text new page (beta)
English (pdf)
Article in xml format
Article references
Automatic translation
Send this article by e-mail
Cited by SciELO 
Similars in
SciELO 
Permalink
From an ethnobotanical perspective, the assemblages of plants have evolved by human selection of local and nonindigenous plants that share certain characteristics, are effective remedies (Linares & Bye 1987) or have better nutritional, forage, and textile properties, among others. The local uses of these plants have constantly evolved due to the combination of biological, environmental, historical, socioeconomic, and cultural conditions. Mexican medicinal herbs are assorted into complexes. Each complex comprises plant species sharing common names, morphological and/or aromatic characteristics, and curative properties (Bye & Linares 2015).
Tamale (Spanish: tamal, singular; tamales, plural) is a traditional Mesoamerican dish made of maize-based dough (sweet, salty, or sour) steamed in different kinds of leaves as a wrapping. In Mexico, the fresh and dry corn husk and the banana leaves are the most popular materials used for tamales. Nonetheless, in the state of Veracruz, other leaves of numerous species from wild origin are traditionally used in the fresh stage for this purpose. For example, Calathea spp., Heliconia spp., Pimenta dioica (L.) Merr. Renealmia mexicana Klotzsch ex Petersen, and Stromanthe macrochlamys (Woodson & Standl.) H. A. Kenn & Nicolson, among others (Lascurain-Rangel et al. 2017). Its use is due to the taste and aroma impregnated in the tamale's dough after cooking, which is highly appreciated by local consumers (Lascurain-Rangel et al. 2022).
In central Veracruz, Mexico, (Figure 1) the leaves of the genus Oreopanax Decne. & Planch. (Araliaceae), are used as wrappers for tamales called “xocos” or “chocos”, with their characteristic smell and taste. The word “xoco” or “choco” means acid or sour, so the local name of this type of tamales may derive from its way of preparation because sometimes the dough is left to ferment slightly. The xocos can be sweet or salty, and the leaf has a characteristic and unique flavor that is neither bitter nor acidic.
So, we have considered that the Oreopanax species form a food complex: Oreopanax capitatus (Jacq.) Decne. & Planch., Oreopanax echinops (Schltdl. & Chm.) Decne., and Oreopanax flaccidus Marchal (Figure 2). Mainly in the municipalities of Coatepec, Xalapa, Naolinco, Tlacolulan, Banderilla, Xico, and Chiconquiaco, they are not considered properly tamales because their dough has no filling; hence they have been considered a side dish that accompanies meals based on moles (a traditional Mexican dish).
Figure 2 Leaves and tamales xocos. A) Oreopanax equinops, B) Oreopanax flaccidus, C) Oreopanax capitatus.
The genus Oreopanax is distributed in Tropical America and has approximately 75 to 80 species, of which 13 have been registered for Mexico (Villaseñor 2016) and seven for Veracruz (Sosa 1979). They are evergreen trees or shrubs, often epiphytic and dioecious. In this work, we selected to study leaves of three species of the Oreopanax complex used in Veracruz to prepare tamales: 1) O. capitatus is commonly known as mata palo, jaboncillo, hoja de caballero, cabellera de palo, chico, and coamatl. It presents a wide altitude range compared to the other species and is distributed from South America, the Caribbean, and Central America. 2) O. echinops known as choco, hoja de queso, and cinco hojas (Sosa 1979, Lascurain-Rangel et al. 2017), is distributed in Mexico and Central America, and 3) O. flaccidus is rarely used and collected from wild conditions. This species is not sold in local markets, is considered rare and endemic to Mexico (Villaseñor 2016) and has recently been assessed for The IUCN Red List of Threatened Species in 2020, listed as Endangered (Fuentes et al. 2020). Table 1 summarizes the distribution and some botanical features of the Oreopanax species included in this study.
Table 1 Taxa, leaves, pubescence, distribution, altitude, and type of vegetation of xoco tamales food complex (Oreopanax spp.) (Sosa 1979).
| Taxa | Leaves | Pubescence | Distribution | Altitude m asl | Type of vegetation |
|---|---|---|---|---|---|
| O. capitatus | Leaves entire, obovate, oblong, oblong elliptical, 6-25 cm long by 6-18 cm wide | Glabrous, rarely with scattered pubescence | Chiapas, Hidalgo, Oaxaca, Puebla, Tabasco, and Veracruz | 125-1,850 | Deciduous forest; high, medium, or low evergreen forest; secondary forest |
| O. echinops | Palmate-compound leaves, the young sometimes trilobate, 3-7 leaflets, sessile, elliptical to obovate, 9-26 cm long by 10-19 cm wide | Upper and lower side with stellate pubescence | Chiapas, Mexico City, Colima, Durango, Guerrero, Hidalgo, Jalisco, Michoacán, Oaxaca, Puebla, Sinaloa, and Veracruz | 1,200-1,650 | Oak; deciduous forest; pine; secondary vegetation |
| O. flaccidus | Simple, ovate, or elliptical ovate leaves, 17-25 cm long by 7-15 cm wide | Upper surface scabrous and papillose, underside densely pubescent | Hidalgo, Oaxaca, Puebla, and Veracruz | 2,320 | Pine and oak forest |
To contribute to the differentiation between the species belonging to a complex of medicinal plants, various studies have been carried out, such as the chemical composition and the characterization of their biological effects. For example, in Mexico, species of the genus Agastache (Lamiaceae) have been evaluated for their toxic and curative effects associated with species recognition (Estrada-Reyes et al. 2004, 2014, Ventura-Martínez et al. 2017). Ethnobotanical, morphophysiological, and phytochemical evidence distinguishes two evolutionary processes in the differentiation of Agastache from the lemon balm complex (Carrillo-Galván et al. 2020). On the other hand, Psacalium radulifolium (Kunth) H. Rob. & Brettell forms the matarique complex, which includes several species of Asteraceae (Linares & Bye 1987) and was studied for its antimicrobial activity by Garduño-Ramírez et al. (2001). Finally, the copalchi complex is used to control glycemia in diabetes, which includes several species of the Rubiaceae and Euphorbiaceae families, particularly the Hintonia and Exostema genera. These species have been studied chemically and pharmacologically to contribute to quality control procedures and to identify botanical products made with these plants (Mata et al. 1990, Cristians et al. 2014, 2018, Rivero-Cruz et al. 2019).
However, the studies referring to the food complexes of Mexico are still scarce compared to those species used in traditional medicine as remedies. Among these, the Quintonil complex of the quelites group stands out, which groups twelve species of Amaranthus throughout the national territory, being the most consumed the quelites: A. hybridus L., A. retroflexus L., A. palmeri S. Watson, A. powellii S. Watson, A. dubius Mart. ex Thell., A. spinosus L., A. leucocarpus S. Watson, A. blitoides S. Watson, and A. watsonii Standl. (Mapes et al. 1997, Mapes Sánchez et al. 2012, Linares & Bye 2020). Depending on the geographical area, the species, and the culture, the quelites have different names such as: quintonil, red quintonil, donkey quelite and water quelite, among others. Quintoniles are highly appreciated and sold in the local markets; as fresh, aged, or dehydrated products in the case of Chihuahua state.
The main goal of this study is to describe the ethnobotanical food complex of xocos (O. capitatus, O. echinops, and O. flaccidus), their properties in terms of the chemical composition of their essential oils, and the anatomical characteristics of leaves used as wrapping for tamales in Veracruz that may contribute to their differentiation from the complex.
Materials and methods
Plant materials. Leaves of O. capitatus and O. echinops species were recollected on January 25 and 26, 2021 in the Clavijero Botanic Garden of the Instituto de Ecología, A. C. in Xalapa municipality, Veracruz; at Latitude 19° 40′ 39″ N; Longitude 97° 01′ 07″ W, elevation 1,400 m asl. Leaves of O. flaccidus were recollected in the locality of Atapalchico, Tlacolulan municipality, Veracruz, 3 km from Tlacolulan town; Latitude 19° 40′ 39″ N; Longitude 97° 01′ 07″ W, elevation 1,813 m asl.
Voucher samples: The botanical materials for O. capitatus (I. Acosta, 4044), O. echinops (I. Acosta, 4045 & 4046), and O. flaccidus (I. Acosta, 4047) were deposited at the Herbarium XAL of the Instituto de Ecología, A. C. (Xalapa, Veracruz, Mexico). Sampled leaves came from four individuals of each species in two localities.
Extraction (essential oils preparation). All the chemicals used in this procedure were purchased directly from Sigma-Aldrich (St. Louis, MO, USA) and were used without additional purification. The essential oil from the freshly aerial parts of each species was obtained by a hydrodistillation process. Fresh plant material was ground briefly in a food processor (Nutribullet®). Afterwards, 250 g were transferred to a 1 L flat bottom flask equipped with a magnetic stirrer and distilled water (600 mL) was added. The material was hydrodistilled for three hours once boiling started. The distilled liquors were transferred to a separatory funnel, and the aqueous phase was extracted with dichloromethane twice (100 mL × 2). The organic layers were collected, combined, and filtered through anhydrous Na2SO4. The solvent was evaporated in a rotatory evaporator (R-II, BÜCHI, Flawil, Switzerland) at 600 mbar, 25-30 °C. The oily residue was weighted, and yield was obtained as follows (Soto-Armenta et al. 2017):
where:
Y: |
yield (%) |
M: |
mass of the obtained essential oil (g) |
M 0: |
initial amount of the plant material (g) |
The essential oils were stored at -20 °C until further analysis; four replicates were prepared for each species.
Analysis by Gas Chromatography Coupled to Mass Spectrometry (GC-MS). One microliter of each essential oil was injected into the GC port, and the chromatographic separation and analysis were carried out in a gas chromatograph coupled to a mass analyzer (Shimadzu, Single Quadrupole QP2010 Ultra) as previously described by Lascurain-Rangel et al. (2018). Briefly, helium gas was used as carrier gas (1.2 mL/min, constant flow), and a ZB-5MSi column (30 length × 0.25 mm inner diameter × 0.25 µm film thickness; Zebron, Phenomenex Inc.) was used as a stationary phase. A split injector at a 16.7 rate and temperature of 250 °C was used to introduce the sample to the GC column. GC oven temperature for compound separation was adjusted at an initial temperature of 50 °C was held for 4 minutes, then it increased at a rate of 15 °C/min up to 250 °C, which was held for 5 minutes. The MS was operated in electron impact (EI, 70 eV) mode with a source temperature of 230 °C, interface temperature of 250 °C, and a continuous scan from 30 m/z to 500 m/z. The mass spectrum data of volatile compounds present in the Oreopanax oils were compared with those in the NIST/EPA/NIH Mass Spectral Library, NIST 11, Software version 2.0 (National Institute of Standards and Technology, www.nist.gov), using a range of 84-100 % similarity values, with the Lab solutions GCMS solutions 2.72 software (Shimadzu, Japan) and by co-elution with authentic standards under the same analytical conditions above described. The analysis of essential oils was carried out in quadruplicate, and all standards used for comparison were purchased in Sigma-Aldrich (St. Louis, MO, USA) at a GC grade purity (> 95 %). With the spectrometric dataset of the three species (m/z_Rt pair values) and NIST identification, a heatmap with hierarchical ordering was constructed using the Metaboanalyst bioinformatics platform (https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml) for comparative purposes. Raw spectrometric data were Log (10) transformed, autoscaled, and normalized by quantiles.
Leaf anatomy study. Sampling.- To study the leaf anatomy of the Oreopanax leaves, sections of approximately 0.5 × 0.5 cm were taken from each leaf, from the center to the left margin, and at the apex. Those sections were fixed in a mixture of formaldehyde, acetic acid, and 70 % alcohol (5:5:90 per volume) for several days. Then, samples were washed in water several times until the smell of formaldehyde became imperceptible.
Dehydration.- Samples were dehydrated in a Tertiary Butyl Alcohol (TBA) series (Ruzin 1999) till reaching pure TBA.
Paraffin embedding.- Samples were transferred to glass vials with pure TBA, and a few paraffin shavings were added. The vials were kept at room temperature. When the TBA dissolved the paraffin shaves, more shaves were added. This procedure was repeated three more times, and then the vials were put in an oven at 60 oC for 24 hours. The mixture of paraffin-TBA was discarded, and fresh, melted paraffin was added to each vial. After two changes in pure paraffin, molds were cast with two or three sections in each mold, following the procedure of Ruzin (1999). After trimming the paraffin molds and mounting them on a wooden support, sections 18-20 µm thick were obtained with a rotary microtome Leica RM 2125RTS using a disposable blade.
Staining.- The paraffin with the tissue sections was removed with 100 % xylol, then, tissues were rehydrated by passing them through a series of decreasing ethanol concentrations down to pure water. Sections were stained with 0.05 % methyl blue in water for 2-3 hours. Afterward, sections were washed in three water changes and dehydrated in increasing series of ethanol, up to 100 %. Then, sections were transferred to a mixture of equal parts of ethanol and methyl salicylate (as a clearing agent). After two changes in pure methyl salicylate, sections were mounted with synthetic resin dissolved in xylene and covered with a glass coverslip of 2.5 × 4 cm. Images of the most remarkable features were taken with a microscope Nikon Eclipse E600, with bright field or ultraviolet light.
Results
Chemical characterization. Given that consumers value traditional xoco tamales because of the characteristic flavor provided by the leaves of Oreopanax, the study of the chemical composition of the leaves included the analysis of the essential oils to identify distinctive metabolites associated with each species contributing to the differentiation of this food complex. The extraction by hydrodistillation allowed us to obtain the essential oil from each species of Oreopanax as colorless oils with a spiced but pleasant odor. In Table 2, it can be observed that the three species analyzed presented a similar yield (%) of essential oil. So, there were no differences in terms of essential oil abundance that could be associated with a given species.
Table 2 Amounts and yields of essential oils obtained from the hydrodistillation of fresh aerial parts of three species of Oreopanax.
| Species | Essential oil (mg) | Appearance | Yield * |
|---|---|---|---|
| O. capitatus | 22.3 | Colorless oil | 0.009 ± 0.0 |
| O. flaccidus | 21.8 | Colorless oil | 0.009 ± 0.0 |
| O. echinops | 19.0 | Colorless oil | 0.008 ± 0.0 |
Notes: *Essential oil yield is expressed as average (n = 4) in percentage w/w ± the standard deviation.
Later, the same essential oils from Oreopanax spp. were analyzed by GC-MS combined with co-elution with a set of authentic standards to increase the accuracy of some identifications. From these analyses, a total of 44 volatile compounds were identified in the leaves of Oreopanax spp. (Table 3), based on their spectrometric fingerprints compared with the reference compounds or with those reported in the NIST database. As expected for those plants used as spices in traditional food, chemical composition was complex in the three studied species and among the compounds found in their leaves as it can be observed in the corresponding chromatograms (Figure 3). We are reporting for the first time the presence of several aliphatic and aromatic alcohols and terpenoids (mono, sesqui, and diterpenes) as some of the major volatiles in xoco leaves.
Table 3 Volatile composition by GC-MS of the essential oils from leaves of Oreopanax spp.
| No. | Chemical name | RT (min) | S (%) | RA (%) | Chemical class | ||
|---|---|---|---|---|---|---|---|
| O. flaccidus | O. echinops | O. capitatus | |||||
| 1 | 3-Methyl-1-butanol | 3.13 ± 0.00 | 100* | 1.95 ± 1.22 | ND | 1.11 ± 0.52 | Aliphatic alcohol |
| 2 | 3-Hexenol | 5.59 ± 0.21 | 100* | 4.59 ± 5.31 | 1.25 ± 0.84 | 6.53 ± 0.99 | Aliphatic alcohol |
| 3 | 2-Hexenol | 5.78 ± 0.01 | 96 | 0.68 ± 0.30 | 0.72 ± 0.31 | 0.67 ± 0.48 | Aliphatic alcohol |
| 4 | 1-Hexanol | 5.87 ± 0.02 | 100* | 3.97 ± 3.17 | 2.08 ± 1.26 | 9.03 ± 2.15 | Aliphatic alcohol |
| 5 | Butyl glicol | 6.50 ± 0.00 | 93 | 0.37 ± 0.26 | 0.23 ± 0.20 | ND | Aliphatic alcohol |
| 6 | 6-Hepten-1-ol | 7.50 ± 0.00 | 93 | ND | 0.90 ± 0.40 | ND | Aliphatic alcohol |
| 7 | Phenol | 7.73 ± 0.00 | 94 | 1.10 ± 0.52 | ND | 0.65 ± 0.47 | Phenol |
| 8 | 1,2-Epoxycyclooctane | 8.02 ± 0.00 | 88 | ND | 0.65 ± 0.28 | ND | Epoxide |
| 9 | 2-Methylenecyclohexanol | 8.36 ± 0.00 | 88 | 0.41 ± 0.40 | ND | 1.43 ± 0.32 | Alcohol |
| 10 | Benzyl alcohol | 8.58 ± 0.00 | 100* | 2.63 ± 0.58 | 3.15 ± 1.70 | 4.17 ± 1.12 | Aromatic alcohol |
| 11 | Benzeneacetaldehyde | 8.73 ± 0.00 | 98 | 1.39 ± 0.27 | 0.88 ± 0.56 | 1.04 ± 0.71 | Aromatic aldehyde |
| 12 | (Z)-4-Decen-1-ol | 8.94 ± 0.00 | 86 | ND | 0.34 ± 0.23 | 0.38 ± 0.26 | Alcohol |
| 13 | trans-Linalool oxide | 9.10 ± 0.00 | 100* | 4.08 ± 1.62 | 1.59 ± 0.26 | 3.96 ± 0.84 | Monoterpene |
| 14 | cis-Linalool oxide | 9.29 ± 0.00 | 100* | 2.86 ± 1.20 | ND | 3.04 ± 0.46 | Monoterpene |
| 15 | Linalool | 9.45 ± 0.01 | 100* | 16.30 ± 3.65 | 8.18 ± 1.76 | 21.13 ± 3.54 | Monoterpene |
| 16 | Phenylethyl alcohol | 9.62 ± 0.00 | 89 | 2.80 ± 0.42 | 1.32 ± 0.98 | 3.12 ± 0.57 | Aromatic alcohol |
| 17 | (E)-(3,3-Dimethylcyclohexylidene)-acetaldehyde | 9.87 ± 0.00 | 85 | 0.58 ± 0.41 | ND | ND | Aldehyde |
| 18 | p-Cymen-8-ol | 10.48 ± 0.00 | 93 | 6.38 ± 0.43 | ND | ND | Monoterpene |
| 19 | α-Terpineol | 10.59 ± 0.00 | 100* | 7.07 ± 1.03 | 2.13 ± 0.83 | 5.79 ± 0.59 | Monoterpene |
| 20 | Bornyl alcohol | 10.65 ± 0.00 | 88 | 2.68 ± 0.71 | ND | ND | Monoterpene |
| 21 | Nerol | 10.85 ± 0.00 | 100* | 5.45 ± 0.68 | ND | 3.51 ± 0.23 | Monoterpene |
| 22 | Geraniol | 11.10 ± 0.00 | 100* | 5.76 ± 0.50 | 1.77 ± 0.85 | 3.94 ± 0.35 | Monoterpene |
| 23 | Eugenol | 12.14 ± 0.00 | 100* | 4.74 ± 2.59 | 20.55 ± 3.90 | ND | Phenylpropanoid |
| 24 | α-Copaene | 12.44 ± 0.00 | 100* | ND | 2.72 ± 2.42 | ND | Sesquiterpene |
| 25 | Dihydrodehydro-beta-ionone | 12.68 ± 0.00 | 88 | 0.68 ± 0.07 | ND | ND | Ketone |
| 26 | Caryophyllene | 12.87 ± 0.00 | 100* | ND | 3.21 ± 3.80 | ND | Sesquiterpene |
| 27 | β-Ionone | 13.31 ± 0.00 | 100* | ND | 2.89 ± 1.98 | 0.88 ± 0.92 | Ketone |
| 28 | 2,4-Di-tert-butylphenol | 13.48 ± 0.00 | 95 | 11.88 ± 3.84 | 13.99 ± 1.54 | 8.50 ± 1.09 | Aromatic alcohol |
| 29 | δ-Cadinene | 13.67 ± 0.00 | 92 | ND | 6.54 ± 4.37 | 1.81 ± 2.66 | Sesquiterpene |
| 30 | trans-Nerolidol | 13.95 ± 0.77 | 100* | ND | ND | 0.81 ± 0.77 | Sesquiterpene |
| 31 | 1-Heptadecene | 14.17 ± 0.00 | 91 | 0.81 ± 0.33 | ND | 0.61 ± 0.41 | Unsaturated hydrocarbon |
| 32 | Spathulenol | 14.21 ± 0.00 | 90 | 2.40 ± 1.17 | 5.48 ± 1.74 | 8.11 ± 1.01 | Sesquiterpene |
| 33 | Caryophyllene oxide | 14.28 ± 0.00 | 100* | ND | 2.58 ± 0.56 | ND | Sesquiterpene |
| 34 | (-)-Globulol | 14.29 ± 0.00 | 92 | 0.93 ± 0.79 | ND | 2.04 ± 1.36 | Sesquiterpene |
| 35 | (-)-Spathulenol | 14.63 ± 0.00 | 85 | 1.25 ± 1.04 | 8.19 ± 1.00 | 4.52 ± 0.93 | Sesquiterpene |
| 36 | (-)-δ-Cadinol | 14.72 ± 0.00 | 84 | ND | 2.51 ± 0.99 | ND | Sesquiterpene |
| 37 | α-Cadinol | 14.80 ± 0.00 | 84 | ND | 0.74 ± 0.52 | 2.00 ± 0.27 | Sesquiterpene |
| 38 | 1-Nonadecene | 15.69 ± 0.00 | 97 | 0.87 ± 0.34 | ND | ND | Unsaturated hydrocarbon |
| 39 | cis-1-Chloro-9-octadecene | 15.70 ± 0.00 | 88 | ND | 2.24 ± 0.86 | 1.16 ± 0.79 | Halogenated hydrocarbon |
| 40 | n-Hexadecanoic acid | 16.82 ± 0.00 | 92 | 0.71 ± 0.64 | ND | ND | Organic acid |
| 41 | Behenic alcohol | 17.07 ± 0.00 | 97 | 0.76 ± 0.34 | 0.75 ± 0.52 | ND | Long-chain alcohol |
| 42 | (-)-Kaurene | 17.74 ± 0.00 | 89 | ND | 2.34 ± 1.99 | ND | Diterpene |
| 43 | 1-Heptacosanol | 18.48 ± 0.00 | 96 | 0.42 ± 0.32 | ND | ND | Long-chain alcohol |
| 44 | 1,3-Benzenedicarboxylic acid, bis(2-ethyl-hexyl) ester | 20.13 ± 0.10 | 92 | 3.42 ± 2.32 | ND | ND | Aromatic ester |
Notes: RT represents the retention time expressed in minutes. RA, represents the relative peak area (relative area concentration) of the different compounds detected for each essential oil, expressed as percentage. Data are presented as the average (n = 4) ± standard deviation (S.D.). S (%), means similarity percentage. The tentative names of detected compounds were annotated according to NIST/EPA/NIH Mass Spectrometry library 2014 (National Institute of Standards and Technology, www.nist.gov), using a range of 84-100 % similarity values, with the Labsolutions GCMSsolutions 2.72 Software. *Compounds identity confirmed by co-elution with authentic standards.
Figure 3 Comparative analysis of volatile profiles obtained by GC-MS with the essential oils of xocos leaves. A) Oreopanax equinops, B) Oreopanax capitatus, C) Oreopanax flaccidus.
To distinguish among the species of the xoco complex, the essential oils obtained by GC-MS were ordered hierarchically in a heatmap (Figure 4). The heatmap revealed two well-defined clusters among species, O. echinops being the most distinct species when compared to O. capitatus and O. flaccidus. O. echinops contained the most significant accumulation (major abundance) of a total of 10 volatiles that were absent in the other species and includes β-ionone, δ-cadinene, (-)-kaurene, α-copaene, caryophyllene, among others. These metabolites were classified as sesquiterpenoids and could be considered as distinctive chemical markers in this species. Interestingly, O. capitatus and O. flaccidus were more similar and share approximately 17 volatiles that were absent in O. echinops, such as acetaldehyde, phenol, 1-heptadecene, nerol, α-terpineol, geraniol, linalool, being some of these metabolites from a monoterpenoid origin. However, although O. capitatus and O. flaccidus were more like each other, they both had also distinctive compounds. For example, the essential oil of O. capitatus contained five compounds [(-)-spathulenol, cis-1-chloro-9-octdecene, trans-neorolidol, α-cadinol and (Z)-4-Decen-1-ol] that does not contained the essential oil of O. flaccidus, and this comprised seven compounds (1-heptacosanol, bornyl alcohol, dihydrodehydro-β-ionone, p-cymen-8-ol,1-nonadecene, n-hexadecanoic acid, and 1,3-benzenedicarboxylic acid) that were not present in the essential oils of the O. capitatus and O. echinops (Figure 4).
Figure 4 Heatmap with hierarchical clustering of Oreopanax spp. The plot was built using the spectrometric dataset obtained by GC-MS. In X-axis the replicates of each essential oil per species are indicated, and Y-axis contains the names of the volatile compounds identified by co-analysis with standards or through NIST database assignments. The three species are colored-coded by the three classes labeled at the top of the heatmap.
Leaf anatomy. Leaves of Oreopanax spp. shared some anatomical features (Table 4): They were hairy (except O. capitatus, which was glabrous) in the abaxial surfaces, with long, three-to-four-branched trichomes (Figures 5A, B). The most remarkable common feature was the presence of resin canals along the veins and in the cortical parenchyma of the main veins (Figures 6A-F). Those canals were lined with six to eight epithelial cells (Figure 6D) in one or two layers and had diameters from 15.5-23 µm in the midvein and 15-25 µm in minor veins. In the lamina, resin canals were formed in connection with the minor veins, on the top and at the bottom of each vein, between the palisade parenchyma (top) or between the spongy parenchyma (bottom) (Figure 6E). In the midvein, the resin canals were formed in the cortex (Figures 6B, D) or between the vascular bundles (Figure 6C). Lamina thickness varied from 430 to 580 µm. O. capitatus was the thickest with 580 µm, O. flaccidus had 520 µm, while the thinner was O. echinops, with only 430 µm of thickness. The thickest parts of each leaf were the midveins of the three species.
Table 4 Comparative anatomical features of three Oreopanax species leaves. Characteristics of the lamina, resin canals in the main vein and venules in the species of xoco tamales (Oreopanax spp.) food complex.
| Epidermis | Lamina | Resin canals in main vein | Resin canals in venules | |||||
|---|---|---|---|---|---|---|---|---|
| Species | Trichomes present | Thickness (µm) | Number of epithelial cells | Diameter 1 (µm) | Diameter 2 (µm) | Number of epithelial cells | Diameter 1 (µm) | Diameter 2 (µm) |
| O. echinops | Yes (articulated) | 436 | 6.2 | 19.2 | 18.2 | 6.0 | 25.3 | 22.5 |
| O. capitatus | No | 581 | 5.9 | 23.0 | 20.8 | 5.6 | 17.0 | 16.3 |
| O. flaccidus | Yes (articulated) | 520 | 6 | 20.0 | 15.5 | 6.6 | 18.3 | 15.4 |
Figure 5 Leaf surfaces of Oreopanax leaves. A) O. echinops, adaxial surface. B) O. flaccidus, adaxial surface. C) O. capitatus, abaxial surface. A) and B) show articulated trichomes (arrows), while C) is glabrous. Scale bars: 10 mm.
Figure 6 A) O. echinops midvein, cross-section. Resin ducts look like dark areas (red arrows) under UV light. Lignin fluorescence is orange/red in the xylem secondary walls; the fibers forming the sheath surrounding the vascular tissue fluoresce in green. B) O. flaccidus cross-section through midvein. Bright field. Most of the canals form in the cortical tissue, both in the abaxial (low arrow) and adaxial (upper arrow) sides (arrows). Smaller canals can be observed in the center of the midvein (yellow arrow). C) O. capitatus midvein in cross-section. Resin ducts are associated with the phloem rays (arrows). D) O. echinops. Detail of two large resin canals formed by six to eight epithelial cells (arrows). E) O. flaccidus. Lamina cross-section through a minor vein. An upper canal can be seen (arrow) in close contact with the palisade parenchyma. F) O. flaccidus. Cross-section of the lamina comprising a portion of the midvein, showing several resin canals (arrow). Scale bars: A) = 300 μm; B) = 500 μm; C) = 200 μm; D) 25 μm; E) = 50 μm; F) = 260 μm.
Discussion
Our chemical analysis and anatomical studies of the Oreopanax of the xoco complex indicate that these three species share volatile compounds. However, they also have important differences in their volatile profiles that could be related to their flavor and distinguishable by local consumers of xoco tamales. The resin canals of O. capitatus and O. flaccidus are formed by one to two layers of epithelial cells. These three species have in common the formation of resin canals along the minor leaf veins, or in the cortex of midveins.
The extraction by hydrodistillation indicates no differences in essential oil content that could be associated with a given species. The essential oils from the leaves of Oreopanax spp. were analyzed by GC-MS, indicating a total of 44 volatile compounds among these species, highlighting the presence of several aliphatic and aromatic alcohols, sesqui and monoterpenoids as some of the most significant volatiles in xoco leaves.
It is well known that essential oils from medicinal and food plants are related to a wide spectrum of biological activities, and industrial and technological applications, this is mainly because of their volatile compounds’ composition. Table 5 summarizes the bioactivities reported for the major distinctive volatiles found in each Orepanax species. This information provides added value to the use of xoco leaves to traditional cuisine since these oils contain compounds that are beneficial for the consumer's health.
Table 5 Biological activities and applications reported for selected volatiles present in Oreopanax species leaves.
| Species | Major compounds detected | Reported uses and applications | Reference |
|---|---|---|---|
| O. echinops | β-Ionone | Attractant, repellant, anti-inflammatory, antifungal, antitumoral | Parella et al. 2021 |
| δ-Cadinene | Anti-inflammatory, anticancer, antiparasitic, antioxidant | Egas et al. 2015, Alves-Silva et al. 2023 | |
| (-)-Kaurene | Antitumor, antibacterial, antiviral, anti-inflammatory | Ding et al. 2017 | |
| α-Copaene | Cytotoxic, cytogenetic, antioxidant, anti-inflammatory, insect attractant | Turkez et al. 2014, Liu et al. 2022 | |
| Caryophyllene | Neuroprotective, anti-inflammatory, antimicrobial, gastroprotective, anticancer | Machado et al. 2018 | |
| O. flaccidus | Dihydrodehydro-β-ionone | Insect attractant, flavor, and fragrance in the food industry | Parella et al. 2021, Qi et al. 2022. |
| 1-Heptacosanol | Hypocholesterolemic | Martínez et al. 1999. | |
| Bornyl alcohol | Drug carrier, antimicrobial, anti-inflammatory, additive in cosmetic and perfume manufacturing | Zielinska-Błajet & Feder-Kubis 2020, Kulkarni et al. 2021 | |
| p-Cymen-8-ol | Antifungal | Kürkçüoglu et al. 2006, | |
| 1-Nonadecene | Antifungal | Khan & Javaid 2021 | |
| O. capitatus | (-)-Spathulenol | Immunomodulatory, antioxidant, anti-inflammatory, antiproliferative, antimycobacterial, antitumoral, larvicidal, analgesic | Dos Santos et al. 2022, Ziaei et al. 2011, do Nascimento et al. 2018, Mathew & Thoppil 2011 |
| t-Neorolidol | Antileishmanial, flavoring agent, antineoplastic, antimalaria, antiulcer, anti-inflammatory, analgesic, antifungal, antioxidant, fragrance ingredient | Arruda et al. 2005, Lee et al. 2007, McGinty et al. 2010, Klopell et al. 2007, Fonseca et al. 2016 | |
| α-Cadinol | Insecticidal, antitumor, antifungal | He et al. 1997, Chang et al. 2001, 2008 |
The odor profiles of essential oils obtained by GC-MS revealed two well-defined clusters among species (Figure 3, O. echinops being the most distinct species when compared to O. capitatus and O. flaccidus. Oreopanax echinops contains the greatest accumulation of 10 volatiles that are absent in the other species and includes β-ionone, δ-cadinene, (-)-kaurene, α-copaene, and caryophyllene, among others. These compounds are classified as sesquiterpenoids and could be considered distinctive chemical markers in this species. Sesquiterpenoids are biosynthesized by the mevalonic acid pathway that occurs mainly at the cytosol in plant cells (Dewick 2009). Interestingly, O. capitatus and O. flaccidus are more similar and share approximately 17 volatiles that are absent in O. echinops, such as acetaldehyde, phenol, 1-heptadecene, nerol, α-terpineol, geraniol, linalool, among others. These volatiles are monoterpenoid-type compounds, whose biosynthetic origin is throughout the 1-deoxy- D-xylulose 5-phosphate (DXP) or non-mevalonate pathway that takes place at the chloroplast level in plants (Dewick 2009), suggesting a noticeable difference in the active biosynthetic and enzymatic machinery among the studied xoco plants. However, although O. capitatus and O. flaccidus are more similar to each other, they both have also distinctive compounds. Perhaps, conducting deeper molecular studies on these xoco species such as transcriptomics targeting the expression levels of gene clusters for key enzymes in the mevalonic acid biosynthesis such as 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase in O. echinops or DXP synthase for O. capitatus and O. flacciuds will allow corroborating these metabolic differences. Untargeted metabolomics using total crude extracts from leaves instead of essential oils could be also useful in distinguishing these species. Nonetheless, to the best of our knowledge, there are no reports of the traditional use of xoco leaves in the form of powder or ground material as occur for other spices.
Leaves of Oreopanax spp. share some anatomical features; for instance, they are hairy (except O. capitatus, which is glabrous) on both surfaces, with long, three-to-four-branched trichomes. The most remarkable common feature is the presence of resin canals along the veins and in the cortical parenchyma of the main veins. In the lamina, resin canals are formed in connection with the minor veins, on the top and bottom of each vein, between the palisade parenchyma or the spongy parenchyma. In the midvein, the resin canals are formed in the cortex or between the vascular bundles. Lamina thickness varies: O. capitatus is the thickest, with 580 µm, O. flaccidus 520 µm, and O. echinops 430 µm.
Mexican xoco tamales food complex (Oreopanax spp.) has O. capitatus as the signature species (Linares & Bye 1987) that characterizes this ethnobotanical complex because it is the most common one in regional markets as well as the preferred species. The taxa O. echinops and O. capitatus are used and traded in the same distribution range, at least in central Veracruz, except for O. flaccidus, which has not been observed for local sale (Lascurain-Rangel et al. 2017). So far, it is unknown if there are substitutes for these species by other local ones that could be included in this complex. Studies will be necessary to identify the uses of Oreopanax leaves in other regions of the country.
