Introduction

Currently, most of the antioxidants used in the food industry are synthetic; the most representative ones are of the phenolic type like 2-tert-butyl-hydroxyanisol (BHA), 2-tert-butyl-hydroxytoluene (BHT) and tert-butyl-hydroquinone (TBHQ). A disadvantage of these compounds is that their consumption above certain levels could be toxic [1]. However, there are contradictory data about such toxicity [2]. For that reason, in the last few years, the interest for natural antioxidants and the consumption of healthy foods such as fruits, vegetables, herbal teas, among others, has increased. Antioxidants reduce oxidative stress in the body by neutralizing free radicals, preventing, attenuating or slowing down several diseases such as cancer and atherosclerosis [2–6].

Plant products have been used in traditional medicine since ancient times. In Asia, for example, ginger (Zingiber officinale) has been used extensively to treat colds, bronchitis, cough, vomiting and nausea, among other physical discomforts [7–11]. Ginger contains different components depending on where it originates and if it is a fresh or a dry rhizome [8]. Some of the chemical components in ginger are of phenolic type (6-gingerol and its related compounds like shogaol) [12–14] which have antioxidant and terpenic properties. The main ones are zingiberene, β-bisabolene, α-farnesene, β-sesquiphellandrene y α-curcumene [9]. However, in spite of its chemical composition, ginger has not frequently been used as a natural source of antioxidant compounds.

Therefore, the purpose of this experiment was to evaluate ginger’s potential use as a source of antioxidants by determining under which physical conditions the highest quantity of antioxidants is liberated in an aqueous solution of Zingiber officiale, by applying a Box-Behnken experimental design. Generally, the antioxidant activity is determined in different matrixes by extracting compounds that make this activity possible, using organic solvents such as ethanol [15], methanol [16] and hexane, or by applying supercritical fluids [14].

Materials and Methods

Sample

Approximately 30 kg of whole ginger rhizomes previously conditioned (washed, crushed and frozen) were provided by “Productos Orgánicos de Blackberry de la Sierra Norte de Puebla S.C. de R.L.”Footnote 1 (State of Puebla, Mexico). The sample was stored in polyethylene bags and was kept at a freezing temperature of −4 °C until its use.

Experimental Design to Obtain Aqueous Extracts of Ginger

To optimize physical conditions of aqueous extraction of ginger, in order to obtain the highest antioxidant activity (AA), a Box-Behnken central fractional factorial design was applied. The independent variables and the ranges for the extraction process (Table 1) were selected based on the normal conditions on which an infusion is prepared and according to some similar data reported in the literature (Table 4).

Table 1 Box-Benhken experimental design for the preparation of aqueous ginger extracts
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From the performed experimental design, 15 experiments were conducted in triplicate. The antioxidant activity of the aqueous extracts was determined through FRAP and DPPH* free radical assay; the content of total phenolics was determined by Folin & Ciocalteu’s method.

A conventional solid–liquid extraction was performed according to the Box-Behnken design, using a jacketed beaker (Schott Duran®), an stirring heater (Nuova Sarrer-Barnstead Thermolyne®) at 600 rpm, a recirculating water bath (VWR®) and distilled water, as extraction solvent.

Antioxidant Activity Determination

DPPH* Free Radical Method

For this determination, the technique proposed by Brand-Williams et al. in 1995 was used with some modifications [23]. A calibration curve was prepared [0 a 33 μM] from a standard solution of Trolox 1 mM in MeOH. To each standard solution, 2.9 mL of DPPH* (2,2-diphenyl-1-picrylhydrazyl) 0.1 mM in MeOH, were added. All standard solutions were taken to a final volume of 3 mL with MeOH. Together with the standards, a control sample was prepared containing only 0.1 mL of MeOH and 2.9 mL of DPPH*. Standards were left to react in darkness for 50 min and the absorbance was measured at 515 nm, using methanol as blank. The AA of the aqueous extracts was measured using the same procedure used for the calibration curve, replacing the Trolox solution with 100 μL of each extract. All determinations were made in triplicate.

Finally, the calibration curve was formed of [Trolox] vs % DPPH*remnant, as of the absorbance obtained values for the control sample and for each standard. The percentage of DPPH*remnant was calculated by using the Eq. 1 [24].

$$ \%\;{\mathrm{DPPH}}_{* remnant}=\left[\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{t} \arg \mathrm{e}\mathrm{t}}}{{\mathrm{A}}_{\mathrm{control}}}\right]\times 100 $$
(1)

Where: Asample = Absorbance obtained for the sample, Atarget = Absorbance obtained for methanol and Acontrol = Absorbance obtained for the control sample.

Ferric Reducing Antioxidant Power Method (FRAP)

This analysis was carried out using the modified Benzie and Strain [25] FRAP method. The reactive FRAP was prepared from an acetate buffer (300 mM to pH 3.6), ferric chloride hexahydrated (20 mM) and TPTZ (4,6-tripryridyl-s-triazine): 10.0 mM, prepared in HCl 40 mM. The three solutions were mixed in proportions of 10:1:1 (v/v/v).

A calibration curve was formed, [0 to 100 mM], from a standard solution of ferric chloride tetrahydrated in HCl 40 mM. To each standard solution of the curve 1 mL of FRAP reactive was added and it was taken to a final volume of 10 mL with distilled water. All the solutions were incubated at 37 °C for 4 min and its absorbance was measured at 593 nm using a blank containing only FRAP reactive. The AA of the aqueous extracts was measured using the same procedure used for the calibration curve, replacing the ferric chloride tetrahydrated with 250 μL of each extract. Every determination was made in triplicate. Equation 2 was used to measure AA since the results followed a linear behavior [Fe2+] vs. absorbance.

$$ A= aX\left[Fe2+\right]+b $$
(2)

Determination of Total Phenolics Using Folin & Ciocalteu’s Method

A calibration curve was formed to estimate total phenolics at a concentration interval of 0 to 15 mg/L, as of a standard solution of gallic acid (GA) 1000 mg/L. The corresponding volume was taken from each standard, 2 mL of Na2CO3 were added to 7.5 %, 2 mL of Folin & Ciocalteu’s reactive and it was diluted to 10 mL with distilled water. The absorbance readings were made at 760 nm. To determine phenols in aqueous extracts of ginger, the same procedure was followed as for the calibration curve, replacing GA with 1 mL of each sample. The determinations were made in triplicate. Since the results of [GA] vs. absorbance followed a linear behavior, the concentration of total phenolics was calculated using Eq. 3.

$$ A=a\;X\left[ Total\; phenols\right]+b $$
(3)

Analysis of Results

From the data obtained using the Minitab 17 program, the polynomial experimental designs were achieved; contour plots and response surface graphs were formed. With the purpose of determining the influence of the analyzed variables (temperature, time and sample quantity) on the liberation of antioxidant compounds from aqueous extracts of ginger, a comparative analysis of the response graphs on the different treatments was performed. Finally, the correlation between the concentration of the antioxidant compounds measured by DPPH* and FRAP methods, and the content of total phenolics in the aqueous extracts of ginger was established. In order to validate the experimental model, a prediction interval was obtained for each assay. The results are presented in Supplementary Material 1B.

Results and Discussion

Experimental Design to Obtain Aqueous Extracts of Ginger

The polynomials obtained from the Box-Behnken experimental design of the antioxidant activity determined by DPPH* and FRAP are presented in Table 2, as well as the total phenolics. Variable A (temperature) showed a positive effect while variable C (sample quantity) showed a negative effect. As opposed to the others, variable B (time) had positive effects on the liberation of phenolic compounds, but negative effects on antioxidant activity. However, the ANOVA of the data showed that interactions between temperature-time and temperature-concentration influenced significantly (p > 5 %) the extraction of antioxidant compounds Supplementary Material 1A.

Table 2 Polynomial and correlation coefficients obtained from the Box-Behnken experimental design of the aqueous ginger extracts
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For the three assays that were used, the determined correlation coefficients were superior to 95 % (Table 2), thus, this experimental design can be used to optimize the extraction conditions under which ginger must be submitted to get the higher extraction of compounds with antioxidant activity and total phenolics. The contour and surface plots obtained from the Box- Behnken experimental design are shown in Fig. 1. Temperature was the main parameter that influenced the liberation of antioxidant compounds (via radical and redox reactions) and the liberation of total phenolics in the aqueous extracts of ginger. Figure 1a shows that the extracts that were tested at 90 °C presented an antioxidant activity equal to or higher than 400 mg Trolox/100 g, regardless of the time of extraction. Figure 1b shows the antioxidant activity by oxidation-reduction where it was observed that by increasing the temperature, levels higher than 640 mg Fe2+/100 g were obtained. The liberation of phenolic compounds into the aqueous solution presented the same trend regarding temperature because levels higher than 250 mg AG/100 g were obtained in the aqueous ginger extracts tested at 90 °C (Fig. 1c).

Fig. 1
figure 1

Contour and surface plots obtained from the Box- Behnken experimental design. a DPPH* free radical, b Ferric reducing antioxidant power method (FRAP) and c Total phenolics

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To determine the influence of physical factors (temperature, time and sample quantity) on the liberation of antioxidant compounds from aqueous extracts of ginger (Fig. 2), a comparative analysis of the response graphs about the different treatments was performed. The results show that there is no need of using high concentrations of the sample (Fig. 2c) to obtain a higher liberation of antioxidant compounds by applying a high temperature (90 °C, Fig. 2a). Furthermore, it was observed that the time of extraction (Fig. 2b) did not influence the liberation of such compounds.

Fig. 2
figure 2

Influence of the physical factors on the liberation of antioxidant compounds from aqueous extracts of ginger

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Determination of the Antioxidant Activity Through DPPH*, FRAP and Total Phenolics

The results of the AA in the aqueous extracts of ginger measured by using DPPH*, FRAP and total phenolics are shown in Table 3. All the aqueous extracts that were analyzed using DPPH* showed an AA of 100.2–555.6 mg Trolox/100 g. The highest values (537.4 and 555.6 mg Trolox/100 g) were obtained from the extractions of 2 grams of the sample extracted at 90 °C and 55 °C during 15 and 5 min, respectively. Regarding the AA determined by using FRAP, the values were around 147.6–1237.4 mg Fe2+/100 g. The highest value corresponded to the extract obtained from 2 g of the sample, extracted at 90 °C for 15 min. Conditions that meet those under which the highest value of AA was obtained using the DPPH* method. The second highest result (813.8 mg Fe2+/100 g) was obtained under the same conditions of sample concentration and time of extraction but at 20 °C. This could indicate that the increase of temperature favors the liberation of compounds that can lead to oxidation-reduction reactions. When the AA of the samples was analyzed by determining the content of total phenolics, it was observed that the conditions under which the highest value was obtained (332.4 mg GA/100) were the same conditions as for the DPPH* and FRAP assays.

Table 3 Antioxidant activity measured by DPPH*, FRAP and total phenolics in aqueous ginger extracts (n = 3 ± SD)
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The results of some studies on the antioxidant activity of ginger mainly measured by DPPH* and total phenolics methods are presented in Table 4. In these experiments, the extraction solvents that were used are ethanol, methanol and water, obtaining very diverse values (20.6–1974.7 mg Trolox/100 g sample); however, these results are comparable to the ones obtained in this research. The analysis of literature (Table 4) indicates that there are many variables (physical conditions of extraction, the part of the plant that was used, type and variety of the solvent, among others) that significantly influence the extraction of compounds with antioxidant activity.

Table 4 Values of antioxidant capacity of ginger samples reported in literature
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Correlation Between the Antioxidant Activity Measured by DPPH*, FRAP and Total Phenolics

Figure 3 shows the correlation between the AA measured by DPPH* and the content of total phenolics, obtaining a value of R2 = 0.52. This value indicates that the compounds responsible for AA in the analyzed aqueous extracts of ginger are not mainly of the phenolic type, which would indicate the presence of other compounds such as terpenes. On the contrary, the correlation between the AA measured by FRAP and the content of total phenolics resulted in a value of R2 = 0.83. This response shows that by using this technique, the compounds extracted from ginger, responsible for AA, are mainly of the phenolic type. Other authors [15, 17] have studied the correlation between antioxidant activity and the content of phenolic compounds in samples of ginger, vegetables and medicinal plants. These authors have obtained R2 values from 0.65 to 0.96.

Fig. 3
figure 3

Correlation between the antioxidant activity measured by DPPH*, FRAP and total phenolics

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Conclusions

The Box-Behnken experimental design that was used allowed the determination of optimal temperature, time and sample quantity for the aqueous extraction of compounds with antioxidant activity from ginger.

By using a conventional aqueous solid–liquid extraction, it was achieved the liberation of compounds with similar or higher antioxidant capacity than those reported in literature using organic solvents and times of extraction ranging from few minutes to 24 h. The obtained results are relevant to the company that provided samples since they are developing non-alcoholic ginger beverages.