Terpenoid profiles of resin in the genus Dracaena are species specific

Phytochemistry 170 (2020) 112197

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Terpenoid profiles of resin in the genus Dracaena are species specific Lucie Vaníčková

a,b,c

b,c

, Antonio Pompeiano , Petr Maděra

d,∗

e

, Tara Joy Massad , Petr Vahalík

f

T

a

Department of Chemistry and Biochemistry, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic International Clinical Research Centre of St.Anne's University Hospital Brno, Brno, Czech Republic d Department of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Brno, Czech Republic e Department of Scientific Services, Gorongosa National Park, Sofala, Mozambique f Department of Forest Management and Applied Geoinformatics, Faculty of Forestry and Wood Technology, Mendel University in Brno, Brno, Czech Republic b c

ARTICLE INFO

ABSTRACT

Keywords: Dracaena ombet heuglin ex kotschy & peyr. (Asparagaceae) Dracaena serrulata baker (Asparagaceae) Dracaena cinnabari Balf.f. (Asparagaceae) Dracaena draco (L.) L. (Asparagaceae) Resin volatile profiles SPME GC×GC-MS PCA

Dragon's blood is the colloquial name for the red resin produced by tree species in the genus Dracaena (Asparagaceae), and the resin is directly involved in plant defensive mechanisms against pathogen and herbivore attack. It is also widely used in traditional folk medicine due to its antiviral, antimicrobial and antitumor activities. In the present work, a method using solid phase microextraction combined with two-dimensional gas chromatography with time-of-flight mass spectrometric detection was developed for the analysis of resin from five Dracaena species, namely Dracaena cinnabari Balf. f., D. serrulata Baker, D. ombet Heuglin ex Kotschy & Peyr., D. draco subsp. draco, and D. draco subsp. ajgal. Twenty terpenoid components in the resins of the five species were identified after comparative study of the volatile metabolite profiles. Monoterpenes were found to be species specific, and the observed differences might be further investigated as a possible means of identifying chemotaxonomic markers. In addition, for the first time, we describe the terpenoid volatile profiles of D. ombet and D. serrulata resins.

1. Introduction Dragon's blood is the red resin, rich in specialised metabolites, produced by the xylem of plants in distinct genera, including Croton, Dracaena, Daemonorops and Pterocarpus (Gupta et al. 2008; Jia-Yi et al. 2014). In Dracaena species (Asparagaceae), dragon's blood is secreted as part of a naturally induced defense mechanism in reaction to pathogen infection, insect attack or mechanical injury (Jura-Morawiec and Tulik, 2016). Specialised metabolites in Dracaena resin have biological activities for clinical applications, and therefore the red resin is widely used in folk medicine due to its analgesic (Peres et al. 1998), anti-inflammatory (Miller et al. 2001), antioxidative (Machala et al. 2001), antiviral (Orozco-Topete et al. 1997), antimicrobial (Gupta and Gupta, 2011), antihemorrhagic (Esmeraldino et al. 2005), antimutagenic (Saffi et al. 2004; Teng et al. 2011), immunomodulatory (Risco et al. 2003), antiulcer (Fischer et al. 2004), and antitumor (Gonzales and Valerio, 2006) activities. Dragon's blood derived from Dracaena species is a phenolic-based resin (Langenheim, 2003) with a well-studied chemical composition (Gonzalez et al. 2004; Gupta et al. 2008; Masaoud et al. 1995; Shen et al. 2007). For example, diverse classes of specialised metabolites

∗

such as flavonoids, triterpenes, steroids and steroidal saponins, lignans, and phenolics were identified in Dracaena cochinchinensis (Lour.) S.C.Chen resin (Jia-Yi et al. 2014). The main chemical constituents of D. cochinchinensis dragon's blood are flavonoids (Jia-Yi et al. 2014). Numerous flavonoids, chalcones, chalconepolymers, stilbenes, and sterol saponins have also been identified in dragon's blood from Dracaena draco (L.) L. and D. cinnabari Balf. f. (Gupta et al. 2008). However, few studies have focused on the volatile oleoresin constituents of these species (Baumer and Dietemann, 2010; Santos et al. 2011; Silva et al. 2011). Foliar volatiles of D. reflexa Lam. have been studied (GuribFakim and Demarne, 1994), but studies of volatiles from the species' resin are still missing. The present work undertakes to investigate the potentially important oleoresin volatiles of five Draceana species, including D. draco, D. draco ajgal, and D. cinnabari. In addition, the resin of two other unstudied species, D. ombet Heuglin ex Kotschy & Peyr and D. serrulata Baker, was also examined. Comparative study of the dragon's blood of different geographical areas and of diverse species might be used to estimate the origin of different sources of resin used medicinally, helping validate the stated medicinal properties of different resins (Teng et al. 2015). In addition, dragon's blood has been used as a red colourant in gold lacquers as well as translucent glazes and paints.

Corresponding author. Zemědělská 3, 61300, Brno, Czech Republic. E-mail address: [email protected] (P. Maděra).

https://doi.org/10.1016/j.phytochem.2019.112197 Received 15 August 2019; Received in revised form 8 October 2019; Accepted 5 November 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

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Understanding the specific volatile signatures of resins from different sites may also aid in the identification of artwork from unknown locations (Baumer and Dietemann, 2010). In the present work we postulate that the chemical composition of dragon's blood is species specific. The few studies available thus far support this hypothesis. Gonzalez et al. (2004) compared D. draco and D. tamaranae Marrero Rodr., R.S.Almeira & M. Gonzales-Martin, concluding that the chemical composition of dragon's blood from Dracaena draco subsp. draco (from the Canary Islands and Cape Verde) and D. draco subsp. ajgal Benadid et Cuzin (from Morocco) were similar, while several flavans found in these two taxa were not present in dragon's blood from D. tamaranae. Sousa et al. (2008) developed a simple and rapid liquid chromatography method with diode-array UV–VIS spectrophotometric detection for the authentication of dragon's blood resins from D. draco and D. cinnabari and Daemonorops trees. Using this method, it was discovered that the flavylium chromophores, which contribute to the red colour of these resins, differ between species and could be used as markers to differentiate among Dracaena species. Key vibrational spectroscopic marker bands were identified in the Raman spectra of the resins, which were suggested for the identification of the botanical and possible geographical sources of modern dragon's blood resins from D. cinnabari, Daemonorops draco (Willd.) Blume (Arecaceae), D. draco and Corymbia terminalis (F.Muell.) K.D.Hill & L.A.S.Johnson (Myrtaceae; Edward et al. 2001). Gas chromatography coupled to mass spectrometric detection (GC-MS) has been applied for identification of specific flavonoid resin markers of D. draco, D. cinnabari, Daemonorops draco, and Daemonorops micracantha (Griff.) Becc. (Baumer and Dietemann, 2010). Here, for the first time, we use a method combining solid phase microextraction (SPME) for resin volatile collection with two-dimensional gas chromatography coupled to mass spectroscopic detection (GC × GC-MS) for characterization of species specific terpeniod markers of D. cinnabari, D. serrulata, D. ombet, D. draco subsp. draco, and D. draco subsp. ajgal.

(Class), the majority of monoterpenes clustered together, while sesquiterpenes formed a second cluster. α-Pinene (M2), camphene (M3), βpinene (M4), δ-3-carene (M6), and limonene (M8) were present at different concentrations in the five Dracaena species. These five monoterpenes were present in high concentrations in the resin of D. draco subsp. draco (C), D. ombet (E) and D. cinnabari (S), while in D. serrulata (O) and D. draco subsp. ajgal (M) resins they were present in lower quantities. To further evaluate the monoterpenes as possible species-specific markers, PCA analyses were performed to detect differences or similarities in the Dracaena species (Fig. 3). The first two dimensions accounted for 79.50% of the total variance. The monoterpenes δ-2-carene (M5), p-cymene (M7), and limonene (M8) were specific to D. serrulata (O), whereas α-pinene (M2), camphene (M3), β-pinene (M4) and δ-3carene (M6) were typical of D. cinnabari (S) (Fig. 3A). Dracaena serrulata (O) formed a distinct cluster, whereas the other two remaining clusters were comprised of D. cinnabari (S) and D. ombet (E), and by D. draco subsp. draco (C) and D. draco subsp. ajgal (M), respectively (Fig. 3B). 3. Discussion 3.1. Methods development The SPME-GC × GC-MS method was developed for the fast detection of terpenoid markers for Dracaena species identification. SPME extraction was compared with solvent extraction of Dracaena resin volatiles in n-hexane and methanol. The resin was insoluble or very slightly soluble in n-hexane but was soluble in methanol as previously reported (Al-Fatimi, 2018). The GC × GC-MS analyses of the hexane and methanol extracts of the resin did not produce volatile profiles as only trace concentrations of compounds were extracted. On the other hand, the SPME method was capable of extraction and pre-concentration of terpenoid compounds. Another advantage of the SPME technique for volatile collection is the solvent-free approach and low sample volume required. Consequently, the analysis is less expensive, less timeconsuming and easily automated when compared to classical methods relying on solvent extractions. SPME was previously used by Santos et al. (2011) and Silva et al. (2011) for the analyses of volatiles from D. draco leaves and fruit, respectively. In general, the GC × GC-MS method used for volatile identification allowed for better separation of coeluting compounds and a significant improvement in detection sensitivity (Vaníčková et al. 2012). Comprehensive two-dimensional gas chromatography (GC × GC) offers enhanced separation efficiency, reliability in qualitative and quantitative analyses, the ability to detect compounds at low concentrations, and provides a complete chemical signature of samples. These features are essential in the analysis of complex samples, such as Dracaena spp. resins, in which the number of compounds may be large or the analytes of interest are present at trace levels (Kallio, 2008). For screening purposes, analyses can be performed with a GC with a flame-ionization detector, which is widely available, and RI reported here can be used for the identification of terpenes.

2. Results 2.1. Method optimization The resin volatile profiles collected on DVB/PDMS, CAR/PDMS and PDMS fibers differed qualitatively and quantitatively (Supplementary Fig. 1). DVB/PDMS fiber was chosen for further analyses because it absorbed a broader range of compounds. Based on optimization of the conditions for the SPME method, we determined the incubation period should be 15 min at 40 °C followed by adsorption of volatiles onto the SPME fiber for 30 min at 40 °C. The comparison of volatiles extracted in n-hexane/methanol with those adsorbed on the SPME fiber (DVB/ PDMS) is shown in Supplementary Fig. 2. SPME collected a more complete array of the volatiles produced by Dracaena resin than did the solvent extraction method. 2.2. Terpenoid profiles In total, 20 terpenoid compounds, including eight monoterpenes and twelve sesquiterpenes, were identified by the SPME-GC × GC-MS technique from the resins of Dracaena (Table 1, Fig. 1). The chemical profiles of the five Dracaena species revealed intraspecific variation differing quantitatively and qualitatively (Fig. 1 and Table 1). GC × GC-MS allowed for detailed structural identification of nineteen terpenoid compounds in the resin volatile mixture. One of the sesquiterpenes (S12) remains unidentified. A heat map was constructed to visualize the relative proportions of the twenty volatiles in each of the species (Fig. 2). Dracaena draco subsp. ajgal from Morocco (M) and D. serrulata from Oman (O) clustered together, indicating similarities in their volatile chemical profiles, while D. cinnabari from Socotra (S), D. ombet from Ethiopia (E), and D. draco subsp. draco from the Canary Islands (C) formed another cluster. In the compound dendrogram

3.2. Volatile compounds in Dracaena resin Our comparison of terpenoid profiles was based on an evaluation of 20 compounds, 13 of which are described in Dracaena resin volatiles for the first time here. These compounds include five monoterpenes, namely α-thujene, α-pinene, camphene, β-pinene, and δ-2-carene, and eight sesquiterepenes, namely (−)-isodauca-6,9-diene, γ-elemene, trans-muurola-3,5-diene, γ-humulene, γ-himachelene, ε- and ω-amorphene, and α-muurolene. Furthermore, this is the first report of the volatile composition of D. serrulata and D. ombet resins. Many terpenoid compounds identified in Dracaena resins are common plant volatiles (El-Sayed, 2012) and have valuable medicinal properties, including anti-carcinogenic, antimalarial, antiulcer, antimicrobial, antiseptic, 2

3

M1 M2 M3 M4 M5 M6 M7 M8 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

α-Thujene α-Pinene Camphene β-Pinene δ-2-Carene δ-3-Carene p-Cymene Limonene (−)-Isodauca-6,9-diene γ-Elemene trans-Muurola-3,5-diene γ-Humulene γ -Himachelene ε -Amorphene α-Muurolene ω-Amorphene α-Calacorene β-Calacorene Caryophyllene oxide Sesquiterpene

Compound 93, 91, 77, 41, 65, 105, 136 93, 91, 77, 79, 41, 121, 136 93, 121, 79, 41, 67, 107, 136 93, 41, 69, 79, 77, 121, 136 93, 121, 91, 77, 79, 41, 136 93, 77, 79, 121, 41, 105, 136 119, 91, 134 68, 67, 93, 79, 53, 107, 121, 136 105, 119, 161, 41, 91, 93, 204 121, 93, 41, 107, 67, 204 105, 161, 119, 41, 81, 91, 204 41, 93, 91, 79, 55, 133, 105, 204 93, 133, 41, 91, 105, 79, 55, 204 161, 81, 41, 91, 105, 93, 120,204 105, 93, 41, 91, 161, 79, 119, 204 119, 161, 105, 41, 91, 81, 93, 204 157, 142, 115, 128, 183, 200 157, 142, 115, 129, 138, 200 41, 79, 93, 91, 69, 55, 109, 220 185, 143, 128, 115, 157, 200

MS fragmentsc 931 941 960 986 1001 1016 1031 1036 1395 1433 1451 1463 1483 1495 1514 1543 1548 1561 1574 1632

RId 928 936f 950f 978f 1003f 1011f 1024f 1029f 1393g 1436f 1451h 1483g 1476f 1496g 1498f 1540g 1540f 1559f 1580f –

f

RIref 574, 1.13 586, 1.15 610, 1.18 640, 1.21 658, 1.23 676, 1.21 694, 1.33 700, 1.24 1084, 1.33 1120, 1.35 1138, 1.38 1150, 1.42 1162, 1.42 1180, 1.42 1198, 1.41 1216, 1.44 1228, 1.54 1240, 1.56 1252, 1.49 1300, 1.63

RTe – 50.85 ± 2.23 0.90 ± 0.04 8.73 ± 0.78 – 12.98 ± 1.04 4.11 ± 0.56 8.75 ± 0.05 9.46 ± 0.34 – 1.93 ± 0.02 2.29 ± 0.10 – – – – – – – –

DD – 5.31 ± 0.13 2.78 ± 0.18 1.43 ± 0.05 3.16 ± 0.47 2.28 ± 0.32 21.94 ± 1.65 4.40 ± 0.64 13.51 ± 1.09 1.18 ± 0.06 2.81 ± 0.21 1.45 ± 0.04 3.37 ± 0.46 12.43 ± 1.35 12.04 ± 1.09 9.16 ± 1.76 tr 2.01 ± 0.08 0.52 ± 0.01 tr

DDA

– 26.62 ± 2.05 1.35 ± 0.07 4.03 ± 0.41 – 7.96 ± 0.29 32.80 ± 2.54 11.19 ± 1.08 3.25 ± 0.13 – 1.29 ± 0.08 1.21 ± 0.03 0.53 ± 0.01 2.39 ± 0.08 5.31 ± 0.10 2.07 ± 0.05 – – – –

DO

2.14 ± 0.02 11.80 ± 0.46 10.53 ± 0.13 2.00 ± 0.09 – 3.31 ± 0.25 2.37 ± 0.14 3.41 ± 0.09 0.64 ± 0.06 1.03 ± 0.02 44.13 ± 1.29 2.08 ± 0.07 15.86 ± 0.34 – – 0.61 ± 0.03 – tr – –

DC

tr ≤ 0.3%. Key: DD - Dracaena draco subsp. draco from Canary Islands, DDA - D. draco subsp. ajgal from Marocco, DO - D. ombet from Ethiopia, DC - D. cinnabari from Socotra, DS - D. serrulata from Oman. a The numbering of the compounds corresponds with Fig. 1. b Abbreviation corresponding to Fig. 2, M1-8 monoterpenes, S1-12 sesquiterpenes. c Typical mass spectrometric fragments confirmed with those published by Adams (2007), and by Joulain and König (1998) for monoterpenes and sesquiterpenes identification, respectively. d Retention indices (RI) on DB-5 column. e Retention time (RT) in seconds on 1st, and 2nd column, respectively. RIref Reference retention indices as published by fBabushok et al. (2011), gJoulain and König (1998), and hAdams (2007).

Abb

Noa

tr 0.92 ± 0.06 0.33 ± 0.01 tr 3.15 ± 0.05 tr 10.73 ± 1.02 1.95 ± 0.06 23.89 ± 1.04 1.27 ± 0.02 9.83 ± 0.45 tr 14.14 ± 0.76 3.34 ± 0.08 12.99 ± 1.03 9.68 ± 0.19 0.70 ± 0.01 5.57 ± 0.23 – 0.82 ± 0.03

DS

Table 1 Terpenic compounds (average ± RSD %) identified by SPME-GC × GC-MS technique in the headspace samples of Dracaena spp. resin. The compounds were identified by a comparison of their MS fragmentation patterns and retention indices (RI) with synthetic standards and values published previously. In the absence of standards, identifications were carried out by a comparison with the reference spectra NIST library, the Wiley/NBS registry of mass spectral data, and published retention indices.

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Fig. 1. Detail of SPME-GC × GC-MS analysis of Dracaena spp. terpenes. (A) Dracaena draco subsp. draco from Canary Islands, (B) D. draco subsp. ajgal from Marocco, (C) D. ombet from Ethiopia, (D) D. cinnabari from Socotra, (E) D. serrulata from Oman. Each dot represents one compound. The numbering corresponds to Table 1. The intensity of the signals is colour-coded from blue (zero) to red (maximum). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4

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Fig. 2. Heat map of 20 terpenic volatiles (columns) identified by SPME-GC × GC-MS analyses in five species (rows) of the Dracaena spp. The dendrograms were created using correlation-based distances and the Ward method of hierarchical clustering (P < 0.05). Columns are coloured according to chemical class (light orange = monoterepenes M1-8, dark orange = sesquiterpenes S1-12, Table 1). Key: C – Dracaena draco subsp. draco from Canary Islands; E − D. ombet from Ethiopia; M – D. draco subsp. ajgal from Marocco; O – D. serrulata from Oman; S – D. cinnabari from Socotra. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

nematicidal, larvicidal, anti-inflammatory and diuretic properties (Schwab et al. 2008). Nevertheless, the majority of studies on Dracaena are focused on the isolation and identification of flavonoids and sterols (Baumer and Dietemann, 2010; Gupta et al. 2008; Masaoud et al. 1995; Yi et al. 2011), while only a limited number of reports describe the composition of terpenoid volatiles from Dracaena resins. Santos et al. (2011) used SPME-GC-MS to analyze leaf extracts of D. draco (from the Azores, Portugal) and detected 31 components, eight of which were terpenes. Both our work and that of Santos et al. (2011) document the presence of limonene, α-calacorene, and caryophyllene oxide in Dracaena spp. resins. Twenty-six volatiles were also identified from D. cochinchinensis resin extracted in hexane and run on a GC-MS; these include three sesquiterpenes (τ-cadinol, τ-muurolon and α-cadinol; Teng et al. 2015). GC-MS analyses of D. reflexa leaf volatiles revealed 16 terpenoid compounds, 13 of which are monoterpenes and three of which are sesquiterpenes (Gurib-Fakim and Demarne, 1994). Two of the D. reflexa monoterpenes, δ-3-carene and p-cymene, were also identified in the present study. In comparison with previous reports on Dracaena volatile organic compounds (VOCs), our method allows for the identification of a wider spectrum of terpenoids. This may be due to

the combination of the analytical approaches we used, including the optimized SPME technique for VOCs collection and our very sensitive GC × GC-MS method. 3.3. Species specificity in Dracaena resins We demonstrated that the composition of terpenoids in Dracaena is species specific. We therefore propose that monoterpene profiles of Dracaena resins may be evaluated in future studies as chemotaxonomic traits that allow for the identification of the species origin of Dracaena resins. In pines, the composition of monoterpenes in cortical oleoresin changes with location and season (Mita et al. 2002). In addition, high variation in the presence/absence of particular volatile compounds was found within Boswellia species (Burseraceae); this variability could have been caused by different environmental features associated with the trees sampled, by differences in the timing of sampling, or by differences in the part of the trees sampled (stem base vs. annual shoots; Maděra et al. 2017). Nonetheless, studies collectively suggest the composition of Dracaena resin is species specific. In previous reports on Dracaena marker determination, Edward et al. (2001) found differences 5

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Fig. 3. Principal component analyses (PCA) of transformed GC × GC-MS data of eight monoterpenes (M) identified in Dracaena spp. resin. (A) Variables factor map represents projection of variables (M1-8, Table 1) on the plane defined by the first two principal components. (B) Hierarchical clustering is score plot describing the species and their clustering. Coloured boxes indicate particular clusters. Key: C – Dracaena draco subsp. draco from Canary Islands; E − D. ombet from Ethiopia; M – D. draco subsp. ajgal from Marocco; O – D. serrulata from Oman; S – D. cinnabari from Socotra.

such as the terpenes studied here, play important ecological and evolutionary roles, most notably in the deterrence of natural enemies. Detailed studies of herbivores and chemical diversity should be undertaken in the genus Dracaena in order to better understand its phytochemical richness and contribute to ecological investigations of chemically mediated plant-insect interactions.

among resin from two species, D. draco and D. cinnabari. Key vibrational spectroscopic marker bands were identified in the Raman spectra of the resins, and they were used for species identification and determination of the geographical origin of samples (Edward et al., 2001). Gonzalez et al. (2004) compared the composition of VOCs in the resin of D. draco and D. tamaranae, finding that the chemical composition of resins from D. draco subsp. draco (Canary Islands and Cape Verde) and D. draco subsp. ajgal. Together with our data and the results of Gonzalez et al. (2004) and Sousa et al. (2008), these findings all demonstrate the chemical composition of Dracaena resin is an accurate and useful species identifier. Currently D. draco is found only in a very restricted area of southern Morocco. It is noteworthy that these Moroccan populations have been designated as their own subspecies, D. draco subsp. ajgal, an indication that the taxon is in the early stages of speciation (Marrero et al. 1998). If chemotypes align with phylogeny, our results would suggest that the most closely related species are D. draco subsp. draco from the Canary Islands and subsp. ajgal from Morocco followed by D. ombet from Ethiopia and D. cinnabari from Socotra. Dracaena serrulata from Oman is the most distinct from the other four. However, Lu and Morden (2014) investigated the phylogenetic relationship among Dracaenoid genera and species using chloroplast DNA loci and have come to different conclusions. According to their results the most closely related species are D. draco and D. serrulata, whereas D. cinnabari and D. ombet form distinct clusters and are also distant from each other. In contrast, morphological work suggests that D. draco is closely related to the Socotra species, D. cinnabari (Marrero et al., 1998). Work with other groups of plants has similarly demonstrated that chemotypes do not always match phylogenies. Becerra (1997) found only a weak relationship between phylogeny and chemical similarity for Bursera species, common trees in the dry forests of Mexico. Likewise, Kursar et al. (2009) found a weak correlation between phylogenetic and chemical distances within the Neotropical tree genus, Inga. Overall chemical similarity between species was also not associated with phylogeny in the genus Protium (Salazar et al., 2018). This lack of phylogenetic signal in the expression of specialised metabolites suggests divergent selection on antiherbivore defences, such that closely related species do not necessarily produce similar defences. This should make it more difficult for herbivores to track hosts over evolutionary time, thereby reducing herbivore pressure on plants and resulting in the evolutionary lability of defensive traits (Endara et al., 2015). Specialised plant metabolites,

4. Conclusions Here we developed a fast and reliable method for the identification of Dracaena resin volatile markers using SPME-GC × GC-MS. The optimal conditions were obtained by heating the Dracaena resin samples in a water bath for 15 min and subsequently extracting volatiles with DVB/PDMS for 30 min at 40 °C. We demonstrated that the composition of terpenoid volatiles is species specific in Dracaena and that the observed differences in monoterpene composition might be further investigated as a possible means of identifying chemotaxonomic markers. 5. Experimental 5.1. Chemicals Methanol, n-hexane and synthetic standards of α-pinene, β-pinene, camphene, δ-2-carene, δ-3-carene, p-cymene, limonene, and caryophyllene oxide in GC-MS quality were purchased from Sigma-Aldrich (Czech Republic). 5.2. Resin collection Resins from the following species were analysed: Dracaena cinnabari - Socotra (Yemen), Dracaena serrulata – Oman, Dracaena ombet – Ethiopia, Dracaena draco subsp. draco – Canary Islands, Dracaena draco subsp. ajgal – Morocco. The data describing the collection sites, GPS locations of individual trees, season, altitude and climate are summarized in Supplementary Table 1, and Supplementary Fig. 3. Resin was collected from adult trees of comparable size. Trees were estimated to be between 150 and 300 years old. It is not possible to collect resin samples from saplings because they are very rare in the environment. The resins of five individuals of each species were collected from tree trunks and placed in glass vials sealed with Teflon covers and stored at −5 °C until analyses. Dracaena species grow primarily in semi-desert 6

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environments and on rocks (Médail and Quézel, 1999). Because of the very dry climate of Dracaena habitats, growth and resin production is extremely slow. Isolated individuals of Dracaena draco are found in parks and gardens on Tenerife, and sample collection requires permission from landowners. Naturally occurring trees are protected by law and sample collection is strictly prohibited.

and columns (terpenes) so that rows (and columns) with similar profiles were closer to one another, causing these profiles to be more visible to the eye. Second, each entry in the data matrix was displayed as a colour, making it possible to view the patterns graphically. The dendrograms were created using correlation-based distances, and the Ward method of agglomeration was used in this analysis. The differences in the chemical composition of the samples from the five species were analysed by principal component analysis (PCA). Prior to PCA, peak areas were subjected to logarithmic transformation; intraspecific scaling was performed by dividing each species score by its standard deviation; the data were centered by species’ scores. In PCA analyses, hierarchical clustering based on Pearson correlations showed that species with similar chemical profiles cluster together. All computations were performed with R 3.5.2 language and environment (R Core Team, 2018), and the R packages FactoMineR (Le et al. 2008) and pheatmap (Kolde, 2019) were used.

5.3. Volatile collection Solid phase microextraction (SPME) coupled with two-dimensional gas chromatography with mass spectrometric detection (GC × GC-MS) was used to characterize the chemical profiles of Dracaena resins. Prior the experiment, different conditions for SPME incubation temperature (25, 35, 40 °C), SPME incubation time (5, 15, 30 min) and SPME fiber coating (polydimethylsiloxane, polydimethylsiloxane, carboxen/polydimethylsiloxane) were tested for optimization. Volatile chemical profiles of Dracaena resins obtained by SPME technique were further compared with those obtained by solvent extraction (n-hexane, methanol). A representative sample of Dracaena cinnabari resin from Socotra island was used for the optimization procedure. Samples (1 mg of resin) were weighted and placed into glass vials and immediately analysed. Three SPME fiber types (Supelco, Bellefonte, PA, USA) were evaluated and conditioned according to the manufacture's recommendation before use. Polydimethylsiloxane (PDMS) 100 μm fiber was conditioned at 250 °C for 30 min, divinylbenzene/polydimethylsiloxane (DVB/PDMS) 65 μm fiber was conditioned at 250 °C for 30 min and carboxen/polydimethylsiloxane (CAR/PDMS) 75 μm fiber was conditioned at 300 °C for 60 min. All fibers were exposed to the sample (1 mg) headspace under the same conditions. The glass vials (2 mL) were capped with a PTFE septum and screw cap. The sample was incubated for 15 min at 40 °C water bath. Then, the fiber was exposed to the headspace at 40 °C for 30 min. The fiber was subsequently inserted into GC injector for thermal desorption of the analytes for 5 min at 250 °C in splitless mode. The solvent extraction was performed as follows: 500 μL of n-hexane (Sigma-Aldrich, Czech Republic) or methanol (Sigma-Aldrich, Czech Republic) were added to a 1 mL glass vial that contained 1 mg of the resin. The sample was than shaken for 2 h at room temperature and subsequently centrifuged for 15 min at 3000 rot. speed. 200 μL were then transferred to glass via inserts and analysed by GC × GC MS by injecting 1 μL in splitless mode.

Declaration of competing interest The authors have declared that no competing interests exist. Furthemore, the authors declare that the presnented work is not under consideration for publication elsewhere, its publication is approved by all authors and the responsible authorities where the work was carried out. The work will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder. Acknowledgements This research has been financially supported by the Internal Grant Agency of Mendel University in Brno, Czech Republic (project No. IGA VT 2017009). AP was supported by the project no. LQ1605 from the National Program of Sustainability II (MEYS CR). We would like to thank Hana Kalivodová and Klára Lengálová for the collection of the resin samples. We acknowledge Dr. Robert Hanus (IOCB, Prague, Czech Republic) and Dr. Pavlína Kyjaková (IOCB, Prague, Czech Republic) for assistance during the experiments and four reviewers for valuable comments on the manuscript. Appendix A. Supplementary data

5.4. Chemical analyses

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112197.

The GC × GC-MS analyses of the volatiles extracted from the resin were performed using a LECO Pegasus 4D instrument (LECO Corp., St. Joseph, MI, USA) equipped with a non-moving quad-jet cryomodulator connecting the 1st and the 2nd columns. The methodology has been described in detail elsewhere (Vaníčková et al. 2012). A series of nalkanes (C6–C40, Sigma-Aldrich, Czech Republic) was used to determine the retention indices for the analytes. The compounds were identified by a comparison of their MS fragmentation patterns and retention indices (RI) with synthetic standards and values published previously (Adams, 2007; Gupta et al. 2008; Joulain and König, 1998; Teng et al. 2015). Five repetitions were analysed for each sample (N = 5) resulting in 125 chromatographic runs.

References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ mass Spectrometry. Allured Publishing Corporation, Illinois, USA. Al-Fatimi, M., 2018. Ethnobotanical survey of Dracaena cinnabari and investigation of the pharmacognostical properties, antifungal and antioxidant activity of its resin. Plants 7, 1–13. Babushok, V.I., Linstrom, P.J., Zenkevich, I.G., 2011. Retention indices for frequently reported compounds of plant essential oils. J. Phys. Chem. Ref. Data 40 (4), 43101–1–47. Baumer, U., Dietemann, P., 2010. Identification and differentiation of dragon's blood in works of art using gas chromatography/mass spectrometry. Anal. Bioanal. Chem. 390, 1363–1376. Becerra, J.X., 1997. Insects on plants: macroevolutionary chemical trends in host use. Science 27, 253–256. Edward, H.G.M., de Oliveira, L.F.C., Quye, A., 2001. Raman spectroscopy of coloured resins used in antiquity: dragon's blood and related substances. Spectrochim. Acta, Part A 57, 2831–2842. El-Sayed, A.M., 2012. The pherobase database of pheromones and semiochemicals. http://www.pherobase.com. Endara, M.-J., Weinhold, A., Cox, J.E., Wiggins, N.L., Coley, P.D., Kursar, T.A., 2015. Divergent evolution in antiherbivore defences within species complexes at a single Amazonian site. J. Ecol. 1107–1118. https://doi.org/10.1111/1365-2745.12431. Esmeraldino, L.E., Souza, A.M., Sampaio, S.V., 2005. Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin. Phytomedicine 12, 570–576.

5.5. Statistics For all species studied, each chromatographic peak was analysed based on its area relative to the sum of the areas of all peaks in the chromatogram. The relative peak areas of 20 compounds (as identified by the GC × GC-MS in the deconvoluted total-ion chromatogram mode) were calculated from five replicates for each species and sample. A heat map was used for visualizing the complex data sets organized as matrices as described in Vaníčková et al. (2017). A heat map performed two actions on a matrix. First, it reordered the rows (species) 7

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Mita, E., Tsitsimpikou, C., Tsiveleka, L., Petrakis, P.V., Ortiz, A., Vagias, C., Roussis, V., 2002. Seasonal variation of oleoresin terpenoids from Pinus halepensis and Pinus pinea and host selection of the scale insect Marchalina hellenica (Homoptera, Coccoidea, Margarodidae, Coelostonidiinae). Holzforschung 56, 572–578. Orozco-Topete, R., Sierra-Madero, J., Cano-Dominguez, C., Kershenovich, J., OrtizPedroza, G., Vazquez-Valls, E., Garcia-Cosio, C., Soria-Cordoba, A., Armendariz, A.M., Teran-Toledo, X., 1997. Safety and efficacy of Virend® for topical treatment of genital and anal herpes simplex lesions in patients with AIDS. Antivir. Res. 35, 91–103. Peres, M.T.L.P., Pizzolatti, M.G., Yunes, R.A., Monache, F.D., 1998. Clerodane diterpenes of Croton ururucana. Phytochemistry 49, 171–174. R Core Team, 2018. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Risco, E., Ghia, F., Vila, R., Iglesias, J., Álvarez, E., Cañigueral, S., 2003. Immuno-modulatory activity and chemical characterisation of Sangre de Drago (dragon's blood) from Croton lechleri. Planta Med. 69, 785–794. Saffi, J., Echeverrigaray, S., Henriques, J.A.P., Salvador, M., 2004. Mutagenic and antioxidant activities of Croton lechleri sap in biological systems. J. Ethnopharmacol. 95, 437–445. Salazar, D., Lokvam, J., Mesones, I., Pilco, M.V., Milagros, J., Zuniga, A., de Valpine, P., Fine, P.V.A., 2018. Origin and maintenance of chemical diversity in a species-rich tropical tree lineage. Nat. Ecol. Evol. 2, 983–990. Santos, R.P., Mendes, L.S., Silva, B.M., de Pinho, P.G., Valentao, P., Andrade, P.B., Pereira, J.A., Carvalho, M., 2011. Phytochemical profiles and inhibitory effect on free radical-induced human erythrocyte damage of Dracaena draco leaf: a potential novel antioxidant agent. Food Chem. 124, 927–934. Schwab, W., Davidovich-Rikanati, R., Lewinsohn, E., 2008. Biosynthesis of plant-derived flavor compounds. Plant J. 54, 712–732. Shen, C.C., Tsai, S.Y., Wei, S.L., Wang, S.T., Shieh, B.J., Chen, C.C., 2007. Characterization and determination of six flavonoids in the ethnomedicine ‘‘Dragon's Blood’’ by UPLC-PAD-MS. Nat. Prod. Res. 21, 377–380. Silva, B.M., Santos, R.P., Mendes, L.S., de Pinho, P.G., Valentao, P., Andrade, P.B., Pereira, J.A., Carvalho, M., 2011. Dracaena draco L. fruit: phytochemical and antioxidant activity assessment. Food Res. Int. 44, 2182–2189. Sousa, M.M., Melo, M.J., Parola, A.J., de Melo, J.S.S., Catarino, F., Pina, F., Cooke, F.E.M., Simmonds, M.S.J., Lopes, J.A., 2008. Flavylium chromophores as species markers for dragon's blood resins from Dracaena and Daemonorops trees. J. Chromatogr. A 1209, 153–161. Teng, Z., Dai, R., Meng, W., Chen, Y., Deng, Y., 2011. Offline two-dimensional RP/RPLC method to separate components in Dracaena cochinchinensis (Lour.) SC Chen xylem containing resin. Chromatographia 74, 313–317. Teng, Z., Zhang, M., Meng, S., Dai, R., Meng, W., Deng, Y., Huang, L., 2015. A comparative study on volatile metabolites profile of Dracaena cochinchinensis (Lour.) S.C. Chen xylem with and without resin using GC-MS. Biomed. Chromatogr. 29, 1744–1749. Vaníčková, L., do Nascimento, R.R., Hoskovec, M., Ježková, Z., Břízová, R., Tomčala, A., Kalinová, B., 2012. Are the wild and laboratory insect populations different in semiochemical emission? The case of medfly sex pheromone. J. Agric. Food Chem. 60, 7168–7176. Vaníčková, L., Nagy, R., Pompeiano, A., Kalinová, B., 2017. Epicuticular chemistry reinforces the new taxonomic classification of the Bactrocera dorsalis species complex (Diptera: tephritidae, Dacinae). PLoS One 12, e0184102. Yi, T., Chen, H.-B., Zhao, Z.-Z., Yu, Z.-L., Jiang, Z.-H., 2011. Comparison of the chemical profiles and anti-platelet aggregation effects of two “Dragon's Blood” drugs used in traditional Chinese medicine. J. Ethnopharmacol. 133, 796–802.

Fischer, H., Machen, T.E., Widdicombe, J.H., Carlson, T.J., King, S.R., Chow, J.W., Illek, B., 2004. A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells. J. Ethnopharmacol. 93, 351–357. Gonzales, G.F., Valerio, L.G., 2006. Medicinal plants from Peru: a review of plants as potential agents against cancer. Anti Cancer Agents Med. Chem. 6, 429–444. Gonzalez, A.G., Leon, F., Hernndez, J.C., Padron, J.I., Sanchez-Pinto, L., Barrera, J.B., 2004. Flavans of dragon's blood from Dracaena draco and Dracaena tamaranae. Biochem. Syst. Ecol. 32, 179–184. Gupta, D., Bleakley, B., Gupta, R.K., 2008. Dragon's blood: botany, chemistry and therapeutic uses. J. Ethnopharmacol. 115, 361–380. Gupta, D., Gupta, R.K., 2011. Bioprotective properties of Dragon's blood resin: in vitro evaluation of antioxidant activity and antimicrobial activity. BMC Complement Altern. Med. 11, 13–17. Gurib-Fakim, A., Demarne, F., 1994. Volatile constituents of Dracaena reflexa Lam. var. angustifolia Baker. J. Essent. Oil Res. 6, 651–652. Jia-Yi, F., Tao, Y., Chui-Mei, S.-T., Lin, Z., Wan-Ling, P., Ya-Zhou, Z., Zhong-Zhen, Z., Chen, H.-B., 2014. A aystematic review of the botanical, phytochemical and pharmacological profile of Dracaena cochinchinensis, a plant source of the ethnomedicine “Dragon's blood”. Molecules 10650–10669. Joulain, D., König, W.A., 1998. The Atlas of Spectral Data of Sequiterpene Hydrocarbons. Verlag Hamburg, Germany. Jura-Morawiec, J., Tulik, M., 2016. Dragon's blood secretion and its ecological significance. Chemoecology 26, 101–105. Kallio, M.K., 2008. Comprehensive Two-Dimensional Gas Chromatography: Instrumental and Methodological Development. Dissertation thesis. University of Helsinky. Kolde, R., 2019. Pheatmap: Pretty Heatmaps. R package version 1.0.12. https://CRAN.Rproject.org/package=pheatmap. Kursar, T.A., Dexter, K.G., Lokvam, J., Pennington, R.T., Richardson, J.E., Weber, M.G., Murakami, E.T., Drake, C., McGregor, R., Coley, P.D., 2009. The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genus Inga. Proc. Natl. Acad. Sci. 106, 18073–18078. Langenheim, J.H., 2003. Plant Resins: Chemistry, Evolution, Ecology and Ethnobotany. Timber Press, Portland, Cambridge. Le, S., Josse, J., Husson, F., 2008. FactoMineR: an R package for multivariate analysis. J. Stat. Softw. 25, 1–18. Lu, P.L., Morden, C.W., 2014. Phylogenetic relationships among Dracaenoid genera (Asparagaceae: nolinoideae) inferred from chloroplast DNA loci. Syst. Bot. 39, 90–104. Machala, M., Kubínová, R., Hořavová, P., Suchý, V., 2001. Chemoprotective potentials of homoisoflavonoids and chalcones of Dracaena cinnabari: modulations of drug-metabolizing enzymes and antioxidant activity. Phytother. Res. 15, 114–118. Maděra, P., Paschová, Z., Ansorgová, A., Vrškový, B., Lvončík, S., Habrová, H., 2017. Volatile compounds in oleo-gum resin of Socotran species of Burseraceae. Acta Univ. Agric. Silvic. Mendel. Brun. 65, 73–90. Marrero, A., Almeida, R.A., González-Martín, M., 1998. A new species of the wild dragon tree, Dracaena (Dracaenaceae) from Gran Canaria and its taxonomic and biogeographic implications. Bot. J. Linn. Soc. 128, 291–314. Masaoud, M., Ripperger, H., Porzel, A., Adam, G., 1995. Flavonoids of dragon's blood from Dracaena cinnabari. Phytochemistry 38, 745–749. Médail, F., Quézel, P., 1999. The phytogeographical significance of S.W. Morocco compared to the Canary Islands. Plant Ecol. 140, 221–244. Miller, M.J., Vergnolle, N., McKnight, W., Musah, R.A., Davison, C.A., Trentacosti, A.M., Thompson, J.H., Sandoval, M., Wallace, J.L., 2001. Inhibition of neurogenic inflammation by the Amazonian herbal medicine sangre de grado. J. Investig. Dermatol. 117, 725–730.

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