2.1 Collection of the plant material

The fresh rhizomes of the Chinese and Saudi ginger were purchased from the local farms of Al Hasa, Saudi Arabia and from the vegetable section of the Carrefour supermarket in Muscat. They were kept in a refrigerator to prevent the drying of the ginger rhizome. The samples were identified by a faculty member in the College of Pharmacy, National University and the plant specimen was deposited in the Pharmacy lab for the future reference.

2.2 Isolation of essential oil

The fresh ginger was cleaned under tap water to remove soil and other impurities. Approximately 349.5 g of Chinese ginger and 462.0 g of Saudi ginger were chopped into small pieces and made into a paste by using domestic blender. The ginger essential oil was isolated by the clevenger apparatus. The oily layer was separated following hydrodistillation for 6 h (Mohammed et al., 2019).

The oil was dried over anhydrous sodium sulphate and then the percentage (%) yield of each essential oil was calculated with respect to the dried weight taken. The oils kept in air tight plastic bottles were stored in a refrigerator until further use.

2.3 GC- MS analysis

The chemical volatile composition of essential oil was analyzed on a Perkin Elmer Clans 600 GC System, fitted with HP-5MS capillary column (30 m X 0.25 mm i.d X 0.25 μm film thickness; maximum temperature, 350 °C), coupled to a Perkin Elmer Clarus 600C MS. Standard operating conditions were used for the analysis viz., carrier gas- helium (99.9999% purity); flow rate- 1.0 mL/min; injection temperature-290 °C, transfer line temperature- 280 °C and ion source temperature- 270 °C; ionizing energy-70 eV; injected sample volume-1 uL; split ratio of 150:1. The oven temperature program was set to 60 °C at a rate of 3 °C/ min −280 °C held for 2 min. Electron multiplier (EM) voltage was obtained from auto-tune. The mass spectra were scanned in the range of 40–550 amu to obtain the data for identification. The identity of the compounds was established by comparing the obtained spectrum with the mass spectrum libraries.

2.4 Antibacterial activity

The antimicrobial activity was determined by the well diffusion method using Muller Hinton Agar nutrient media. Four previously sterilized nutrient agar plates were used to culture the Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 8739) bacteria. Two culture plates each were swabbed with the S. aureus and E. coli. Five holes of uniform diameter were made in each plate with the help of a cork borer under aseptic conditions. Three different concentrations of the each oil (2.5, 5.0, and 10 μL) were tested for the antibacterial activity. A volume of 10 µL of each concentrations of the Chinese and Saudi ginger essential oils were added to the holes in Gram negative and Gram positive cultured petri plates. Dimethyl sulfoxide (DMSO) was used as a diluent to make up the volume to 10 μL. A disc of Ampicillin (25 µg) was added as a reference antibiotic and DMSO (10 µL) was used as a negative control. Afterwards, the cultured plates were incubated for 24 h at 37 °C. Finally the zone of inhibition for each concentration of ginger volatile oil was measured the next day.

2.5 In silico prediction of bioactivity and molecular docking studies

The five major chemical constituents of the ginger volatile oils were identified with the help of gas chromatogram. The simplified molecular-input line-entry system (SMILES) notations of selected chemical constituents were fed in to the PASS online software to predict their biological activity spectrum. The X- ray crystal structure of mycobacterial protein enoyl reductase (PDB: 4TZK) was downloaded from RCSB protein data bank. The molecular docking studies of the major constituents on to the mycobacterial protein was performed using Pyrex with Vina Wizard tool to find out the binding energy and to know the various ligand–receptor interactions responsible for the antibacterial activity of ginger volatile oil. Drug discovery studio (DDS) was used for the visualization of docking poses.

3.1 Percentage (%) yield of isolated ginger volatile oil

Hydrodistillation method is one of the common and easiest methods employed to extract the essential oil. The Chinese and Saudi ginger produced the 0.14% and 0.2% v/w of essential oil and clearly indicates low yield as well as variation in % yield. The fresh ginger contains 1–2% of volatile oil, the low yield suggest either the ginger rhizomes were not mature enough, either dried or extraction method was not appropriate. The Chinese oil was noticed to be of dark yellow in colour while the Saudi ginger oil was of light yellowish- green tint. The variation in the yields of oils could be due to the difference in geographical conditions, cultivation time, harvesting time, the origin of the ginger, and other environmental factors (Onyenekwe and Hashimoto, 1999).

3.2 Chemical composition of ginger volatile oils by GC–MS analysis

Chemical composition of volatile oils isolated from the Chinese and Saudi ginger cultivars were analyzed with the help of GC–MS chromatogram (Figs. 1 and 2). The volatile compounds were identified by comparing the retention time and molecular weight with the reference compounds in the NIST library.

Close

Fig. 1

Chromatogram of Chinese ginger oil.

Export to PPT

Close

Fig. 2

Chromatogram of Saudi ginger oil.

Export to PPT

The Chinese and Saudi varieties were found to contain 43 and 30 volatile components and the results are presented in the Table 1. The volatile components of ginger oil belong to monoterpenoid, sesquiterpene and sesquiterpene alcohol, etc. The five major volatile constituents identified in the Chinese and Saudi oils include α-zingiberene (17.94% & 7.68%), β-phellandrene (10.81% & 7.11%), ar-curcumene (10.31 & 15.78%), β-sesquiphellandrene (7.69% & 6.99%) and β-bisabolene (7.59% & 7.33%) respectively (Figs. 3 and 4). As it can be seen from the Table 1, α–zingiberene (monocyclic sesquiterpene hydrocarbon) is the major constituent of the Chinese rhizome variety while Saudi ginger was noted to be of ar-curcumene chemotype. Our results of the Chinese chemotype are in agreement with the study of Toure and Xiaoming. They detected 48 volatile constituents in the Chinese ginger oil and identified α-zingiberene as the major constituent with a little higher content than obtained in the current study (Toure and Xiaoming, 2007). Some of the volatile constituents such as α-pinene, L-camphene, β-myrcene, β-linalool, L-borneol, citral, geraniol, β-farnesene, β-elemene, elemol and trans-nerolidol, etc., are common in both the volatile oils.

Close

Fig. 3

Five major volatile constituents identified in the Chinese and Saudi ginger rhizome varieties.

Export to PPT

Close

Fig. 4

Chemical structure of five major volatile constituents found in Chinese and Saudi ginger oil.

Export to PPT

Results of the previous studies carried out on ginger from the Chinese, Guinean and Sub Himalayan region have reported that variation in chemical compositions of different ginger cultivars are influenced by geographic origin, climatic conditions, maturity at harvest, age of plant, time of harvest and method of extraction, etc (Toure and Xiaoming, 2007; Nampoothir, 2012; Onyenekwe and Hashimoto, 1999). Several studies demonstrated α–zingiberene to be the major constituent in the ginger rhizomes from the Ghana, Thailand, Poland, Nigeria, Australia and India whereas the oil from the Cuba and Brazil contained ar-curcumene as the major component (Menon et al., 2007; Nogueira de Melo et al., 2011).

3.3 Antibacterial activity of ginger essential oil

The results of the antibacterial effect at different concentrations (2.5, 5.0 and 10 µL) of ginger oil against Gram positive bacteria (S. aureus) and Gram negative (E. coli) by using well diffusion method are presented in Table 2 (Al-Aamri et al., 2018). Ginger oil showed the dose dependant zone of inhibition against both the Gram positive and Gram negative bacteria. Results indicate that ginger oil failed to inhibit the growth of Gram negative bacteria at the lowest dose of 2.5 µL. In general, ginger volatile oil was found to exhibit better activity against Gram positive bacteria (9–13 mm zone of inhibition) in comparison to Gram negative strain (7–10 mm zone of inhibition). This difference in activity could be because of the presence of the lipopolysaccharide layer in the structure of Gram negative bacteria which reduces the activity of the ginger oil. Though, the oil differs in their chemical composition but interestingly no difference in their antibacterial spectrum against Gram positive bacteria was observed. Both the oils at the dose of 10 µL exhibited 13 mm of zone of inhibition against gram positive bacteria. Saudi ginger oil exerted better effect than the Chinese oil against E. coli (8 and 10 mm respectively). The results of ginger oil antibacterial activity are comparable to the standard drug Ampicillin (25 µg). Our study also proves that the Chinese and Saudi ginger oil exhibits slightly better antibacterial activity than the Vietnamese ginger oil (Stoyanova et al., 2006). It could be proposed that the antibacterial activity of the oil is related to its chemical composition, especially sesquiterpenes. It can be stated that the major constituent does play a role in antibacterial activity but minor components also help in exerting the synergistic action.

3.4 In silico prediction of bioactivities and molecular docking studies

Prediction of activity spectra for substances (PASS) was used to predict the antimicrobial, antineoplastic/chemopreventive and anti-inflammatory activities of the five major volatile constituents of the ginger volatile oil. The PASS software predicts the bioactivity of molecules based on the structural similarity to the large data base of known active substances. Pa and Pi values (bioactivity score) predict the probability of compound to be active. A compound with a bioactivity score of 0.5 or above has higher chances to be active. The Pa values for predicted bioactivities lies in between 0.856 and 0.369 (Table 3). The predicted score for antineoplastic/chemopreventive activity (0.856–0.644) was found in the following order; β–bisabolene > β-phellandrene > β-sesquiphellandrene > ar–curcumene > α–zingiberene. All the five compounds are predicted to possess antifungal activity (0.638 – 0.491) while only β–bisabolene is observed to have anti-inflammatory activity (0.723). Only four constituents are found to possess antibacterial activity (0.369–0.441).

Molecular docking studies were also performed using mycobacterial acyl carrier protein reductase enzyme (PDB ID: 4TZK) to corroborate the experimental results of antibacterial activity. Enoyl acyl carrier protein reductase is considered as a potential target to inhibit bacterial growth (Alsaraf et al., 2020). The binding energies of the major constituents of ginger oils were found to be in the range −8.4 to −6.9 kcal/mol and indicate moderate to good inhibition of the enzyme (Table 4). Ar-curcumene and α–zingiberene were observed to fit and bind strongly in the enzyme pocket. The binding energy of the major volatile constituents was found in the following order; ar-curcumene < α–zingiberene < β–bisabolene < β–sesquiphellandrene < β–phellandrene. Interactions of ar-curcumene and α–zingiberene with the protein chain, the polar receptor surface and their 2D interactions are illustrated in Figs. 5 and 6. 2D interaction diagram of ar-curcumene with the protein showed only π - σ (PHE-41, ILE-95), π -alkyl (ILE-122, VAL-65) and alkyl (ILE-16) hydrophobic interactions with the protein residues (Fig. 5c). The two endocyclic double bonds of α–zingiberene form π -alkyl interactions with ILE-95, ILE-122 and VAL-65 while the exocylic double bond in the side chain forms the similar bonding with ILE-16. Docking pose of α–zingiberene also revealed π-π and π-σ interactions with the protein residues (Fig. 6c).

Close

Fig. 5

Molecular docking of ar-curcumene with enoyl acyl carrier protein reductase PDB:4TZK) (a) ar-curcumene in the pocket of protein (b) Hydrogen bond receptor surface of enzyme interacting with ar-curcumene and (c) 2D interactions of ar-curcumene with enzyme.

Export to PPT

Close

Fig. 6

Molecular docking of α–zingiberene with enoyl acyl carrier protein reductase PDB:4TZK) (a) α–zingiberene in the pocket of protein (b) Hydrogen bond receptor surface of enzyme interacting with α–zingiberene and (c) 2D interactions of α–zingiberene with the enzyme.

Export to PPT