The white incense plant (English name: Basil) belongs to the genus Ocimum, family Lamiaceae, one of the most abundant essential oil plant families, and is herbaceous, branched, and has small leaves, symmetrical leaf blades, velvety leaves 3-4 cm long, about 1-2 cm wide, clustered flowers of 6-10 flowered verticillasters (Monga et al. 2017), widely found in tropical regions such as India, West Africa, and Nigeria, and other coastal areas, like in provinces of Vietnam.

In Vietnam, Ocimum gratissimum L. is a spice, food preservative, and medicinal herb that treats various diseases such as respiratory infections, diarrhoea, headache, fever, ophthalmic diseases, skin diseases, and pneumonia. This is the most valuable and comprehensive plant used in traditional medicine because of its therapeutic properties. In addition, the antioxidant potential of these plants is due to their abundance of bioactive compounds, also known as secondary metabolites, such as alkaloids, flavonoids, phenols, and saponins (Wu et al. 2016). The main component of O. gratissimum essential oil (OgEO) collected in the Thai Binh province (Vietnam) is eugenol (59.448%), along with other bioactive natural compounds, including trans-β-ocimene (10.382%), β-cubebene (11.783%), caryophyllene (6.966%), and copaene (2.479%) (Huong et al. 2020).

In some other studies, eugenol has resistant effects against several bacteria, such as Staphylococcus aureus and Escherichia coli (Melo et al. 2019). OgEO is also resistant to some pathogenic fungi in humans and plants (Sandeep 2017). In addition, studies on Ocimum EOs’ antimicrobial ability to evaluate certain microorganisms' inhibitory ability to cause food spoilage create the potential to develop preservatives/seasonings from natural compounds to control food spoilage (Trang and Quynh 2018). From there, it can be seen that the potent antibacterial activity of essential oil can be applied in food and medicine.

Currently, this plant is grown widely in the Dak Lak province (Vietnam) for use as spices and collected essential oils; however, its chemical composition and biological activity depend on soil and weather conditions. Therefore, oils may have more distinctive biological properties than elsewhere. Although there have been many studies on

OgEO with diverse bioactivity and many applications for humans, there are no studies to evaluate the properties of OgEO like its physical properties, chemical composition, and antimicrobial ability. Therefore, the implementation of this study is aimed to clarify the properties of OgEO.

Material preparation

The aerial parts of O. gratissimum were collected in Dak Lak province (Vietnam) and cut by a sharp knife at the time of blooming. The fresh material is harvested early at 5-6 am and distilled immediately afterward (a maximum of 4 h after harvest). The EOs are isolated using the steam distillation method at 100°C for 2.5 h. On average, the per-batch yield was 40 kg material/batch. The efficiency of the distillation process accounted for 0.7% (v/w), and the obtained EO was stored at 4°C in a tightly sealed glass vial until analysis.

Bacterial strains

The foodborne bacteria strains were selected to identify antibacterial activity, including Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Bacillus cereus (ATCC 11778), and Staphylococcus aureus (ATCC 25923). They were provided by the Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City.

Determination of the EO’s physical properties

The freezing point (FP), relative density (RD), and absolute density (AD) were determined according to International Organization for Standardization (ISO), including ISO 1041 (1973) and ISO 279 (1998), respectively.

Gas chromatography-mass spectrometry (GC-MS) analysis

The chemical composition of RoEO was analyzed using the GC-MS method according to the procedure described by Quyen and Quoc (2023). 1 µL of EO was injected into a gas chromatograph (Shimadzu Nexis GC-2030, Janpan) with a versatile capillary column (Rtx-5sil-MS, 30 m × 0.25 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 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. Mass spectra were recorded at the ionization energy of 70 eV in EI mode.

Evaluation of the antibacterial activity (AA) of the EO

The AA of the oil was determined by agar disc diffusion according to the procedure described by Quoc (2022) with minor changes. The test was performed in a petri dish containing solid, sterile Mueller Hinton agar (MHA) media. The EOs are impregnated on sterile paper plates with a diameter of 6 mm (5 µL EO/disc) and placed on the surface of a medium spread of 0.1 mL of bacterial suspension (0.5 McFarland standard, approximately 1.5 × 108 CFU/mL. Ampicillin (10 µg/disc) and dimethyl sulfoxide (DMSO) (5%, v/v) were used as positive and negative controls, respectively. Finally, all discs were incubated for 24 h at 37°C and the antimicrobial activity of the essential oil was assessed by the diameter of the inhibition zone in mm.

Statistical data analysis

All the experimental results were analyzed using Statgraphics Centurion software (Version 15.1.02). Every assay was done in triplicate, and the obtained results were expressed as mean ± standard deviation, except for the GC-MS analysis, which was done once. Analysis of variance (ANOVA) with Fisher’s least significant difference procedure was used to determine the significant differences (P < 0.05) between means.

Physical properties of OgEO

Table 1 shows that the pH value of OgEO is 3.9233 ± 0.0058. The study outcome was lower than that of the EO of Ceratonia siliqua L. pulp (pH = 4.3) (Ouis and Hariri 2018) and Mentha arvensis leaves (pH = 4.67) (Quoc 2022). This result suggests that the chemical components of the EO strongly affect the pH, and the EO may contain an amount of volatile free acids.

Table 1 . Physical properties of OgEO.

No.	Physical properties	Value
1	Freezing point (FP, °C)	- 38±0.0000
2	pH at 20°C	3.9233±0.0058
3	Absolute density at 20°C (AD, g/mL)	1.0115±0.0116
4	Relative density at 20°C (RD)	1.0254±0.0021

For the AD of EO, there are two scenarios: lighter than water (AD: 0.9982071 g/mL), such as the EO of allspice berries (AD: 0.978 g/mL) and basil leaves (AD: 0.912 g/mL) according to results of Martinez-Velazquez et al. (2011), and heavier than water, such as the EOs of O. basilicum (1.196 g/mL) and O. sanctum (1.552 g/mL) (Khair-ul-Bariyah 2013). In this case, the AD of OgEO was 1.0115 ± 0.0116 g/mL. With a value greater than 1, this finding is similar to that of other Ocimum and differs from other materials due to various EO components. Similarly, the RD value is higher than 1, so the obtained EO easily submerged under water in the separation process.

According to our knowledge, each EO had a different FP, and the FP of OgEO in this study is quite low (-38°C), lower than that of the EOs of Eucalyptus camaldulensis leaves (0-1°C) and M. arvensis leaves (-7.33°C) (Abdul-Majeed et al. 2013; Quoc 2022). This difference can also be explained due to the difference in the chemical composition of EOs. Until this point, no studies have reported the FP of OgEO from other regions. Therefore, we cannot compare them, which is also an interesting issue to study.

Chemical profile of OgEO

The GC-MS analysis revealed that 23 volatile compounds in oil accounted for 100% of the oil (Table 2). Among them, eugenol has the highest content (76.01%), followed by some compounds in the group of sesquiterpenes, including D-germacrene (5.28%), β-caryophyllene (3.60%), and D-cadinene (1.13%), and some other low content substances. In addition, some compounds of the monoterpenes group do not account for a high proportion but are also indispensable components in OgEO with typical substances, such as trans-β-ocimene (5.99%), β-myrcene (0.39%), 4-δ-carene (0.38%), and others.

Table 2 . Chemical profile of OgEO.

No.	Compounds	Molecular Formula	Rt. (min)	Content (%)
1	β-phellandrene	C10H16	6.158	0.47
2	3-ethyl-4-hydroxy-dihydro-furan-2-one	C6H10O3	6.246	0.33
3	β-myrcene	C10H16	6.432	0.39
4	4,4-dimethylcyclohex-1-ene carboxylic acid	C9H16O2	6.754	0.31
5	4-δ-carene	C10H16	6.945	0.38
6	trans-β-ocimene	C10H16	7.247	5.99
7	cis-β-ocimene	C10H16	7.446	0.37
8	γ-terpinene	C10H16	7.676	0.36
9	Cyclohexene	C10H16	8.168	0.49
10	β-linalool	C10H18O	8.361	0.38
11	2H-indene, 3,3a,4,5,6,7-hexahydro-	C9H14	8.753	0.33
12	2,4,6-octatriene	C8H12	8.851	0.38
13	3-cyclohexenol	C6H10O	9.764	0.72
14	Ocimenol	C10H16O	10.135	0.44
15	Eugenol	C10H12O2	12.372	76.01
16	α-copaene	C15H24	12.795	0.94
17	β-bourbonene	C15H24	12.923	0.40
18	β-elemene	C15H24	12.964	0.43
19	β-caryophyllene	C15H24	13.445	3.60
20	D-germacrene	C15H24	14.275	5.28
21	γ-muurolene	C15H24	14.681	0.40
22	D-cadinene	C15H24	14.727	1.13
23	4-methyl-2-phenyloxane	C12H16O	20.444	0.47
Total				100

Eugenol content (76.01%) in oil originated from the Dak Lak province is higher than that of OgEO collected from other regions; for example, the eugenol content in OgEO from State of Maranhão (Brazil) is 74.83% (Melo et al. 2019), 68.81% in OgEO from eastern Eastern Kenya (Matasyoh et al. 2007), and 54% in OgEO from India (54%) (Prabhul et al. 2009). Various distillation methods, soils, climates, and source of material can explain this difference. According to Kamatou et al. (2012), eugenol is an aromatic substance with a pleasant smell and a spicy, pungent taste; that is slightly soluble in water and soluble in organic solvents. It has beneficial health effects thanks to its antimicrobial, anti-inflammatory, analgesic, antioxidant, and anticancer activities. In addition, it was also used as a pesticide and fumigant. Other chemical compounds also possess biological properties that positively affect human health, so the findings about the ingredients contained in OgEO create potential applications in different fields, such as food preservation, medicine, and cosmetics.

Antibacterial activity (AA) of OgEO

The antimicrobial ring diameter obtained from OgEO is presented in Table 3. The results demonstrated that OgEO had significant antimicrobial activity against Gram-negative (E. coli and S. typhimurium) and Gram-positive (B. cereus and S. aureus) bacteria.

Table 3 . Antibacterial zones of OgEO.

No.	Microorganisms	Diameter of EO inhibitory zones (mm)	Diameter of ampicillin inhibitory zones (mm)	Diameter of DMSO inhibitory zones (mm)
1	E. coli	25 ± 0.000Ab	24.333 ± 1.155Ac	-
2	S. aureus	19 ± 1.000Ba	9.667 ± 0.577Ab	-
3	B. cereus	19.333 ± 0.577Ba	7.333 ± 1.155Aa	-
4	S. typhimurium	28.667 ± 2.082Bc	24.333 ± 0.577Ac	-

Different letters within a row (A-B) or column (a-c) denote significant differences (P < 0.05) between samples or microorganisms, respectively. Note: “-” indicates the absence of antibacterial activity of OgEO against the tested bacterial strains..

According to Sebei et al. (2015), the antimicrobial ring diameter (D) is used to classify the susceptibility of EOs to microorganisms as follows: not sensitive (D < 8 mm), sensitive (D: 9-14 mm), very sensitive (D: 15-20 mm), and extremely sensitive (D > 20 mm). The bacterial inhibitory diameter of ampicillin (positive control) is listed in the order: S. typhimurium/E. coli > S. aureus > B. cereus, of which S. typhimurium and E. coli are extremely sensitive to ampicillin. In contrast, S. aureus and B. cereus belong to the sensitive and not sensitive groups, repectively. For negative control (DMSO), they have no antibacterial ability against the tested bacteria. This also shows that the results obtained from the positive and negative controls are reliable. The OgEO’s bacterial inhibitory diameter is arranged in the order: S. typhimurium > E. coli > B. cereus/S. aureus. The AA of OgEO is better than that of ampicillin, with E. coli and S. typhimurium belonging to the extremely sensitive group (D ≥ 25 mm), while S. aureus and B. cereus belong to the very sensitive group (D = 19 mm).

Compared to results obtained by Matayoh et al. (2008), the bacterial inhibitory diameters of OgEO in various places in Kenya against S. aureus, E. coli, S. typhi, and Bacillus spp. were 13.5-26.6, 10.5-32.5, 10-20.5, and 16-30 mm, respectively. These results are very different from those of this study because the AA of OgEO strongly depends on the chemical composition of the EO.

The AA of OgEO was ascribed to the presence of eugenol; it is a bioactive compound with strong antibacterial, antiviral, and antifungal properties (Ulanowska and Olas 2021). In particular, in this study, the proportion of eugenol in OgEO is relatively high (76.01%), so the AA is higher than that in the positive control. This is also a highlight of this study. The hydroxyl group on eugenol is thought to alter the membrane fatty acids, change cell morphology, disrupt the cytoplasmatic membrane, affect the transport of ions and ATP, and inhibit some bacterial enzymes. All of these effects lead to bacteria cell death (Marchese et al. 2017). In addition, the synergistic abilities of bioactive compounds in EOs also play an important role in bacterial inhibition; it can enhance AA compared to one compound.

In general, this study evaluated some physical properties of OgEO originating from the Dak Lak province (Vietnam), and identified its chemical composition, including 23 volatile compounds, with eugenol accounting for the highest content (76.01%). The obtained oil also possesses a strong AA against bacteria strains, such as S. aureus, E. coli, S. typhimurium, and B. cereus. From the obtained results, OgEO has outstanding advantages that have potential in various fields, such as food, cosmetics, and medicine.

This research was performed at the Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City (Vietnam). The authors would like to thank Ho Truong Bao Trung and Nguyen Ngoc Tin for their helpful advice on various technical issues examined in this paper.