GC/MS Profiling of Becium grandiflorum Essential Oil and Evaluation of Its Antiviral Activity

Genus Becium of the Lamiaceae family encounters numerous medicinally and economically valued aromatic plants. The hydrodistilled essential oil of Becium grandiflorum (Lam.) Pic. Serm. aerial part was analyzed using GC/MS. A total of 32 compounds amounting to 99.68% of the essential oil were identified. Oxygenated diterpenes constituted the largest share (43.45%) in the studied oil followed by sesquiterpenes hydrocarbons (34.24%). Pimara-7,15-dien-3-ol (20.58%), 3-α -hydroxymanool (14.07%), caryophyllene (8.17%), sandaracopimaradiene (7.98%), Selina-3,7 (11)-diene (5.61%), and germacrene D (4.95%) are the chief identified compounds in B. grandiflorum essential oil. The essential oil was assessed for its antiviral potential against herpes simplex virus 1 (HSV-1) at a non-cytotoxic concentration using a cytopathic effect (CPE) inhibition assay on the Vero cell line. B. grandiflorum essential oil exhibited a pronounced in vitro cytotoxicity against Vero cells with 50 % cytotoxic concentration (CC 50 ) of 7.75 ± 0.84 µg/mL and weak anti-HSV-1 potency with percent inhibition of 21.36 ± 2.13 at a maximum non-toxic concentration of 1 µg/mL.


Introduction
Lamiaceae (Labiatae) traditionally named as the mint family, is a family of flowering plants that encompasses more than 3000 species. Salvia, Scutellaria, and Stachys are among the largest genera of Lamiaceae. Moreover, thyme, oregano, mint, basil, and rosemary are the most prevalent culinary spices of this family. These plants are native to the Mediterranean region. They are herbs or shrubs with a characteristic aroma on account of the presence of a considerable percentage of essential oil [1,2]. Essential oil is a complex combination of liquid, volatile chemical constituents that are mainly obtained from plant material through distillation using water and/or steam or mechanical processes [3]. Moreover, essential oils are composed of phenylpropanoids, low molecular weight hydrocarbon derivatives, or terpenoids. The later encounters the major class of chemical constituents of essential oils. In addition to their antimicrobial, antifungal, antiviral, cytotoxic, and insecticidal potency, they are broadly used in the cosmetics industry, perfumery, and aromatherapy [4]. Worldwide, there has been a notable surge in the use of plant extracts or essential oils for the treatment and prevention of numerous ailments due to their inestimable safety and efficacy profiles [5]. Literature review revealed that various plants of the genus Becium which is a synonym of Ocimum [6,7] are a rich source of essential oils with valuable biological activities [8]. Ocimum basilicum essential oil along with some identified monoterpenes such as camphor, thymol, and 1, 8cineole exhibit antiviral potential against bovine viral diarrhea virus (BVDV) [9]. Moreover, eugenol, one of the chief constituents of different Ocimum species essential oils, possesses antiviral power against herpes simplex viruses type 1 and 2 (HSV-1 & 2) and human immunodeficiency virus (HIV) by inhibiting viral replication [8].
HSV, or Herpes Simplex Viruses, are viruses with double-stranded DNA that are categorized into two antigenic types: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). HSV-1 is one of the primary causes of human infections affecting more than 60% of the global population. It can induce orofacial infections (herpetic labialis), herpetic keratitis, and lifethreatening encephalitis [10]. It is mainly transmitted through oral-to-oral contact and to less extent through oral-genital contact [11]. The virus can establish a latency period and can be reactivated in certain conditions leading to significant complications in immunocompromised patients [12].
Becium grandiflorum (Lam.) Pic. Serm. is an aromatic perennial woody shrub belonging to the family Lamiaceae that grows up to one meter tall. It is cultivated on the rocky slopes of the altitudes of Ethiopia and is commonly known as Tebeb. In traditional medicine, it was commonly used against several ailments such as malaria, spider bites, swellings, influenza, and respiratory depression. Moreover, citizens of the Tigray region usually apply crushed leaves on the wound area to aid in wound healing which was recently evidenced by a research article [13]. Literature review revealed that B. grandiflorum ethanolic extract exhibits blood sugar-lowering potential [7] in addition to its antimicrobial activity against Staphylococcus aureus and S. pneumonia [14].
This study aimed to explore the chemical profile of the essential oil of B. grandiflorum cultivated for the first time in Egypt using gas chromatography coupled to mass spectrometry (GC/MS) along with evaluation of its in vitro antiviral activity against HSV-1 using cytopathic effect (CPE) inhibition assay on African green monkey kidney (Vero) cell line.

Plant Collection and Authentication
The aerial part of B. grandiflorum (Lam.) Pic.Serm.
[15] was collected at the flowering stage in July 2021 from Eng. Khalid El Haddad's farm, Giza, Egypt. The plant material's identity was verified by Eng. Therese Labib, Botanical Consultant and Specialist at Orman and Qubba Botanical Gardens. An authentic specimen (PHG-P-BG-325) was preserved in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Hydrodistillation of the Essential Oil and GC/MS Analysis
The aerial part of B. grandiflorum (250 g) was hydrodistilled for 4 h in Clevenger-type apparatus. In accordance with the Egyptian pharmacopeia, anhydrous sodium sulfate was utilized to dehydrate the obtained essential oil that was reserved at 4 °C for subsequent analysis [16]. Based on the fresh plant weight, the yield was calculated (yield % v/w). For separation and identification of the chemical constituents, analytical GC/MS was used (Shimadzu GCMS-QP2010, Koyoto, Japan) supplied with fused bonded Rtx-5MS column (30 m x 0.25 mm i.d. x 0.25 µm film thickness, Restek, USA) and supported with a split/splitless injector. The column's initial temperature was maintained (isothermal) for 3 min at 50 °C followed by linear temperature increase at a rate of 5 °C/min to reach 300 °C then the temperature was maintained (isothermal) at 300 °C for 10 min. The temperature of the injector was held constant at 280 °C. Helium was employed as a carrier gas, flowing at a rate of 1.37 mL/min. All the mass spectra were captured under the following settings: The filament emission current was 60 mA; the ionization voltage was 70 eV; and the ion source temperature was 220 °C. With split mode, diluted samples (1% v/v) were injected (split ratio 1:15). Essential oil chemical constituents were identified by comparing the Kovats index (KI) on the Rtx-5 column to the available literature [17], research articles, and computer library (NIST-11 mass spectral library) [18-20].

Cell Culture and Virus Propagation
Vero cells were collected from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) was supplied for the cultivation of Vero cells which was provided with 1% L-glutamine, HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) buffer, 50 μg/mL Gentamycin and 10% heat-inactivated fetal bovine serum (FBS). The cell cultures were kept in a humidified environment at 37 °C containing 5% CO 2 and were subcultured twice a week [21]. The cytopathogenic HSV-1 virus was cultured and assessed in confluent Vero cells [22]. By utilizing The Spearman-Karber technique, the number of infectious viruses was counted by calculating the 50% tissue culture infectious dose (TCID 50 ) with 20 µl of inoculum in each well and eight wells for each dilution [23].

Cytotoxicity Assay
Cytotoxicity assay was conducted at the Regional Center for Mycology and Biotechnology (RCMB, Al-Azhar University, Cairo, Egypt). Cellular vitality and proliferation were assessed by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay [24]. Vero cells were grown in 96-well plates containing 100 μL of the growth medium at a concentration of (2 × 10 5 cells/mL) and incubated for 24 h to allow cellular adherence. After incubation, Vero cells were treated with different concentrations of the tested essential oil dissolved in a fresh medium. Using a multichannel pipette, two-fold dilutions in the row of the tested sample at a concentration range (3000 µg/mL to 2 µg/mL) were supplied to the flat-bottomed multiwell plates (Falcon, Jersey, NJ, USA). For 48 h, the 96-well plates were kept at 37 °C in a humidified atmosphere supplied with 5% CO 2 [24]. After completion of the incubation period, the culture medium was eliminated and the multiwell plates were refilled with 100 µL of fresh culture medium. 10 µL of 5 mg/mL MTT dissolved in phosphate buffer saline (PBS) was introduced to each well including untreated control and incubated for 4 h at 37 °C. Following the incubation period, the medium was eliminated and 50 µL of dimethyl sulfoxide (DMSO) was introduced to each well and properly stirred using a pipette before incubation under the same conditions for 10 min. The absorbance was recorded at 590 nm using a microplate reader (SunRise, TECAN, Inc, USA) [24]. The percentage of cell viability was computed as [(ODt/ODc)] x 100% where ODt represents the mean optical density of the test sample while ODc is the mean optical density of control cells. A relationship was plotted between the number of viable cells and the concentrations of the tested essential oil to draw the survival curve of Vero cells. The 50% cytotoxic concentration (CC 50 ) was estimated using GraphPad Prism software (San Diego, CA. USA) by plotting the dose-response curve for each concentration. The sample's maximum non-toxic concentration (MNTC) was measured to be utilized in the assessment of antiviral potency.

Screening for Antiviral Activity
Cytopathic effect (CPE) inhibition assay was used to assess the antiviral activity of the tested essential oil at the Regional Center for Mycology and Biotechnology (RCMB, Al-Azhar University, Cairo, Egypt). This method was employed to demonstrate certain suppression of cellular activity. CPE in susceptible Vero cells was measured by the MTT dye uptake method [25,26]. In a multiwell microtiter plate, Vero cell monolayers at a cellular concentration (2x10 5 cells/mL) were seeded. This was followed by 24 h incubation period at 37 °C in a humidified chamber supplied with 5% CO 2 . The medium was eliminated and replaced with fresh DMEM before being inoculated with 10 4 doses of HSV-1. Various concentrations of the tested essential oil maintained in a fresh medium were supplied to the wells and kept at 37 °C for 48 h. As a control, non-infected and infected untreated Vero cells were utilized. The suppression of the CPE and the cellular protection provided by the tested essential oil relative to the control were used to measure antiviral potency. Three independent tests with four replicates per treatment were evaluated. In this experiment, Acyclovir was used as a positive control. Cells' vitality was evaluated using a colorimetric MTT assay after the completion of the incubation period.

Chemical Composition of B. grandiflorum Essential Oil
The aerial part of B. grandiflorum yielded 0.08% v/w essential oil (EO). The essential oil was lighter than water with pale yellow color. The GC/MS chromatogram is provided in Fig. 1. The identified components of B. grandiflorum essential oil were listed in Table 1  . Diterpene hydrocarbons come in the third subclass with 12.31% followed by fatty acid-derived volatiles (4.97%) and oxygenated sesquiterpenes (4.43%). The major identified constituents were Pimara-7,15-dien-3-ol (20.58%), 3-α-Hydroxy-manool (14.07%), Caryophyllene (8.17%), Sandaracopimaradiene (7.98%), Selina-3,7(11)diene (5.61%), and Germacrene D (4.95%). Fig.  2. represents the major identified chemical components. Analysis of B. grandiflorum EO cultivated in Ethiopia demonstrated that the major constituents are (Z)-p-ocimene (17.6%), myrcene (8.4%), limonene (8.0%), and βcaryophyllene (7.6%) [18]. The qualitative and quantitative variations in the EO content of B. grandiflorum cultivated in Ethiopia vs. that newly cultivated in Egypt may be attributed to variations in the collection time and cultivation conditions e.g., the soil type, pH and content of organic matter which can affect the chemical constituents of the plant [32]. It is supported by the fact that Ethiopian B. grandiflorum natively grows on the sandy soil of the altitudes and slopes [13] in contrast to the newly cultivated in Egypt that grows in muddy soil.

Cytotoxicity Evaluation
Assessment of cytotoxicity is a crucial step before the evaluation of a potential antiviral agent by CPE inhibition assay. To establish the maximum non-cytotoxic concentration of B. grandiflorum EO to be employed for possible antiviral use, an MTT assay was conducted against Vero cells. MTT assay is one of the most widely used colorimetric assays for the assessment of cell viability that depends on the reduction of water-soluble tetrazolium dye (MTT) by mitochondrial dehydrogenase enzyme in metabolically active cells [33]. Vero cell proliferation was dose-dependently suppressed by EO of B. grandiflorum as illustrated in Fig. 3. An in vitro cytotoxicity study reported that herbal extract having IC 50 value < 20 µg/mL is considered cytotoxic [34]. In the present study, the oil exhibited significant cytotoxic activity against Vero cells with CC 50 as low as 7.75±0.84 µg/mL. The high cytotoxic activity of the B. grandiflorum EO could be related to the dominance of oxygenated diterpenes that provide powerful cytotoxicity [35, 36].

Screening for Antiviral Activity
The potential antiviral action of B. grandiflorum EO was performed by viral CPE inhibitory method on Vero cells and observed using MTT assay. Table 2 shows the anti-HSV-1 activity of B. grandiflorum EO compared to acyclovir as a reference antiviral agent. At a maximum non-toxic concentration of the EO (1 µg/mL), the mean CPE inhibition % was 21.36±2.13, while that of acyclovir was 96.03±1.14. This result may indicate a weak anti-HSV-1 potential of B. grandiflorum EO. These findings are in agreement with another report [37] that evaluated the antiviral activity of O. campechianum EO against the Human herpes virus (HSV-1& HSV-2) and found that the oil has no activity against HSV-1 but showed inhibitory activity against HSV-2 with EC 50 74.33±10.9 μg/mL. According to the literature, none of the major identified compounds except β-Caryophyllene has any reported anti-HSV-1 activity. Interestingly, one study supported the potential of β-Caryophyllene against HSV-1 with IC 50 of 0.25 μg/mL [38]. While B. grandiflorum EO did not show remarkable anti-HSV-1 potential, other species of the same genus have shown antiviral activities. Previous reports demonstrated that O. gratissimum leaves aqueous extract showed anti-HIV-1 with IC 50 of 1.1 mg/mL [39]. On the other hand, it was reported that the methanol and dichloromethane extracts of O. americanum L. exhibited antiviral potency against HSV-1 with IC 50 of 67.51 and 25.29 μg/mL, respectively [40]. Although various members of the genus Ocimum display antiviral properties, surprisingly B. grandiflorum showed weak anti-HSV-1 activity which may be attributed to the qualitative and quantitative diversity in the chemical constituents of B. grandiflorum EO.

Conclusion
The current investigation is the first to report the volatile composition of B. grandiflorum newly cultivated in Egypt via GC/MS. Oxygenated diterpenes and sesquiterpene hydrocarbons are the chief classes of chemical components in B. grandiflorum EO. In addition to EO characterization, the anti-HSV-1 activity was evaluated. B. grandiflorum EO showed minor anti-HSV-1 power that may be attributed to variations in the chemical composition of the EO as a result of discrepancies in plant harvesting age, genetic makeup, sunlight, place