Effects of solvents on total phenolic content and antioxidant activity of ginger extracts

Ginger (Zingiber o�cinale) is a popular spice which used for the treatment of different gastrointestinal and in�ammatory discomfort. In the present study, the total phenolic content (TPC) and antioxidant activity of ginger extract using four solvents (ethanol, methanol, acetone and ethyl acetate) were determined. Among the four solvents, methanol extract showed that the maximum phenolic (1183.813 mg GAE/100 g at Ayikel and 1022.409 mg GAE/100 g at Mandura) and the least were found in acetone extract (748.865 mg GAE/100 g at Ayikel and 690.152 mg GAE/100 g at Mandura). In addition, the highest DPPH radical scavenging activity (84.868% at Ayikel and 82.883% at Mandura) was observed in methanol. However, acetone showed the least DPPH radical scavenging activity (73.864% at Ayikel and 70.597% at Mandura). Antioxidant activities of ginger extracts were also expressed as IC 50 values and acetone extract has maximum IC 50 value (0.654 and 0.812 mg/mL) followed by ethyl acetate and ethanol, while the lowest for methanol extracts (0.481 and 0.525 mg/mL). The result of this study showed that extraction solvents signi�cantly affected the total phenolic content and antioxidant activities of ginger. Thus, ginger can be regarded as promising candidates for natural sources of antioxidants with high value of phenolic contents.


Introduction
Natural bioactive compounds especially plant sources have been investigated for their characteristics and health effects [1].Many spices like cardamom, long pepper, black cumin, ginger, bishops weed and coriander are highly cultivated in Ethiopia over many years.However, ginger (Zingiber o cinale Roscoe) is cultivated in many places of the country than any other spices [2].The refreshing aroma and pungent taste makes ginger an essential ingredient in most world cuisine and food processing industry [3].Besides, ginger has been employed as an alternative medicine around the world for anti-arthritic [4], protects against gastrointestinal ulcers, improves blood circulation, lowers blood glucose in the treatment of diabetes [5] and diarrhea [6].
Numerous active ingredients are present in ginger such as terpenes (sesquiterpene hydrocarbons), alkaloids and polyphenols [7].
Phenolic compounds are associated with a high number of biological activities and one with special interest is their antioxidant capacity [8] and may help to protect the cells against the oxidative damage caused by free radicals [9,10].Antioxidant activities of ginger have been identi ed by many researchers [11,12].Several studies revealed that ginger has showed antioxidant activity against lipid oxidation and oxidative stress [13,14].Due to simplicity and low economic outlay, classical extraction methods are most commonly used for isolating these compounds in many samples.For successful separation and determination of biologically active compounds from plant material is mainly depends on the type of solvent used in the extraction procedure.Extraction with water alone was not as effective as extraction with aqueous solution of organic solvents such as ethanol, methanol, diethyl ether, chloroform, ethyl acetate and nbutanol [15][16][17].
Taking into account all these aspects, the present study was undertaken with the purpose of determining the effects of aqueous solution of different solvents (ethanol, methanol, acetone, and ethyl acetate) on the total polyphenol and antioxidant capacity of ginger extracts collected from local markets in Ayikel and Mandura towns, Ethiopia.

Materials And Methods
Chemicals DPPH (2,2-diphenyl-1-picrylhydrazyl), ascorbic acid, gallic acid, sodium carbonate, Folin-Ciocalteu reagent were purchased from Sigma-Aldrich (Mumbai, India).The solvents acetone, ethanol, methanol and ethyl acetate were ere obtained from Merck (Darmstadt, Germany).All the reagents and chemicals used were of analytical reagent grade and were acquired from commercial sources.Deionized water was used for sample preparation, dilution, and rinsing apparatus prior to analysis.

Sample collection and preparation
Three kilograms of fresh ginger rhizome samples (n=6) with no apparent physical or microbial damages were collected randomly from local markets of Ayikel and Mandura town, Ethiopia.The collected samples from each study area was pooled together and mixed well to have one bulk sample from each site.The ginger samples were washed with tap water and distilled water; and nally were peeled.The peeled samples were then sliced separately into pieces using scalpel and dried at room temperature for several days.Finally, the dried samples were ground to a ne powder using grinder and then sieved using mesh and stored in until required for extraction.

Extraction of ginger samples
Solvent extractions are most commonly used procedures to extract polyphenol from plant materials due to their ease of use, e ciency and wide applicability.For the present study, an aqueous solution of methanol, acetone, ethanol and ethyl acetate (1:4, v, v) were used as solvent to extract total polyphenol contents from the samples.One gram (1.0 g) of ginger was weighed and mixed with 20 mL of organic solvents (acetone, methanol, ethanol, and ethyl acetate) into different 100 mL conical asks and covered with aluminum foil.The solution magnetically stirred at 900 rpm for 24 h at room temperature.The supernatant was collected, filtered and nally kept in the refrigerator at 4 until further analysis.

Total phenolic content
The concentration of total phenol present in ginger extracts was determined by Folin-Ciocalteu (FC) reagent method described by Munro et al. [18].Brie y, 0.5 mL of solvent extracts of each ginger sample was mixed with 2.5 mL of 10% Folin-Ciocalteu reagent.After 5 min in the dark, 2 mL of 7.5% sodium carbonate was added.The solution was agitated with a vortex mixer for a min before incubated in the dark for 1 h at room temperature.The absorbance was determined using UV-Vis spectrophotometer at 760 nm.The calibration curve was established using gallic acid (5-150 mg/L).The phenolic content was expressed as milligram gallic acid equivalents per 100 g dry extract (mg GAE/100 g).All determinations were performed in triplicate.

Antioxidant capacity
The antioxidant activity of ginger extracts were evaluated using the method described by Koleva, et al. [19].Brie y, 1.0 mL of sample extracts at various concentrations (0.2-1.5 mg/mL) was added to 2 mL of 0.040 g/L DPPH in methanol solution.The test tube was incubated in the dark for about 30 min at room temperature.Ascorbic acid was used as positive control.Different concentrations range from 0.2-1.5 mg/mL of ascorbic acid were used for constructing calibration curve and IC 50 values were calculated.
The antioxidant activity was recorded spectrophotometrically at an absorbance of 517 nm and the percentage inhibition of radicals was calculated using the following formula: Where A bl is the absorbance of the blank DPPH solution without ginger extract, and A sa is the absorbance of sample extracts with DPPH.

Statistical Analysis
All the experiments were carried out in triplicate and the values were expressed as mean ± Standard deviation and the data were analyzed statistically using the IBM SPSS software (version 20).An analysis of variance was performed by one-way ANOVA and signi cant differences between the means due to composition of extraction solvent were determined by Tukey's HSD (homogeneous subset difference) test at the signi cance level p = 0.05.

Results And Discussion
Total Phenolic Content The total phenolic contents were determined by plotting standard calibration curve of different concentration of gallic acid using spectrophotometer at 760 nm.The values of TPC were calculated as gallic acid equivalents (GAE) per 100 gram of dry weight.
The amount of total phenolic content in ginger samples collected from Ayikel and Mandura were in uenced signi cantly by extracting solvent (p < 0.05), and the contents were varied within the range of 690.152 to 1183.813 mg of GAE/100 g of dry weight for acetone and methanol, respectively.Among the solvents, methanol was the most e cient extracting solvent for TPC, followed by ethanol, ethyl acetate and acetone, indicating that the TPC extracted in ginger were higher in polar solvents compared with less polar solvents (Table 1).The variations in the extract yields from ginger using different solvents might be explained by the difference in polarity of different compounds in the samples [20,21].
Between the two study areas, the higher TPC were found in a ginger sample collected from Ayikel in all extracts.The difference in the quantity of TPC may be attributable to different intrinsic and extrinsic factors, including cultivars, type of soil and growing conditions, maturity state and harvest conditions [22,23].

Antioxidant Activity
Antioxidant activity of ginger extract was evaluated using ascorbic acid as standard.It is one of the greatest antioxidant compound known by scavenging the stable radical of 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH).
The antioxidant values obtained are presented in Table 2. From the data it is evident the Ayikel ginger samples showed higher value of DPPH (% inhibition) and TPC as compared to Mandura.The value for DPPH (% inhibition) activity of the extracts can be ranked as: methanol extract > ethanol extract > ethyl acetate extract > acetone extract.In addition, the current nding revealed that antioxidant activity was signi cantly correlated with the phenolic content.Except ethanol and ethyl acetate extracts, there were signi cant differences (p<0.05) of DPPH radical scavenging abilities between all extracts.As with TPC value, it was observed that methanol extract owned the highest DPPH radical scavenging ability, followed by ethanol, ethyl acetate and acetone.The radical scavenging activities of ginger were close to the positive control, i.e., ascorbic acid with % inhibition of 89.75±0.361).
The result was in agreement with those ndings reported in literature, where the phenolic content and antioxidant activity were in uenced by extracting solvents.In addition, the current nding revealed that highest DPPH radical scavenging activities of ginger extract was obtained when methanol was used for extracting solvent [1,24,25].
The half maximal inhibitory concentration (IC 50 ) is de ned as the amount of antioxidant that causes decrease the DPPH concentration by 50%, [26].IC 50 value was calculated from the linear regression plots of percentage inhibition (% DPPH scavenging activity) against concentration of ginger extracts.As depicted in Table 3, the IC 50 values of Ayikel were ranged from 0.481 to 0.654 mg/mL for methanol and acetone extracts, respectively.Similarly, it was found that acetone extracts owned the highest IC 50 value followed by ethyl acetate, ethanol and methanol extracts in Mandura ginger.This implies that the concentration of acetone extract required decreasing the initial concentration of DPPH solution by 50% is 0.654 mg/mL, whereas for methanol extract is 0.481 mg/mL.The result showed that IC 50 value is inversely related to its antioxidant capacity.
It was elucidated that the methanol extracts showed highest antioxidant activities than the other solvents.However, the IC 50 values with regards to different solvents used for extraction was as follows: acetone > ethyl acetate > ethanol > methanol.The results are similar to that reported by [17,27], where by a lowest DPPH radical-scavenging activity of a plant extract had the highest IC 50 .
In our study ascorbic acid was used as the positive control; with an IC 50 value estimated at 0.239 mg/mL while the IC 50 values of the ginger extracts ranged from 0.481 to 0.812 mg/mL.This indicates that the extracts are slightly potent inhibitors in comparison with ascorbic acid.

Comparison of current study with results from other countries
There are some reports from different countries on the analysis of the phenolic contents and antioxidants activities of ginger.It is important to compare the results obtained in this study with the values reported in other countries.This comparison helps to identify the differences in composition of samples between countries.
As shown in Table 4, the total phenol contents of ginger extract obtained in this study is higher than that of the results reported by Sharif and Bennett [28] and Adel and Prakash [29].
However, methanol extract reported by Ghorab et al. [30] was found to be higher than the results of this study.The total polyphenol of methanol extract was found to be comparable with the results reported by Mohd and Muhd [31].Besides, total phenol content of acetone and ethyl acetate extract reported by Mohd and Muhd [31] and Ghasemzadeh et al. [32], respectively were found to be higher than the results obtained in this study at both study sites.
The antioxidant properties of ginger were also compared with the reports from other countries.As shown in Table 5, the antioxidant activities were found to be slightly higher than reported by Ghasemzadeh et al, [1,32], Mohd and Muhd, [31].However, the results of present study were in agreement with the reported values by Ghorab et al. [30] and Sharif and Bennett [28].The differences in the total phenol contents and antioxidant activity of this study with previously reported values were attributed to several factors such as the difference in plant variety, the method and conditions of extraction (temperature and time), environmental conditions, degree of ripeness, plant variety and sun exposure [17,33,34].For instance, the ginger studied by [28] and [29] were extracted 8 h and 3 h respectively, while in study the ginger was extracted for 24 h.

Conclusion
According to the results, the yield and e ciency of the phenolic content extraction depends on the type and kind of the solvent which is being isolated.The highest concentration of phenolic compounds in the extracts were obtained using solvents of high polarity relative to the other solvents, methanol extract manifested greater power of extraction for phenolic compounds from ginger rhizome.
The highest total phenolic content is 1183.813±0.418mg GAE/100 g DW for methanol extract, and 1009.917±0.140mg GAE/100 g DW for ethanol extract for Ayikel samples and 1022.409±0.265mg GAE/100 g DW for methanol extract, followed by 941.847±0.177mgGAE/100 g DW for ethanol extract for Mandura samples.For total phenolic extraction from ginger methanol was more e cient than ethanol, ethyl acetate and acetone.Methanol extract has maximum antioxidant activity than all other solvents followed by ethanol extract.

Declarations
There are many techniques to extract total polyphenols from plants, such as Soxhlet extraction, maceration, supercritical fluid extraction, subcritical water extraction, and ultrasound assisted extraction.

Table 2 :
% inhibition of ginger extractFor each plant sample, values in same row for each solvent followed by a different letter (x, y) are significantly different (p < 0.05) by Tukey's multiple range tests.

Table 3 :
50 (mg/mL) values of ginger by different solvent extract *Values represented mean ± S.D. of three parallel measurements.

Table 4 .
Comparison of total phenol content of ginger with that reported in the rest of the world (mg GAE/100 g)

Table 5 .
Comparison of % inhibition of ginger in this study with that reported in the rest of the world