ABSTRACT:

The semiarid region of Brazil holds a great richness of medicinal and aromatic plants with considerable potential for pharmaceutical, food, cosmetic and biopesticide industries. Martianthus leucocephalus (Mart. Ex Benth.) J. F. B. Pastore is endemic to this region, and its essential oils contain a principle compound, isobornyl formate, which demonstrates antimicrobial activity against Bacilus cereus, Staphylococcus aureus and Candida albicans. In spite of its significant pharmacological potential, little is known about its growth. In light of the influence of seasonality on plant growth, development, and secondary metabolism, the present study evaluated the growth and essential oil content of M. leucocephalus grown and harvested during different months of the year in the edaphoclimatic conditions of Feira de Santana, Bahia State, Brazil. The experimental design was entirely randomized, with twelve harvesting periods and five replicates. The study acquired monthly data of mean temperatures, relative humidity, rainfall, irradiance, and photoperiod from the National Institute of Meteorology (INMET) and quantified the fresh and dry weights of leaves, flowers and branches, as well as leaf area, and essential oil content. The data were submitted to Spearman correlation analysis and the means were compared using the Scott-Knott test. Total leaf masses and oil contents were higher during periods with longer photoperiods and higher solar irradiance. Rainfall and relative humidity reduced plant growth and essential oil content. Higher total mean dry masses were recorded from September to January (except October), while oil content was higher in March.

Key words:
semiarid region; terpenes; biomass; endemic medicinal plant; Hyptis leucocephala

RESUMO:

O semiárido brasileiro apresenta grande riqueza de espécies medicinais e aromáticas, com intenso uso na indústria farmacêutica, alimentícia, de cosméticos e de biopesticidas. A espécie Martianthus leucocephalus (Mart. ex Benth.) J. F. B. Pastore é endêmica dessa região. Seu óleo essencial apresenta como composto majoritário o formiato de isobornila, com atividade antimicrobiana frente à Bacilus cereus, Staphylococcus aureus e Candida albicans. Devido à grande influência da sazonalidade sobre o crescimento e produção vegetal, sobretudo quando envolve o metabolismo secundário de plantas, o objetivo deste trabalho foi avaliar o crescimento e a produção de óleo essencial de M. leucocephalus cultivada e colhida em diferentes meses do ano, nas condições edafoclimáticas de Feira de Santana, BA. O experimento foi conduzido em delineamento inteiramente casualizado (DIC), com doze tratamentos, constituídos dos diferentes meses de colheita, com cinco repetições. Mensalmente, foram obtidos os valores médios de temperatura, umidade relativa do ar, precipitação pluviométrica, irradiância e fotoperíodo, através do Instituto Nacional de Meteorologia (INMET), quantificando-se também a massa fresca e seca das folhas, flores e caule, a área foliar e o teor de óleo essencial das plantas. Os dados foram submetidos à análise de correlação de Spearman e as médias foram comparadas pelo teste de Scott-Knott. A massa seca total e o teor de óleo aumentaram em função da irradiância e reduziram em função da precipitação pluviométrica e umidade relativa do ar. As maiores médias de massa seca total foram encontradas durante os meses de setembro a janeiro, exceto outubro, e de teor de óleo no mês de março.

Palavras-chave:
região semiárida; terpenos; biomassa; planta medicinal endêmica; Hyptis leucocephala

INTRODUCTION:

The Brazilian semiarid region, dominated by "Caatinga" vegetation, has many medicinal and aromatic plants, including the Lamiaceae family with many endemic taxa (GIULIETTI et al., 2004GIULIETTI, A.M. et al. Diagnóstico da vegetação nativa do bioma Caatinga. Brasil: Ministério do Meio Ambiente, 2004. 44p. Available from: <Available from: http://ainfo.cnptia.embrapa.br/digital/bitstream/item/18267/1/Biodiversidade_Caatinga_parte2.pdf >. Accessed: Nov. 06, 2014.
http://ainfo.cnptia.embrapa.br/digital/b... ). Several species of this family have proven pharmacological capabilities, including antimicrobial, cytotoxic, anti-inflammatory, anti-HIV, and insecticide activities (FALCÃO & MENEZES, 2003FALCÃO, D.Q.; MENEZES, F.S. The Hyptis genus: an ethnopharmacological and chemical review Revista Brasileira de Farmacognosia, v.84, n.3, p.69-74, 2003. Available from: <Available from: http://www.rbfarma.org.br/files/pag_69a74_vjml00dk.pdf >. Accessed: Nov. 06, 2014.
http://www.rbfarma.org.br/files/pag_69a7... ).

Martianthus leucocephalus (Mart. ex Benth.) J. F. B. Pastore (Lamiaceae), formerly recognized as Hyptis leucocephala Mart. ex Benth., is an endemic medicinal plant of the Brazilian semiarid region (HARLEY & PASTORE, 2012HARLEY, R.M.; PASTORE, J.F.B. A generic revision and new combinations in the Hyptidinae (Lamiaceae), based on molecular and morphological evidence. Phytotaxa, v.58, p.1-55, 2012. Available from: <Available from: http://www.mapress.com/phytotaxa/content/2012/f/p00058p055f.pdf >. Accessed: Nov. 06, 2014. doi: 1179-3163.
http://www.mapress.com/phytotaxa/content... ). It is a decumbent aromatic herb, up 20cm tall, with chartaceous leaves, flowers with a green calyx and purple corolla (OLIVEIRA et al., 2011OLIVEIRA, L.M. et al. Propagação vegetativa de Hyptis leucocephala Mart. ex Benth. e Hyptis platanifolia Mart. ex Benth. (Lamiaceae). Revista Brasileira de Plantas Medicinais, v.13, n.1, p.73-78, 2011. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-05722011000100011 >. Accessed: Nov. 06, 2014. doi: 10.1590/S1516-05722011000100011.
http://www.scielo.br/scielo.php?script=s... ). Leaves, flowers and branches produce essential oils (0.2% average yield, with isobornyl formate being the principal compound) with proven antimicrobial activity against Bacillus cereus, Staphylococcus aureus, and Candida albicans (LUCCHESE et al., 2005LUCCHESE, A.M. et al. Óleos essenciais do gênero Hyptis da região do semi-árido da Bahia. In: SIMPÓSIO BRASILEIRO DE ÓLEOS ESSENCIAIS, 3., 2005, Campinas, SP. Anais... Campinas: Instituto Agronômico de Campinas, 2005. p.118. ).

Essential oils are volatile compounds, consisting mainly of mono- and sesquiterpenes, with wide applications in the pharmaceutical, food, cosmetic, and bio-pesticide industries. These oils derive from the secondary metabolism of plants, and accumulate in trichomes or secretory cavities (TAIZ & ZEIGER, 2013TAIZ, L.; ZEIGER, E. Fisiologia vegetal. Porto Alegre: ArtMed, 2013. 954p.). Their production depends mainly on genetic and environmental factors, although they can be influenced by cultivation techniques (CASTRO et al., 2002CASTRO, D.M. et al. Composição fitoquímica dos óleos essenciais de folhas da Lippia alba (Mill). N.E.Br em diferentes épocas de colheita e partes do ramo. Revista Brasileira de Plantas Medicinais, v.4, n.2, p.75-79, 2002. Available from: <Available from: http://www.ibb.unesp.br/Home/Departamentos/Botanica/RBPM-RevistaBrasileiradePlantasMedicinais/artigo_13_v4_n2.pdf >. Accessed: Nov. 06, 2014. doi: 1516-0572.
http://www.ibb.unesp.br/Home/Departament... ), with the harvest season appearing to have a marked effect, as oil volumes and active constituents are not constant throughout the year (GOBBO-NETO & LOPES, 2007GOBBO-NETO, L.; LOPES, N.P. Medicinal plants: factors of influence on the content of secondary metabolites Química Nova, v.30, n.2, p.374-381, 2007. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-40422007000200026 >. Accessed: Nov. 06, 2014. doi: 10.1590/S0100-40422007000200026.
http://www.scielo.br/scielo.php?script=s... ).

Environmental factors such as temperature, rainfall, relative humidity, irradiance, and photoperiod can affect the production of these secondary compounds. In Lippia alba (Mill), for example, the yields of essential oil were higher in the summer when the temperature was elevated (BARROS et al., 2009BARROS, F.M.C. et al. Seasonal variability and terpenoid biosynthesis of the essential oil of Lippia alba (Mill.) N. E. Brown (Verbenaceae). Quimica Nova, v.32, n.4, p.861-867, 2009. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0100-40422009000400007&script=sci_arttext >. Accessed: Nov. 06, 2014. doi: 10.1590/S0100-40422009000400007.
http://www.scielo.br/scielo.php?pid=S010... ). BOTREL et al. (2010BOTREL, P.P. et al. Teor e composição química do óleo essencial de Hyptis marrubioides Epl., Lamiaceae em função da sazonalidade. Acta Scientiarum Agronomy, v.32, n.3, p.533-538, 2010. Available from: <Available from: http://www.scielo.br/pdf/asagr/v32n3/a22v32n3.pdf >. Accessed: Nov. 06, 2014. doi: 10.4025/actasciagron.v32i3.3415.
http://www.scielo.br/pdf/asagr/v32n3/a22... ) came to similar conclusions in their research with Hyptis marrubioides Epl., observing that metabolite production was greater during the summer (when day length was longer). Although these results demonstrate the influence of the growth environment, each species tends to respond in a unique fashion, with different levels of essential oil production throughout the year.

Plant growth analysis has been widely used to quantify the rate of assimilation and utilization of organic compounds, and can be very useful in understanding how environmental factors influence the growth and production of essential oils in aromatic species.

The present study sought to evaluate the growth and production of essential oils by M. leucocephalus during a twelve month period and correlate these parameters with climatic factors to determine the most favorable season for cultivation and for obtaining essential oils under the edaphoclimatic conditions of Feira de Santana, Bahia State, Brazil.

MATERIALS AND METHODS:

The experiments were conducted in the experimental unit (Horto Florestal) of the State Universidade Estadual de Feira de Santana (UEFS). This region is under the influence of a semiarid climate (KÖPPEN, 1931KÖPPEN, W. Climatologia. México. Fundo de Cultura Econômica, 1931. 478p.; THORNTHWAITE, 1948THORNTHWAITE, C.W. An approach towards a rational classification of climate. Geographical Review, v.38, p.55-94, 1948. Available from: <Available from: http://www.jstor.org/discover/10.2307/210739?sid=21105704264061&uid=2&uid=4 >. Accessed: Mar. 19, 2015. doi: 10.2307/210739.
http://www.jstor.org/discover/10.2307/21... ) in Bahia State, Brazil, (12°16' S x 38°58' W) at 234 meters above sea level, with a mean annual temperature of 24°C and a mean precipitation rate of 848mm per year. The test plants were obtained through the propagation of stocks kept in the Medicinal and Aromatic Plant Collection of the University; they were identified at the UEFS Herbarium (HUEFS, Voucher 25 322).

Apical cuttings were grown in polypropylene cups filled with Biomix^(r) commercial substrate, with added organic material (1:1) following the methodology described by OLIVEIRA et al. (2011OLIVEIRA, L.M. et al. Propagação vegetativa de Hyptis leucocephala Mart. ex Benth. e Hyptis platanifolia Mart. ex Benth. (Lamiaceae). Revista Brasileira de Plantas Medicinais, v.13, n.1, p.73-78, 2011. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-05722011000100011 >. Accessed: Nov. 06, 2014. doi: 10.1590/S1516-05722011000100011.
http://www.scielo.br/scielo.php?script=s... ). Soil was chemically analyzed and fertilized before planting, using 6.160g of limestone powder, 732.6g of phosphorus (simple-superphosphate), and 10 liters of organic material per plot. After 60 days, the plants were replanted in eight rows per plot (1.2 x 10.0m), in full sunlight. The plots were weeded monthly and irrigated using a micro-sprinkler system. After 120 days of further cultivation, it was initiated random harvesting each month in 1m² subplots (five repetitions).

It was evaluated the following growth parameters on a monthly basis for one year: leaf area, fresh and dry masses of stems, leaves, and flowers, as well as the essential oil content. After weighing, the fresh material was placed in paper bags and dried by forced air circulation at 60°C to a constant weight. Leaf area was estimated from sub-samples, composed of three repetitions of 20 leaves randomly collected from each plot. Leaves were scanned in a HP4C deskjet scanner and their areas were quantified using QUANT software (VALE et al., 2003VALE, F.X.R. et al. QUANT A software for plant disease severity assesmsment. In: INTERNATIONAL CONGRESS OF PLANT PATHOLOGY, 8., 2003, Christchurch, New Zealand. Proceedings... Christchurch, New Zealand, 2003. p.105.). Their corresponding dry weights of each sub-sample were obtained and the mean values were used to estimate the total leaf area of the samples.

Essential oil extractions from the dried leaves and flowers from each subplot were performed by hydrodistillation in a Clevenger apparatus, with 3 hours of distillation. After distillation, the oil was collected and anhydrous sodium sulfate was added to remove excess water. The essential oil content was quantified based on its mass, as determined using an analytical balance, being expressed as a percentage (grams of oil per 100g of dry matter).

Monthly temperature, relative humidity, precipitation, photoperiod, and irradiance data were obtained from the Instituto Nacional de Meteorologia (INMET) in the years 2012 INSTITUTO NACIONAL DE METEOROLOGIA (INMET). Monitoramento das estações automáticas. Available from <Available from http://www.inmet.gov.br/sonabra/maps/automaticas.php >. Accessed: 2012 and 2013.
http://www.inmet.gov.br/sonabra/maps/aut... and 2013, and their means were used to correlate growth parameters with essential oil production.

The variables obtained were calculated using descriptive statistics: minimum and maximum values, means and standard deviations, coefficients of variation (%), and the Shapiro-Wilk normality test. It was also calculated the Spearman correlation coefficients using the CORR procedure of SAS. The correlation coefficients were tested by the Student t test, at a 5% level of significance. The analyses were performed using Statistical Analysis System - SAS software (SAS Institute Inc, 2004SAS INSTITUTE. SAS user's guide: statistic: version 9.1.3. Cary, 2004. 1CD. ). Means were compared using the Scott-Knott test (P≤0.05) using the SISVAR statistical program (FERREIRA, 2010FERREIRA, D.F. Sisvar, version 5.3 (Build 75). Lavras, 2010. 1 CD.).

RESULTS AND DISCUSSION:

Descriptive statistics showed high variability in the growth rate of M. leucocephalus, especially in terms of the dry masses of the flowers (Table 1), with relatively high coefficients of variation (CV), indicating that this species was strongly affected by climatic factors during the different months of the year, and even within each harvest period. The oil content varied greatly (from 0.10% to 0.36%), with a yearly mean of 0.20%, similar to the results reported by LUCCHESE et al. (2005LUCCHESE, A.M. et al. Óleos essenciais do gênero Hyptis da região do semi-árido da Bahia. In: SIMPÓSIO BRASILEIRO DE ÓLEOS ESSENCIAIS, 3., 2005, Campinas, SP. Anais... Campinas: Instituto Agronômico de Campinas, 2005. p.118. ). Among the climatic variables, descriptive statistics demonstrated higher CV for precipitation and irradiance (Table 1), reflecting their irregularity during the year. The data demonstrated normality at a 5% level of significance by the Shapiro-Wilk test.

Growth parameters varied significantly during the twelve months of observation (Table 2), even under irrigated growing conditions. It is noteworthy that M. leucocephalus is a wild species, still in the process of domestication. According to TAIZ & ZEIGER (2013TAIZ, L.; ZEIGER, E. Fisiologia vegetal. Porto Alegre: ArtMed, 2013. 954p.), plants at this stage adopt survival strategies of flexible growth patterns that allow their adaptation to locations that may differ from ideal, particularly with respect to solar radiation.

The total dry matter (TDM) per area was higher during the months of September, November, December, and January (Table 2) when day length and irradiance levels were higher (Figure 1). Correlation analysis demonstrated that these climatic variables tend to positively influence TDM growth, while high humidity tends to decrease this metric (Table 3); the same correlations were observed with stem dry mass (SDM) and flower dry mass (FDM). Higher temperatures increased flower production (Table 3). Total leaf area (TLA) also showed a positive correlation with temperature, irradiance, and photoperiod, while negatively correlated with relative humidity, following the same general pattern as TDM. The leaf dry mass (LDM) was not correlated with these same climatic variables, showing high production during most of the study period indicating that this species can grow well throughout the year if regularly watered.

Figure 1
Irradiance levels and precipitation in relation to increases in total dry matter (TDM) of Martianthus leucocephalus (Mart. Ex Benth.) JFB Pastore, exposed to the edaphoclimatic conditions of Feira de Santana, Bahia State, Brazil.

Essential oil content (OC) showed a positive correlation with irradiance and a negative correlation with precipitation and relative humidity, with its production being highest during months of low rainfall and high levels of solar radiation. The highest essential oil content of Otacanthus azureus (Linden) Ronse was observed in treatments with 100% irradiance, with decreasing production as light levels decreased (SERUDO et al., 2013SERUDO, R.N. et al. Acúmulo de matéria seca e rendimento de óleo da planta Otacanthus azureus em função da luminosidade e adubação nitrogenada. Scientia Plena, v.9, n.11, p.1-5, 2013. Available from: <Available from: http://www.scientiaplena.org.br/sp/article/view/1644/916 >. Accessed: Nov. 06, 2014. doi: 110201-1.
http://www.scientiaplena.org.br/sp/artic... ). Similarly, higher levels of oil production were observed in Lippia citriodora Lam. (GOMES et al., 2009GOMES, P.A. et al. Influência do sombreamento na produção de biomassa, óleo essencial e quantidade de tricomas glandulares em cidrão (Lippia citriodora Lam.). Revista Biotemas, v.22, n.4, p.9-14, 2009. Available from: <Available from: https://periodicos.ufsc.br/index.php/biotemas/article/view/2175-7925.2009v22n4p9/17631 >. Accessed: Nov. 06, 2014. doi: 10.5007/2175-7925.2009v22n4p9.
https://periodicos.ufsc.br/index.php/bio... ) and Hyptis marrubioides Epl. (SALES et al., 2009SALES, J.F. et al. Influência do nível de irradiância no crescimento, produção e composição química do óleo essencial de hortelã-do-campo (Hyptis marrubioides Epl.) Ciências Agrárias, v.30, n.2, p.389-396, 2009. Available from: <Available from: http://www.uel.br/revistas/uel/index.php/semagrarias/article/view/2594 >. Accessed: Nov. 06, 2014. doi: 10.5433/1679-0359.2009v30n2p389.
http://www.uel.br/revistas/uel/index.php... ) when grown in full sunlight. The negative correlation of essential oil production with rainfall and humidity has been observed in other studies, such as that undertaken by PRAVUSCHI et al. (2010PRAVUSCHI, P.R. et al. Efeito de diferentes lâminas de irrigação na produção de óleo essencial do manjericão (Ocimum basilicum L.). Acta Scientiarum. Agronomy, v.32, n.4, p.687-693, 2010. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S1807-86212010000400017&script=sci_arttext >. Accessed: Nov. 06, 2014. doi: 10.4025/actasciagron.v32i4.3160.
http://www.scielo.br/scielo.php?pid=S180... ) with basil (Ocimum basilicum L.). These authors established that excessive irrigation can negatively affect development, resulting in low oil productivity. Studies with Plectranthus amboinicus (Lourr.) Spreng. likewise showed increased essential oil contents in months with low rainfall, indicating that excess water can reduce oil production (CARNEIRO et al., 2010CARNEIRO, F.B. et al. Variação da quantidade de β-cariofileno em óleo essencial de Plectranthus amboinicus (Lour.) Spreng., Lamiaceae, sob diferentes condições de cultivo. Revista Brasileira Farmacognosia, v.20, n.4, p.600-606, 2010. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0102-695X2010000400021&script=sci_abstract&tlng=pt >. Accessed: Nov. 06, 2014. doi: 10.1590/S0102-695X2010000400021.
http://www.scielo.br/scielo.php?pid=S010... ).

The results presented here established that higher essential oil content can be obtained from Martianthus leucocephalus under the edaphic and climatic conditions of Feira de Santana, Bahia State (with irrigation) by planting in May and June (the period in which essential oil production is low, but vegetative growth is higher), with harvesting between September and March when essential oil production is high.

CONCLUSION:

Essential oil content and dry mass accumulation in Martianthus Leucocephalus is influenced by the edaphoclimatic condition of Feira de Santana, and is especially favored by the high irradiance levels during the Austral summer months.

ACKNOWLEDGEMENTS

The authors thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship granting fund of first author, the Fundação de Amparo à Pesquisa do Estado de Bahia (FAPESB) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research financial support.

REFERENCES:

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