Chemical composition of essential oil of Melissa officinalis L. and antioxidant activity from Boa Vista-RR, Brazil

1 Department of organic and Inorganic Chemistry, University of Extremadura, Caceres, Spain. 2 Post-graduate Program in Biodiversity and Biotecnology,State Coordination of Roraima, Federal University of Roraima,Campus Paricarana,Brazil. 3 Faculty of Earth Sciences and Conservation, National University of Agriculture, Catacamas, Olancho, Honduras. 4 Chromatography Laboratory, Institute of Exact Sciences,Department of Chemistry,UFMG,Belo Horizonte-MG Brazil. 5 Department of Food Science, Louisiana State University, United States. 6 Faculty of Technological Sciences, National University of Agriculture,Catacamas, Olancho, Honduras. 7 Higher Tecnological Institute National Autonomous University of Honduras,Tela, Honduras.


INTRODUCTION
Mellissa officinalis L. popularly known as lemon balm, sweet balm or common balm, belongs to the Laminaceae family (Awad et al., 2009). This plant is of Asian and European origin; it is cultivated in Brazil for more than a century; a perennial and can vary from 20 to 80 cm and 30-100 cm in height, with its membranous leaves dark green in the upper and light green. On the underside, it has a large size, petiolate, opposite, lanceolate, oval, hairy and well protruding (Couto, 2006). Authors such as Osbaldeston (2000) point out that M. officinalis was used *Corresponding author. E-mail: ismonterof@unex.es.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License for medicinal purposes more than 2000 years ago. It is known in traditional European medicine aslemonbalm. Saad and Said (2011) point out that lemon balm was used in the Middle Ages to stop bleeding, treat toothache, earache, bent neck and baldness.
M. officinalis L. is a plant used as sedative, and tonic properties are attributed to the nervous system (Aghilikhorasani and Makhzan, 2008) as well as being implicated in relaxation processes and anti-anxiety, insomnia, anti-diarrheal, anti-ulcer properties (Lin et al., 2012;Shakeri et al., 2016;Ghsemi-dehkordiet al., 2002); it presents other functions such as improving benign palpitation and sexual dysfunction (Alijaniha et al., 2015).
In the food industry, according to Bisset and Wichtl (2001), it is a plant used to give fragrance to different foods and beverages; it is also used in the pharmaceutical industry. The essential oil is obtained from flowers with a light yellow color and citric odor, especially the presence of citral, geranial, neral and citronel acetate in their chemical composition (Dawson et al., 1988). Essential oil is responsible for the antibacterial and antifungal properties of the plant (Mimica-dukic et al., 2004). Essential oil of M. officinalis has antioxidant activity, mainly attributed to phenolic acids such as hydrocinamic acid and rosmarinic acid (Caniova and Brandsteterova, 2001). The objective of this work is to study the chemical composition of the essential oil of M. officinalis in the Roraima region (Brazil) in the northern Amazon as well as its antioxidant activity and total phenolic compounds

Preparation of samples
Samples were collected in the Boa Vista-Roraima city (Brazil) and taken to the Laboratory of Environmental Chemistry of the Nucleus of Research and Postgraduate Studies in Technology. The essential oil is separated from the hydrolate and assembled in the amber and refrigerated bottle; it is made in different analyses where a part of the essential oil is sent to the Department of Chemistry of the Federal University of Minas Gerais (UFMG). It is carried out with chromatography analyses. Another part of the oil and hydrolatewere used to carry out the antioxidant activity and total phenolic compoundswere analyzed.

Total phenolic compounds
The determination of total phenolic compounds was performed by the FolinCiocateau method, where a stock solution of 250 g mL -1 of essential oil in methanol was initially prepared. Subsequently, 0.1 mL of this solution was transferred to a test tube and 0.1 mL of methanol was added. Then 2.5 mL FolinCiocateau and 2.0 mL 7.5% sodium carbonate were added to the test tube.The formed solution was taken to a 50°C water bath for 5 min and read on a 760 nm spectrophotometer. The calibration curve was made with a gallic acid standard at concentrations of 5, 10, 20, 40 and 80 g mL -1 of each of the standard solutions was removed and placed in a test tube to which 2.5 mL of FolinCiocateau reagent and 1.5 mL of sodium carbonate solution were added and the absorbance readings at 760 nm (Nakashima, 1993).

Antioxidant activity
For the determination of antioxidant activity, the 2,2-diphenyl-2picrylhydrazine (DPPH) reduction method was used, according to the methodology proposed by Kondo (2002), where primarily the crude samples of essential oil were solubilized in ethanol at concentrations of8, 20, 40 and 80 g mL -1 and subsequently a 60uM DPPH ethanol solution was prepared. Samples were prepared by mixing 50 L of DPPH solution and a control solution, where the sample volume was replaced with 50 uL of ethanol, and absorbance readings at 517 nm were taken. 30 min.
The determination of total phenolic compounds was performed by the FolinCiocateau method, where a Stock solution of 250 g mL -1 of essential oil in methanol was initially prepared. Subsequently, 0.1 mL of this solution was transferred to a test tube and 0.1 mL of methanol was added. Then 2.5 mL Folin Ciocateau and 2.0 mL 7.5% sodium carbonate were added to the test tube. The formed solution was taken to a 50 C water bath for 5 minutes and read on a 760 nm spectrophotometer. The calibration curve was made with a gallic acid standard At concentration of 5, 10, 20, 40 and 80 g mL -1 of each of the standard solutions was removed and placed in a test tube to which 2.5 mL of FolinCiocateau reagent and 1.5 mL of sodium carbonate solution were added and the absorbance readings at 760 nm (Nakashima, 1993). Table 1 presents the main constituents in the identified M. officinalis essential oil as well as the Retention time and the identified substances in total 22 compounds.A total of 22 chemical constituents were identified in M. officinalis essential oil (Figure 1), with GC-FID: geranial (34.6%), neral (26.0%), caryophyllene (7.5%), caryophyllene oxide (5.3%), -pinene (5.3%) and sabinene (3.6%).

RESULTS AND DISCUSSION
Compared to the values determined byKhalili et al. (2018), by hydrodistillation, the geraniale and neral values were 19.53 and 16.39%, respectively; they were lower than those found in this study, being the major constituent found by these authors for the study (caryophyllene oxide with 23.71%). Other authors such as Pirbalouti et al. (2019) studied the composition of the essential oil of M. officinalis by hydrodistillation; geranium values of 38.34% and neral of 31.93% were obtained; values close to those obtained in this work. The differences in the chemical composition of the different constituents of the essential oil influence environmental and genetic factors, as well as post-harvest plant processing factors (Lemos et al., 2017).
The main constituents were later identified by GC-MS ( Figure 2). Table 2 shows the results of total phenolic Compounds and antioxidant activity for M. officinalis L. essential oil. Queiroz et al. (2014) evaluated the phenolic compounds in different extracts of M. officinalis L. where the aqueous extract presented total phenolic compounds concentrations of 817 mgmL -1 . Authors such as deMorais and Nascimento (2016) studied the total phenolic compounds in different phytotherapics from M. officinalis L. where the concentrations obtained from total phenolic compounds ranged from 18.541 to 75.16 mg AGEgrams of sample.

Antioxidant activity
Essential oils are rich in phenolic compounds, with reducing properties that play an important role in free radical sequestration as well as chelation of transitionmetals (Sousa et al., 2007). The percentage of inhibition evaluated with DPPH increases with the concentration of essential oil. M. officinalis L. essential oil can be used as a pharmaceutical and nutritional   product as a natural antioxidant source (Koksal et al., 2011). The inhibition percentage for the major concentration is 68.12% high compared to the quercetin standard used as positive standard with the inhibition percentage of 88.92%

Conclusion
The present work provides information on the chemical profile of 22 constituents of the M. officinalis L. essential oil by the hydro distillation method. It is a simple, fast and free of any residual solvent, being a method used to quantitatively determine constituents, volatile foods and medicine.