz, H-12), 3.89 (1H, ddd, J =8.8, 4.6, 2.4 Hz, H-6), 3.99 (1H, ddd, J =11.5, 4.9, 2.4 Hz, H-7), 5.53 (1H, m, H-3), 5.55 (1H, m, H-13), 5.68 (1H, m, H-14), 5.69 (1H, m, H-9), 5.90 (1H, dd, J =18.6, 8.3 Hz, H-10), 6.06 (1H, m, H-4); 13C-NMR (CDCl3, 150 MHz) δ: 82.8 (d, C-1), 80.8 (s, C-2), 110.8 (d, C-3), 142.5 (d, C-4), 36.0 (t, C-5), 79.7 (d, C-6), 66.0 (d, C-7), 35.2 (t, C-8), 129.2 (d, C-9), 131.8 (d, C-10), 35.5 (t, C-11), 82.3 (d, C-12), 132.8 (d, C-13), 127.0 (d, C-14), 18.4 (q, C-15).
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Fig 4 Structural diversity of diterpenes (15–19) and a triterpene (20) isolated from L. nangii collected from the coastal waters of North Borneo Island
(+)-3Z,6R,7R-obtusenyne (10): C15H20OBrCl, yellow oil, [α]D28 +10.0° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 0.86 (3H, t, J =7.3 Hz, H3-15), 1.90 (2H, m, H-14), 2.46 (1H, m, H-8), 2.58 (1H, m, H-11), 2.83 (1H, m, H-5), 2.87 (1H, m, H-5), 3.15 (1H, d, J =2.1 Hz, H-1), 3.23 (1H, br s, H-8), 3.23 (1H, br s, H-13), 3.40 (1H, m, H-11), 3.59 (1H, br s, H-6), 4.07 (1H, m, H-7), 4.27 (1H, m, H-12), 5.56 (1H, d, J =11.0 Hz, H3), 5.60 (1H, m, H-9), 5.62 (1H, m, H-10), 6.04 (1H, dd, J = 11.0, 7.8 Hz, H-4); 13C-NMR (CDCl3, 150 MHz) δ: 84.5 (d, C-1), 82.1 (s, C-2), 112.0 (d, C-3), 141.0 (d, C-4), 35.3 (t, C-5), 80.8 (d, C-6), 62.9 (d, C-7), 34.2 (t, C-8), 129.3 (d, C-9), 130.8 (d, C-10), 35.0 (t, C-11), 55.3 (d, C-12), 83.2 (d, C-13), 28.5 (t, C-14), 10.4 (q, C-15). Z -dihydrorhodophytin (11): C15H20OBrCl, white needles, [α]D28 +69.5° (CHCl3; 1.0), 1H-NMR (CDCl3, 600 MHz) δ: 1.09 (3H, t, J =7.6 Hz, H3-15), 1.92 (1H, dq, J =15.2, 7.6 Hz, H-14), 2.02 (1H, m, H-14), 2.26 (1H, dd, J =10.8, 6.8 Hz, H11), 2.58 (1H, m, H-8), 2.58 (1H, m, H-11), 2.76 (1H, ddd, J = 14.5, 7.6, 6.9 Hz, H-8), 2.81 (1H, ddd, J =14.4, 8.3, 5.5 Hz, H-5), 2.87 (1H, ddd, J =14.4, 6.9, 5.5 Hz, H-5), 3.13 (1H, s, H-1), 4.04 (1H, ddd, J =14.7, 4.8, 4.1 Hz, H-13), 4.05 (1H, dd, J =8.3, 3.5 Hz, H-7), 4.12 (1H, m, H-12), 4.31 (1H, ddd, J =8.3, 5.5, 5.5 Hz, H-6), 5.56 (1H, d, J =11.0 Hz, H-3), 5.78 (1H, dd, J = 10.4, 7.6 Hz, H-9), 5.85 (1H, ddd, J =10.4, 6.9, 3.5 Hz, H-10), 6.07 (1H, ddd, J =11.0, 8.3, 6.9 Hz, H-4). 13C-NMR (CDCl3, 150 MHz) δ: 83.2 (d, C-1), 80.7
(s, C-2), 111.6 (d, C-3), 141.7 (d, C-4), 35.9 (t, C-5), 74.5 (d, C-6), 64.9 (d, C-7), 34.4 (t, C-8), 127.9 (d, C-9), 130.7 (d, C-10), 32.1 (t, C-11), 79.7 (d, C-12), 62.5 (d, C-13), 29.6 (t, C-14), 13.1 (q, C-15). Neolaurallene (12 ): C15H20O2Br2, colourless needles, [α]D28 +74.0° (CHCl3; 0.5), 1H-NMR (CDCl3, 600 MHz) δ: 1.08 (3H, d, J =7.6 Hz, H3-15), 1.61 (1H, m, H-14), 2.03 (1H, m, H-5), 2.03 (1H, m, H-14), 2.30 (1H, m, H-5), 2.31 (1H, m, H-8), 2.76 (1H, m, H-8), 2.76 (1H, m, H-11), 3.13 (1H, br s, H-11), 3.72 (1H, m, H-13), 3.76 (1H, m, H-7), 3.78 (1H, m, H-12), 4.03 (1H, br s, H-6), 4.52 (1H, dd, J =13.8, 7.6 Hz, H-4), 5.59 (1H, m, H-9), 5.62 (1H, m, H-3), 5.76 (1H, m, H-10), 6.07 (1H, d, J =6.2 Hz, H-1); 13C-NMR (CDCl3, 150 MHz) δ: 75.4 (d, C-1), 202.3 (s, C-2), 102.9 (d, C-3), 75.8 (d, C-4), 40.0 (t, C-5), 73.7 (d, C-6), 81.6 (d, C-7), 27.8 (t, C-8), 128.3 (d, C-9), 129.7 (d, C-10), 35.2 (t, C-11), 53.7 (d, C-12), 84.5 (d, C-13), 24.2 (t, C-14), 11.9 (q, C-15). Itomanallene B (13): C17H23O3Br, colourless oil, [α]D28 + 84.0° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 0.96 (3H, t, J =7.3 Hz, H3-15), 1.30 (1H, m, H-14β), 2.00 (1H, m, H-14α), 2.08 (3H, s, H3-16), 2.18 (1H, ddd, J =13.7, 7.3, 4.9 Hz, H-5α), 2.24 (1H, ddd, J =13.7, 5.4, 1.5 Hz, H-5β), 2.39 (2H, m, H-8), 2.74 (2H, m, H-11), 3.97 (1H, ddd, J =7.3, 7.3, 3.4 Hz, H-7), 4.81 (1H, dddd, J =7.3, 5.8, 5.4, 2.0 Hz, H-4), 5.31 (1H, m, H-6), 5.33 (1H, m, H-12), 5.38 (1H, m, H-9), 5.47 (1H, dd, J =5.8, 5.8 Hz, H-3), 5.47 (1H, m, H-13),
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5.49 (1H, m, H-10), 6.07 (1H, dd, J =5.8, 2.0 Hz, H-1); 13CNMR (CDCl3, 150 MHz) δ: 74.3 (d, C-1), 201.3 (s, C-2), 102.0 (d, C-3), 73.5 (d, C-4), 39.0 (t, C-5), 74.6 (d, C-6), 80.6 (d, C-7), 27.1 (d, C-8), 124.7 (d, C-9), 130.6 (d, C-10), 30.5 (d, C-11), 126.8 (d, C-12), 132.8 (d, C-13), 25.6 (t, C-14), 13.8 (q, C-15), 170.4 (s, C-16), 21.1 (q, C-17). Pannosallene (14 ): C15H20O2Br2, colourless needles, [α]D28 +64.3° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ: 1.09 (3H, t, J =7.3 Hz, H3-15), 1.78 (1H, ddq, J =14.7, 6.8, 7.3 Hz, H-14), 1.88 (1H, ddd, J =13.7, 6.4, 3.9 Hz, H-5), 2.01 (1H, ddq, J =14.7, 2.4, 7.3 Hz, H-14), 2.34 (1H, ddd, J =13.7, 6.4, 4.4 Hz, H-8), 2.43 (1H, ddd, J =13.7, 7.8, 6.4 Hz, H-5), 2.63 (1H, ddd, J =13.7, 11.2, 3.9 Hz, H-8), 2.69 (1H, m, H11), 2.72 (1H, ddd, J =15.6, 7.3, 2.0 Hz, H-11), 3.96 (1H, ddd, J =11.2, 4.4, 4.4 Hz, H-7), 4.09 (1H, m, H-12), 4.09 (1H, m, H-13), 4.21 (1H, ddd, J =6.4, 4.4, 3.9 Hz, H-6), 4.50 (1H, m, H-4), 5.56 (1H, dd, J =7.8, 5.9 Hz, H-3), 5.77 (1H, ddd, J = 10.7, 7.3, 7.3 Hz, H-10), 5.81 (1H, ddd, J =10.7, 6.4, 6.4 Hz, H-9), 6.06 (1H, dd, J =5.9, 1.0 Hz, H-1); 13C-NMR (CDCl3, 150 MHz) δ: 73.3 (d, C-1), 201.1 (s, C-2), 102.4 (d, C-3), 74.7 (d, C-4), 39.8 (t, C-5), 73.7 (d, C-6), 83.9 (d, C-7), 29.7 (t, C-8), 129.6 (d, C-9), 127.3 (d, C-10), 30.7 (t, C-11), 79.8 (d, C-12), 58.1 (d, C-13), 28.1 (t, C-14), 11.3 (q, C-15). Neoirieone (15): C20H30O3Br2, colourless needles, [α]D28 −5.9(CHCl3; 0.5), 1H-NMR (CDCl3, 600 MHz) δ: 0.87 (3H, s, H3-20), 1.11 (3H, s, H3-19), 1.29 (3H, s, H3-18), 1.33 (3H, s, H3-16), 1.42 (3H, m, H3-17), 1.53 (1H, m, H-12), 1.56 (1H, m, H-3), 2.05 (1H, m, H-2), 2.08 (1H, m, H-13), 2.13 (1H, m, H-3), 2.39 (1H, m, H-2), 2.39 (1H, m, H-9), 2.43 (1H, m, H-13), 2.44 (1H, m, H-12), 2.79 (1H, d, J =17.9 Hz, H-9), 4.32 (1H, t, J =8.9 Hz, H-14), 4.57 (1H, dd, J =13.1, 4.1 Hz, H-1), 6.98 (1H, s, H-6); 13C-NMR (CDCl3, 150 MHz) δ: 60.3 (d, C-1), 30.1 (t, C-2), 38.3 (t, C-3), 74.5 (s, C-4), 76.3 (s, C-5), 144.3 (d, C-6), 149.4 (s, C-7), 199.8 (s, C-8), 52.1 (t, C-9), 45.5 (s, C-10), 49.8 (s, C-11), 36.2 (t, C-12), 31.6 (t, C-13), 64.4 (d, C-14), 48.1 (s, C-15), 26.7 (q, C-16), 20.5 (q, C-17), 25.5 (q, C-18), 22.7 (q, C-19), 23.9 (q, C-20). (−)-Angasiol (16 ): C20H30O3Br2, colourless needles, [α]D28 −10.1° (CHCl3; 0.5), 1H-NMR (CDCl3, 600 MHz) δ: 1.02 (3H, s, H3-20), 1.22 (3H, s, H3-19), 1.25 (1H, m, H-16), 1.29 (1H, m, H-10), 1.33 (1H, m, H-12), 1.41 (1H, m, H-7), 1.45 (3H, s, H3-17), 1.60 (1H, m, H-12), 1.61 (1H, m, H-3), 1.65 (1H, d, J =9.6 Hz, H-5), 1.72 (1H, m, H-10), 1.76 (1H, m, H-16), 1.80 (1H, m, H-8), 1.93 (1H, dd, J =14.1, 6.2 Hz, H-3), 2.03 (1H, m, H-13), 2.10 (1H, m, H-2), 2.12 (1H, m, H-7), 2.18 (1H, m, H-6), 2.33 (1H, m, H-13), 2.37 (1H, m, H-8), 2.41 (1H, m, H-2), 3.90 (1H, dd, J =12.7, 4.1 Hz, H-14), 4.10 (1H, dd, J =11.7, 6.2 Hz, H-1); 13C-NMR (CDCl3, 150 MHz) δ: 52.1 (d, C-1), 33.4 (t, C-2), 38.7 (t, C-3), 84.2 (s, C-4), 64.0 (d, C-5), 36.4 (d, C-6), 32.6 (t, C-7), 32.2 (t, C-8), 62.2 (s, C-9), 51.9 (t, C-10), 73.1 (s, C-11), 40.4 (t, C-12), 30.9 (t, C-13), 65.9 (d, C-14), 37.2 (s, C-15), 50.5 (t, C-16), 21.8 (q, C-17), 177.2 (s, C-18), 23.3 (q, C-19), 33.2 (q, C-20).
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11-Deacetylpinnaterpene C (17): C20H32O3Br2, colourless oil, [α]D28 +6.6° (CHCl3; 0.5), 1H-NMR (CDCl3, 600 MHz) δ: 1.01 (3H, s, H3-20), 1.23 (3H, s, H3-19), 1.27 (1H, m, H-10), 1.30 (1H, m, H-16), 1.33 (3H, s, H3-17), 1.35 (1H, m, H-5), 1.35 (1H, m, H-7), 1.40 (1H, m, H-3), 1.43 (1H, m, H-12), 1.63 (1H, m, H-8), 1.65 (1H, m, H-3), 1.65 (1H, m, H-12), 1.72 (1H, d, J =13.8 Hz, H-10), 1.78 (1H, dd, J =14.4, 3.4 Hz, H-16), 2.00 (1H, m, H-2), 2.03 (1H, m, H-13), 2.10 (1H, m, H-8), 2.15 (1H, m, H-7), 2.22 (1H, m, H-2), 2.37 (1H, m, H-13), 2.48 (1H, m, H-6), 3.92 (1H, dd, J =12.7, 3.8 Hz, H-14), 4.12 (1H, m, H-1), 5.33 (1H, s, H-18); 13C-NMR (CDCl3, 150 MHz) δ: 59.4 (d, C-1), 33.1 (t, C-2), 41.3 (t, C-3), 83.8 (s, C-4), 64.1 (d, C-5), 36.3 (d, C-6), 34.3 (t, C-7), 32.8 (t, C-8), 62.8 (s, C-9), 52.9 (t, C-10), 73.3 (s, C-11), 40.3 (t, C-12), 31.1 (t, C-13), 66.6 (d, C-14), 37.3 (s, C-15), 50.8 (t, C-16), 22.6 (q, C-17), 99.8 (d, C-18), 23.3 (q, C-19), 33.3 (q, C-20). Neoirietetraol (18 ): C20H34O4Br2, colourless needles, [α]D28 −43.0° (CHCl3; 1.0), 1H-NMR (CDCl3, 600 MHz) δ: 0.95 (3H, s, H3-18), 1.13 (3H, s, H3-20), 1.23 (3H, s, H3-16), 1.26 (3H, s, H3-17), 1.34 (1H, m, H-3), 1.50 (1H, m, H-6), 1.54 (3H, s, H3-19), 1.75 (1H, m, H-8), 1.91 (1H, m, H-9), 2.02 (1H, m, H-2), 2.02 (1H, m, H-6), 2.04 (1H, m, H-9), 2.13 (1H, m, H-13), 2.14 (1H, m, H-8), 2.15 (1H, m, H-3), 2.40 (1H, m, H-2), 2.93 (1H, m, H-13), 4.19 (1H, t, J =9.6 Hz, H-14), 4.27 (1H, ddd, J =7.6, 2.8, 2.8 Hz, H-12), 4.68 (1H, dd, J =13.1, 4.1 Hz, H-1); 13C-NMR (CDCl3, 150 MHz) δ: 65.7 (d, C-1), 30.8 (t, C-2), 38.1 (t, C-3), 75.7 (s, C-4), 78.8 (s, C-5), 32.1 (t, C-6), 82.9 (s, C-7), 30.0 (t, C-8), 32.7 (t, C-9), 43.4 (s, C-10), 53.8 (s, C-11), 81.8 (d, C-12), 45.2 (t, C-13), 62.4 (d, C-14), 48.4 (s, C-15), 26.7 (q, C-16), 18.6 (q, C-17), 21.3 (q, C-18), 24.8 (q, C-19), 24.8 (q, C-20). 10-Hydroxykahukuene B (19): C20H32O2Br2, colourless needles, [α] D 28 +8.1° (CHCl 3; 0.1), 1 H-NMR (CDCl 3 , 600 MHz) δ: 0.91 (1H, dd, J =12.4, 2.0 Hz, H-8α), 0.96 (3H, s, H3-16), 1.16 (3H, s, H3-20), 1.21 (3H, s, H3-17), 1.21 (3H, s, H3-19), 1.31 (1H, m, H-14α), 1.65 (1H, m, H-14β), 1.67 (1H, m, H-15α), 1.74 (1H, m, H-15β), 1.82 (1H, dd, J =12.6, 12.4 Hz, H-7α), 2.04 (1H, dd, J =12.6, 2.0 Hz, H-7β), 2.08 (1H, m, H-3α), 2.10 (1H, m, H-4α), 2.26 (1H, m, H-11α), 2.28 (1H, m, H-3β), 2.28 (1H, m, H-4β), 2.30 (1H, m, H-11β), 3.31 (1H, dd, J =10.9, 5.3 Hz, H-10), 3.80 (1H, dd, J =12.4, 4.2 Hz, H-12), 4.64 (1H, dd, J = 13.0, 4.6 Hz, H-2), 4.79 (1H, br s, H-18α), 5.13 (1H, br s, H-18β); 13C-NMR (CDCl3, 150 MHz) δ: 44.1 (s, C-1), 65.2 (d, C-2), 36.1 (t, C-3), 33.9 (t, C-4), 146.6 (s, C-5), 49.9 (s, C-6), 23.7 (t, C-7), 45.3 (d, C-8), 73.7 (s, C-9), 74.8 (d, C-10), 38.5 (t, C-11), 63.1 (d, C-12), 39.8 (s, C-13), 38.0 (t, C-14), 24.0 (t, C-15), 17.6 (q, C-16), 23.7 (q, C-17), 114.5 (t, C-18), 25.1 (q, C-19), 14.2 (q, C-20); HRMS m /z 487.0654 [M+Na]+ (calc for C20H32Br2O2Na, 487.0647). Intricatetraol (20 ): C 30 H 54 O 6 Br 2 Cl 2 , colourless oil, [α]D28 +53.0° (CHCl3; 0.3), 1H-NMR (CDCl3, 600 MHz) δ:
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1.17 (6H, s, H3-27(28)), 1.26 (6H, s, H3-26(29)), 1.40 (2H, m, H-12(13)), 1.52 (2H, m, H-5(20)), 1.53 (2H, m, H-9(16)), 1.68 (6H, s, H3-1(24)), 1.78 (2H, m, H-4(21)), 1.78 (2H, m, H-5(20)), 1.78 (2H, m, H-12(13)), 1.78 (6H, s, H325(30)), 1.92 (2H, m, H-8(17)), 2.00 (2H, m, H-8(17)), 2.16 (2H, m, H-9(16)), 2.46 (2H, m, H-4(21)), 3.58 (2H, d, J = 10.3 Hz, H-11(14)), 3.85 (2H, t, J =7.6 Hz, H-7(18)), 4.07 (2H, d, J = 11.0 Hz, H-3(22)); 13 C-NMR (CDCl 3 , 150 MHz) δ: 28.2 (d, C-1(24)), 72.7 (s, C-2(23)), 67.7 (d, C-3(22)), 29.5 (t, C-4(21)), 37.8 (t, C-5(20)), 74.4 (s, C-6(19)), 84.8 (d, C-7(18)), 27.2 (t, C-8(17)), 32.3 (t, C9(16)), 86.8 (s, C-10(15)), 78.2 (d, C-11(14)), 30.4 (t, C12(13)), 33.6 (q, C-25(30)), 24.9 (q, C-26(29)), 24.7 (q, C-27(28)). Populations of L . nangii from TARMP and DINA yielded a total of five sesquiterpenes, six acetogenins and five diterpenes. TARMP specimens contained two non-chamigranes (α-snyderol (1 ) (3.0 %) and (6R ,10S ,11S)-6-(3-hydroxy-3methyl-pent-2-enyl)-7,10,11-trimethylcyclo-hex-7-enol (2 ) (0.6 %)); three acetylene-type C-15 acetogenins (Z pinnatifidenyne (8) (4.4 %), (3Z)-laurenyne (9) (2.0 %), (+ )-3Z ,6R ,7R -obtusenyne (10) (2.4 %)); and three diterpenes (neoirieone (15) (2.3 %), (−)-angasiol (16) (2.1 %) and 11deacetylpinnaterpene C (17) (2.2 %)). As for the DINA population, three non-chamigrane sesquiterpenes (α-snyderol (1) (5.4 %), (6R,10S,11S)-6-(3-hydroxy-3-methyl-pent-2-enyl)7,10,11-trimethylcyclo-hex-7-enol (2) (1.0 %) and (6R ,10R, 11S )-6-(3-hydroxy-3-methyl-pent-2-enyl)-7,10,11trimethylcyclo-hex-7-enol (3) (1.0 %)); three acetylene type C-15 acetogenins (Z -pinnatifidenyne (8 ) (17.7 %), (3Z )laurenyne (9 ) (6.7 %) and (+)-3Z ,6R ,7R -obtusenyne (10 ) (5.8 %)); and two diterpenes ((−)-angasiol (16 ) (5.2 %) and 11-deacetylpinnaterpene C (17) (4.0 %)) were identified. Two populations from TMMP collected from Carrington Island and Banggi Island showed the same chemical composition. There were two chamigrane-type sesquiterpenes (deoxyprepacifenol (6) and cycloelatanene B (7)), two acetylene type C-15 acetogenins ((+)-3Z,6R,7R-obtusenyne (10) (1.5 %) and Z-dihydrorhodophytin (11) (16 %)) and two diterpenes (neoirietetrol (18) (11.3 %) and 10-hydroxykahukuene B (19) (0.3 %)). The remaining two populations from TSMP, Selakan Island and Lohok Butun (Bum-Bum Island), showed similar chemical composition to each other, but different from those from TARMP, DINA and TMMP. A mixture of non-chamigraneand chamigrane-type sesquiterpenes—dactyloxene A (4 ) (0.5 %), 2,10-dibromo-3-chloro-α-chamigrene (5) (1.0 %), deoxyprepacifenol (6 ) (1.0 %) and cycloelatanene B (7 ) (3.5 %)—were found to be present in these populations. In addition, three bromoallene-type C-15 acetogenins (neolaurallene (12) (10.4 %), itomanallene B (13) (1.3 %) and pannosallene (14) (1.7 %)) were found to be present together
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with one triterpene (intricatetraol (20) (2.5 %)). The relative composition of halogenated secondary metabolites in these populations is described in Table 1.
Discussion More than 100 species of the red algae genus Laurencia are widely distributed globally. Most members of this genus can be distinguished based on their morphological features. However, detailed identification of these species is rather complex due to a high degree of morphological variation within individual species (Masuda et al. 1998). Findings of research by Erickson (1983), Masuda et al. (1998) and Suzuki and Vairappan (2005) have led to the observation that at least one, or a set of specific secondary metabolites, could characterize most of the populations of Laurencia . Therefore, the presence of halogenated secondary metabolites in this genus has been suggested to assist in their taxonomical classification. A classical case could be seen in the two Japanese seaweeds, Laurencia composita Yamada and Laurencia okamurae Yamada, that have often been confused with each other due to their similarity in gross morphology; however, these two species could be distinguished on the basis of their secondary metabolites. L. composita is characterized by chamigrene-type sesquiterpenoids, whilst L . okamurae is marked by cuparane-type sesquiterpenoids (Masuda et al. 1996). In addition, Laurencia nipponica Yamada and Laurencia japonensis Masuda are other examples of Laurencia with a similar gross morphology which has lead to complications in their identity. However, both these species can be distinguished based on several internal features, such as the branching pattern and the position of nucleus as well as their different sets of halogenated secondary metabolites (Abe and Masuda 1998). As described above, red algae of the genus Laurencia are unique in their ability to produce halogenated secondary metabolites belonging to a specific group of chemical skeletons: sesquiterpenes (non-chamigrane and chamigrane), C-15 acetogenins (acetylene and bromoallene types), diterpenes, triterpenes and bromoindoles. Molecular and transcriptomic study has identified the genes that are involved in the important steps during the biosynthesis of the building blocks of terpene precursors, such as dimethylally diphosphate, isopentenyl diphosphate, and the higher-order building blocks, such as geranyl diphosphate (de Oliveira et al. 2012). These compounds are produced from primary metabolites through specific biogenesis pathways, and the halogenation of the biosynthesized compounds is catalyzed by lactoperoxidase and bromoperoxidase (BPO; Suzuki et al. 2009). Recent transcriptomic investigation on Laurencia dendroidea J. Agardh reported the increase on the bromination activity of red algae in response to infection signals, such
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Table 1 Relative distribution of halogenated secondary metabolites in populations of L. nangii from the coastal waters of North Borneo Island No. Compound type
Compound name
Locations of seaweed populations TARMP DINA TMMP TSMP
4 5 6 7 8 9 10 11 12 13 14 15 16
Sesquiterpene (NC) (Howard and Fenical 1976) α-Snyderol Sesquiterpene (NC) (Findlay and Li 2002) 6R,10S,11S)-6-(3-hydroxy-3-methyl-pent-2-enyl)-7,10, 11-trimethylcyclo-hex-7-enol Sesquiterpene (NC) (Findlay and Li 2002) 6R,10R,11S)-6-(3-hydroxy-3-methyl-pent-2-enyl)-7,10, 11-trimethylcyclo-hex-7-enol Sesquiterpene (NC) (Schmitz et al. 1978) Dactyloxene A Sesquiterpene (C) (Suzuki et al. 1979) 2,10-Dibromo-3-chloro-α-chamigrene Sesquiterpene (C) (Ireland et al. 1976) Deoxyprepacifenol Sesquiterpene (C) (Dias and Urban 2011) Cycloelatanene B C-15 Acetogenin (A) (González et al. 1982) Z-pinnatifidenyne C-15 Acetogenin (A) (Takahashi et al. 2002) (3Z)-laurenyne C-15 Acetogenin (A) (Manzo et al. 2005) (+)-3Z,6R,7R-obtusenyne C-15 Acetogenin (A) (Norte et al. 1989) Z-dihydrorhodophytin C-15 Acetogenin (BA) (Suzuki et al. 1984) Neolaurallene C-15 Acetogenin (BA) (Suzuki et al. 2002) Itomanallene B C-15 Acetogenin (BA) (Suzuki et al. 1996) Pannosallene Diterpene (Howard et al. 1982) Neoirieone Diterpene (Pettit et al. 1978) (−)-Angasiol
17 18
Diterpene (Ji et al. 2007a) Diterpene (Takahashi et al. 2002)
11-Deacetylpinnaterpene C Neoirietetrol
19 20
Diterpene (Ji et al. 2007b) Triterpene (Suzuki et al. 1993)
10-hydroxykahukuene B Intricatetraol
1 2 3
as agar oligosaccharide, indicating an important role of this enzyme in the chemical defence of Laurencia (de Oliveira et al. 2012). In addition, the fact that these compounds are biosynthesized in the “corps en cerise” that are present in the outer cortical cells and have been shown to be present even in their early stages of trichoblast cells seems to auger well with the suggestion of their importance as defence metabolites. The presence of secondary metabolites as a defence mechanism in Laurencia is also suggested to be evolutionarily selected, not induced by the colonization of the surface bacteria. The transcriptomic profile of L. dendroidea and its associated microbiome revealed that the microbial community was similar regardless of the seaweed population’s species or origin; hence, suggesting the absence of seaweed surface colonization selectivity in the microbiome population that forms biofilm on Laurencia. This eliminates the role of surface bacteria in the production of any halogenated secondary metabolites in the different species of Laurencia. In addition, the absence of BPO in seaweed surface microbial population would further reduce their possible role in the production of halogenated secondary metabolites. In addition to their roles as inherently synthesized seaweed defence metabolites, these compounds have also been shown to exhibit potent biological activities against important clinical assay. The halogenated secondary metabolites produced by Laurencia have also been shown to
X X
X X X
X X X X X
X X X
X X X X
X X X X X
X X X
X X X X X
exhibit antimicrobial activities against human and seaweed pathogens, anti-inflammation activity against RAW 267.4 macrophage cells, anticancer activities against several cancer cell lines and anti-fouling activities (Vairappan 2003; Vairappan et al. 2010, 2013). In recent years, the chemotaxonomic approach has taken a centre stage since phylogenetic reconstruction based on the genes of terpene synthases has been proven to be difficult to infer a phylogenetic relationship among the taxonomic groups due to their low bootstrap support. In the coastal waters of North Borneo Island (Sabah), there are three other major Laurencia species: Laurencia snackeyi (Weber van Bosse) Masuda, Laurencia majuscula (Harvey) Lucas and Laurencia similis Nam et Saito. Each of these species is characterized by the presence of halogenated metabolites belonging to the sesquiterpenes (snyderane type), chamigrane type and bromoindole type, respectively. To date, L. nangii has been reported as a prolific producer of C-15 acetogenins with the acetylene and bromoallene functionality (Masuda et al. 2002; Vairappan and Tan 2009; Kamada and Vairappan 2012). Those findings suggest that these halogenated secondary metabolites are beneficial in the taxonomic study of this genus as they could act as chemotaxonomical markers. In this study, we reported the discovery of three different sets of halogenated secondary metabolites from L . nangii
J Appl Phycol (2014) 26:1189–1198
populations in North Borneo Island. Therefore, through this finding, we could suggest the presence of three chemical races in the populations that we have investigated. The first race is grouped by the production of non-chamigrane sesquiterpenes, acetylenes and diterpenes in the populations of Tunku Abdul Rahman Marine Park and Dinawan Island. The second race is grouped by the production of chamigrane-type sesquiterpenes, acetylenes and diterpenes in the populations of Tun Mustapha Marine Park. The third race is suggested to contain a mixture of non-chamigrane- and chamigrane-type sesquiterpenes, bromoallenes and triterpenes in the populations of Tun Sakaran Marine Park. Differences in the halogenated secondary metabolites from the populations of L. nangii in Borneo might be caused by the different locations of each population and perhaps some minor variation in the genomic content of these populations. Tunku Abdul Rahman Marine Park and Dinawan Island, which are located at the northwestern coast of Borneo Island, are covered by the South China Sea, whilst Tun Mustapha Marine Park in the north coast of Borneo Island is located at the boundary of South China Sea and Celebes Sea. On the other hand, Tun Sakaran Marine Park is located in the northeastern coast of the Borneo Island and covered by the Celebes Sea. Populations from Tunku Abdul Rahman Marine Park and Dinawan Island produced similar groups of compounds, which might be because of their very close distance to each other. Hence, L. nangii in both populations were exposed to the same environmental factors. Due to the placement of Tun Mustapha Marine Park in the central location between South China Sea and Sulu Sea, water parameters such as the current flow, salinity and temperature in this area might be slightly different from the populations in the South China Sea (TARMP and DINA). This might lead to the production of a slightly different group of compounds from the first group. In addition, the population in Tun Sakaran Marine Park is the farthest from the other three populations, and the compounds produced also are very different. This might be because the abiotic factors in Celebes Sea water are very much different due to the volcanic influence of the “ring of fire” at its vicinity as compared to that of the South China Sea. Therefore, the compounds produced also are very different. In addition, there are also some reports that attempt to correlate the concentration of terpenoids in marine organisms to be influenced by the nutrient regime and light intensity at their respective geographical area (Sudatti et al. 2011). The optimal defence model suggested that a higher concentration of secondary metabolites could be produced if plants are exposed to a more vulnerable environment (Rhoades 1979). As all three chemical races were isolated from three different locations, this situation might be influenced by the differences of the physical (salinity and temperature), chemical (biological oxygen demand, dissolved oxygen and dissolved nitrogen) and biological parameters (microbes and frequent harmful algae bloom) of the seawater in each location.
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The presence of chemical race among species in Laurencia is reported in several reports published to date (Masuda et al. 1996; Kamada and Vairappan 2012). Masuda et al. (1997) first suggested the presence of chemical races in L. nipponica Yamada populations in Japan. Chemical races were described as the existence of a particular Laurencia species with similar morphology features but from a different geographical area (either close or distant) and that produces a different type profile of halogenated secondary metabolites. In 2002, Masuda et al. suggested the presence of two chemical races in North Borneo populations of L. nangii. The first race was characterized by the production of C-15 acetogenins by the L. nangii population in Kota Kinabalu and Kuala Penyu (northwestern coast), whilst the second race was characterized by the production of acetylene-type C-15 acetogenins and diterpenes by the L. nangii population in Sandakan (northeastern coast). However, Kamada and Vairappan (2012) reported that upon reexamination, it was found that the presence of the diterpene-type compound in the Sandakan population as reported by Masuda et al. (2002) was due to contamination from Laurencia sp. that grow between the thallus of L. nangii. In addition, a report by Kamada and Vairappan (2012) suggested bromoallenes as a new chemical race in the population of L. nangii in North Borneon Island. In conclusion, three different sets of halogenated secondary metabolites from different populations of L. nangii in North Borneo Island have been discovered. Based on this finding, we propose the presence of three chemical races in the populations that we have investigated. The first race is grouped by the production of non-chamigrane sesquiterpenes, acetylenes and diterpenes in the populations of Tunku Abdul Rahman Marine Park and Dinawan Island. The second race is grouped by the production of chamigrane-type sesquiterpenes, acetylenes and diterpenes in the populations of Tun Mustapha Marine Park. The third race contains a mixture of non-chamigrane- and chamigrane-type sesquiterpenes, bromoallenes and triterpenes in the populations of Tun Sakaran Marine Park. This is also the first report pertaining to the presence of non-chamigrane- and chamigrane-type sesquiterpenes and triterpenes in L. nangii. Hence, it could be suggested that these could be new chemical races in L. nangii.
Acknowledgment The authors would like to thank the International Foundation for Science (IFS) and Organization for the Prohibition of Chemical Weapons (OCPW) for research grant IFS 4836/2 and Sabah Parks for the research permit and assistance during sample collection.
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