Chemical Composition and Antimicrobial Activity of the Crude Extracts Isolated from Zingiber Officinale by Different Solvents

Ginger (Zingiber officinale), a member of the Zingiberaceae family, is a well-known spice used in the daily diet in many Asian countries [1]. It is a rhizomatous plant grown throughout South‐eastern Asia, China and in parts of Japan, Austria, Latin America, Jamaica and Africa. It has been used as a spice and medicine in India and China since ancient times. It was known in Germany and France in the ninth century and in England in 10th century for its medicinal properties [2].


Preparation of extraction
Soxhlet extraction (SE): The extraction of soluble compounds from Zingiber officinale by the soxhlet method was performed by using methanol or n-hexane as a solvent. The soxhlet procedure consisted of ground ginger rhizomes (30 g) placed inside a thimble loaded into the soxhlet extractor (Gerhardt/Germany). The total extracting time was (10 hrs.), and the total amount of solvent was (300 ml) maintained continuously refluxing over the sample. The solvent assays were performed at solvent boiling temperature. After the extraction the solvent was removed from the solute mixture by reduced pressure with rotary evaporator (Heidolph/Germany).
In order to obtain a completing dry extract, the resultant extracts were transferred to glass dishes and were left in 40°C oven for 24 hrs. , then they were left at 4°C until assessments of their antimicrobial activities.

Antimicrobial activity
Antibacterial activities of Zingiber officinale extracts were evaluated by means of agar-well diffusion assay [14] with some modifications. Fifteen milliliters of the molten agar (45°C) were poured into sterile petri dishes (Ø90 mm). Working cell suspensions were prepared and 100 μl was evenly spreaded onto the surface of the agar plates of Mueller-Hinton agar (HIMEDIA, India). Once the plates had been aseptically dried, 6 mm wells were punched into the agar with a sterile Pasteur pipette. The residual extracts were dissolved in their extracting solvents to yield the final concentration: 50, 25, 12.5, 6.25 and 3.1 mg/ml and sterilized by filtration (filter pore size 0.45 µm). Thus, 100 μl were placed into the wells and the plates were incubated at 37°C for 24 hrs. Solvents were used as negative control while antibiotic of streptomycin at the concentration of 10 (µg/disk) was used as positive control. Antimicrobial activity was evaluated by measuring the diameter of circular inhibition zones around the well. A positive result was defined as an inhibition zone (halo size) of 9 mm or more appearing around the holes, indicating presence of antibacterial substance in the extracts tested [10]. Tests were performed in triplicate.

Analysis of main constituents of ginger extracts using high performance liquid chromatography (HPLC)
The main compound were separated on FLC (Fast Liquid Chromatographic) column under the optimum condition, column was phenomenex C-18, 3 µm particle size (50×4.6 mm I.D) column. Mobile phase was THF (tetrahydrofuran), deionized water acidified with 0.1% acetic acid (80:20 v/v) gradient program from 0% B to 100% B for 12 minutes. Detection UV set at 282 nm, and flow rate 1.2 ml/ min. The separation occurred on liquid chromatography Shimadzu 10 AV-LC equipped with binary delivery pump model LC-10 A-SPD spectrophotometer.

Antimicrobial activity of extracts
The in vitro antimicrobial activity of the two extracts against the tested bacteria and fungi was assessed in both cases of presence and absence of inhibition zone diameters. We found that the antimicrobial activity of the two extracts i.e., diameter of inhibition zone mainly depend on their concentration and on the strain of tested bacteria and fungi.
So when the methanol used as a solvent, the antimicrobial activity for Zingiber officinale rhizomes extract shown in table 2. Gram-positive bacteria: Staphylococcus epidermidis shows sensitivity to methanol extract concentration ranges from 50-25 (mg/ml), and Staphylococcus aureus show sensitivity to concentration ranges from 50-12.5 mg/ml. While, for Gram negative bacteria: Klebsiella sp. and Enterococcus sp. show sensitivity to concentration ranges from 50-25 mg/ml, Proteus sp., Escherichia coli and Pseudomonas florescent both shown sensitivity to concentration of 50 mg/ml. Moreover Candida albicans fungi show sensitivity to concentration of 50 mg/ml. While, the antimicrobial activity for the extract by using n-hexane as a solvent is shown in table 3, Gram-positive bacteria: Staphylococcus epidermidis shows sensitivity to concentration of 50 mg/ml, while Staphylococcus aureus show sensitivity to concentration ranges from 50-25 mg/ml. This extract is also show inhibition activity against Gram-negative bacteria: Proteus sp., Escherichia coli, Enterococcus sp., Pseudomonas florescent all show sensitivity towards 50 mg/ml concentration. Moreover, this concentration also shows inhibition action against Candida albicans fungi.
But as it is clear from the results when the n-hexane used as a solvent its extract is less effective than methanol extract against the same tested bacteria and fungi.
While, for negative control (solvents) used for preparation different concentrations show no activity against any tested bacteria and fungi. As shown in *Peak area was calculated from area of (sample/total area)×100

Discussion
Our present study design to obtain preliminary information on the in vitro antimicrobial activity of Zingiber officinale rhizomes on eight pathogenic micro-organisms, the agar-well diffusion method was preferred to be used in this study, and the two crude extracts were analyzed by HPLC.
The results show that the extraction of ginger by using either methanol or n-hexane has an antimicrobial activity against both bacteria and fungi. This may be caused as a result of the presence of gingerol and shogaol as active ingredient within ginger. Since many studies indicated that the antimicrobial potency of ginger mainly caused by the presence of oxygenated mono-and sesquiterpenes, phenolic compounds (shogaol, gingerol) [15][16][17][18], which are lipid-soluble phenol compounds primarily isolated from the root of ginger [10,19].
Bajpai and his colleague also support our explanation that as a result of the presence of mono-and sesquiterpenoids within plant extract, which consider main cause for their antimicrobial mode of action. Since these compounds have different ways of effect since these compounds not only attack cell walls and cell membranes i.e., affecting their permeability and release of intracellular constituents (e.g. ribose, Na glutamate) but they also interfere with membrane functions (electron transport, nutrient uptake, protein, nucleic acid synthesis and enzyme activity). Thus, these compounds might have several invasive targets which could lead to the inhibition of bacterial pathogens [20].
Moreover, our results show that there is a noticeable difference in the antimicrobial activity of the extract of ginger when the methanol is the solvent than the extract when n-hexane is the solvent as seen in       table 2 and table 3. There are two points which can help in explanation of this the first point one that as a result of variations in the chemical composition which found between the two ginger extracts i.e., variation related with presence of α-curcumene which is monoterpene hydrocarbon within methanol extract but not within n-hexane (Table  1). Chemical variation leads to difference within the antimicrobial activity [18]. While the other point is that because of the difference in concentration of this material i.e., α-curcumene between the two extracts this may also leads to differences in the antimicrobial potency, this difference in concentration could be due to the nature of solvent used for extraction [18].
Also the results for both extracts were more effective against the Gram-positive bacteria compared to the results for the Gram-negative ones. The higher resistance of the Gram-negative bacteria could be due to the complexity of the cell wall of this group of microorganisms. Indeed, the external membrane of Gram-negative bacteria renders highly hydrophilic surfaces whereas the negative charge of the surface of the Gram-positive wall may reduce their resistance to antibacterial compounds [15].     In addition, the results of antifungal activity assays showed that the two extracts of Zingiber officinale had inhibitory effects on the growth of candeda albicans fungi. Our results are in agreements with previous study in literature mentioned above. The inhibitory effect observed may be a result of monoterpene which is reported to have a wide range of antifungal activity. Since the main mechanism by which will produce its anti-fungal and antimicrobial action through the disruption of bacteria or fungal membrane Integrity [21].

Conclusion
From the obtained results of our present study we can indicate that ginger extracts have exhibited wide spectrum of antimicrobial properties. Therefore, they can be used for preserving various foodstuffs against microbial spoilage and it can be incorporated into medications for topical antifungal or antibacterial therapy.