Chemical Constituents, Antibacterial Activity and Mode of Action of Elephant Ginger (Zingiber officinale var. officinale) and Emprit Ginger Rhizome (Zingiber officinale var. amarum) Essential Oils

Ginger (Zingiber officinale Rosc) is a spice plant, which is extensively used worldwide, and is morphologically classified into three types, including the red, gajah or elephant and emprit ginger.1 These tend to differ from one another in terms of shape, color, odor, and chemical constituents. Specifically, the elephant variety possesses a huge rhizome, less odor and fiber, while the red has a relatively smaller rhizome, red skin, sharp odor and more fiber. Conversely, Emprit ginger has small rhizome, beige skin color with sharp odor and more fiber.1


INTRODUCTION
Ginger (Zingiber officinale Rosc) is a spice plant, which is extensively used worldwide, and is morphologically classified into three types, including the red, gajah or elephant and emprit ginger. 1 These tend to differ from one another in terms of shape, color, odor, and chemical constituents. Specifically, the elephant variety possesses a huge rhizome, less odor and fiber, while the red has a relatively smaller rhizome, red skin, sharp odor and more fiber. Conversely, Emprit ginger has small rhizome, beige skin color with sharp odor and more fiber. 1 Traditionally, ginger has been used in the treatment of cold, digestion disorder, pain, fever, inflammation, and others. 2 However, the extract scientifically possesses pharmacological activities, including antidiarrheal, based on the inhibitory effect of B-FITC toxin, on the receptor binding mechanism at a concentrations of 25 and 50 µg/ ml. 3 In addition, antidiabetic, 4 anticonvulsion, 5 antinausea, 5 antibacterial, 6,7 lipid decreasing, 8,9 and anti-inflammatory 10 effects have also being exploited with the essential oils of ginger, e.g., Zingiber striolatum. Specifically, the antimicrobial activity recorded in prior studies show a minimal inhibition concentration (MIC) value of 0.78-3.12 mg/mL, anticancer effect was reported for leukemia (K562), cervical (A549), and prostate (PC-3) cancers, with IC 50 value of 29.67; 48.87; 86.05 µg/ ml, respectively. 11 Pharmacology activities of ginger essential oils depend on the inherent chemical composition. The aim of this research, therefore, was to analyze the chemical constituents, test antibacterial activities, and observe the mode of action of elephant and emprit ginger rhizome essential oils.

Plant material
The rhizomes of elephant and emprit ginger were collected from Balai Penelitian Tanaman Rempah dan Obat (BALITTRO), Bogor, Jawa Barat, and determined at Jatinangor Herbarium, Taxonomy laboratory, Biology faculty, Padjadjaran university, with specimen number 123/HB/03/2018. Therefore, soil and others were cleaned from the fresh specimen, using water flow, and chopped to 2 mm thickness after draining.

Isolation of essential oil
The isolation of essential oils was conducted using water and steam distillation method for 3 hours, and both samples were then stored in the refrigerator prior to further analysis.

Gas chromatography-mass spectrometry (GC-MS) analysis
The chemical constituent of elephant and emprit ginger essential oils were analyzed with GC (Shimadzu GCMS-QP2010 Ultra). Furthermore, the system temperature was initiated at 60 º C, and subsequently raised to 280 º C at the injector. The detector used helium as eluent, at a 1.31 ml/minutes flow rate, 80.2 kPa pressure, and 1.7 cm/sec linear speed. Moreover, the MS system had a source temperature of 230 o C and a 0.80 KV detector.

Antibacterial activity
Antibacterial tests was conducted by the microdilution methods, using Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC6633 Bacillus cereus ATCC11778, Methicillin-resistant Staphylococcus aureus (MRSA) as positive gram bacteria and Pseudomonas aeruginosa ATCC9027, Escherichia coli ATCC8939 as negative bacteria. Furthermore, all microorganisms were obtained from the Microbiology Laboratory, Bandung Technology Institute, and the procedures were performed according to the National Committee for Clinical Laboratory Standard.

Mode of action
The mode of action for both essential oils were observed using Scanning Electrons Microscopy (SEM) at the Mineral Faculty Laboratory, Bandung Technology Institute, using the procedure stipulated by Anam. 12,13

Antibacterial activities
The test for antibacterial activity in both samples provided positive results, as shown by the MIC and MBC values, which was more potent in the elephant essential oil. This variety inhibited the growth of 5 bacteria, including Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Escherichia coli, and MRSA, while the emprit ginger prevented the growth of 3 species, encompassing Staphylococcus aureus, Escherichia coli, and MRSA. Table 2 shows MIC and MBC of both samples. Table 2 demonstrates that the strongest inhibition of growth by both essential oils occurred with Staphylococcus aureus at 250 µg.mL -1 MIC. Therefore, the mode of bacteria damage action was observed using Scanning Electron Microscopy (SEM). Figures 1 and 2 show SEM results.

DISCUSSION
There was a difference in the components and concentrations between elephant and emprit ginger essential oil. The main component also varied between the samples evaluated and as seen in study by Noori (2018) 14    Citral. These differences are possibly influenced by the location of the growing plant, time of harvest, plant genetics and other factors. Furthermore, the statement is supported by the research of Bhattarai, 16 which stipulated the existence of variation in components amongst three types of ginger grown in different location.
The chemical component is the determinant of antibacterial activity, exhibited by the sabinen, α-pinen, β-pinen, β-phellandrene, γ-elemene, δ-elemene, β-caryophyllene, γ-caryophyllene, and germacrene B content. [17][18][19][20][21] These were all identified in the elephant ginger variety, making it a comparably better antibacterial agent, due to the ability to inhibit the growth of all test bacteria, including the Gram positive. Conversely, emprit ginger only restrained the growth of 2 microorganisms (E. coli and S. aureus), while a research by El-baky (2010) 22 showed the specific inhibitory activity of β-sesquiphellandrene, caryophyllene and zingiberene against the growth of Gram positive bacteria. These components, however, tend to be more available in the elephant ginger essential oil.
SEM micrograph (Figure 1 and Figure 2 A-C) showed the normal Staphylococcus aureus cell as circular in shape with smooth surface, which were modified after growth in the presence of elephant and emprit ginger essential oils, as well as Amoxicillin. This drug is known to act by binding to penicillin-binding proteins, which inhibit a transpeptidation process, therefore leading to the activation of autolytic enzymes in the bacterial cell wall, followed by lysis. 23 Based on previous research, damage of bacteria has been affiliated with interactions between essential oil and enzyme, as well as the protein membrane, [24][25][26] as the chemical constituents penetrate the cellular bilayer membrane. This leads to increased permeability, and the subsequent leakage. [24][25][26] (Zingiber officinale var. amarum) Essential Oils Pharmacognosy Journal, Vol 12, Issue 2, Mar-Apr, 2020

CONCLUSION
Based on the results and discussion, elephant and emprit ginger essential oils possess antibacterial activity against Gram positive and negative bacteria, at a strength that is determined by the chemical constituents. In addition, SEM micrograph showed modification in bacteria morphology, following a treatment with both samples.