ABSTRACT

Lippia alba (Mill.), popularly known bushy matgrass, is considered an aromatic and medicinal plant with physiotherapeutic characteristics, leading to its use in the chemical and pharmaceutical industries. Therefore, the study aimed to evaluate the chemical composition and yield of the essential oil of Lippia alba (Mill.) after the occurrence of frost in southern Brazil. The study was carried out in an experimental area located at the Federal University of Santa Maria, Frederico Westphalen Campus, in July 2014. The essential oil was extracted using the hydrodistillation technique for three hours after the start of boiling. The chemical composition of Lippia alba (Mill.) essential oil was identified through chromatographic analysis. The chemical composition of Lippia alba (Mill.) essential oil was not altered by frost. As for the yield, it was 0.311% before the frost and 0.363% after the frost.

Keywords
bushy matgrass; winter; linalool; temperature

INTRODUCTION

Essential oils are the most important raw materials in the pharmaceutical, food and agricultural industries, due to their therapeutic, antimicrobial, antioxidant, and antifungal activities (Burfield & Reekie, 2005Burfield T & Reekie SL (2005) Mosquitoes, malaria and essential oils. International Journal of Aromatherapy, 15:30-41.; El Tamer, 2002El Tamer MK (2002) Molecular regulation of plant monoterpene biosynthesis in relation to fragrance. Netherlands, Wageningen University and Research. 151p.). In plants they are related to defense and attraction of pollinators among other important ecological functions (Prins et al., 2010Prins CL, Vieira IJ & Freitas SP (2010) Growth regulators and essential oil production. Brazilian Journal of Plant Physiology, 22:91-102.). Like other secondary metabolic groups, these compounds play a crucial role in plant acclimatization to environmental conditions (Taiz et al., 2021Taiz L, Zeiger E, Møller IM & Murphy A (2021) Fundamentos de Fisiologia Vegetal. 6ª ed. Porto Alegre, Artmed Editora. 584p.).

Lippia alba (Mill.) from the Verbenaceae family is an example of these plants. Native to South America, it can be found in tropical and subtropical climates, in sandy soils on the banks of lakes and rivers (Silva et al., 2006Silva NA, Oliveira FF, Costa LCB, Bizzo HR & Oliveira RA (2006) Caracterização química do óleo essencial da erva cidreira (Lippia alba (Mill.) NE Br.) cultivada em Ilhéus na Bahia. Revista Brasileira de Plantas Medicinais, 3:52-55.). It is a plant widely used in folk medicine due to its analgesic, sedative, mildly spasmolytic, anxiolytic, and mildly expectorant properties (Linde et al., 2016Linde GA, Colauto NB, Albertó E & Gazim ZC (2016) Quimiotipos, extracción, composición y aplicaciones del aceite esencial de Lippia alba. Revista Brasileira de Plantas Medicinais, 18:191-200., Aziz et al., 2019Aziz MA, Mehedi M, Akter MI, Sajon SR, Mazumder K & Rana MS (2019) In vivo and in silico evaluation of analgesic activity of Lippia alba. Clinical Phytoscience, 5:01-09.), besides having different aromas. The aroma is associated with the constituents present in the essential oil, which can vary both qualitatively and quantitatively in relation to factors such as time of year, phenology, rainfall, plant age, geographic location, and meteorological elements (da Silva Junior et al., 2019Da Silva Junior AQ, da Silva DS, Figueiredo PLB, Sarrazin SLF, Bouillet LEM, de Oliveira RB, Maia JGS & Mourao RHV (2019) Seasonal and circadian evaluation of a citral-chemotype from Lippia alba essential oil displaying antibacterial activity. Biochemical Systematics and Ecology, 85:35-42.).

In the state of Rio Grande do Sul, Brazil, the most favorable temperature for the growth and development of Lippia alba (Mill.) occurs between October and April, where the mean air temperature varies from 12 to 25 °C. As in several species, during winter, there is a general reduction in plant growth rate, probably related to a decrease in mean daily temperatures (Schmidt et al., 2016Schmidt D, Caron BO, Prochnow D, Cocco C, Elli EF, Stolzle J, Altissimo B & Heinzmann BM (2016) Effect of frost on yield and composition of Aloysia triphylla essential oil. Journal of Medicinal Plants Research, 10:88-92.), along with the total loss of leaves with the occurrence of frost, causing losses and making it impossible to harvest the leaves for essential oil production.

There are two mechanisms that can cause damage to cell structure and decrease in plant tissue firmness. First, perforation of the cell membrane can occur caused by intracellular ice crystals, which contribute to the reduction of turgor pressure. The second is associated with damage to the cell wall structure, caused by ice crystals formed in the extracellular environment, causing cell collapse (Alves & Piccoli, 2012Alves JA & Piccoli RH (2012) Qualidade de pequis fatiados e inteiros submetidos ao congelamento. Ciência Rural, 4:904-910.).

As water is more diluted in the apoplast, freezing occurs first, as the freezing point is higher than that of the symplast, which is more concentrated. Extracellular crystals cause cellular dehydration, due to the water potential that forms, moving water from the symplast to the apoplast. Dehydration caused by freezing leads to plasmolysis in the membrane, and this damage is irreversible; however, there is no rupture of the plasmatic membrane. For example, at a temperature of -10 °C, about 90% of the symplast water is osmotically lost to the apoplast. In this process, the plasma membrane is contracted and moves away from the cell wall, becoming stiffer due to the low temperature, and may be damaged (Taiz et al., 2021Taiz L, Zeiger E, Møller IM & Murphy A (2021) Fundamentos de Fisiologia Vegetal. 6ª ed. Porto Alegre, Artmed Editora. 584p.).

Thus, the following hypothesis was generated: the yield and chemical composition of Lippia alba (Mill.) essential oil extracted after the occurrence of frost is altered. Therefore, the study aimed to evaluate the chemical composition and yield of the essential oil of Lippia alba (Mill.) after the occurrence of frost in southern Brazil.

MATERIAL AND METHODS

The study was carried out in an experimental area located at the Federal University of Santa Maria, Frederico Westphalen Campus, in Rio Grande do Sul, Brazil, at latitude 27° 23’ S; longitude 53° 25’ W, altitude of 461 m in the city of Frederico Westphalen, during the month of June 2014. The climate, according to Köppen’s climate classification, is Cfa, with distinct seasons throughout the year and temperatures varying from -2.3 to 36.3 °C and, for the coldest months (June, July and August), an average temperature of 15.1 °C, resulting in possible formation of frost during this period. Rainfall is well distributed throughout the year, ranging from 2103.16 to 1578.6 mm (Caron et al., 2024). The predominant type of soil at the site is Neossolo Litólico Eutrófico Típico (Entisol) (Cunha et al., 2011Cunha NG, Silveira RJC, Koester E, Oliveira LD, Alba JMF, Terres VC & Lopes RT (2011) Estudos de Solos do Município de Frederico Westphalen, RS. Pelotas, Embrapa. 32p. (Circular Técnica, 116).).

Lippia alba (Mill.) seedlings were produced from cuttings measuring an average of 20 cm in length, collected from mother plants. The cuttings were added to trays with 90 cm³ tubes filled with a mixture of commercial substrate and vermiculite, after which these trays were placed in a greenhouse until they reached the point of transplantation. The cuttings were irrigated using a sprinkler system.

The seedlings were transplanted into the field on November 23, 2011, with a spacing of 0.8 m between plants and 1.0 m between rows, comprising 0.8 m² of area per plant. Each plant was considered to be an experimental unit. Weed management was carried out manually with weeding, and irrigation, when necessary, was carried out using a drip system. No chemical product was applied to the plants as it could alter the composition of the essential oil extracted.

The study was carried out in a completely randomized design (CRD), with three repetitions. The assessments were carried out in July 2014, considering the coldest month of the year, obtaining an average of 13.9 °C, according to Caron et al. (2024). Collections were carried out in two different situations: 1) on a typical winter day with low temperatures (4.6 ºC), without frost formation; and 2) with the occurrence of frost (minimum temperature of 2.6 ºC).

After completion of extraction, the oil was measured in a glass beaker. To determine the effect of frost on the essential oil [yield (%) and chemical composition], care was taken in relation to the weather forecast from the previous day, regarding the probability of frost occurring on the day of collection. After the formation of frost, it was not possible to collect a large volume of plant material due to the accelerated occurrence of leaf senescence present in the plant.

Data on the meteorological element air temperature were collected from the automatic meteorological station linked to the National Institute of Meteorology (INMET), located 50 m away from where the study was conducted. In the collections of plant material prior to the formation of frost, the average minimum air temperature was 4.2 °C with a minimum of 2.2 °C; for collection after the presence of frost, the average maximum was 3.5 °C and the average minimum was 1.9 °C, with a minimum temperature recorded of -1.8 °C. The mean, maximum and minimum temperatures recorded during the month of July can be seen in Figure 1.

Figure 1
Maximum, mean and minimum air temperature for the month of July 2014 recorded by the meteorological station registered by INMET.

The essential oil was extracted using the hydrodistillation technique for three hours after the start of boiling. Each sample had 200 g of green leaves. These leaves were removed from the apical, middle and basal regions of the plant to obtain homogeneity. Collection was carried out at 2 pm, which allows the leaves to dry after the occurrence of frost and dew in the morning. The yield (%) of essential oil was determined by the following equation 1:

The chemical composition of Lippia alba (Mill.) essential oil was identified through chromatographic analysis using an Agilent Technologies 6890N GC-FID gas chromatograph equipped with a DB-5 capillary column (30 x 0.25 mm; film thickness of 00.25 µm) and connected to a FID detector, and the temperature was adjusted as described by Schmidt et al. (2016)Schmidt D, Caron BO, Prochnow D, Cocco C, Elli EF, Stolzle J, Altissimo B & Heinzmann BM (2016) Effect of frost on yield and composition of Aloysia triphylla essential oil. Journal of Medicinal Plants Research, 10:88-92..

The chemical compounds identified in essential oils were determined through the method described by Adams, (2009)Adams RP (2009) Identification of essential oil components by gas chromatography/mass spectrometry. 4º ed. Illinois, Allured Business Media. 804p.. To calculate the concentration of chemical compounds, the relationship between the total area of their peaks and the total area of all constituents of the sample shown in the gas chromatography analysis was established, with a flame ionization detector, and the result was expressed as percentage.

The data were initially analyzed regarding the adherence of residuals to the normal distribution and homogeneity of the residual variances, using the Shapiro-Wilk (p < 0.05) and Bartlett (p < 0.05) tests, respectively, which indicated that the statistical assumptions were met. Subsequently, data were subjected to an analysis of variance for evaluating possible treatment effects. When a significant effect was verified by the F test (p < 0.05), the necessary complementary analyses were carried out, with the help of Genes software (Cruz, 2013Cruz CD (2013) Genes: a software package for analysis in experimental statistics and quantitative genetics. Acta Scientiarum. Agronomy, 35:271-276.).

RESULTS AND DISCUSSION

The oil contents according to the analysis of variance did not differ statistically between the treatments; before the occurrence of frost, the plants had a content of 0.311% and, after the frost, a small increase was observed, reaching 0.363%, which may be associated with dehydration that occurred in the leaf cells due to frost damage. The situations before and after the occurrence of frost can be seen in Figure 2.

Figure 2
Situation of Lippia alba plants at the moment (a) and after (b) the occurrence of frost.

According to Barros et al. (2009)Barros FMCD, Zambarda EDO, Heinzmann BM & Mallmann CA (2009) Variabilidade sazonal e biossíntese de terpenóides presentes no óleo essencial de Lippia alba (Mill.) NE Brown (Verbenaceae). Química Nova, 32:861-867., variations in the yield of Lippia alba (Mill.) essential oil occur on the order of 0.1-1.0% due to the influence of the different collection times on the levels. In winter, the yield reaches minimum values and increases again in spring and summer. In winter, the conditions comprise low temperatures, short periods of insolation, and high levels of relative humidity and rainfall, thus causing a decrease in essential oil contents (Barros et al., 2009Barros FMCD, Zambarda EDO, Heinzmann BM & Mallmann CA (2009) Variabilidade sazonal e biossíntese de terpenóides presentes no óleo essencial de Lippia alba (Mill.) NE Brown (Verbenaceae). Química Nova, 32:861-867.). In addition, depending on the season (cold or hot), plants may have a smaller healthy leaf area conducive to essential oil extraction, directly affecting production.

In the essential oil extracted from Lippia alba (Mill.) leaves, 22 constituents were identified from the GC-MS analysis and are listed by retention time (Table 1). The main compounds identified in the essential oil were Myrcene, eucalyptol, β-trans-Ocimene, and linalool, which make up 64.9% of the oil before frost and 64.2% of the oil after frost. Oxygenated monoterpenes constituted the majority of the essential oil, with 45.8% before frost and 46.7% after frost (Table 1). The compound linalool varied between 35.7% before and 36.2% after frost, myrcene between 14.2% before and 14.0% after frost, eucalyptol varied between 7.7% before and 7.5% after frost and β-trans-Ocimene 7.2% before and 6.4% after frost.

Most essential oil storage in plants occurs in glandular and peltate trichomes, located on both sides of the leaves: adaxial and abaxial (Gattuso et al., 2008Gattuso S, van Baren CM, Gil A, Bandoni A, Ferraro G & Gattuso M (2008) Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora Palau) from Argentina. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7:190-198.; Taiz et al., 2021Taiz L, Zeiger E, Møller IM & Murphy A (2021) Fundamentos de Fisiologia Vegetal. 6ª ed. Porto Alegre, Artmed Editora. 584p.). The essential oil, due to its characteristics, does not freeze, and it is likely that the cells of these trichomes are not damaged, as they have high concentrations of oil and low concentration of water, which helps to keep the structure of their biomembranes intact in the presence of frost (Schmidt et al., 2016Schmidt D, Caron BO, Prochnow D, Cocco C, Elli EF, Stolzle J, Altissimo B & Heinzmann BM (2016) Effect of frost on yield and composition of Aloysia triphylla essential oil. Journal of Medicinal Plants Research, 10:88-92.).

The formation of intracellular ice does not occur, unless the freezing point of the cells is below -10 °C, and in general, when intracellular freezing occurs, cells die due to the mechanical destruction of the biomembranes (Mazur, 1969Mazur P (1969) Freezing injury in plants. Annual Review of Plant Physiology, 20:77-109.; Arora, 2018Arora R (2018) Mechanism of freeze-thaw injury and recovery: A cool retrospective and warming up to new ideas. Plant Science, 270:301-313.). This explains why there was no leakage of essential oil from the cells of the evaluated plants, even with the damage caused by frost, since no temperatures capable of causing intracellular freezing and degradation of biomembranes were recorded.

After the occurrence of frost (July 23rd, 2014), tissue necrosis and subsequent senescence and death of leaves were visually observed, with temperatures ranging from 0.9 to -1.8 °C for a total of eight hours, characterizing the species as sensitive to frost (Figure 2). With this, we can observe the importance of knowing the response of species to adverse production conditions and the importance of crop management on a commercial scale during the winter. Under conditions of low temperatures and frost formation, the occurrence of leaf necrosis and senescence is common. From the results obtained, it appears that it is possible to collect the plants and extract the essential oil after the occurrence of frost, as described for Aloysia triphylla by Schmidt et al. (2016)Schmidt D, Caron BO, Prochnow D, Cocco C, Elli EF, Stolzle J, Altissimo B & Heinzmann BM (2016) Effect of frost on yield and composition of Aloysia triphylla essential oil. Journal of Medicinal Plants Research, 10:88-92.; however, it was observed that complete leaf senescence occurs within two days, requiring rapid collection of plant material to avoid total necrosis and leaf fall.

CONCLUSION

The essential oil content of Lippia alba (Mill.) was not affected by the occurrence of frost. The total yield obtained from the leaves was 0.311% before frost and 0.363% after frost. Therefore, the leaves can be harvested immediately after the phenomenon, but before necrosis and total senescence of the leaves.

The components remained stable after the occurrence of frost, maintaining the standard and quality of production expected for the winter. Thus, the hypothesis generated in the work was rejected.

ACKNOWLEDGMENTS, FINANCIAL SUPPORT AND FULL DISCLOSURE

The authors would like to thank the scientific initiation scholarships provided by the following funding bodies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). They also thank CNPq for the productivity grants level 1D to the author Denise Schmidt and 1C to the author Braulio Otomar Caron.

REFERENCES

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