Prospecting for non‑timber forest products by chemical analysis of four species of Lauraceae from the Amazon region of Colombia

Lauraceae is a family of woody plants of economic importance mainly for their commercial exploitation as timber, as well as spices/food. Nonetheless, overexploitation is causing a decline in both the population and the associated ecosystems due to the lack of sustainability strategies and knowledge of alternative ways of utilization. The focus of this research was to determine if the secondary metabolites found/identified in the volatile fractions/ethyl acetate extracts of Aniba panurensis , Nectandra cuspidata , Ocotea cymbarum and O. myriantha from the Amazon region of Colombia (Departamento de Caquetá) would be promising/interesting for industry, so that uses/exploitation other than timber could be recommended. In this work, the chemical compositions by GC–FID/MS of the volatile fractions/ total extracts (by HS–SPME/SDE/maceration) of the trunk wood of these trees were determined. The results were: (i) the volatile fractions/extracts of A. panurensis were composed of 88–94% benzenoid‑type aromatic esters (benzyl salicylate and benzoate); (ii) N. cuspidata contained 95% sesquiterpenes (α‑copaene and α‑cubebene/germacrene D) by HS–SPME, 89% oxygenated and hydrocarbonated sesquiterpenes (τ‑cadinol and δ‑cadinene) by SDE, and 87% sesquiterpenes and aporphine alkaloids (α‑copaene/germacrene D/δ‑cadinene/α‑cubebene and dicentrine/dehyd‑ rodicentrine) by solvent extraction; (iii) O. cymbarum contained mainly 63% sesquiterpenes and monoterpene ethers (α‑copaene/ trans ‑calamenene and eucalyptol) by HS–SPME, 63–85% of monoterpene alcohols (α‑terpineol/borneol)/ hydrocarbons (α‑/β‑pinenes)/ethers (eucalyptol) and phenylpropanoid ethers (methyleugenol) by SDE/solvent extrac‑ tion; and (iv) for O. myriantha , the constituents per family were 91% sesquiterpenes (bicyclogermacrene/germacrene D)—HS–SPME, 72% sesquiterpene alcohols and sesquiterpenes/monoterpenes (spathulenol and bicyclogermacrene/ δ‑3‑carene)—SDE, and 69% benzenoid‑type aromatic esters and sesquiterpene hydrocarbons/alcohols (benzyl salicylate and bicyclogermacrene/α‑cadinol)—solvent extraction. In conclusion, the main constituents identified in the


Introduction
The term "non-wood forest products" (NWFP) refers to those biological products (tangible goods) obtained from forests, other wooded lands and trees outside forest that are not timber or fuelwood; such products must be for human use/benefit.Derived products could include whole plants (herbs, shrubs/trees; medicinal or not) or their parts (fruits, leaves, bark, trunks, roots, seeds) for applications in medicine (drugs), food (edible products-fruits/seeds/roots/rhizomes)/spices/dyes, pharmaceutical/cosmetic/perfumery (essential oils/gums/ resins/extracts/fractions), as well as sources of natural ingredients/raw materials, among others.NTFP harvesting/processing would offer great employment/economic opportunities for the poor rural populations surrounding the forest areas containing the biological resource but would also contribute to tropical forest conservation processes [1][2][3].
An important fact is that Colombia has a wide diversity of species (ca.255 spp.) of the Lauraceae family, represented by the genera Ocotea (75 spp.) and Nectandra (28 spp.), the most diverse and widely distributed in the country according to the Herbario Nacional de Colombia [16].Meanwhile, the genus Aniba has only 20 spp. in the entire national territory.In the southern region of the country (Departamento de Caquetá) where tropical rainforest predominates, the Yarí-Caguán Reserve (municipalities of San Vicente del Caguán/Cartagena del Chairá) has an abundance of species of the Lauraceae family, which are exploited for commercialization, because they are timber, e.g., Aniba panurensis, Nectandra cuspidata, Ocotea cymbarum [syn.Mespilodaphne cymbarum (Kunth) Trofimov], and O. myriantha.Regardless of this use, these four species have ethnobotanical uses and fragrances that could suggest other applications besides timber harvesting [17,18].
Therefore, in this work, the chemical compositions of the volatile fractions (by HS-SPME/SDE) and ethyl acetate extracts (by simple maceration) of trunk woods of the Aniba panurensis, Nectandra cuspidata, Ocotea cymbarum and O. myriantha from the Amazon region of the departamento de Caquetá (Colombia) were determined by GC-FID/MSD, to establish if their secondary metabolites would be promising/interesting for the industry, so that other applications/uses/exploitations than timber would be recommended.

Text mining
The text mining analysis related to the bibliometric exploration on the topic of interest of this manuscript was based on two search equations (Supplementary Materials) which were run on the Scopus database (Elsevier, BV, 2023); the search found 1262 indexed entries.The graphs of the co-occurrence analysis generated by the bibliometric analysis for each search equation through the Scopus database are displayed in Fig. 1.As can be seen therein, many specific words correlated with the keywords/search equations were found in the database; nonetheless, the words non-timber forest products/forestry/forest mana gement/livelihood/conservation, in that order, had the greatest occurrence for each search equation.In contrast, the scientific dynamics from 2010 to 2023 indicated that 2022/2020/2021/2014/2019 were the years with the highest science activity with 142/141/140/125/111 records, respectively.Considering the areas of knowledge, Agricultural/Biological Sciences, Environmental Science and Social Sciences were the most important for the keyword non-timber forest products; whereas for the names of the four species were Agricultural/Biological Sciences, Pharmacology/Toxicology/Pharmaceutics and Biochemistry/ Genetics/Molecular Biology.Finally, the top five of countries according to the number of records  based on the equation were E.E.U.U., Brazil, India, Indonesia and United Kingdom.The top five Latin American countries were Brazil, Mexico, Colombia, Peru and Argentina with 162-10 records.
When a search equation (Supplementary Materials) was used, which included the terms non-timber forest products and the four names of the species of interest, no records were found.This result is relevant because it revealed the "gap" of information on this topic, creating an opportunity for the generation of new knowledge.In other words, based on the information that would be gathered on the chemical compositions of the four species under study and their potential to be used as natural ingredients/raw material for the pharmaceutical/ perfumery/cosmetic industries or medicine, the species could be prioritized for sustainable exploitation as nonwood forest products.

Plant material
Samples (trunk woods consisting of sapwood/heartwood) from Aniba panurensis, Nectandra cuspidata, Ocotea cymbarum and O. myriantha were collected in the Unidad de Ordenación Forestal Yarí/Caguán (UOF), Amazon Forest Reservation, Cartagena del Chairá (Caquetá-Colombia) in August-September 2015.The UOF is a protected forest reserve (ca.840,000 ha), containing non-stable (ca.26%) and stable (ca.49%) forest zones.On the other hand, the taxonomic identification of the species was carried out by Francisco Rojas-Triana at the Herbario Forestal UDBC-Univ.Distrital Francisco José de Caldas, and the collection of plants was made under Resolution No. 738 of July 8, 2014, conferred by the Agencia Nacional de Licencias Ambientales (ANLA).
In addition, trunk woods were sampled as reported by Bárcenas Pazos [51] with the main objective of establishing the physical-mechanical properties of the timbers; for this, 3-4 individuals (plants) of each species were randomly selected (distanced ca.5-10 km, in a stable forest zone), from which three sections were isolated (lower, middle and upper parts, in triplicate) and rectangular blocks (62.5 cm 3 ) were taken (in triplicate) for each of them.For the determination of the physical-mechanical properties, a set of representative samples of the wood of each species was considered; as well as another set of samples was used for the respective chemical analysis.From the latter group, the wood samples (blocks of each section/tree, dehydrated/stored (on shelves in paper bags, not stacked) at room temperature (humidity controlled) until extraction process) were chipped (very thin and short slices), mixed/homogenized and subjected to three extraction methods (headspace solid-phase microextraction, simultaneous distillation-extraction and simple maceration) to obtain the secondary metabolites.

Obtaining the volatile fractions/total extracts Headspace-solid-phase micro-extraction
Vapor phase constituents of the trunk wood were trapped using a SPME device (Supelco), with a polydimethylsiloxane (PDMS)-coated fiber (100 μm), sampling the headspace.The plant samples (1 g) were thermally preconditioned (50 °C) for 10 min, and then, the fiber was exposed to the headspace of each sample (separately) at 50 °C during 30 min (non-equilibrium conditions according to Muñoz [52] and Field et al. [53]).After sampling was completed, the SPME fiber was desorbed (5 min, 250 °C) with the analytes inside the GC-MS inlet port [54].All samples were prepared/analyzed in duplicate.

Simultaneous distillation-extraction
In addition, the volatile fractions of the trunk wood (10 g) of A. panurensis, N. cuspidata, O. cymbarum, and O. myriantha were isolated by the Likens & Nickerson microscale apparatus, modified by Godefroot et al. [55], using CH 2 Cl 2 (2 mL) as the extraction solvent for 2 h.The extracts were dehydrated with anhydrous sodium sulfate, and 1 μL of each extract (individually) was analyzed by GC-MS.All samples were prepared/analyzed in duplicate.

Maceration process
Total extracts of the four wood samples were obtained by maceration using ethyl acetate as the solvent.The plant material (30 g) was in contact with the solvent (100 mL) under stirring for 7 days at 25 °C.The extracts were concentrated to 1 mL, dehydrated with anhydrous sodium sulfate, and analyzed by GC-MS [54].All samples were prepared/analyzed in duplicate.

Chemical analysis by GC-FID/MSD
Each volatile fraction/total extract was analyzed using a Trace 1310 gas chromatograph with a flame ionization detector (FID, 250 °C) along with an ISQ Series mass spectrometer (Thermo Fisher Scientific), with a split/ splitless inlet (split ratio, 10:1), liquid autosampler (AI/AS 1310 Series, Thermo Fisher Scientific), or manual injection (SPME).A Rxi ® -1 ms column (crossbond PDMS, 30 m × 0.25 mm ID × 0.5 µm df, Restek) was useful for the separation by individual constituents.The GC oven temperature setting was adjusted as described by Muñoz-Acevedo et al. [54].Chromatographic and spectroscopic data were processed and analyzed through Thermo Xcalibur ™ (Version 2.2 SP1.48, Thermo Fisher Scientific) and Automated Mass Spectral Deconvolution and Identification System software (AMDIS ® , Build 130.53, Version 2.70).Linear temperature-programmed retention indices were calculated from a homologous series of C 7 -C 35 aliphatic hydrocarbons and analyzed under the same conditions as the samples by GC-FID/MSD.Then, the constituents were identified by comparing their mass spectra with those of the available databases/libraries (NIST11, NIST Retention Index, and Wiley9) along with the certified reference standards of some terpenes, and the linear retention indices reported and consulted in the existing literature [56][57][58][59][60].As an important point, some constituents that could not be unequivocally identified, information about their molecular ions (M +⋅ ) and base peaks (BP-100% intensity) are reported in Table 1.

Cluster analysis of data
To carry out Cluster analysis with the data, Statistica software (version 10, data analysis software system, StatSoft, Inc.) was used to establish the best correlation between chemical compositions, methods of analysis and the samples of plant material used.

Uses for timber/non-timber forest products
The trunk woods of the four species from the Amazon Region, which are light-to-medium-heavy (0.613-0.666 g/cm 3 ) woods, have been used to make rafts, furniture and the core of lathed boards.In addition, the three species called amarillos/laurel (yellows/bay), A. panurensis, N. cuspidata and O. cymbarum, are exploited for flooring, finishing, carpentry and construction, according to Solórzano et al. [18] and León [61].On the other hand, the results of the mechanical properties (low-to-medium strength values), reported by Solórzano et al., determined the promising uses of these woods, i.e., for the manufacture of handicrafts, packaging, stowage, exterior/interior finishes, stairs, post/beams, roofing, musical instruments, toys, pencils, etc.
Even so, the promising uses of timber mentioned above, based on uncontrolled/excessive anthropogenic extractive activity on these large trees, could put at risk the populations of these four species, the forest cover of this region, as well as the associated ecosystems.For these reasons, it is necessary to find other alternative uses that allow sustainability but also provide them with high/ greater value (e.g., sources of new/known chemical substances that could be useful in medicine, food, flavorings/ perfumes/cosmetics, and pharmaceutical/agrochemical industries).

Chemical compositions of volatile fractions and extracts
An important aspect to consider in this work was the possibility that the volatile profile could be significantly changed/lost from the plant material over time.Nonetheless, the plant material used was dry wood (sapwood/ heartwood) composed mainly of lignocelullose and vegetable cells, which would influence the biosynthesis/ preservation/retention/release/function of some secondary metabolites (termed extractives and varying between 2% and 10%) over time and ecological environment; subsequently, they can be extracted and used.These metabolites include terpenoids (volatile and fragrant components), benzenoids (ester derivatives) and alkaloids in nature [62,63].Thus, the structural complexity (biopolymers) of the sapwood/heartwood does not allow the complete and fast release of secondary metabolites (volatile, semivolatile, and non-volatile); only those constituents (volatile) found at the surface level of the wood (or in the bark) could be partially lost.For the exhaustive extraction of all of them from wood, it is necessary to increase the surface area of the sample (transforming  it into very small particles, chips or pellets).This type of matrix ensures that secondary metabolites could be isolated/obtained later (days, weeks, and months) by different extraction methods.Some well-known examples from the scientific literature on the use of woods in commercial applications/uses (inner/outer bark, chips, shavings/flakes, sawdust or small pieces of wood) for the isolation of bio-ingredients or raw material of fragrant products for the perfumery and condiments/spices industries are cinnamon (C.zeylanicum), sandalwood (Santalum album), cedarwood (Juniperus virginiana), guaiac-wood (Bulnesia sarmienti), laurel (L.nobilis), balsam of Peru (Myroxylon pereira), camphor (C.camphora), ishpingo (Ocotea quixos), sassafras (O.cymbarum) and rosewood/bois de rose (Aniba rosaeodora, A. duckei) [5,[64][65][66].Furthermore, it is very important to clarify why ethyl acetate was chosen as the extraction solvent, as well as the non-equilibrium conditions used for the isolation of volatile fractions by HS-SPME.Thus, ethyl acetate was chosen as the solvent because of its intermediate polarity, low boiling point (77 °C, easy removal by distillation), ability to isolate both non-polar (preferably)/polar (with less preference) and low to relatively high-molecular weight molecules, low cost, low log P (log P 0.71-hydrophobicity/hydrophilicity), low water solubility (8.7%w/w), and low toxicity (non-toxic to the environment) [67,68].For HS-SPME, non-equilibrium conditions [69] were chosen and carried out due to the complexity of volatile mixture (very common circumstance for flavor and fragrances fields, > 20 constituents) with which the partition equilibrium varies constituent-to-constituent (different chemical nature) and therefore, there are different equilibria [70], and the occurrence of the competitive and displacement processes of terpenes during absorption when the temperature and time are higher than 50 °C and 30 min, respectively [52,53].
In the case of the chemical constituents (volatile/nonvolatile) of the wood of Colombian A. panurensis, they were different from those identified/recognized in the Brazilian species according to the reports mentioned above.However, Gottlieb and Kubitzi [75] reviewed/ studied the chemomarkers found in Aniba spp.from the Amazon area; the metabolites were styryl(aryl)-pyrones and benzyl (derivatives) benzoates, without neolignans.However, other authors [26,[76][77][78][79] reported that trunk wood of Aniba sp., A. burchellii and A. terminalis, besides containing esters derived from benzoic acid, had several piperonylbenzofuran neolignans.
Another interesting report was carried by Courtois et al. [82], who studied (by HS-SPME/GC-MS) the volatile organic compounds released by tropical trees (including Lauraceae) from French Guiana; these authors stated that the bark/leaves of A. panurensis mainly emitted α-/β-pinenes, p-cymene, limonene, β-caryophyllene/αhumulene, α-copaene/δ-cadinene and germacrene D, as volatile organic compounds [this chemical composition was also different from that of this work, but some monoterpenes coincided although in very low relative amounts (< 0.3%)].This type of compounds (terpenes) would have a defensive function against fungi/bacteria/ insects [83].
Cluster analysis applied to the data set allowed drawing a hierarchical tree (Fig. 3) with two principal groupings according to the similarities/differences between the chemical compositions of the four Lauraceae species.The figure showed that the chemical profiles for any extraction method used for A. panurensis trunk were similar, with some differences for EAE (Group I); in this case, the chemically predominant family was the benzenoid-type aromatic esters (88-94%).While the remaining compositions of the other species (N.cuspidata, O. cymbarum and O. myriantha) were closer to each other, which were gathered in Group II (where sesquiterpenoids predominated in their great majority).This result is very interesting/important because it agrees with what was reported by Rohwer [50], who stated that the genera Nectandra and Ocotea are closer and their names are interchangeable (as synonyms) in biological descriptions due to their similar morphological/botanical characteristics (they have four locules in the anthers), as well as certain chemomarkers.
Finally, as one of the most relevant aspects of this manuscript are the prospective uses of the main chemical constituents identified in the volatile fractions/extracts of the four Lauraceae species from the Colombian Amazon region, these were determined/compiled according to the reviewed science literature.Thus, some constituents have been mainly used as fragrance and flavoring ingredients (for perfumery, cosmetic and oral care products), food flavoring (for beverages), fixatives/solvents (for perfumery or chemical industry).Detailed information on these uses is included in Table S1, which contains the names of the 18 major components identified in the samples analyzed along with the type of compound, biological activities, toxicity, maximum permissible levels, industrial applications and interest for the industry.It is very important to highlight the potential that these individual constituents could have for uses/exploitation other than the industries mentioned above, but also, that the biological properties demonstrated by an individual compound could be altered when interacting with other constituents in a mixture (e.g., extracts) producing synergistic or antagonistic effects.As demonstrated, benzyl salicylate (1) was a good antifungal (MIC < 40-160 µg/mL for six dermatophytes) and insecticidal (non-/arthropods) substance, as well as anti-inflammatory and phytotoxic [110][111][112].Just as benzyl salicylate is widely used in the cosmetic/perfumery industries as an active ingredient/raw material, so is benzyl benzoate (2).In addition, this molecule has been applied topically as a treatment for scabies and lice; it presented antifungal, antimite, pediculicidal (dogs), repellent (ticks/chiggers/mosquitoes), oestregonic and phytotoxic (herbicidal) activities, and as an arthropod/non-arthropod pest control.This constituent was also a semiochemical against Euglossa spp./Euplusia spp./Megalopta spp.(attractant), Nematus prasinus (pheromone), Calindoea trifascialis (allomone) and Anomala octiescostata (kairomone) [110,111,[113][114][115].

Conclusions
It was reported that, (i) for the first time, the chemical composition of the volatile fractions and ethyl acetate extract of O. myriantha trunk; (ii) the high content of benzenoid-type aromatic esters in the wood of Colombian A. panurensis; (iii) the unusual presence of the aporphine alkaloids dicentrine and dehydrodicentrine in the N. cuspidata trunk.In addition, (iv) chemical similarity was found/evidenced between the wood of the trunks of Nectandra sp. and Ocotea spp.under study.Finally, (v) the most abundant chemical constituents identified as secondary metabolites (particular/uncommon) in the four species, which would have promising bioactivities and uses in the industry (based on the scientific literature reviewed), would allow recommending other non-timber use/exploitation for these trees, e.g., fragrance ingredients/raw materials, semiochemicals, insecticides, anticancer/antitumoral/antiparasitic/antimicrobial/antiviral/ anti-AChE/antinociceptive/vasorelaxing substances, etc., with which some sustainability strategies could be created along with productive projects (including closing the cycle-orange or circular economy).

Fig. 1
Fig. 1 Co-ocurrence diagrams according to the search equations used for the scientometric analysis.A non-timber forest products; B four species under study

a
Area normalization based on GC-FID analysis; R I-Cal : Retention index calculated; R I-Lit : Retention index of literature; SPME: Headspace-solid-phase micro-extraction; SDE: simultaneous distillation-extraction; SM: Simple maceration (ethyl acetate extraction); b Tentatively identified; c Compared to the respective certified reference standard; M +⋅ : Molecular ion; BP: Base peak (100% intensity); NI: not identified

100 Fig. 3
Fig. 3 Hierarchical tree acquired by Cluster analysis showing four groupings based on similarities/differences of the secondary metabolites identified in the trunks of A. panurensis (AP), N. cuspidata (NC), O. cymbarum (OC) and O. myriantha (OM)

Table 1
Chemical composition of secondary metabolites (> 1.0%) isolated/analyzed by HS-SPME/SDE/SM/GC-MS from wood trunks of four Lauraceae species