Rapid assessment of chemical compounds from Phyllogorgia dilatata using Raman spectroscopy

Abstract

The gorgonian Phyllogorgia dilatata is endemic to the Brazilian coast which is listed as threatened with extinction. This species is known to produce sterols, mono- to tetra-terpenes, conjugated polyenals and peptides. The main objective of this study is to present an alternative method for identification of different classes of compounds based upon a Raman mapping technique using FT-Raman spectroscopy. The Raman analysis performed directly on the tissues (in situ) revealed the occurrence of peridinin, diadinoxanthin, conjugated polyenal and linoleic acid, that were also confirmed by Raman analysis of partitioned crude extracts. We have demonstrated that the technique has potential for use in guiding chromatographic separations and in providing information with respect to the early stages of a tissue necrosis through “purpling”. It may become a valuable non-destructive technique for monitoring the accumulation or production of metabolites during a biological interaction.

Keywords
Phyllogorgia dilatata; Octocoral; Raman spectroscopy; Carotenoid; Polyenal; Fatty acid

Introduction

The gorgonian Phyllogorgia dilatata is endemic to the Brazilian coast and is listed as being threatened with extinction (MMA, 2005). The leaf-like form is unique among octocorals, in which branches of axes anastomose to form a network filled by coenenchyme (Castro et al., 2010). The morphology of the colonies resembles an elephant ear when in movement underwater. The beauty of these colonies makes them attractive to tourists and divers exploit them commercially. From a chemical point of view, this species is known to produce sterols, mono- to tetra-terpenes, conjugated polyenals and peptides (Almeida et al., 2014).

The first chemical study of P. dilatata and its associated zooxanthellae revealed the presence of 23,24ɛ-dimethylcholesta-5,22-dien-3β-ol (Kelecom et al., 1980). Subsequently, the identification of sesqui-, di- and tetra-terpenes: two nardosinanes (Kelecom et al., 1990; Fernandes and Kelecom, 1995) and three germacranes sesquiterpenes (Maia, 1991; Martins and Epifanio, 1998), one diterpene (Martins and Epifanio, 1998) and two carotenoids (Martins and Epifanio, 1998; Maia et al., 2012), were reported. In addition to terpenes, P. dilatata produces non-methylated conjugated polyenes in damaged tissues (Maia et al., 2012, 2013), and besides secondary metabolites, a 5 kDa peptide has been identified (De Lima et al., 2013). Ecological and biological activities of crude extracts and pure compounds have also been described. Crude extracts have been demonstrated to be deterrent against fish in field assays (Epifanio et al., 1999), to have antifoulant properties against balanids (Pereira et al., 2002) and mussels (Epifanio et al., 2006) and to possess antibacterial activity (De Lima et al., 2013). The feeding deterrence and antifouling (mussel Perna perna) properties were ascribed to the diterpene 11β,12β-epoxycembranolide (Epifanio et al., 1999, 2006). The antimicrobial activity, active against bacteria known to cause hospital infections, was attributed to a 5372.66 Da peptide against bacteria known to cause hospital infections (De Lima et al., 2013). The carotenoids, peridinin and diadinoxanthin produced by the symbiotic zooxanthellae, are known to possess antioxidant activities (Pinto et al., 2000; Lavaud et al., 2002; Dambeck and Sandmann, 2014), which may be crucial for corals that live in the fotic zone.

In an attempt to perform a rapid identification of chemical constituents from P. dilatata, we have used Raman spectroscopy as a technique for analyzing tissues in situ (prior to extraction) and after extraction with organic solvents. Raman spectroscopy is based on the inelastic scattering of photons from molecules irradiated with a monochromatic light (a laser, for instance), and is related to populations of the molecular vibrational and rotational energy levels. Raman spectroscopy and the combination with microscopy (microspectroscopy) provide information about a particular chemical bond, as well as the molecular environment and the overall molecular structures. It is suitable for qualitative and quantitative analysis of the individual compounds. It has been extensively applied to terrestrial plants and animals for the identification of carotenoids (Schulz et al., 2005a; de Oliveira et al., 2010; Baranska et al., 2013), terpenes (Schrader et al., 1999; Talian et al., 2010), essential oils (Schrader et al., 1999; Schulz et al., 2005b), fatty acids, sterols (Baeten et al., 1998; Yang et al., 2005; Schulz and Baranska, 2007), flavonoids (Schrader et al., 1999; Baranska et al., 2006; Schulz and Baranska, 2007), alkaloids (Schulz and Baranska, 2007; Baranska and Schulz, 2009; Baranska et al., 2013), polyacetylenes (Schrader et al., 1999; Schulz et al., 2005b; Baranska et al., 2013), and wood resins (Edwards et al., 2004), among other analytical uses. It is also readily obvious that the technique is a non-destructive analytical method, which is very important in the field of natural products.

The use of Raman spectroscopy for the identification of natural products of ecological significance from corals has been previously reported (Maia et al., 2014b). Additionally, Raman analysis has been applied to animals, algae, and dinoflagellates for the identification of carotenoids, sterols, nonsubstituted conjugated polyenals, chromophores of green fluorescent proteins (GFP), chlorophylls, melanins, mycosporine-like amino acids (Maia et al., 2014b) and polysaccharides (Bansil et al., 1978; Ellis et al., 2009; Pereira et al., 2009). Raman spectroscopy may become a valuable non-destructive technique for monitoring the accumulation or production of metabolites during a biological process. The technique can also be used to obtain information with respect to the distribution and concentration of molecular species in a very short period of time compared with the usual time and solvent consuming separation procedures.

In the present study we describe the distribution of compounds within Brazilian coral tissues by the use of macro, micro-Raman and Raman imaging techniques. By using these methods we seek to understand the relationship between the presence of such compounds and color properties, which could be related to different stages of tissue infection and/or induced inflammation in coral species.

Materials and methods

Animal material

Samples of Phyllogorgia dilatata Esper, 1806 (Alcyonacea, Gorgoniidae) were collected in September 2009 by scuba divers at a depth of 3 m at Saco dos Cardeiros, Arraial do Cabo, Rio de Janeiro coast (23°44 S-42°02 W) and identified by Clovis B. Castro (UFRJ).

Extraction and fractionation

After collection, the colonies were immediately frozen in dry ice. Raman spectroscopic analyses were performed in situ with cream or off-white, gray and purple tissue samples and using the crude extracts methanol/dichloromethane (MeOH:CH₂Cl₂) 1:1, hexane, ethyl acetate (EtOAc) and methanol (MeOH) extracts according to Maia et al. (2012). For the extraction, mixtures of MeOH/CH₂Cl₂ 1:1 (in the first washing procedure) and CH₂Cl₂ (for the following washing procedures) were used. The extracts were combined and partitioned between hexane/MeOH (nonpolar) and EtOAc/water (medium polarity) furnishing three further extracts: hexane, EtOAc and MeOH. The EtOAc extract was submitted to silica gel (TLC grade) vacuum chromatography employing a gradient of 0-100% of ethyl acetate in hexane, followed by methanol, affording 13 fractions (Maia et al., 2012). Extraction to detect the presence of carotenoids in animal tissues was made according to McGraw et al. (2005), where a small piece of the tissue was extracted and partitioned between aqueous pyridine and hexane-tert-methyl butyl ether (TBME). Sclerites, which are the mineral content of skeleton and composed of calcite (CaCO₃), were isolated according to the methodology described by Leewis and Janse (2008).

Spectroscopic analysis and Raman data processing

Fourier transform Raman measurements were carried out using a Bruker RFS 100 instrument and a Nd:YAG laser operating at 1064 nm. The instrument with 4 cm⁻¹ of spectral resolution was equipped with a Ge detector cooled with liquid nitrogen and coupled to a RamanScope III microscope system. Good signal-to-noise ratios for macro-Raman spectroscopy were obtained using 300 and 1000 scans and a range of laser powers at the sample (100-300 mW). The scattered radiation was collected at 180° geometry. The collected spectra were processed by the Bruker Opus software package version 6.0. In samples submitted to micro-spectroscopy, the laser is focused on the tissue by an Olympus BX 40× microscope objective. Micro-spectroscopy Raman imaging was performed by consecutive Raman measurements that were spatially resolved point by point on different places in a sample selected after visual inspection through the microscope. A collection of Raman spectra across a defined area of the coral tissue generated a map recorded on a RamanScope III Bruker microscope with an Olympus BX 40× objective and a motorized stage coupled to a MultiRAM FT-Raman spectrometer equipped with a 1064 nm laser line and a germanium detector. All spectra were collected in a 50-4000 cm⁻¹ range with 4 cm⁻¹ resolution, 300 scans per point and the laser power set at 300 mW. An area of approximately 2 cm² was analyzed. The Raman images were constructed in Matlab software 7.10.0(R2010a); the Simplisma (Windig et al., 1990) algorithm was employed to separate the pure variables in the data set.

Results and discussion

Predation, competition (Coll, 1992; Chiappone et al., 2003; Lesser, 2004; Paul and Ritson-Williams, 2008; Paul et al., 2011), diseases (Rosenberg et al., 2007; Francini-Filho et al., 2008; Mydlarz et al., 2008), thermal stress (Hughes et al., 2003), and current flux (Lin and Dai, 1996) are biotic and abiotic agents that influence growth, form, healthiness and population density of coral colonies (Thompson et al., 2015). The octocoral P. dilatata has been found to be chemically defended against diverse marine organisms (Almeida et al., 2014). However, the agent that causes the purpling disease inducing tissue necrosis has not been identified (Alves et al., 2010). Alves et al. (2010) have demonstrated that apparently healthy and diseased coral (P. dilatata) possess a vibrio microbiota associated in the mucus; this octocoral could be a reservoir of potentially virulent strains of vibrio. On the other hand, it might be a normal component of the holobiont. In our study, purpling has been observed in areas with necrosis and in areas with no apparent tissue damage. Samples were analyzed by means of macro, micro-Raman and Raman imaging spectroscopy.

In situ analysis using macro-Raman spectroscopy

Samples from different parts of the colonies as well as from different colonies submitted to macro and micro-Raman analysis have revealed variations of chemical constituents. In Fig. 1 the in situ analysis of apparently healthy colonies showed major bands at 1525, 1184, 1145, 1128, 1020 and 1005 cm⁻¹ addressed to carotenoids and bands attributed to CaCO₃ (calcite) at 1752, 1089, 715 cm⁻¹ (Maia et al., 2012). Deconvolution of the sharp band centered at 1525 cm⁻¹ and broad bands at 1159 and 1005 cm⁻¹ confirmed the presence of the carotenoids peridinin 1 and diadinoxanthin 2 (Fig. 1B and C and Table 1), which were unambiguously identified in fractions eluted with a mixture of hexane and ethyl acetate (1:1) and (4:6), respectively from the EtOAc extract (Maia et al., 2012, 2013). The identification of peridinin 1 was performed by analysis of the fingerprint bands at 1929 cm⁻¹ assigned to the symmetrical vibration of the allene functional group ν(CCC), together with bands at 1750 ν(CO), 1616 ν(CC), 1525 ν(CC), 1184 δ(CH), 1145 δ(CH), 1021 δ(CCH₃) (Dietzek et al., 2010; Maia et al., 2012). The diadinoxanthin 2 spectrum showed a characteristic key acetylene band at 2173 cm⁻1 assigned to the ν(CC) and distinct bands at 1537 ν₁(CC), 1159 ν₂(CC) and 1018 cm⁻¹δ(CCH₃) (Maia et al., 2013).

Fig. 1
Macro Raman analysis: Panel A – in situ FT-Raman spectrum from P. dilatata and deconvolution for vibrational modes ranging from 1560 to 1500 cm⁻¹, from 1200 to 1140 cm⁻¹ and from 1040 to 980 cm⁻¹; Panel B – FT-Raman spectrum from diadinoxanthin; Panel C – FT-Raman spectrum from peridinin.

Table 1
Main observed Raman bands (in cm⁻¹⁾ and tentative vibrational assignments from in situ analysis of tissues, crude extracts and fractions fromPhyllogorgia dilatata excitation at 1064 nm in macroscopic mode.

Off-white tissue	Purple tissue	Hexane extract	Hex/TBME extract	EtOAc extract	Peridinin	Diadinoxanthin	Tentative assignment
						2173 m	ν (CC)
				1930 w	1929 w		ν (CCC)
1750 w	1750	1743 w		1754 w	1752 w		ν (CO)
1662 m		1658 s	1658 s	1656 w		1654 w	ν (CC)/(CO)
1612w				1615 w	1616 w		ν (CC)
1525s	1502 s	1530 m		1525 s	1525 s	1537 s	ν (CC)
1445 m		1440 s	1440 m	1448 m	1450 w	1446 m	δ (CH)
		1301 m	1301 m				δ (CH2)
1266 w		1266 m	1266 m				δ (CH)
1184 m				1184 m	1184 m		δ (CH)
1159 m	1118 s						ν (CC)
1147 m				1145 m	1145 m	1159	δ (CH)
1089 s	1089 s						ν 1(CO3) a
		1073 w	1068 w				ν (CC)
1020 w	1014 w			1020 w	1021 w	1018 w	δ (CCH3)
1005 w						1004 w	δ (CCH3)
716 m	715 m						ν 4(CO3) a

• s: strong; m: media; w: weak.
• ^a
  Calcite vibrational modes.

In situ analysis of purple tissues and purple sclerites (Fig. 2 and Table 1) showed bands at 1502 ν(CC), 1116 ν(CC), 1018 cm⁻¹δ(CCH₃) attributed to conjugated polyenals 3 and additional bands related to calcite (Maia et al., 2012, 2014a; Fernandes et al., 2015). Although carotenoids and polyenals are conjugated polyenes, they present distinct features (Fernandes et al., 2015).

Fig. 2
Macro Raman analysis: (a) FT-Raman spectrum from purple sclerites; (b) purple tissue; (c) colorless sclerites; (d) P. dilatata tissue in situ.

The observation of the band at 1658 cm⁻¹ together with bands at 1301 and 1266 cm⁻¹ suggested fatty acids in the tissues (Figs. 2 and 3), which were confirmed by analysis of the hexane extract showing a collection of bands at 3014 ν(CH), 2925 ν(CH₂), 2898 ν(CH₃), 1743 ν(CO), 1658 ν(CC) cis, 1440 δ(CH₂), 1301 δ(CH₂), 1266 δ(CH) cis, 1073 ν(CC) that could be assigned to linoleic acid 4 by comparison with literature data (Meurens et al., 2005; Afseth et al., 2006; Schulz and Baranska, 2007).

This spectral pattern was also observed in the hexane/TBME extract (Fig. 3 and Table 1) and the fraction was eluted with hexane/EtOAc 7:3 and 6:4 from the EtOAc crude extract (Fig. 4). The hexane/TBME extract was prepared in order to determine the presence of carotenoids in the tissues by a simple chemical test as proposed by McGraw et al. (2005); however, this procedure did not yield carotenoids but resulted in the isolation of fatty acids. The fraction eluted with hexane/EtOAc 2:8 showed major Raman bands at 1650-1611 ν(CC), 1445 δ(CH₂), 1380 δ(CH), which is characteristic of terpenes (Daferera et al., 2002; Schulz and Baranska, 2007). Increasing the gradient of the hexane/ethyl acetate to 1:9, and to 100% ethyl acetate, resulted in the observation of Raman bands at 2171 ν(CC), 1930 ν(CCC), 1747 ν(CO), 1544 ν(CC), 1451 δ(CH), 1211, 1187 δ(CH), 1157 δ(CH₃), 1020 δ(CH), 941 ν(CO) (Dietzek et al., 2010; Maia et al., 2012, 2013) that could be assigned to a mixture of peridinin and diadinoxanthin (Fig. 4). However, the presence of these bands could also be attributed to a mixture of different xanthophylls with similar chemical structures or a compound that bears allene and acetylene functional groups common to dinoflagellates (Johansen et al., 1974; Maoka et al., 2002; Dembitsky and Maoka, 2007).

Fig. 3
Macro Raman analysis: Panel A – (3100–2700 cm⁻¹); Panel B – (1800–800 cm⁻¹): FT-Raman spectra of the (a) linoleic acid; (b) TBME:hexane extract; (c) hexane extract; (d) Phyllogorgia dilatata tissue in situ.

Fig. 4
Macro Raman analysis: FT-Raman spectra of the (a) ethyl acetate fraction; (b) hex/ethyl acetate 1:9 fraction; (c) hexane/ethyl acetate 2:8 fraction; (d) hexane/ethyl acetate 3:7; (e) hexane/ethyl acetate 6:4 fraction; (f) hexane/ethyl acetate 7:3 fraction; (g) ethyl acetate extract; (h) hexane extract and (i) MeOH/CH₂Cl₂ 1:1 extract.

In situ analysis using micro-Raman spectroscopy

Micro-Raman spectroscopy was used to obtain the in situ spectra of specific regions of the tissues. Colonies presenting dark (gray) and clear (off-white) coloration showed distinct Raman spectra: the dark regions revealed major bands that were assigned to the carotenoid peridinin 1 (1525, 1616, 1446, 1180 and 1145 cm⁻¹) and bands attributed to CaCO₃ (calcite) (1752, 1089 and 715 cm⁻¹). The off-white portion revealed calcite vibrational modes together with major Raman bands attributed to fatty acids (1658, 1450, 1300-1200 cm⁻¹) and minor bands characteristic of carotenoids (1527, 1186, 1159, 1016, 1020 cm⁻¹). Tissues containing injuries and purple pigmentation were also investigated (Fig. 5). In situ irradiation on site presenting original color (off white or cream) showed typical bands of peridinin 1 at 1525, 1446, 1180, 1145 cm⁻¹. Injured purple tissue showed bands at 1500, 1118 and 1017 cm⁻¹ that were identified as the conjugated polyenal 3. The regions of tissue where the purple pigment shows up as a purple shadow showed bands attributed to both class of compounds (carotenoids and conjugated polyenals) (Fig. 5). Polyenals and carotenoids are linear conjugated polyenes that have been characterized by the analysis of vibrational bands attributed to ν(CC), ν(CC) and δ(CCH₃) modes. There are three main differences between polyenals and carotenoids observed by Raman spectroscopic analysis: (1) the red-shifted wavenumber position of the ν(CC) stretching mode of polyenals by ca. 30 cm⁻¹ when compared with carotenoids, (2) the absence in polyenals at resonance conditions of the deformation mode related to the δ(CCH₃) group at ca. 1000 cm⁻¹, and (3) the presence of δ(CCH₃) at ca. 1020 cm⁻¹ (Maia et al., 2014a; Fernandes et al., 2015). The wavenumber position of the (CC) stretching vibrations may be influenced by the length of the conjugated carbon-carbon chain, by the substitution pattern in the chain, and by the interaction with other chemical constituents present in the biological matrix (Schulz et al., 2005a; de Oliveira et al., 2010, 2011).

Fig. 5
Micro Raman analysis: Panel A: in situ Raman spectra from Phyllogorgia dilatata tissues. (a) Dark region; (b) white region and (c) Raman spectrum of linoleic acid. Panel B: in situ Raman spectra from P. dilatata tissues. (a) Purple tissue; (b) purple and off-white tissue and (c) off-white tissue.

Discriminating compounds by Raman micro-imaging

The Raman mapping was applied for the assessment of carotenoids and polyenals distribution across the surface of coral tissues presenting or not variations in coloration pattern (Fig. 6). For this purpose, measurements were done point by point in an area of 160 μm × 120 μm and the Raman images were generated by means of Raman intensities from carotenoids and polyenals. As can be seen in Fig. 6, gradient of coloration in chemical maps is correlated with the intensity of Raman bands indicating the relative content of each substance present in the tissue. The Raman image of the area A obtained from off-white colonies apparently healthy showed Raman major bands at 1526, 1446, 1184, 1145 cm⁻¹ characteristic of peridinin 1 colored according to band intensities (Fig. 6A); the dark orange color represents the region with highest amount of the carotenoid and the yellow color represents the region with lowest content of carotenoid. Raman images from the area B, where purple pigmentation co-occurs with undamaged off-white tissues, and showed the presence of both carotenoids and polyenals (Fig. 6B) due to bands at 1526, 1184, 1145 cm⁻¹ and 1502, 1118 cm⁻¹, respectively. The map showed areas with gradients of orange to yellow colors addressed to carotenoids and areas with pink to purple colors addressed to polyenals. The Raman image of the area C belongs to the damaged tissue, which showed Raman bands at 1502 and 1118 cm⁻¹ typical of polyenals, represented by purple color (Fig. 6C).

Fig. 6
In situ Raman imaging analysis from Phyllogorgia dilatata tissues: (A) off-white tissue; (B) purple and off-white tissue and (C) purple tissue. The chemical maps are colored according to the intensity of Raman bands characteristic for carotenoid (1 and 2), polyenal (3) and carotenoid and polyenal compounds (1, 2 and 3).

The micro-Raman and mapping analysis demonstrated that necrotic areas presented bands predominantly of conjugated polyenals (Figs. 5 and 6C) and the apparently healthy region furnished Raman spectra addressed to carotenoids (Figs. 5 and 6). Analysis of slightly purple undamaged tissue showed bands belonging to carotenoids and polyenals (Figs. 5 and 6). Polyenals are inserted into the calcium carbonate sclerites, which are structures embedded in the connective tissue produced by skeleton-forming cells (Sorauf, 1980). It could be hypothesized that polyenals are endogenously synthesized locally, explaining the facultative occurrence. In analogy to octocorals, it has been suggested that polyenals from parrots (psittacofulvins) are synthesized at growing feathers, within the maturing feather follicle (McGraw and Nogare, 2004). On the other hand, carotenoids are produced by the photosynthetic endosymbionts which provide nutrients to the host and enable them to survive in oligotrophic environment (Fujise et al., 2014). The reduction or absence of carotenoid Raman bands in P. dilatata tissues may indicate expulsion of the symbiont from the host or/and a decrease in production of these compounds in the symbiont; both hypotheses may compromise the maintenance and survival of the colony. Studies performed with the phytoplanktonic coccolithophorid Emiliania huxleyi have shown the activity of the diadinoxanthin 2 cycle (conversion of diatoxanthin in diadinoxanthin) occurring in response to viral infection (Llewellyn et al., 2007). Variation in the concentration of individual carotenoids was observed, such as an increase of diatoxanthin relative to chlorophyll-a versus a decrease in diadinoxanthin and β-carotene content (Llewellyn et al., 2007). Differences in pattern of coloration and chemical composition observed in tissue samples presenting dark (gray) and non pigmented (white) areas (Fig. 5) may suggest that the apparent bleached colony contained the endosymbiont due to the presence of minor bands of carotenoids; the predominance of fatty acid bands in the non-pigmented portion maybe attributed to the animal metabolism or dietary input.

It has been straightforwardly to noticed that Raman maps obtained from damaged tissue have shown bands attributed exclusively to polyenals, according to Fig. 6C; this can be rationalized as follows: coral produces pigments as polyenals into the sclerites as an inflammatory response to an invading agent; purple sclerites may be positioned on or underneath colorless sclerites which in P. dilatata have no pigment since carotenoids are not embedded in calcite matrix (Fig. 2). Mapping performed in off-white tissues with purple shadows clearly showed the purple sclerites emerging from the surface of coral prior to the necrosis.

In the experiment performed with double colored tissue, dark region showed the presence of carotenoid bands, whereas the white region showed the same compounds with different spectral pattern. The conclusion that can be achieved from this fact is very clear, healthy tissues present carotenoid in its composition, whereas damaged or non-pigmented tissues do not show such chemicals analyzed by Raman spectroscopy despite the agent: biotic or abiotic.

These data have indicated that Raman spectroscopy is suitable to detect variations in the chemical composition of P. dilatata tissues and the technique is able to provide information about early stages of a tissue infection and/or any other agent that induce inflammation. The analysis of the fractioned crude extract may have a potential use in guiding chromatographic and spectroscopic studies. It is expected that different Raman spectroscopic approaches (individual Raman spectra, Raman-mapping, the combination of chromatographic separation and Raman) can be applied to simultaneous and rapid study in situ of chemical compounds from marine organism. Summing up, Raman data, mainly based on micro-spectroscopy Raman imaging, have clearly shown that healthy tissues of P. dilatata present very strong bands assigned to carotenoids indicating the association with the dinoflagellate symbiont. Damaged tissues do not present Raman bands addressed to carotenoids, meaning that the host has lost the source of nutritional input from the symbiont leaving coral colonies more susceptible to disease and mortality. It could be understood as a typical fingerprint of the coral health condition: healthy tissues present Raman spectrum containing carotenoids bands, in bleached or damaged tissues, and Raman spectrum is devoid of typical symbiont compounds.

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  » http://dx.doi.org/10.3389/fcimb.2014.00176
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Publication Dates

• Publication in this collection
  Dec 2015

History

• Received
  20 Apr 2015
• Accepted
  7 Sept 2015