Chemical profile and biological properties of the essential oil of Rosemary leaves

Rosemary leaf essential oil (RoEO) is extracted using steam distillation. The aim of study is 6 to determine physicochemical characteristics of essential oil (EO), such as acid/saponification/esterification index, relative/absolute density, freezing point, and fragrance retention. The chemical composition of EOs was analyzed by gas chromatography-9 mass spectrometry (GC-MS) method and identified 50 volatile compounds, of which α-Pinene (33.76%), 1,8-Cineole (18.47%), and Levoverbenone (6.11%) constituted the highest proportions in EO. The antioxidant capacity (AC) of the EO was evaluated by 2,2-diphenyl-1-12 picrylhydrazyl (DPPH) free radical scavenging method with a half-maximum inhibitory concentration (IC 50 ) of 425.473 mg/mL. In particular, antibacterial activity (AA) by the paper plate diffusion method for susceptibility testing to essential oil showed that RoEO strongly 15 inhibited the growth of four tested bacterial strains ( Staphylococcus aureus , Bacillus cereus , Salmonella typhimurium , and Escherichia coli ). Perhaps, it is possible to apply RoEO in the food industry and other fields owing to the good properties of 18 RoEO.

Rosemary (Rosmarinus officinalis L.) is a genus of the Lamiaceae family; it is a flowering plant with a very fragrant and pleasant smell.This plant is native to the Mediterranean, but is widely cultivated worldwide [1,2].Rosemary has only been grown in Vietnam in recent years, 24 and the capital of rosemary thrives in the tropics.At the same time, Bac Giang province is located in the tropical monsoon climate region of Northeast Vietnam.The annual average temperature is approximately 23°C, with the relative humidity ranging from 74% to 87%.The 27 amount of surface water, rainfall, and groundwater is enough to supply agriculture [3].Bac Giang province has a mild climate compared to other regions in Vietnam and it is entirely favorable for cultivating and developing various crops, especially rosemary.

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The major compounds common in rosemary leaf essential oil (RoEO) are terpenes which are divided into monoterpenes, sesquiterpenes, diterpenes, and triterpenes, depending on the number of carbon atoms.The terpenes in rosemary can be found as carnosol, epirosmanol, 33 ursolic acid, carnosic acid, and oleanolic acid (triterpenes) [4].The essential oil (EO) gained from the leaves had a major component of 1,8-cineole (35.8%), while caryophyllene (16.7%) was predominant in the EO extracted from the stem.For EO extracted from flowers, the main 36 compound is caryophyllene oxide (11.9%) [5].Of note, rosemary leaves are most commonly used for essential oil extraction [6].Rosemary has the highest antioxidant activity among the natural antioxidants from plants [7].According to pharmacological studies performed by 39 extracting EO from rosemary, EO has some special biological effects, such as anti-diabetic, anti-infective, and anti-inflammatory [4].According to research by Andrade et al., the yield of RoEO using steam distillation is up to 2.5%; the obtained RoEO is a pale yellow to colorless 42 liquid, insoluble in water, and has a characteristic camphor aroma.The main components of RoEO are 1,8-cineole (15-55%), α-pinene (9.0-26%), camphor (5.0-21%), camphene (2.5-12%), and β-pinene.(2.0-9.0%); this ratio varies depending on the growth stage and local 45 climatic conditions [4].In addition, the composition of EO may vary according to the extraction method; the quality or quantity of the chemical composition of EO may vary according to the parts of the plant used for extraction, soil conditions, changes in climate, and the growth cycle 48 of raw materials [8].
Many studies related to Rosmarinus officinalis L. already exist; however, to date, there are no studies on the chemical composition, physicochemical properties, antioxidant activity, and 51 antibacterial potential of the RoEO grown in Bac Giang province.Therefore, the primary aim of this study was to evaluate all of the above properties of RoEO from Vietnam because it

EXPERIMENTAL
Plant material: Rosemary leaves were harvested in Bac Giang province (Vietnam) at 57 maturity about 6-8 months after planting.RoEO was isolated using the steam distillation method (250 kg of fresh leaves/batch).The yield of EO was about 0.82% (v/w), and the RoEO was kept in a dark bottle at 4°C until analysis.

Evaluation of fragrance retention (FR) of the RoEO:
Fragrance retention (FR) was determined by EO concentration and scent duration, according to Mahajan with some small adjustments [13].The EOs were prepared at concentrations at 10, 20, 30, 40, and 50% (v/v) 69 in ethanol 96%.Then, samples were prepared by adding 3 drops to the odor test paper, allowing a few seconds to penetrate the paper.The time it took to lose the EO scent entirely under normal conditions was recorded.

Gas chromatography-mass spectrometry (GC-MS) analysis:
The constituents of RoEO were investigated by the GC-MS. 1 μL of EO was injected into a gas chromatography machine (Shimadzu Nexis GC-2030, Japan) with a versatile capillary column (Rtx-5sil-MS, 30 m × 0.25 75 mm × 0.25 μm, Restek Technologies, USA) equipped with a quadrupole mass analyzer (Shimadzu GC-MS-QP2020 NX, Japan).Helium was used as a carrier gas at a constant flow rate of 3.0 mL/min, and a split ratio of 10:1.The injection temperature was 250°C and the 78 temperature program was set as follows: initial temperature of 50°C, held for 2 min, increased until 250°C at a rate of 10°C/min, and held for 5 min, increased until 280°C at a rate of 10°C/min, and held for 3 min.The chemical compositions of the RoEO were identified based 81 on the comparison between their mass spectra with NIST 2017 library.

Evaluation of the antioxidant capacity (AC) of the RoEO:
The determination of the antioxidant capacity (AC) of EO using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging of 84 free radicals (DPPHRSC) assay was performed as described by Gounder and Lingamallu with some minor corrections [14].RoEO was dissolved in ethanol (96%, v/v) to achieve different concentrations (100, 200, 300, 400, 500, and 600 mg/mL).Then, 0.3 mL of RoEO was mixed A c c e p t e d m a n u s c r i p t with 3.7 mL of 0.1 mM DPPH solution in ethanol.This solution was kept in the dark for 30 min at 26-28°C and the absorbance of mixture was recorded at 517 nm.Ascorbic acid was used as a standard to compare with the AC of the RoEO.Percent inhibition plotted against EO 90 concentration to reckon concentration gives 50% inhibition (IC50).The AC was calculated as the following formula:

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where A0 is the absorbance of control and As is the absorbance of RoEO samples in the presence of DPPH solution at 517 nm.

Evaluation of the antibacterial activity (AA) of RoEO:
The antibacterial activity (AA) of 96 RoEO was evaluate based on the paper disc diffusion assay with some minor modifications [15].100 µL of bacterial suspension (0.5 McFarland standard, approximately 1.5 × 10 8 CFU/mL) was inoculated into Mueller Hinton Agar medium.Then, EO impregnated the sterile 99 paper discs placed on the agar surface (5 μL EO/disc).In this study, ampicillin (10 μg/disc) and dimethyl sulfoxide (DMSO) solution (5%, v/v) were considered as positive and negative antibacterial control.The petri dishes were incubated for 24 h at 37°C and the AA was  In fact, most of the components in RoEO possess biological properties.Some chemical 153 compounds account for a small proportion of RoEO, but they also have some specific characteristics.For example, Myrcene (1.44%) was used as a aroma agent in the food industry, as well as cosmetic products [22], while Limonene exhibited anti-inflammatory, 156 antioxidant, antinociceptive, anticancer, antidiabetic, antihyperalgesic, and antiviral effects in health [23].Therefore, these ingredients contribute to the RoEO potential in medicine and the food and cosmetic industries.higher than the control sample (ascorbic acid) with IC50 = 27.73 (µg/mL) (Fig. 1).This finding also proves that the AC of RoEO is very weak.Compared to RoEOs from different places, the IC50 in this study was much higher than that of RoEO in Lam Dong province, Vietnam 165 (IC50 = 75.7 µg/mL) [24] and in Belgrade, Serbia (IC50 = 77.6 µL/mL) [6].In addition, the AC of RoEO is also lower than that of some EOs from other materials, such as A. conyzoides leaves (IC50 = 8 mg/mL) [15], C. griffithii (IC50 = 82.4µg/mL) and C. macrocarpum leaves (IC50 168 = 99.3 µg/mL) [25].From this, it can be seen that the difference in AC can be caused by different chemical composition.Although the low AC of RoEO is a disadvantage, RoEO can apply and study more in typical cases.

A c c e
p t e d m a n u s c r i p t may possess some unique properties when compared to RoEO from various regions of the 54 world.

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evaluated for the bacterial inhibitory zone by measuring the diameter of the antibacterial ring, including the disc diameter of 6 mm.Data analysis:All the experimental results were analyzed using Statgraphics Centurion 105 software (Version 15.1.02).Every assay was done in triplicate.Analysis of variance with Fisher's least significant difference procedure was used to determine the significant differences (p < 0.05) between means.108RESULTSAND DISCUSSIONPhysicochemical properties of RoEO:RoEO is a light yellow, very clear liquid with a characteristic aroma and a slightly sharp taste.The pH value of EO is about 4.713 (

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Antioxidant capacity (AC) ofRoEO: Fig. 2 displays that the antioxidant capacity (AC) depends on EO concentrations.RoEO decolorises the DPPH solution, and the AC increases with increasing concentration of EO.The IC50 value of RoEO was 425.473 (mg/mL), much 162

Table 1 .
Physicochemical properties of the RoEOTable 2 shows the volatile compounds of RoEO.Many chemical RoEO from Campestre da Serra province, Brazil, possessed 20 volatile compounds, such as 144 α-Pinene (40.55-45.10%),1,8-Cineole (17.40-19.35%),Camphene (4.73-6.06%),and Verbenone (2.32-3.86%)[19].This difference in chemical components may be explained due to various climatic conditions, soils, extraction methods, as well as experimental conditions, [18]f different studies.The RD and AD are a characteristic and important index for each type of EO.In this study, RD (0.896) and AD (0.895 g/mL) of RoEO were similar to those in previous studies; for example, RoEO from Lam Dong province -Vietnam (AD: 0.8978 g/mL)[18]and 123 Campestre da Serra province -Brazil (AD: 0.8887 g/mL)[19].The AI, SI, and EI are three important indices used to assess the quality of an EO, and their values in RoEO were 1.852, 14.259, and 12.407 mg KOH/g, respectively.The AI low value 126 indicates that the essential oil is of good quality, with little change or oxidation over time.The AI in this study is slightly higher than that of RoEO originating from Lam Dong province (AI:1.122mgKOH/g),whereas the EI is smaller than that of RoEO originating from Lam Dong 129 province (EI: 15.708 mg KOH/g)[18].Thereby, it can be seen that these indices will depend on many factors, such as chemical composition, harvest time, age of material, distillation method, etc.132The FR of RoEO is quite long, reaching 16.5 h at 50% concentration but only 4 h at 10% concentration.The results demonstrate that RoEO offers high potential for applications in the perfume and food technology.135Chemicalprofile of RoEO:Levoverbenone (6.11%).The results were different from rosemary in different regions, for 141 example, for RoEO in Lam Dong province, Vietnam, 23 compounds were found, including α-Pinene (35.54%),Eucalyptol (20.902%),Bicyclo[3.1.1]hept-3-en-2-one(7.794%)[18], while

Table 2 .
Chemical profile of the RoEO

Table 3 .
Table3shows the antibacterial activity (AA) of RoEO 174 against four strains of foodborne bacteria.The AA of RoEO were arranged in the following order: B. cereus and S. typhimurium < S. aureus < E. coli, while the AA of the positive control (Ampicillin) was listed in order: B. cereus and S. aureus < E. coli and S. typhimurium.With 177 the obtained antibacterial diameter of RoEO, it can be concluded that the AA of RoEO is stronger than that of Ampicillin for three tested bacteria, excluding S. typhimurium.These results prove that RoEO is a very promising material that can be applied in food preservation Antibacterial diameter of RoEO