Plant Materials and Extraction

Zingiber officinale rhizomes used were obtained from local market in Baghdad Province, Iraq. The plant was identified and authenticated in by the Iraqi National Herbarium, Directorate of Seed Testing and Certification, the Iraqi Ministry of Agriculture (certification number 2860 on April 2022).

The rhizomes were initially chipped and then ground using an electrical blender into a fine powder and left to air-dry. After that, 50 g of the powder was soaked in 200 mL of 70% ethanol (Alpha Chemika, India) and allowed to sit at room temperature in a conical flask overnight. A fine muslin cloth was then used to filter the mixture in order to get rid of any solid particles. The resultant combination was then filtered through the Whatman filter paper (No. 1) after the filtering residue was removed twice more using the same method.

A vacuum rotary evaporator (Heidolph, Germany) was used to concentrate the mixture at 40°C. Ultimately, the concentrated extract was heated to 45 °C in an oven until the solvent evaporated. Before being employed for phytochemical investigations, the resultant extract was kept in a sterile container [5]. The yield was computed using the following formula:

Analysis of Phytochemicals

Gas Chromatography-mass Spectrometry (GC-MS)

A Trace GC Ultra coupled to a Data System Quadrupole II (DSQ II) mass spectrometer ‎‎(Thermo Scientific, Bremen, Germany) was used for the GC-MS analysis. For the separation, a Solid Phase ‎Microextraction (SPME) fiber was used, and a 10-minute desorption ‎process was carried out in the GC injection port to achieve the desired results.

The column ‎used was a Stabilwax-DA-Crossband-Carbowax-polyethylene-glycol (Restek, Bellefonte, PA)‎ measuring 30 mm long, 0.32 mm internal diameter, and 1 µm film thickness. The oven temperature program was as follows: initial temperature (40 °C/3 minutes), ramped to 220 °C (at 15 °C/minute), and held (1 minute). Helium was used as ‎the carrier gas, with a constant flow rate of 1.5 mL/minute.

The MS ion source ‎temperature was held at 225 °C, and the electron impact (EI) mode was set with an energy of 70 electron Volts (eV). The mass range was set from 33 to 250 mass/charge (m/z) ratio. The method was ‎adjusted in light of the research conducted by [6].

High Performance Liquid Chromatography (HPLC)

In addition to GC-MS phytochemical analysis, ginger ethanolic extract was examined further by HPLC (Sykamn, Germany). The system was equipped ‎with ‎a ‎C18-Octadecylsilane (ODS) ‎column measuring 250 × 4.6 ‎mm with a particle size of 5 ‎‎µm. ‎‎A 10 µL sample was introduced ‎into the system for ‎analysis.

The mobile phase ‎consisted of ‎two ‎solvents: solvent A (95% ‎acetonitrile + 0.01% ‎trifluoroacetic acid) ‎and solvent B (5% ‎acetonitrile ‎‎+ 0.01% ‎trifluoroacetic acid), flowing at ‎a rate of 1 ‎mL/minute. The gradient ‎program started ‎with 10% ‎solvent A for the first 5 ‎minutes, ‎increased to 25% solvent A ‎between 5-7 minutes, and ‎then to ‎‎40% solvent A ‎between ‎‎7-15 minutes before returning to ‎the initial conditions.

Phenolic ‎‎compounds were ‎‎detected using a UV-visible detector set at ‎a wavelength of 278 nm [7].

Antioxidant Activity, ‎2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

‎The DPPH assay was carried out based on the method of [8] with some modifications. DPPH (ALPHA AESAR, Thermo ‎Fisher, USA) and vitamin C ‎‎(ascorbic acid) (Avonchem Ltd, UK) were used as the free radical and the standard ‎antioxidant, respectively.

The ‎ethanol was of analytical grade and obtained from a local ‎supplier. The ‎absorbance of the ‎‎samples was recorded using a digital UV-spectrophotometer ‎‎(ALS Lab, Germany).

A stock solution of vitamin ‎C (1 mg/mL) and a stock solution of ‎DPPH (0.1 mM) were made in ‎ethanol. Different ‎concentrations of ‎vitamin C (10 to 100 ‎‎µg/mL) and ginger ethanolic ‎extract (10 to 100 µg/mL) were ‎prepared by diluting the stock ‎‎solutions with ethanol. Then, ‎‎100 µL of each sample or standard was ‎added into the ‎DPPH (100 ‎‎µL) in ‎a 96-well microplate and for 30 ‎‎minutes was kept in dark at room temperature.

The ‎‎absorbance  was measured at 517 nm against a blank ‎of methanol ‎using a ‎microplate reader. ‎The DPPH radical scavenging activity ‎was calculated as percentage using ‎the ‎following formula:

Where A control is the absorbance of the control (DPPH solution + ethanol), and A ‎sample is the ‎absorbance ‎of the sample or ‎vitamin C (standard) + DPPH. The IC₅₀ value, ‎a ‎concentration of a sample or ‎a standard that reduces fifty percentage of  DPPH ‎radicals, ‎was ‎obtained from the linear regression equation of the ‎percentage of scavenging activity ‎‎versus ‎the concentration‎.

The activity of antioxidant of the samples was ‎evaluated based on ‎‎their IC₅₀ values according to [9] as follows : very strong: <50 µg/mL, strong: ‎‎‎50-100 µg/mL, medium: 100-250 µg/mL, weak: 250-500 µg/mL, and not active: >500 ‎‎µg/mL.

Results

Figures & Tables

Discussion

Yield

The yield of ginger extract is an important parameter that reflects the efficiency of the ‎‎extraction process. The extraction process in current study of ginger root yielded 18.2% of a ‎thick paste dark-brown paste-like thick ‎sauce ‎texture, with a pungent bitter taste Similar ‎result was reported by [10], who reported a black thick extract‎ ‎with 19.405% yield mango ginger extracted by 70% ethanol in a maceration method. In a ‎study conducted by [11] the acetone ginger extracts obtained from two ‎different geographically sourced ginger, Iraqi ginger and Jamaican ginger, were in the form ‎of a yellowish-brown concentrate, with a lower yield for Iraqi ginger (0.841%) compared to ‎Jamaican ginger (1.069%). The authors suggested this difference could be related to the ‎differences in cultivation conditions [12]. Factors such as the type of solvent used, drying method, extraction ‎time, temperature, and particle size of grinded ginger can ‎all affect the yield [13-16].

Phytochemicals

GC-MS

The GC-MS analysis identified various phytochemicals in the ginger ethanolic ‎extract, including fatty ‎acids, alkenes, aldehydes, and indene derivatives, all falling into five ‎‎distinct groups. Most significantly, fatty acids emerge as the preeminent category, constituting ‎‎45.45% of the identified compounds. These findings align harmoniously with prior ‎research ‎highlighting ginger's rich repository of bioactive components, notably fatty acids, and hydrocarbons [17,18&19] zingiberene, curcumene, α-‎Bergamotene, gingerol, zingerone, Caryophyllene and elemene [20-22]‎.

Among the identified fatty acids are n-Hexadecanoic acid (palmitic acid), ‎‎9,12-‎octadecadienoic acid (Z,Z)- (linoleic acid‎), oleic acid, and 9-octadecenoic acid (E)- (elaidic acid‎). According to [17], ginger oil analyzed by GC-MS exhibited the presence of linoleic acid (79.3%), 9,12-Octadecadienoic acid (Z,Z) (15.22%), ‎‎palmitic acid (12.3%), and 12-hydroxy-oleic acid (2.8%). Such fatty acids have ‎been documented for their various bioactivities, involving antioxidative, anti-inflammatory, and ‎antimicrobial attributes [22, 23]. Notably, oleic ‎acid, characterized as a monounsaturated fatty acid, has garnered recognition for ‎its potential ‎cardiovascular benefits and facilitation of improved insulin sensitivity [24]. With these fatty acids, ginger ethanolic extract may have beneficial health effects and effective therapeutic applications.‎

Additionally, alkenes and aldehydes, both accounting for 9.09% of the ginger ethanolic extract, appear as ‎prominent ‎constituents within the ginger ethanolic extract. The established anti-inflammatory ‎and ‎antioxidant attributes of (+/-)-(Z)-1,9-Hexadecadiene and 9,17-Octadecadienal (Z-) [25] add a ‎layer ‎of significance to these findings. Hence, the ginger ‎ethanolic extract holds promise as a potential source of antioxidants to ‎alleviate oxidative ‎stress and inflammation, aligning with previous in vivo studies [26-31].

One of the noteworthy components of the ginger ethanolic extract, as found by GC-MS ‎analysis, is 1H-indene,2-butyl-5-hexyloctahydro-. This indene derivative accounted for 9.09% ‎of the compounds that were identified. Indenes have been found to possess several biological ‎properties, like anti-cancer, anti-inflammatory, and antibacterial actions ‎‎[32]. The existence of this indene derivative in ginger extract provides support for ‎the putative medicinal properties of the spice.

Further constituents such as zingiberene, curcumene, α-Bergamotene, gingerol, ‎zingerone, Caryophyllene, ‎and elements have also been previously reported [20,21&2] ‎ ‎However, in the current study, such compounds were not ‎detected. There are several possible reasons for ‎this discrepancy, as abundant published works ‎suggest. The composition of phytochemicals in ginger can ‎vary depending on various factors, ‎such as the ginger variety, conditions of growing, cultivation, storage, and processing ‎‎methods [1].

Collectively, and most importantly, it has been reported that the composition of ginger ‎rhizomes fluctuates ‎in metabolite, elemental, pharmacological, and nutritional value ‎composition depend on the original ‎geographical area [22, 17]. It was suggested that ginger grown on ecological plantations exhibits enhanced nutritional and pharmacological properties, accompanied by reduced concentrations of toxic elements, in comparison to ginger from conventional cultivars [17]. The study further demonstrated that ginger cultivars from various regions differ markedly in both their elemental and metabolite profiles. Ginger cultivated on ecological plantations in Japan was found to contain significantly higher levels of macro- and trace elements essential for human nutrition, while also exhibiting lower concentrations of heavy metals—specifically cadmium, lead, and nickel—when contrasted with samples from Nigeria, Australia, and China.

Additionally, the yield of ginger extract is an important parameter that reflects the ‎efficiency of the ‎extraction process. The extraction process in the current study of ginger root ‎yielded 18.2% of a thick, ‎dark-brown paste-like sauce with a pungent bitter taste. A similar ‎result was reported by [10], who reported a black thick extract with a ‎‎19.405% yield of mango ginger extracted by 70% ‎ethanol in a maceration method. In a study ‎conducted by [10], the acetone ginger ‎extracts obtained from two different ‎geographically sourced ginger, Iraqi ginger, and Jamaican ginger, ‎were in the form of a ‎yellowish-brown concentrate, with a lower yield for Iraqi ginger (0.841%) compared ‎to ‎Jamaican ginger (1.069%). The authors suggested that this difference could be related to the ‎differences ‎in cultivation conditions [12].‎

Factors such as the extraction method (type of solvent used, form of ginger [fresh, ‎drying method], ‎extraction time, temperature, and particle size of ground ginger), part of the ‎plant used (root, leaves, ‎stem), and its age can all affect the yield [13,14,15,16; 33&34]

Overall, the wide variety of chemical substances found in ginger, including fatty acids, alkenes, ‎aldehydes, and indene derivatives, indicates the broad potential of ginger for various biological ‎activities. The aforementioned findings provide additional support for the recognized ‎significance of ginger as a rich botanical resource in both traditional and contemporary ‎therapeutic applications.

HPLC

The phytochemical results in the current study of ginger ethanolic extract showed a different ‎range of bioactive compounds. The presence of phenolic compounds in the ginger extract was measured in HPLC. These compounds (caffeic, ellagic, ferulic, gallic, and hydroxybenzoic acids) are known for their antioxidant, anti-inflammatory, and antidiabetic properties [35,36]. Caffeic and ferulic acids (a hydroxycinnamic derivative), and ellagic (a polyphenolic compound) have demonstrated antioxidant, anti-inflammatory, and antidiabetic activities [35,37,38]. The most abundant compound, gallic acid (71.25 ppm), a phenolic acid, is widely documented for its antioxidant and antidiabetic effects [36]. The hydroxybenzoic acid (49.25 ppm), an aromatic acid, is primarily noted for its antioxidant properties [37].

Identification phenolic components in ginger rhizomes have been reported in different studies. For example, benzoic acid (33.31 mg/100 g), ferulic acid (11.41 mg/100 g), and caffeic acid (6.71 mg/100g) as key phenolic constituents were reported by [ 38]. Tohma et al. (36) detected gallic acid at 39.6 mg/kg, caffeic acid at 91.2 mg/kg, ferulic acid at 224.7 mg/kg, and p-hydroxybenzoic acid (29.4 g/kg), while ellagic acid was not found in their analysis. These findings highlight the diverse phytochemical profile of ginger and its potential therapeutic properties [38]. Neither study detected ellagic acid, ‎however, other phenols were detected in both studies were not detected in the current one, for ‎example vanillin (89.4 g/kg, Tohma et al., 92020) vs 11.83 mg/100 g). These variations ‎highlight the potential influence of factors such as sample preparation, ‎geographical origin, ‎and analytical methods on the measured phenolic content in ginger ‎rhizomes. According to [39], it was observed that normal and back plants, despite belonging to the same family and sharing similar morphological characteristics, exhibit a discernible biosynthetic pathway for the synthesis of their primary chemical constituents. Therefore, gingerol-associated phenolics were absolutely recognized in regular ginger, whereas methoxyflavones were obtained in black ginger.

Antioxidant

The DPPH assay is a well-fixed method for assessing the antioxidant activity of different substances, involving plant extracts. The method acts by measuring the reduction of the steady DPPH radical into its corresponding hydrazine form when antioxidants donate hydrogen or electrons, subsequent in a decrease in absorbance at 517 nm. This reduction means causes the DPPH radical, a stable nitrogen-centered free radical, to alter the color from violet to yellow. Antioxidants, or radical scavengers, are compounds that assist this reaction [8].

The effectiveness of antioxidants is determined by the percentage of inhibition at different concentrations, where higher inhibition values indicate stronger antioxidant activity. The antioxidant capacity is further evaluated using the IC50, a concentration required at which 50% of DPPH radicals are inhibited. A lower IC50 value reflects a stronger antioxidant potential.

The results of this study showed antioxidant activity of ginger extract and vitamin C increased with concentration, where the IC50 for the extract and vitamin C were 65.11 µg/mL and 52.54 µg/mL, respectively, indicating that vitamin C exhibits slightly stronger antioxidant activity compared to the ginger extract. This suggests that, under the experimental ‎‎conditions, vitamin C, as it has a higher amount of hydroxyl ‎groups [40], ‎has a stronger antioxidant activity than the ginger extract [This indicates the ginger extract ‎has antioxidant activity similar to vitamin C]. Comparing these results with the literature, ‎[41] reported a maximum ‎DPPH assay result of 65.30% for the ethanolic ‎extract of ginger, which is close to the ‎IC50 value reported here for the ginger extract. This ‎suggests a similar antioxidant capacity ‎in these two studies. Additionally,‎ [21] ‎reported that the DPPH assay of ginger in ethanol extract ranged up to 79%, which is close ‎to the highest percentage of inhibition (83.904%) observed in the current study. [39] conducted antioxidant activity by DPPH assay of methanolic and aqueous ‎extracts of ‎‎5 medicinal plants‎ lemon balm, Asian pigeonwings, lemongrass, turmeric, and ginger. The authors ‎reported that ginger aqueous extract had the highest ‎antioxidant activity ‎(88.05±0.31%). Although the authors in their study did not mention at ‎which concentration this reduction was, neither using vitamin C as a positive control, the ‎results were in line to ones reported in this study.‎ In contrast, the work of [42&43] reported an extremely high ‎DPPH assay result of 90.1% ‎for ginger, although the concentration used (9 mg/mL) is ‎considerably higher than the ‎ranges tested in the current study (10-100 µg/mL).‎ [36] reported that the ‎ethanol and water extract of ginger scavenged 43.8% and 16.2% of DPPH free radicals at  30 ‎‎µg/mL concentration, respectively. Using the same concentration, in this study, 20.21% DPPH free radicals were ‎scavenged. In a study conducted by [44], ‎scavenging ‎‎activities against free radical in DPPH assay of ginger methanolic extract were found to be ‎‎39.6% at 0.25 mg, 64.7% at 0.5 mg, 77.6% at 0.75 mg, ‎and 84.4% at 1 mg. Such results were within the range ‎of our results ‎‎(5.993% – 83.904%).‎ [45] reported that ‎essential oil of ginger in fresh form significantly inhibited 83.03% DPPH radicals, reaching up to ‎at ‎‎240 μg/mL, which is higher than that reported in our study. Similarly, ‎[46] found that a ginger oil extract inhibited 50% of DPPH radicals ‎at 200 µg/mL concentration, a much higher concentration than the IC50 for the ginger ‎‎extract in the present study. This suggests that the ginger oil may have a lower antioxidant ‎‎activity than the ginger extract tested here, which was from dried ginger.‎ On the lower end ‎of the scale, [47] reported a DPPH assay result of just 7.8% ‎for ginger ‎rhizome, suggesting a considerably lower antioxidant activity than observed in our results. [48] ‎used DPPH radical scavenging assay to evaluate ginger antioxidant activity (as ethanolic ‎extract) and reported 12.43±3.84% DPPH free radical scavenging at 100 µg/mL increased to 58.52±0.21% at 1000 µg/mL. Similarly, lower antioxidant activity compared to current results was reported in a study conducted by [34], in which the ethanolic, methanolic, acetone, and ethyl acetate extracts of ginger ‎exhibited 50% ‎DPPH ‎free scavenging of radicals activity at 0.499, 0.481, 0.654, and 0.501 ‎mg/mL, ‎respectively. ‎These values were compared to the IC50 value of vitamin C, which was found to be ‎‎0.239 ‎mg/mL‎. ‎

While there is some variability in the reported DPPH assay results for ‎ginger extracts ‎in the literature, the study results of were generally in line with those of ‎other studies, ‎particularly when considering studies that used ethanolic extracts.‎ Some variations in the ‎results can be attributed to differences in extraction methods, plant ‎parts used, and ‎concentrations tested in different studies. The information confirms the antioxidant potential of ginger root ethanolic extracts, with them proving effective in inhibiting DPPH radicals. Antioxidant activity could be present in phenolic compounds [36].

Acknowledgement

The authors declare that there is no conflict of interest regarding the publication of this paper. 

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