Phytochemical Profile, Antioxidant and Antibacterial Activity of the Essential Oil of Luma Chequen (Molina) A. Gray from Peru

. ABSTRACT Background: Luma chequen belongs to Myrtaceae family and it is known as “arrayan”. In the traditional medicine from Peru, L chequen is used as aromatic plant, anti-inflammatory and hypocholesterolemic. Objective: To determine the phytochemical profile, evaluate the antioxidant and the antibacterial activity of L. chequen essential oil. Material and Methods: In the analysis of the volatile components a Gas Chromatography coupled to Mass Spectrometry (GC-MS) was used to identify the content of terpenes and sesquiterpenes. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was the method used to determine the antioxidant activity and obtain the half inhibitory concentration (IC 50 ). For the antibacterial activity, a colorimetric macrodilution method was carried out to evaluate the effect of the essential oil of L. chequen against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Results: The analysis by GC-MS showed two major components alpha-pinene (62.89%) followed by 1,8-cineole (11.94%), and propanoic acid, 2-methyl-, 2-methylpropyl ester with 8.67%. In the antioxidant activity against DPPH radical, the essential oil of L. chequen showed an IC 50 equivalent to124.60 ± 2.0 µg/mL. In the antibacterial activity, L. chequen had an MIC (minimum inhibitory concentration) for Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 of 4.35 ± 0 µg/mL and 8.71 ± 0 µg/mL respectively. Conclusion: L. chequen presented monoterpene compounds as main phytoconstituents as well as antioxidant and antibacterial activity in vitro . The essential oil might be used as antimicrobial agent in the future overall against S. aureus .


INTRODUCTION
Aromatic plants are the natural sources of essential oils (EO), which are chemical compounds of low molecular weight and volatile nature.On the other hand, essential oils generate interest in the scientific community due to the presence of promising bioactive metabolites, which have been reported antibacterial, antiviral, antifungal, insecticidal, and repellent effects, among others. 1However, its antibiofilm capacity is the one that has generated the most interest due to resistance to multiple drugs and essential oils could be an alternative from a natural source. 2,3aphylococcus aureus (a gram-positive bacteria) is one of the leading causes of bacteremia, endocarditis, cutaneous, osteoarticular, and respiratory infections. 4On the other hand, Escherichia coli (a gram-negative bacteria) typically colonizes the gastrointestinal tract.Typically, E. coli and its human host coexist for decades in excellent health and mutual benefit.These commensal E. coli strains rarely cause disease unless the host is immunocompromised or in episodes like in peritonitis. 5ma chequen is a perennial tree located in South American Andes between 2500 and 4000 masl.It belongs to Myrtaceae family, in Peru is known as "arrayan", within its medicinal uses are for gastrointestinal and respiratory disorders, migraine, and muscular pain.L. chequen growths in the departments of Junin, Ayacucho, Cusco, Lima, Ancash and Pasco. 6The essential oil of leaves and twigs of L. chequen presented two major compounds known as α-pinene and 1,8-cineole, 7 and some biological activities have been demonstrated such as its antioxidant, antibacterial and fungicide activities. 8dditionally, the essential oil had antimicrobial effect against Cladosporium cladosporioides, Cryptococcus neoformans and Proteus vulgaris. 9rrently, searching new antibacterial bioactive compounds from natural sources is still in preclinical phase but some of them are being tested in medical trials.Hence, the aim of this study was to investigate the antioxidant and antibacterial activity of the essential oil of L. chequen against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.

Plant material and essential oil obtention
Five kilograms of Luma chequen of the aerial parts were collected in the province of La Mar, department of Ayacucho, Peru (2 660 masl).The plant was authenticated in the herbarium of the Universidad Nacional Mayor de San Marcos (081-USM-2017).The aerial parts of L. chequen were selected and washed with a 0.1% sodium hypochlorite solution and dried until they were incorporated into a Clevenger apparatus, after two hours, the essential oil was separated by decantation and a few milligrams of anhydrous Na 2 SO 4 were added to purify remaining water essential oil.Finally, the essential oil was stored in sealed amber vial at 4°C until further use.

Gas chromatography analysis of the essential oil of L. chequen
The filtered sample was diluted in the proportion of 1:100 (V/V) in filtered acetone and placed in a vial.The vial was immediately positioned in the autosampler of the GC-MS system (SHIMADZU, GC-2010 Plus).1.0 µL of the work solution was injected into the equipment in splitless mode (Split: 20:1).The sample was run on a RESTEK.RTX-5MS, 30m x 0.25 mm ID x 0.25 µm.The work conditions were the followings: the temperature program was 50 °C starting with increments of 3 °C/ min up to 150 °C for 10 min; and followed by increases of 3°C/min up to 250 °C for 20 min.The helium flow rate was 0.80 mL/min.Volatile chemicals were based on computer matching with the mass spectra from the NIST20 library. 10

Antioxidant activity against DPPH radical
To carry out the antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used as organic radical according to Rojas-Armas et al 11 with slight modifications.Our conditions to measure the antioxidant capacity were: Different conditions of the essential oil of L. chequen (10-500 µg/mL) diluted with dichloromethane were mixed with DPPH solution (0.01mM) prepared with methanol, 300 µL of each sample dilution reacted with 2700 µL of DPPH.Control was performed between 300 µL of methanol and 2700 µL of DPPH.Trolox at 250 mM was used as antioxidant control.After 30 minutes of reaction under dark conditions, absorbances were measured at 517 nm in an UV spectrophotometer.All procedures were done by triplicated.The formula to calculate the percentage of antioxidant activity was: where X 0 is the absorbance of the control (Absorbance must be between 0.6 ± 0.05) and A 1 is the absorbance of the essential oil reacted with DPPH and corrected by the absorbance of blank.Half inhibitory concentration (IC 50 ) was determined by linear regression.

Antibacterial activity of the essential oil of L. chequen
To assess the antibacterial activity, a colorimetric macrodilution method was carried out to evaluate the effect of the essential oil of L. chequen against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922.Both microorganisms were kept on Trypticase Soya agar.Then each dilution was made in Müeller Hinton broth, and then was mixed with resazurin solution.The final concentrations of the essential oil of L. chequen ranged from 0.05 to 40 μL/mL.The positive control was ciprofloxacin following the CLSI recommendations.

Statistical analysis
Results were determined in triplicate, percentages and IC 50 were calculated using GraphPad Prism v6 program.P values less than 0.005 is considered statistically significant.

Chemical profile of the essential oil of L. chequen
According to our results showed in Table 1, we identified 25 compounds, the analysis identified to alpha-pinene (monoterpene) as the main component of the volatile constituents with 62.89% followed by eucalyptol (11.94%), and propanoic acid, 2-methyl-, 2-methylpropyl ester with 8.67% (figure 2).

Antioxidant profile of the essential oil of Luma chequen
The main volatile phytochemicals were responsible of the antioxidant activity against DPPH.The essential oil of L. chequen exhibited a strong antioxidant activity as is shown in Table 2. Trolox also showed better antioxidant activity than essential oil with an IC 50 of 5.4 μg/mL (P=0.3040) and an TEAC of 25 ± 0.01 mg ET/ g essential oil.On the other concentrations there was significant differences between EO and Trolox (P < 0.001).The equation to determine the antioxidant equivalent to Trolox is showed in the Figure 3.

DISCUSSION
Currently, the industry of essential oils extracted from aromatic plants has gained great importance due to its usefulness in various fields such as the manufacture of perfumes, cosmetic products, flavorings, in the food and pharmaceutical industry. 12Essential oils are concentrated, aromatic and volatile hydrophobic liquids obtained from plants, they are made up of terpenoids, sesquiterpenes, alcohols, acids, acyclic esters, aldehydes and lactones. 13garding the phytochemical analysis by GC-MS of L. chequen essential oil, our result showed in Table 1  showed values for alpha-pinene of 57.3% and 1,8-cineole of 7.5%, this essential oil was obtained by steam distillation at 0.72 atm and 91°C for 75 min.According to these values in some cases also depends on its extraction using conventional techniques, the temperature, soil, climate factors and other factors might be influencing in the composition of the volatile constituents. 14In our study, the major component and representative molecule in the essential oil of L. chequen was alphapinene with 62.89% followed by eucalyptol (1,8-cineole) with 22.08%.
In another study reported by Alvarado-Garcia et al, the essential oil of L. chequen from Ancash (Peru) had as main components to α-pinene (56.5%), and 1,8-cineole (8.5%). 6garding the DPPH activity, the mechanism is focused on the sample's ability to donate hydrogen to the DPPH radical, which results in the bleaching of the stable DPPH free radical from the purple color of the DPPH cation to the yellow color of diphenylpricryhydrazine.The antioxidant activity is reflected by a lower half inhibitory concentration IC 50 .The recognized antioxidant activity of its components raised the investigation of the influence of this plant in diseases characterized due to its etiology and complications related to oxidative stress such as diabetes, dyslipidemia and atherosclerosis. 15 the other hand, the main microorganisms related to urinary tract infections are enterobacteria, especially Escherichia coli, considered the most prevalent etiological agent, responsible for 80% of infections.Due to the problems associated with the treatment of various infections, especially antibiotic resistance, herbal substances have acquired new perspectives, such as the growing interest in their use in the search for antimicrobial compounds.According to the World Organization Health around 65 to 80% of the population does not have access to primary health care and resort to traditional medicine in search of relief for many diseases.Previous studies have shown that L. chequen produces active metabolites that have antibacterial properties that are capable of destroying or stopping the growth or multiplication of bacteria. 7 a study for both Gram-negative and Gram-positive bacteria, 1,8-cineole altered the morphology and size of bacterial cells.In addition, these bacteria treated with this monoterpene compound induced apoptosis (S. aureus) because they exhibited a strong condensation of nuclear chromatin in the central nucleoplasm and necrosis (E.coli) because there was a clear reduction of nucleoplasm and nuclear chromatin accumulated in the nuclear membrane.Furthermore, 1,8-cineole is more effective against E. coli than S. aureus because, unlike S. aureus, the cell walls and membranes of E. coli cells were already compromised. 16

CONCLUSION
We concluded that essential oil of L. chequen obtained by steam distillation had as main component to alpha-pinene and the antioxidant activity against DPPH radical showed a good inhibitory capacity similar to Trolox standard.Furthermore, it had better antibacterial effect against S. aureus ATCC 25923 than E. coli ATCC 25922 in the colorimetric method.

Figure 2 :
Figure 2: Main volatile components of the essential oil of L. chequeen.

Figure 3 :
Figure 3: Regression analysis of the antioxidant activity with the standard Trolox.

Table 1 : Volatile compounds of the essential oil of L. chequen.
7s similar with Vallverdú et al,7which