Glandular trichome density and essential oil composition in leaves and inflorescences of Lippia origanoides Kunth ( Verbenaceae ) in the Brazilian Cerrado

The essential oils from leaves and inflorescences of Lippia origanoides Kunth present aromatic and medicinal potential and have been used to treat several diseases, including melanoma. In Brazil, L. origanoides is commonly found in campo cerrado and cerrado stricto sensu, physiognomies featured mainly by the differential light conditions to which short and medium-sized plants are subjected. Our aim was to investigate the glandular trichome density and the yield and chemical composition of the essential oils in leaves and inflorescences of L. origanoides from campo cerrado and cerrado stricto sensu. For glandular density analysis, leaves and inflorescences were processed according to conventional techniques for scanning electron microscopy. The essential oils of leaves and inflorescences were obtained by hydrodistillation and identified with gas chromatography. Bracts and sepals showed the highest glandular density, followed by petals and leaves. The glandular density in the abaxial leaf surface was higher in individuals from the campo cerrado. In both populations the essential oil yield was higher in inflorescences than in leaves. The chemical composition of the essential oils varied among individuals from different areas and inside a same population. Our results demonstrated the chemical plasticity of L. origanoides suggesting the importance of monitoring its popular use.


INTRODUCTION
Species of Verbenaceae are known for the presence of glandular trichomes that secrete essential oils.Many of these species are widely exploited by drug manufacturers and used in popular medicine (Judd et al. 2009, Pascual et al. 2001, Souza and Lorenzi 2008).Lippia origanoides Kunth, a native shrub of the Brazilian Cerrado, is widely distributed in South America from Guiana to northern Argentina, and also occurs in Central America (O' Leary et al. 2012).The essential oil extracted from leaves of L. origanoides shows antimicrobial, antiviral, acaricidal, antioxidant, antibacterial and antiinflammatory activities (Cavalcanti et al. 2010, LUIZ R.S. TOZIN, MARCIA O.M. MARQUES and TATIANE M. RODRIGUES Gomes et al. 2012, Oliveira et al. 2007, Rocha-Guzmán et al. 2007, Stashenko et al. 2010, 2013, Veras et al. 2013).Different chemotypes of L. origanoides were reported in literature and p-cimene, thymol and carvacrol are the major components most commonly found (Cavalcanti et al. 2010, Oliveira et al. 2007, Stashenko et al. 2010, 2013).
In the Brazilian Cerrado, L. origanoides is commonly found in areas of campo cerrado and cerrado stricto sensu.These Cerrado physiognomies are featured mainly by the differential light intensity that reaches the short and medium-sized plants.The cerrado stricto sensu is characterized by the occurrence of medium-sized trees, scattered shrubs and some grasses.In this area the individuals of L. origanoides are shaded by nearby trees in the early hours of the morning and in late afternoon.In the campo cerrado, grasses and small shrubs are predominant, with some sparse trees (Maroni et al. 2006), and individuals of L. origanoides are exposed to full sunlight throughout the day.
Studies indicate that both yield and chemical composition of the essential oils and the glandular trichome density, can be influenced by environmental factors, including herbivory, temperature, water availability, altitude, circadian cycle, seasonality, and light intensity, among others (Argyropoulou et al. 2007, Gianfagna et al. 1992, Gobbo-Neto and Lopes 2007, Gonzáles et al. 2008, Juliani Jr et al. 2002, Martínez-Natarén et al. 2011, Pérez-Estrada et al. 2000, Viljoen et al. 2005, Werker 2000).Whether the different organs in a same plant respond similarly to the environmental fluctuations concerning the essential oil composition is still unknown.
Since leaves and inflorescences of L. origanoides are exhaustively exploited by people living in Cerrado areas in Brazil for medicinal purposes, knowledge on the occurrence of variation in the essential oil composition in plants living in different environmental conditions can be important in order to avoid it being misused.
We investigated the density of the glandular trichomes and the yield and chemical composition of essential oils in leaves and inflorescences of L. origanoides from campo cerrado and cerrado stricto sensu.
Data on the temperature and relative humidity from November 2012 through March 2013 were obtained from meteorological stations in the cerrado stricto sensu and campo cerrado areas.The photosynthetic photon flux density (PPFD) was measured with a PAM fluorometer on successive days.The altitude data were recorded with a GPS.
Table I shows the data for temperature, relative humidity, PPFD and altitude in both areas.

DENSITY OF GLANDULAR TRICHOMES
To evaluate the density of glandular trichomes, two fully expanded leaves were collected from each plant in both populations.To estimate the density of glandular trichomes in reproductive organs, two inflorescences were collected from each individual in the campo cerrado.
Samples of flowers and the middle part of leaf blades were fixed in glutaraldehyde (2.5% with 0.1 M phosphate buffer, pH 7.3, overnight at 4 °C), dehydrated in a graduated acetone series, criticalpoint dried, mounted on aluminum stubs, goldcoated (Robards 1978), and examined with a Fei Quanta scanning electron microscope.
The glandular density in leaves and flowers was calculated in 1 mm 2 using the Scandium software with an image-capture system coupled to the scanning electron microscope.We compared the glandular density between a) the adaxial and abaxial leaf surfaces in individuals from the same population; b) leaves from different populations; and c) leaves and floral parts (bracts, sepals, petals) from the same population.The data were submitted to ANOVA followed by Tukey test, at a 5% probability level.

YIELD AND CHEMICAL COMPOSITION OF THE ESSENTIAL OILS
For the essential oil analyses, leaves and inflorescences were collected from four individuals growing in the cerrado stricto sensu (numbered 1 to 4) and from three individuals growing in the campo cerrado (numbered 1 to 3).Individual number 3 in the campo cerrado was located on an ant colony.
The samples were dried at 40 °C and were subjected to hydrodistillation in a Clevenger-type apparatus (Craveiro et al. 1981) for 2 h.
The qualitative analysis of the essential oil was performed on a gas chromatograph coupled to a mass spectrometer (CG-MS, Shimadzu, QP-5000), with an OV-5 fused silica capillary column (30 m x 0.25 mm x 0.25 μm, Ohio Valley Specialty Chemical, Inc.), operating at an MS ionization voltage of 70 eV, with helium as the carrier gas (1.0 mL/min.).The following chromatography conditions were used: injector at 240 °C, detector at 230 °C, injection volume: 1 µL of solution (1 mg of essential oil/1 mL of ethyl acetate), split 1/20, and the temperature program: 60 °C -240 °C, 3 °C/ min.The compounds were identified by comparison of the acquired mass spectra with those stored in the GC/MS database of the system (NIST 62 lib.) and retention indices (Adams 2007).The retention indices (RI) were obtained from the injection of a mixture of n-alcanes (Sigma-Aldrich, C 9 -C 24 ), employing the same temperature program conditions described above for GC/MS, applying the equation of Van den Dool and Kratz (1963).
The yield of essential oil was analyzed using factorial analysis, and compared among the plant organs and populations by ANOVA, followed by Tukey test at a 5% probability level.

DISTRIBUTION AND DENSITY OF GLANDULAR TRICHOME
Glandular trichomes were observed in both leaf (Fig. 1A, B) and bract (Fig. 1C) surfaces, as well as the abaxial side of sepals (Fig. 1D) and petals (Fig. 1E) of L. origanoides.In the petals, these glands occurred exclusively in the distal region.
In the individuals from the campo cerrado, the glandular density was higher in the inflorescences than in the leaves (F (4,55) =144.3919;P<0.00001) LUIZ R.S. TOZIN, MARCIA O.M. MARQUES and TATIANE M. RODRIGUES (Table II).Bracts and sepals were the organs with the highest glandular density (Table II).The density of glandular trichomes was three to four times higher on the abaxial surface of the leaf (Fig. 1A) than on the adaxial (Fig. 1B) for individuals from both the campo cerrado and the cerrado stricto sensu (Table II).
The abaxial leaf surface of individuals from the campo cerrado showed a higher density of glandular trichomes in comparison to individuals from the cerrado stricto sensu (F (1,22) =15.6354;P=0.0009).Differences in the glandular density were not observed for the adaxial leaf surfaces of individuals from the different populations (F (1,22) =1.1297; P=0.2998) (Table II).

YIELD AND CHEMICAL COMPOSITION OF THE ESSENTIAL OIL
The yield of the essential oil of L. origanoides was higher in inflorescences than in the leaves (Fig. 2) in individuals from both cerrado physiognomies (F (3,8) =22.7906;P<0.0001) (Fig. 2).No significant differences were observed in the oil yield between individuals from the cerrado stricto sensu and the campo cerrado, for both leaves (P=0.390) and inflorescences (P=0.233).
During the analysis of the essential oil yield, the samples from individual 3 (the one that was located on an ant colony in the field) from the campo cerrado were not used, due to the discrepant values obtained for its inflorescences (1.98) and leaves (3.97).We identified 49 substances in the essential oil extracted from leaves and inflorescences of L. origanoides.The amount of substances found in the inflorescences was higher than in the leaves, in both populations.The major compounds varied between the populations, among the individuals from the same population, and between the organs of the same individual (Table III).

DISCUSSION
We have found a higher density of glandular trichomes on the abaxial leaf surface of L. origanoides plants growing in the campo cerrado; in this area the relative humidity was lower and the altitude and PPFD were higher.(Gianfagna et al. 1992, Horgan et al. 2009, Pérez-Estrada et al. 2000).In drier environments, the density of trichomes on leaves can be higher than in plants of the same species growing in wetter situations (Pérez-Estrada et al. 2000).Similarly, light intensity can positively influence the glandular trichomes (Pérez-Estrada et al. 2000, Yamaura et al. 1989).According to Pérez-Estrada et al. (2000), in addition to playing a defensive role against biotic agents, glandular trichomes can help to reflect sunlight and minimize water loss.
Although the PPFD was similar between the areas, plant density in the cerrado stricto sensu is higher, and at certain periods of the day the individuals can be shaded.This does not occur in the campo cerrado, where shorter herbaceous species predominate.The shading of L. origanoides plants in the cerrado stricto sensu may be related to the lower glandular density of their leaves, since plants under more intense radiation tend to have higher glandular density as a protective mechanism (Gianfagna et al. 1992, Pérez-Estrada et al. 2000).In addition, the higher density of glandular trichomes on leaves of L. origanoides plants in the campo cerrado may be related to the higher altitude of this area.
The influence of altitude on the abundance of glandular structures was evaluated by Sheue et al. (2003), who found a higher density of internal glands in individuals of Pinus taiwanensis in middle altitudes (1000 to 2500 m), in comparison with plants at lower (700 m) or higher (3000 m) altitudes.According to the authors, variation in the number of glands can result from genetic and environmental interactions.This suggests that variations in the glandular density in individuals of L. origanoides growing in the campo cerrado and the cerrado stricto sensu may result from interactions between environmental or microenvironmental factors and genetic factors.
Although the glandular density differed in leaves from individuals of L. origanoides living in the campo cerrado and in the cerrado stricto sensu, the yields of essential oil from plants living in the two environments were similar.This may be related to the mixed chemical nature of the LUIZ R.S. TOZIN, MARCIA O.M. MARQUES and TATIANE M. RODRIGUES secretion produced by the glandular trichomes of L. origanoides.Besides lipid substances, Tozin et al. (2015), using histochemical methods, detected the presence of polysaccharides, phenolic compounds, proteins, and alkaloids, among others, in the glandular trichomes of this species.The mixed nature of the secretion produced by the trichomes is an aspect common to other Lippia species (Argyropoulou et al. 2010, Combrinck et al. 2007).Tozin et al. (2015) demonstrated that not all morphotypes of glandular trichomes of L. origanoides produce essential oils.Therefore, the more-abundant glandular trichomes in leaves in individuals from the campo cerrado are probably not secretors of essential oils.We can still suggest that under the environmental conditions of the campo cerrado there is a higher investment in the production of other types of substances by the glandular trichomes, and this provides greater protection against high light intensity and lower relative humidity.Hydrophilic substances can act as a lubricant in dry environments and facilitate leaf expansion (Ascensão et al. 1999).In Lippia species, phenolic compounds are abundant and variable in constitution (Pascual et al. 2001), and may help to protect against intense UV-B radiation resulting from greater exposure to sunlight (Liakoura et al. 1997).
Our results also showed that L. origanoides inflorescences had a higher density of glandular trichomes and produced a higher yield of essential oils in comparison to the leaf blades of individuals in the same environment.These data contrast with studies on other species, which reported a low yield of essential oils in flowers and inflorescences in comparison to leaves (Bassole et al. 2005, Parra-Gárces et al. 2010), and can be related to the different roles of these glands in reproductive organs (Ascensão et al. 1999, Paiva andMartins 2011).We suggest that in L. origanoides, the trichomes in the inflorescences are mainly involved in the production of large amounts of essential oils that play an important role in protecting the reproductive organs against herbivores and pathogens (Stökl et al. 2010).In addition, several terpenes can be reused in the plant metabolism (Harborne 1988), acting as precursors in the synthesis of hormones such as flowering hormones (Danilova andKashina 1987, Roshchina andRoshchina 1993).
Chemical analysis of the essential oils in leaves and inflorescences of L. origanoides revealed a wide variation in the chemical composition of the essential oil among the individuals in each area.This may be related to genetic variability among individuals from these populations.Catalan and Lampasona (2002) found that intraspecific variations in the chemical composition of essential oils are common in species of Lippia.
Studies with L. origanoides (sensu lato) individuals have presented widely divergent chemical profiles.Thymol, carvacrol and pcymene have been reported as the majority compounds (Cavalcanti et al. 2010, Oliveira et al. 2007, Stashenko et al. 2010, 2013).However, in this study, thymol and carvacrol were not found, while pcymene was detected in small quantities.These findings are in accordance with the information reported by Rodrigues et al. (2011) andStashenko et al. (2010), who demonstrated very divergent chemical profiles in this species.The wide variety of chemotypes found in L. origanoides may be a factor of the synonymization proposed by O' Leary et al. (2012); in a revision of the Goniostachyum section of Lippia, the authors synonymized 41 species into only four.The taxon that is presently known as L. origanoides results from the synonymization of 28 taxa that were formerly considered to be separate species (O'Leary et al. 2012).
In some individuals of L. origanoides, αpinene was found in large quantities in the inflorescences.This may be associated with the known role of this substance in attracting pollinators (Stökl et al. 2010).However, the inflorescences of some L. origanoides individuals did not contain this compound in large quantities, perhaps because of TRICHOMES AND ESSENTIAL OIL IN L. origanoides phenological differences among the individuals.Studies indicate that some compounds, such as carvone, limonene and ß-pinene (Bicas et al. 2008) can volatilize or be utilized as precursors in the formation of other substances after the anthesis (Parra-Garcés et al. 2010, Stökl et al. 2010).
Transcaryophyllene and αhumulene were among the major compounds in leaves and inflorescences from several individuals.These substances show several important biological activities, including antiinflammatory, analgesic, and others (Fernandes et al. 2007, Sabulal et al. 2006), and they are used to manufacture soaps, detergents and food products (Standen et al. 2006).
Individual number 3 from the campo cerrado, situated on an ant colony, diverged most widely from the others in oil yield and chemical composition; only this individual contained 1,8-cineole as the major compound, in both the inflorescences (52.2%) and the leaves (58.2%).The abundant production of this substance can be related to ant attacks on this individual, since 1,8-cineole is toxic to herbivorous insects (Prates et al. 1998, Sukontason et al. 2004).This toxic substance penetrates the insect body via the respiratory or digestive system, and is lethal to several animal species (Sukontason et al. 2004).
These findings evidenced the occurrence of variability in the glandular density in leaves of L. origanoides plants living in different areas.Our data demonstrated the chemical plasticity of this species, which can produce essential oils with differential chemical composition in distinct environments and evidenced the differential answers between leaves and inflorescences in a same individual.In view of this plasticity, the use of this species should be better monitored, particularly in popular medicine.the review service of the manuscript; the technical team of the Centro de Microscopia Eletrônica (CME), IBB, UNESP, for helping in the sample preparation; and Dr. Luiz Fernando Rolim de Almeida, IBB, UNESP, for assistance with statistical analyses.

Figure 2
Figure 2 Yield of essential oil of leaves and inflorescences of Lippia origanoides Kunth from two populations in the Cerrado (Means followed by different letters indicate statistical differences in the essential oil yield between organs inside a same population and between different populations by Tukey test at 5% significance level).

TABLE II Glandular density (mm 2 ) in leaf blades and inflorescences of Lippia origanoides Kunth from two physiognomies of the Brazilian Cerrado.
Means followed by different capital letters indicate statistical differences in the glandular density among organs and different lowercase letters indicate statistical differences in the glandular density between populations (Tukey test p < 0.05).

TABLE III Chemical composition of the essential oils (%) from inflorescences and leaves of Lippia origanoides Kunth from cerrado stricto sensu and campo cerrado in São Paulo state, Brazil.
TRICHOMES AND ESSENTIAL OIL IN L. origanoides RI*= Retention index calculated; IR**= Retention index (Adams 2007); nd = not detected; tr = trace (tr ≤0.13).*** Retention index obtained on the website: www.pherobase.com about the influence of exogenous factors on the origin of secretory structures in plants.Studies have reported on the effects of light, temperature, altitude, and availability of nutrients, among others, on the glandular density in different plant species