Composition of the Essential Oils from Flowers and Leaves of Vervain [Aloysia triphylla (L’Herit.) Britton] Grown in Portugal
Santos-Gomes, Paula C
A total of 63 compounds were identified in hydrodistillates from flowers and leaves of vervain plants [Aloysia triphylla (L’Herit.) Britton] grown in Portugal. The three main essential oil compounds present either in flowers or in leaves were geranial (26.8-38.3%), neral (20.8-29.6%), and limonene (5.7-20.6%). 1-Octene, 1-octen-3-ol, p-cymene, (Z)-β-ocimene, and trans-carveol, identified in the flower oil, were not found in the leaf oil of the same plant. However, p-cymene and trans-carveol were found in the leaf oils of young cultivated plants. On the other hand, β-citronellene, β-pinene, neryl acetate, and trans-calamenene, present in the leaf oils, were not found in the flower oils. The main compound groups and percentages found in oils were monoterpene hydrocarbons (29.9 and 22.3%), oxygen-containing monoterpenes (56.9 and 59.0%), sesquiterpene hydrocarbons (6.5 and 6.8%), and oxygen-containing sesquiterpenes (3.0 and 4.8%) (flowers and leaves, respectively). The percentage of the total oxygen-containing monoterpenes, especially aldehydes, in the leaf oils of young cultivated plants, increased from July to December in inverse correlation with that of monoterpene hydrocarbons. The comparison with previous studies performed by other authors points to a significant variation in the chemical composition of vervain oil depending from the origin of the plants.
Key Word Index
Aloysia triphylla, Verbenaceae, lemon verbena, vervain, essential oil composition, limonene, neral, geranial.
Vervain- or lemon verbena [Aloysiatriphylla (L’Herit.) Britton, A. citriodora (Cav.) Ort.,Lippia citriodora (Ort.) HBK, L. triphylla O. Kze., Verbena citriodora Cav., V. triphylla L’Herit.] ( Verbenaceae)- grows spontaneously in South America (Chile, Argentina, Uruguay and Peru) and was introduced in Europe at the end of the 17th century. Since then, it has been cultivated in some countries of southern Europe, namely Spain and Italy, and countries in North Africa, namely Morocco and Tunisia, and India as well. In these areas, the leaves are largely used in herbal teas for their aromatic, digestive and antispasmodic properties. In the United States, vervain is listed as Generally Regarded As Safe (GRAS) for human consumption in alcoholic beverages.
The traditional ethnobotanical applications of vervain include the respective uses as folk remedy in treatments of asthma, spasms, cold, fever, flatulence, colic, diarrhoea, indigestion, insomnia, and anxiety (1-3). In a recent review on the traditional uses, chemistry and pharmacology of the Lippia genus, vervain was reported as being used as analgesic, antiinflammatory and/or antipyretic remedies (4). Other applications of vervain include the uses of the respective leaves for flavoring of beverages and as seasoning for food preparations (4). The analgesic activity of vervain was confirmed by pharmacological assays and the respective active compound was isolated by methanol extraction, followed by partitioning with ethyl acetate, and identified as being acteoside (verbascoside) which additionally exhibited a weak sedation by the oral administration (5). This compound was found as one of the habitual main polyphenolic constituents from the vervain tea, together with some essential oil constituents (6). However, the sedative and anxiolytic activities of vervain infusions were not confirmed in clinic trials (4 and references therein).
The Good Manufacturing Practices on Medicinal Plants requires higher and higher quality control, based on chemical characterization, either the raw materials or the finished products. The chemical characterization of vervain is made, in some Pharmacopoeias (7), by the TLC detection of citral (mixture of the isomers neral and geranial). However, neral and geranial are widespread in medicinal and aromatic plants and are among the major compounds in essential oils of different plant species. On the other hand in some European countries, medicinal and aromatic plants are often sold as mixtures. Therefore, to guarantee the genuine vervain origin of a given sample of plant biomass or respective products, the search for citral is insufficient. A suitable quality control requires that raw materials of each species and respective products must be characterized not by one chemical marker but by the highest possible number of chemical compounds or a genuinely unique combination of several marker compounds.
Flavonoid compounds are good candidates as chemical markers and some of them have been described (2) and identified in vervain raw material and derived products (6,8-10). However, as vervain is cultivated and commercialized mainly to be used as aromatic in herbal teas, due to their agreeable scent, the most suitable chemical markers may continue to be based in the composition of the essential oils obtained from the respective raw material, such as it is used in the preparation of the infusions. The chemical composition of the oils from A. trlphylla has been studied and reviewed (11-13). However, notwithstanding the repeated reference to the presence of some major compounds namely, neral, geranial, limonene, 1,8-cineole, geraniol, β-caryophyllene, and spathulenol (6) different lists of oil compounds from this species have been reported (6,14,15) making difficult the definition of a standard quality profile. Most of the composition studies have been done with oils isolated from dried leaves as mentioned in the French Pharmacopoeia (7). However, according to some authors, other than leaves vervain parts can be used, namely flowering tops (2).
Aloysia trtphylln was included in a Portuguese integrated programme of research and exploitation of aromatic and medicinal plant species in order to meet the needs of the essential oil and extract industries and the need of finding new agrarian cultures as alternatives to the surplus traditional ones. In Portugal many rural people maintain in small farms or backyards vervain shrubs for own consumption and a growing number of those people prefer the use of fresh vervain biomass, namely leaves and flowers, or flowering tops, in the preparation of tisanes just after the respective harvest from the mother vervain shrub plant. That prompted us to characterize the essential oils obtained by independent hydrodistillation of fresh vervain flowers and leaves. To our knowledge this is the first time that this type of approach was followed. The respective results are here reported in comparison with other ones obtained by other authors, from dried vervain raw material of different origin.
Plant material: Green branches (current season’s growth) of vervain shrubs growing in the DRAEDM/ERCA experimental farm (Merelim, Portugal) were randomly harvested on the first day of August and the flowers and leaves from the respective distal parts (-20-30 cm) were separated. Leaves were chopped into smaller pieces, and the respective fresh biomass, as well as those of flowers, were independently processed in the hydrodistillation and essential oil analysis. Voucher specimens are maintained in the same experimental field under the control of the DRAEDM/ERCA from the Portuguese Agriculture Office.
Cuttings obtained from vervain stems of two mature plants growing in a land at Viana do Castelo (around 40 Km from Merelim, NW) were firstly rooted in 2-L pots and then transferred, by December, to an experimental farm from DRAEDM/ERCA located at Arcos de Valdevez (around 40 Km from Merelim, North) where they were cultivated. Seven months later (July), some branches of these young cultivated plants were randomly harvested and the respective leaves were subjected to hydrodistillation and analysis of the respective oils. This procedure was repeated in the following months of September and December of the same year, and June of the following year.
Hydrodistillation and analysis procedure: Samples from fresh flowers and leaves of the mature vervain plant shrubs growing at Merelim as well as leaves from young plants cultivated at Arcos de Valdevez were submitted to hydrodistillation in a Clevenger type apparatus over 1 h, using volumes of 1.0 mL of hexane for retention of the hydrodistillate components. The hydrodistillates from all samples were analysed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC analyses were performed using a Perkin Elmer Autosystem gas Chromatograph equipped with a fused silica DB-5 column (30 m × 0.25 mm, 0.25 µm film thickness composed by 5%-phenyl methylpolysiloxane [J & WScientific]). Temperature programme: 60°-180°C at 3°C/min, 300°C for injector and 320°C for flame ionization detector (FID). H^sub 2^ was used as carrier gas at flow rate of 1.49 mL/min under a column head pressure of 12.5 psi. Injections (0.5 µL) were performed in a split/splitless injector, in the split mode, with the splitter opened at the 1:13 split ratio. Three replicates of each sample were processed in the same way. Percentage values from the listed compounds correspond to the values given in the GC report without correction factors.
GC/MS analyses were performed with a Perkin-Elmer 8500 gas Chromatograph equipped with a fused silica DB-5 as that of GC, connected with a Finnigan MAT Ion Trap Detector (ITD; software version 4.1) operating in EI mode at 70 eV. Injector, interface and ion-source temperatures were 300°C, 260°C and 220°C, respectively. The oven temperature program and injection conditions were as described above for GC. Helium was used as carrier gas with a column head pressure of 12.5 psi.
Compounds were identified by comparison of the respective mass spectra and retention times with those in computer libraries and by comparison of their DB-5 retention indexes with those published in the literature (16). The retention indexes were determined relatively to n-alkanes of a complete series, from the η-octane to the n-tetratriacontane, eluted in the same conditions as the essential oil samples with the exception for the final column temperature, 285°C, which stood for 15 min.
Results and Discussion
The analyses of the hydrodistillates from fresh flowers of a mature vervain shrub grown at Merelim (DRAEDM/ERCA experimental farm) allowed the identification of 59 compounds, corresponding to around 98% of the respective total essential oil. A similar number of compounds (58) were identified in the leaf oils of the same vervain plant, corresponding to around 94%. From the compounds present in the flower oils, 1-octene, l-octen-3-ol mixed with an unidentified compound, p-cymene, (Z)-β-ocimene, and trans-carveol were not found in the leaf oils of the same plant. However, p-cymene and frans-carveol were found in leaf oils of the young plants cultivated at Arcos de Valdevez (Table I). On the other hand, β-citronellene, β-pinene, neiyl acetate, and irens-calamenene, present in the leaf oils of all vervain samples, were not found in the flower oil. A total of 61 compounds were also identified in the oil obtained by steam distillation followed by extraction with ethyl acetate from vervain air-dried plants (small stems, leaves and flowers) of Moroccan origin and cultivated near Agadir (14). On the other hand, a total of 69 compounds were identified in oils obtained by hydrodistillation from dried leaves and leafy branches of plants of the same species grown in Turkey (15). Comparing the list of the oil composition from the vervain plants grown in Morocco with that from the vervain plants grown in Turkey, only 34 compounds are common to both lists. Comparing the list of the compounds identified by us (Table I) with those reported by those authors we found that 33 compounds are common to that reported by Bellakhdar et al. (14), and 45 are common tothat reported by Özek et al. (15). Notwithstanding we have searched for the constituents previously reported, only 29 compounds from the oils of vervain plants studied by us were common to both lists reported by the referred authors (14,15). On the other hand, from the 15 compounds identified by Carnat et al. (6) in the oils from dried vervain leaves of commercial origin (Chile) we found only 10, lacking the alcohols citronellol, nerol, geraniol, and (Z)-nerolidol, and a-terpinyl acetate, reported by these authors.
From of the 63 compounds identified in this work, 57 distribute into four compound groups: monoterpene hydrocarbons (MH), oxygen-containingmonoterpenes (MO), sesquiterpene hydrocarbons (SH), and oxygen-containingsesquiterpenes (SO) (Table II). The highest level of MH was recorded in flowers which, in compensation, had the lowest level of MO. The main constituents that most accounted for the differences in the levels of these compound groups were limonene (MH), neral and geranial (MO). The lowest levels of SH were found in flowers and leaves of mature plants, while the highest level of SO was found in leaves of these plants (Table II). Apart these small differences in the relative percentages of MH, MO, SO, and respective main compounds, besides the above referred few compounds restricted to flowers or leaves, the composition of the oils from these two vervain organs were similar.
The relative contents of the main compound groups, especially those of M H and M O, in the oils from young vervain plant leaves varied over the year. From July to December, the levels of MO increased from 59.4% to 72.6% in inverse correlation with the relative levels of MH, which decreased from 20.8% to 9.1% (Table II). The compounds that most contributed to these variations were the aldehydes neral and geranial (MO) and limonene (MH). However, the seasonal variations were not so drastic as it occurred with other species, namely Salvia officinalis, cultivated at the same site (17). This means that the leaves of vervain can be consumed over the year with low variation in their aromatic properties. Usually, in Portugal, it is recommended that the vervain leaves may be harvested for consumption two times over the year during the months June/July and September/October.
As vervain is a perennial species cultivated essentially by vegetative propagation, namely by cuttings, the genetic background tend to be maintained at local and regional levels being expected that under the same environmental conditions, the composition of the respective oils are maintained stable overthe time. That is favorable to the definition of standard profiles of quality for the oils of vervain based in composition parameters which may include other compounds besides citral (neral and geranial). In our view, the top ten ranking, that is, the ten most representative compounds, would be a good compromise for definition of a standard profile of quality of the oil.
Table III shows the top ten ranking of compounds, defined by the respective percentages, of vervain oils studied in this work and vervain oils from previous studies performed by other authors. As can be seen, the oils of the flowers andleaves of the same mature plants studied in this work had the same top 10 ranking, although not by the same order in what respects the respective less representative compounds. However, three of the compounds of the top 10 ranking from flower oils and leaf oils ofthose mature plants (1,8-cineole, α-pinene, spathulenol), although present in the oils from the young plants cultivated at Arcos de Valdevez, were out from the respective top 10 ranking in change with zingiberene, germacrene D, and caryophyllene oxide or geranyl acetate (Table III). These small percentage differences may be due to different microclimate or soil conditions (not controlled) or different age.
Eight compounds from the top 10 ranking of the oils of mature plants grown in Merelim, match with the top 10 ranking of the compound list reported by Özek et al., (15) and the other two are reciprocally represented. Larger differences were found on the comparison with the top 10 ranking of the compound lists reported by Bellakhdar et al. (14) and by Carnat et al. (6). Two compounds from the top 10 list of the mature vervain plants, as well as two other ones from the top 10 list of the young cultivate plants grown in Portugal, are not represented at all in the lists reported by these authors (Table III). Although our work had been performed with fresh vervain biomass (flowers and leaves) and the authors referred in Table III had performed the respective studies with dried leaves, and apart from the environmental, age, and possible physiological differences, we interpret the major discrepancies in the oil composition reported by the different authors as being essentially due to some genetic diversity, i.e. chemotypes. Some authors believe that the chemical composition of vervain oils is subject to more variation than is generally accepted (14). Considering the top 10 ranking of the oil composition, the vervain plants studied by us resemble more closely with that ones growing in Turkey (15) followed by those ones grown in Chile (6) and finally those ones grown in Morocco (14). In view of such variation, the determination of an essential oil fingerprint of commercialized vervain products from each region gains particular importance for the respective control of quality. The top ten ranking of the vervain oil composition could be used as the main marker in the respective certificate of origin. In this sense, and considering the seasonal variability, the leaf oil of A. triphylla cultivar grown in Arcos de Valdevez, Portugal, would be certified by the presence of geranial (30-38%), neral (23-30%), limonene (6-15%), zingiberene (2-4%), germacrene D (1-4%), β-caryophyllene (1-3%), caryophyllene oxide (2-3%), (E)-β-ocimene (1-3%), sabinene (0.5-3%), and ar-curcumene (1-2%).
This work was supported by the Programme PRAXIS XXI/AdI/ project P-1304 and by the Programme AGRO/8/key action 8. I/Project 338.
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Paula C. Santos-Gomes and Manuel Fernandes-Ferreira*
Urimersidade do Minho, Departamento de Biologia, Escola de Ciências, Largo do Paco, 4709 Braga Codex, Portugal Ana M.S. Vicente
Direcção Regional de Agricultura de Entre Douro e Minho, Quinta de S. José, S. Pedro de Merelim, 4700-859 Braga, Portugal
* Address for correspondence
Received: December 2002
Revised: March 2003
Accepted: May 2003
Copyright Allured Publishing Corporation Jan/Feb 2005
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