Effect of drying temperature on the yield and phytochemical quality of the essential oil of pepper rosemary (Lippia origanoides Kunth) and of clove basil (Ocimum gratissimum L.) / Efeito da temperatura de secagem no rendimento e na qualidade fitoquímica d

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Braz. J. of Develop.,Curitiba, v. 6, n. 8, p. 57107-57120 aug. 2020. ISSN 2525-8761

Effect of drying temperature on the yield and phytochemical quality of the

essential oil of pepper rosemary (Lippia origanoides Kunth) and of clove basil

(Ocimum gratissimum L.)

Efeito da temperatura de secagem no rendimento e na qualidade fitoquímica

do óleo essencial de alecrim-pimenta (Lippia origanoides Kunth) e de alfavaca

(Ocimum gratissimum L.)

DOI:10.34117/bjdv6n8-208

Recebimento dos originais: 25/07/2020 Aceitação para publicação: 14/08/2020

Maira Christina Marques Fonseca

Doutora em Fitotecnia pela Universidade Federal de Viçosa (UFV) Pesquisadora - Empresa de Pesquisa Agropecuária de Minas Gerais Vila Gianeti, casa 46. Campus da UFV, Viçosa, MG, Brasil. CEP: 36570-900

E-mail: mairacmf@yahoo.com.br

Cláudia Lúcia de Oliveira Pinto

Doutora em Microbiologia Agrícola pela Universidade Federal de Viçosa (UFV) Pesquisadora aposentada - Empresa de Pesquisa Agropecuária de Minas Gerais Rua dos Estudantes, 120, apto 202, Centro, Viçosa, MG, Brasil. CEP: 36570-081

E-mail: caudia.epamig@gmail.com

Maria Aparecida N. Sediyama

Doutora em Fitotecnia pela Universidade Federal de Viçosa (UFV) Pesquisadora aposentada da Empresa de Pesquisa Agropecuária de Minas Rua Vinícius de Moraes, 157, Bairro de Fátima, Viçosa, MG, Brasil. CEP: 36572-164

E-mail: mariasediyama@gmail.com

Adilson Sartoratto

Doutor em Química pela Universidade Estadual de Campinas (Unicamp)

Pesquisador do Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas da Unicamp

Rua Alexandre Cazelatto, 999 - Vila Betel, Paulínia, SP, Brasil. CEP: 13148-218 E-mail: adilson@cpqba.unicamp.br

Túlio I. Machado

Mestrando em Biologia pela Universidade Federal de Viçosa (UFV)

Av. Peter Henry Rolfs, s/n - Campus Universitário, Viçosa, MG, Brasil. CEP: 36570-900 E-mail: tulioiglesias96@gmail.com

Sérgio Maurício L. Donzeles

Doutor em Engenharia Agrícola pela Universidade Federal de Viçosa Pesquisador da Empresa de Pesquisa Agropecuária de Minas Gerais Vila Gianetti, Casa 46, Campus da UFV, Viçosa, MG, Brasil. CEP: 36570-900

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Estermary P. Bitencourt

Farmacêutica pela Univiçosa – Centro Universitário e Viçosa

Av. Maria de Paula Santana, 3815 - Silvestre, Viçosa, MG, Brasil, CEP: 36576-340 E-mail: estermarypb@hotmail.com

Maria Regina de M. Souza

Doutora em Fitotecnia pela Universidade Federal de Viçosa (UFV) Pesquisadora da Empresa de Pesquisa Agropecuária de Minas

Vila Gianeti, casa 46. Campus da UFV, Viçosa, MG, Brasil. CEP: 36570-900 E-mail: reginas.epamig@gmail.com

Andréia F. Silva

Mestre em Botânica pela Universidade Federal de Viçosa Pesquisadora da Empresa de Pesquisa Agropecuária de Minas

Av. José Cândido da Silveira, 1.647, União, Belo Horizonte, MG, Brasil. CEP: 31170-495 E-mail: andreiasilva@epamig.br

Yonara P. Neves

Doutora em Fitotecnia pela Universidade Federal de Viçosa (UFV) Pesquisadora da Empresa de Pesquisa Agropecuária de Minas Gerais Vila Gianeti, casa 46. Campus da UFV, Viçosa, MG, Brasil. CEP: 36570-900

E-mail: yonarapoltronieri@hotmail.com

ABSTRACT

Among medicinal species, Lippia origanoides Kunth and Ocimum gratissimum L. stand out for its recognized therapeutic value associated with the medicinal properties of their essential oils. Drying is a critical point in post-harvest processing of medicinal plants for the conservation of bioactive compounds. This work aimed to evaluate the effect of drying temperature on the essential oil’s yield and phytochemical quality extracted from leaves of O. gratissimum and L. origanoides. The medicinal species were grown in an organic system and their leaves were harvest and immediately dried at 40, 50 and 60 °C. The essential oils were extracted from the leaves by hydrodistillation in Clevenger apparatus. There was detected a difference (P <0.01) in the essential oil yield of O.

gratissimum with the increase in the drying temperature, being 1.35% (40 °C), 0.83% (50 °C) and

0.45% (60 °C) ). In L. origanoides there was detected a difference (P <0.01) in the essential oil yield only between 40 °C (3.4%) and 60 °C (2.84%). The increase of drying temperature reduced the levels of the major constituents of O. gratissimum (eugenol, germacrene D and caryophyllene oxide), but did not change the levels of the major constituents of L. origanoides (thymol, trans-caryophyllene and para-cymene). Considering that the increase of the drying temperature promote a reduction in the essential oil yield of both medicinal species and also in the major constituents concentration in O. gratisssimum, it is essential to select the correct drying temperature to guarantee the best yield and the phytochemical quality of the essential oil to ensure therapeutic efficacy. Here, we conclude that the best drying temperature to the essential oil’s yield and phytochemical quality is 40 °C for O. gratissimum and 40 or 50 °C for L. origanoides.

Keywords: medicinal plants, post-harvest, volatile compounds RESUMO

Entre as espécies medicinais, Lippia origanoides Kunth e Ocimum gratissimum L. se destacam por seu reconhecido valor terapêutico associado às propriedades medicinais de seus óleos essenciais. A

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secagem é um ponto crítico no processamento pós-colheita de plantas medicinais para a conservação de compostos bioativos. Este trabalho teve como objetivo avaliar o efeito da temperatura de secagem no rendimento do óleo essencial e na qualidade fitoquímica extraída das folhas de O. gratissimum e L. origanoides. As espécies medicinais foram cultivadas em sistema orgânico e suas folhas foram colhidas e imediatamente submetidas à secagem a 40, 50 e 60 °C. Os óleos essenciais foram extraídos das folhas por hidrodestilação em aparelho Clevenger. Detectou-se diferença (P <0,01) no rendimento de óleo essencial de O. gratissimum com o aumento da temperatura de secagem, sendo 1,35% (40 °C), 0,83% (50 °C) e 0,45% (60 °C). Em L. origanoides foi detectada diferença (P <0,01) no rendimento de óleo essencial apenas entre 40 °C (3,4%) e 60 °C (2,84%). O aumento da temperatura reduziu os níveis dos principais constituintes do óleo essencial de O. gratissimum (eugenol, germacreno D e óxido de cariofileno), mas não alterou os níveis dos principais constituintes de L. origanoides (timol, trans-cariofileno e para-cimeno). Considerando que o aumento da temperatura de secagem promoveu redução no rendimento de óleo essencial de ambas as espécies medicinais e também na concentração de constituintes principais em O. gratisssimum, é essencial selecionar a temperatura de secagem adequada para cada espécie medicinal para garantir melhor rendimento e qualidade fitoquímica do óleo essencial, e garantir sua eficácia terapêutica. Concluímos que a melhor temperatura de secagem visando o rendimento e a qualidade fitoquímica de óleo essencial é de 40 °C para O. gratissimum e entre 40 e 50 °C para L. origanoides.

Palavras-chave: Compostos voláteis, plantas medicinais, pós-colheita

1 INTRODUCTION

According to the World Health Organization (WHO), medicinal plant is considered as any vegetable that has, in one or more organs, substances that can be used for therapeutic purposes or that are precursors of semi-synthetic drugs (WHO, 1998). In Brazil, the National Program for Medicinal Plants and Herbal Medicines was approved and the National Committee for Medicinal Plants and Herbal Medicines was created (BRAZIL, 2008). The main guiding instruments for the development of actions and Programs for Medicinal Plants and Phytotherapy in public health are the National Policy on Medicinal Plants and Phytotherapics (NPMPP), which provides guidelines for the development of the productive chain of medicinal and phytotherapic plants. The second is the National Policy of Integrative and Complementary Practices (NPICP) in the Unified Health System (UHS), which includes guidelines, actions and responsibilities of federal, state and municipal entities for the insertion of services in the public network, among other practices, medicinal plants and phytotherapy (RODRIGUES, 2008).

Despite Brazil's great biodiversity, there are some obstacles to the production and safe use of medicinal plants in the country, namely: extraction, irregular supply and quality control of vegetable raw materials (BARATA, 2005). To reverse this situation, it is essential to develop applied researches focused on the production and post-harvest of medicinal species with the use of appropriate technologies aiming at the conservation of the active principle of interest and,

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consequently, the achievement of the desired therapeutic effect. Among the 71 medicinal species listed in the National List of Medicinal Plants of Interest to the SUS (BRAZIL, 2009), there are pepper rosemary (Lippia origanoides Kunth) and clove basil (Ocimum gratissimum L.), which stand out for their recognized medicinal value associated with essential oils extracted from their leaves and flowers.

L. origanoides belongs to the Verbenaceae family and is popularly known as pepper

rosemary and native to the semiarid region of northeastern Brazil. (CAVALCANTI et al., 2010). The extracted essential oil and its leaves are composed mainly of thymol and carvacrol, compounds that are associated with their biological activity (BRASIL, 2009). In folk medicine, the infusion of its leaves is used for gastrointestinal disorders and respiratory diseases (LINDE et al., 2010). Its essential oil is of great importance in the pharmaceutical, food and cosmetic areas considering its biological activities such as insecticide, larvicide, antimicrobial (CAVALCANTI et al., 2004) and anti-inflammatory (VERAS et al., 2014).

O. gratissimum belongs to the Lamiaceae family, known as clove basil, has its origin in the

east and is naturalized in practically all Brazilian territory (OCIMUM, 2020), presenting different chemotypes (LORENZI & MATOS, 2008). The essential oil extracted from its leaves can mainly contain eugenol, according to the chemotype, being the eugenol most common chemotype (BIASIL et al., 2009). Clove basil essential oil is used to treat respiratory infections, diarrhea, headache, fever, eye problems, skin diseases and pneumonia, in addition to being a potent antidiabetic and antimicrobial agent (MATASYOH et al., 2007; BRASIL, 2015).

Several factors influence the phytochemical quality of essential oils extracted from medicinal species, including climatic variations, the soil, harvest time, the genetic characteristics of the plant, as well as drying and storage conditions (CASTRO & FERREIRA, 2001; BARBOSA et al ., 2008; BIASIL et al., 2009; MARTINAZZO et al., 2009; ROCHA et al., 2012).

In post-harvest processing, drying is a critical point for maintaining plant quality, conserving bioactive compounds and oil yield, considering that these constituents, in general, are very sensitive to high temperatures (MATTOS, 2000). One of the critical points to be studied in the drying process is the determination of the ideal drying temperature for each medicinal species in order to preserve the bioactive compounds of the plants (RADÜNZ et al., 2002).

During the drying process of medicinal plants, there is a reduction of humidity and, consequently, of the enzymatic action, which allows its conservation for longer periods and reduces the multiplication of contaminating microorganisms. Thus, the color and aroma are preserved, characteristics that indicate the quality of the plant material (CORREA JUNIOR et al., 1994). Thus,

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studies on the influence of the drying temperature of medicinal species on the yield and phytochemical quality of essential oils are essential for better preservation of the chemical constituents of interest present in essential oils, factors that are critical to guarantee the therapeutic effect and its economic value. The objective of this study was to verify the effect of drying temperature on the yield and phytochemical quality of essential oils extracted from leaves of L.

origanoides and O. gratissimum. 2 MATERIAL AND METHODS

2.1 GEOGRAPHIC COORDINATES, SOIL CHARACTERISTICS, FERTILIZATION AND IRRIGATION

The research was carried out at the Vale do Piranga Experimental Field of the Minas Gerais Research and Agriculture Company (EPAMIG), in Oratórios-MG. The geographical coordinates are: latitude 20° 30” S, 43° 00” and altitude 500 m, with the following meteorological variables: average annual maximum temperature of 21.8 °C and annual minimum of 19.5 °C and precipitation average annual rainfall of 1,250 mm. The predominant climatic type in the place is the Cwa, humid tropical, the Aw, semi-humid of hot summers, being the original vegetation constituted of subcaducifolia tropical forest, according to Köppen's classification (CUNHA et al., 2000). The loam-clay-sandy soil presented the following chemical characteristics: pH (water 1: 2.5) = 6.2; P (Mehlich) = 49.8 mg dm-3_{; P (remainder) = 40 mg L}-1_{; K = 220 mg dm}-3_{; Ca = 2.6 cmol dm}-3_{; Mg}

= 1.3 cmol dm-3; Al = 0 cmol dm-3; H + Al = 2.8 cmol dm-3; SB = 4.5 cmol dm-3; t = 4.5 cmol dm

-3_{; m = 0%; T = 7.3 cmol dm}-3_{; V = 61%; Organic matter = 2.63 dag kg}-1_{. Fertilization was carried}

out using tanned bovine manure, with the following chemical characteristics (%): N (1.4), P (0.39), K (0.88), Ca (1.54), Mg (0, 27), S (0.23), CO (10.45); C / N (7.46). The drip irrigation system was used and, for the control of spontaneous plants, manual weeding was performed when necessary.

2.2 BOTANICAL IDENTIFICATION

Samples of both species were sent to EPAMIG's PAMG Herbarium, where the botanical identification was confirmed. Exsiccates were assembled (herborization), according to usual botanical standards and incorporated into the collection of the PAMG / EPAMIG Herbarium: Lippia

origanoides Kunth (Verbenaceae), PAMG 57975 and Ocimum gratissimum L. (Lamiaceae), PAMG

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Braz. J. of Develop.,Curitiba, v. 6, n. 8, p. 57107-57120 aug. 2020. ISSN 2525-8761 2.3 SEEDLING PRODUCTION, PLANTING, CULTIVATION AND HARVESTING

The production of L. origanoides and O. gratissimum seedlings was carried out by cuttings, in a greenhouse. The planting and cultivation of medicinal species were carried out in the experimental field, in an area of 2 x 12 m, using the spacing of 0.5 x 1.0 m between plants. For extraction of essential oil, analysis of yield and phytochemical quality, 16 plants located in the central part of the production area were harvested. The branches were harvested during the fall of 2019, between April and May, in the morning.

2.4 DRYING OF LEAVES

Immediately after harvesting, the leaves were detached from the branches and submitted to drying, in an oven with forced air circulation, at 40, 50 and 60 ºC, until reaching constant weight. Subsequently, they were packed in sealed polyethylene bags and packed in kraft paper packaging until the moment of essential oil extraction.

2.5 EXTRACTION OF ESSENTIAL OIL, CALCULATION OF YIELD AND STORAGE The extraction of essential oils was done in a Clevenger apparatus, adapted to a round-bottomed flask with a capacity of 1000 mL. Distilled water (500 mL) and 50 g of dry leaves of the medicinal species were added to the flask, beginning the hydrodistillation process. The extraction was carried out by dragging the essential oil through water vapor, for 2h 30min for both species. Afterwards, the percentage of oil yield was calculated. The oil samples were stored in an amber glass bottle with a screw cap, under refrigeration (4 °C) for further chromatographic analysis.

2.6 IDENTIFICATION OF VOLATILE CONSTITUENTS

The analysis of volatile constituents was performed in an Agilent gas chromatograph, model HP-6890, equipped with an Agilent mass selective detector, model HP-5975 and an HP-5MS capillary column (30 mx 0.25 mm x 0.25 µm). The analysis was performed in splitless injection mode, with the following temperature conditions: injector at 220 °C, column at 60 °C, with heating ramp of 3 °C min-1 and final temperature of 240 °C and detector at 250 °C. Helium was used as carrier gas at a flow rate of 1 mL min-1. A sample of essential oil was dissolved in ethyl acetate (20 mg ∙ mL-1_{) for analysis. The identification of the chemical constituents of essential oils was carried}

out by comparing the retention indices (IR) obtained by the injection of hydrocarbon standards (C-8 to C-24), through the equipment database (NIST-11 library) and with literature data (ADAMS, 2007).

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Braz. J. of Develop.,Curitiba, v. 6, n. 8, p. 57107-57120 aug. 2020. ISSN 2525-8761 2.7 STATISTICAL ANALYSIS

For the analysis of oil yield, the Tukey test was used to compare the averages at 5% probability, with the aid of the SAEG statistical analysis program (RIBEIRO JR., 2001).

3 RESULTS AND DISCUSSION

3.1 YIELD OF ESSENTIAL OIL EXTRACTED FROM O. gratissumum AND L. origanoides LEAVES

There was a significant reduction (P <0.01) in the yield of essential oils with an increase in the drying temperature of the leaves of O. gratissimum (Figure 1A). The yield obtained (1.4%) at 40 °C was higher than that found by Borges et al. (2012) in dry leaves of commercial clove basil (1.02%), by Brada (2011), of 0.8 ± 0.1% and Njoku et al. (2017), of 0.59%. Possibly, temperatures above 40 °C caused damage to leaf trichomes, where essential oils are stored in the clove basil, contributing to the volatilization of oil constituents. This type of damage was reported by Santana et al. (2014) who observed damage to the trichomes of O. gratissimum leaves when subjected to drying in an oven with forced ventilation at 60 ºC. Therefore, it is recommended to dry the leaves of clove basil at 40 °C, for greater yield and phytochemical quality of the essential oil.

In L. origanoides there was a difference (P <0.05) only between the yield of essential oil extracted from leaves subjected to 40 °C and 60 °C (Figure 1B). These results differ from those obtained by Radünz et al. (2002) who found no significant difference in the yield of oils extracted from the leaves of L. sidoides dried at 40, 50, 60 and 70 °C. Moreover, the oil yields observed in this work were higher (Figure 1B), regardless of the drying temperature, than those of Radünz et al. (2002), which was, on average, 2.865%.

The differences found in the yields of the essential oils of O. gratissimum and L. origanoides may be related to the time of harvest (FIGUEIREDO et al., 2009), soil, climate, plant development stage, drying method, irrigation blade ( ALVERENGA et al., 2010), among others (GOBBO-NETO & LOPES, 2007). Thus, it can be inferred that all stages, from production to the extraction of the active ingredient of interest, are important and must be adequate to optimize the performance and quality of essential oils, for each medicinal species.

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Figure 1. Yields of essential oils extracted from leaves of O. gratissimum (A) and L. origanoides (B) submitted to drying in an oven with air circulation at 40, 50 and 60 ºC.

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3.2 IDENTIFICATION OF CHEMICAL CONSTITUENTS IN O. gratissimum ESSENTIAL OILS Eugenol remained as a major constituent in the essential oil extracted from O. gratissimum leaves at all drying temperatures (Table 1), similar results to those was observed in other studies (DUBE, UPADHYAY & TRIPATHI, 1989; SARTORATTO et al., 2004; DAMBOLENA et al., 2010). The highest eugenol content was observed in dry leaves at 40 °C (83.25%), higher than those found by Biasil et al. (2009), of 80.8% in leaves also harvested in autumn, by Matasyoh et al. (2007), 68.8%, and by Brada et al. (2011), of 54.8%.

In terms of the majority compounds, these results differ from those observed by other authors (NJOKU et al., 2017; KÉITA et al., 2000; PRAKASH et al., 2011). What can be associated with drying methods (SHALABY et al., 1995; SILVA et al., 2004) and edaphoclimatic conditions (SIMÕES & SPIT)

A B C 0 1 2 3 4 40 50 60 Esse nti al oi l y ield (% ) Drying temperature (°C) A _AB B 0 1 2 3 4 40 50 60 Esse nti al oi l y ield (% ) Drying temperature (°C)

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Table 1. Identified analytes and their content in essential oils extracted from O. gratissimum leaves submitted to drying in an oven with forced air circulation at 40, 50 and 60 ºC

3.3 IDENTIFICATION OF CHEMICAL CONSTITUENTS IN ESSENTIAL OILS OF L.

origanoides

The constituents identified in the essential oil extracted from the leaves of L. origanoides are shown in Table 2, in which thymol is the major constituent. There was no reduction in thymol content (average content of 81.08%) with increasing drying temperature, which was also observed by Radünz et al. (2002), whose average thymol content was 84%.

The average thymol content found was similar to that observed by Cavalcanti et al. (2004), in which thymol was also the majority (80.8%) and by Brito et al. (2015) (84.95% thymol), and, higher than the levels observed by Cavalcanti et al. (2010) (68.81%), Botelho et al. (2007) (56.7% thymol) and Fontenelle et al. (2007) (59.65% thymol).

As for the levels of carvacrol, the results obtained in this research 0.26% (40o_{C), 0.27%}

(50o_{C) and 0.30% (60}o_{C) differ from those observed by Guimarães et al. (2014) who found carvacrol}

(26.44%) and 1.8-cineol (22.63%) as major compounds; and by Oliveira et al. (2007), where the major compounds were oxygenated monoterpenes (66%), carvacrol (38.6%) and thymol (18.5%). Although the medicinal species (L. origanoides), the season, the time of harvest (morning) and the method of extraction (hydrodistillation) are common, this difference can be attributed to several factors such as: origin of the plant material, genetic variation or chemotypes (STACHENKO et al., 2010), post-harvest treatment, among others (RUIZ et al., 2007).

tR (min) RI Identification Drying temperatures

40 °C 50 °C 60 °C 8.26 1036 cis-beta-ocimene 2.10 3.23 --- 9.32 1067 cis-sabinene hydrate --- 2.18 3.23 13.54 1177 terpinen-4-ol 2.33 1.15 2.04 21.22 1362 eugenol 83.25 74.33 50.89 21.75 1375 alpha-copene 1.00 1.47 1.95 22.11 1384 beta-bourbonene 1.10 1.13 1.90 23.50 1418 trans-karyophylene 2.78 1.87 2.14 25.99 1480 germacrene D 4.26 1.69 --- 27.68 1522 delta-cadinene 0.71 --- --- 29.92 1581 karyophylene oxide 3.24 6.74 17.36 30.33 1591 salvial-4 (14) -en-1-one --- 1.53 --- 33.79 1684 eudesma-4 (15), 7-dien-1beta-ol --- 1.60 4.64

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Table 2. Identified analytes and their content in essential oils extracted from L. origanoides leaves submitted to drying

in an oven with forced air circulation at 40, 50 and 60 ºC

4 CONCLUSIONS

The temperature of 40 °C is recommended for drying O. gratissimum leaves, considering that the increase in drying temperature significantly reduces the yield of essential oil and the eugenol content of 11% (50 °C) and 39% (60 °C).

For L. origanoides leaves, temperatures of 40 °C or 50 °C can be used without significant reduction in the yield and phytochemical quality of the essential oil.

tR (min) RI Identification Drying temperatures 40 °C 50 °C 60 °C 5.15 925 alpha-thujene 0.45 0.30 0.18 5.32 932 alpha-pinene 0.14 --- --- 6.79 990 beta-mycrene 0.78 0.66 0.62 7.59 1016 alpha-terpinene 0.78 0.73 0.70 7.86 1024 para-cimene 4.89 4.67 4.91 7.98 1027 limonene --- 0.23 0.23 8.05 1029 1,8-cineole (eucalyptol) 0.38 0.23 0.25 8.99 1057 gamma-terpinene 2.35 2.14 1.65 12.26 1145 ipsdienol 0.64 0.66 0.59 13.52 1176 terpin-4-ol 0.56 0.53 0.54

15.88 1233 thymol, methyl ester 0.72 0.64 0.70

18.76 1302 thymol 80.29 82.41 80.53 18.95 1306 carvacrol 0.26 0.27 0.30 21.73 1373 alpha-copene 0.36 0.32 0.42 23.52 1416 trans-karyophylene 4.68 4.24 5.25 24.28 1435 aromadendrene 0.36 0.37 0.49 24.86 1450 alpha-humulene 0.30 0.28 0.34

26.34 1486 hydroxy anisol butylate 0.46 0.46 0.50

26.54 1491 M = 204 0.47 0.38 0.47

27.66 1520 delta-cadinene 0.31 0.33 0.36

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ACKNOWLEDGMENTS

We would like to thank the Minas Gerais State Research Support Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) for the financial support.

FUNDING

This work was supported by the Minas Gerais State Research Support Foundation (FAPEMIG) process (CAG - APQ-01358-16) and by the National Council for Scientific and Technological Development (CNPq).

DECLARATION OF INTEREST

The authors declare that there is no conflict of interest.

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