Protective Effect Against Hydroxyl Radical-induced DNA Damage and Antioxidant Mechanism of [6]-gingerol: A Chemical Study

Abstract

[6]-Gingerol is known as the major bioactive constituent of ginger. In the study, it was observed to effectively protect against ${\bullet}OH$-induced DNA damage ($IC_{50}$ $328.60{\pm}24.41{\mu}M$). Antioxidant assays indicated that [6]-gingerol could efficiently scavenge various free radicals, including ${\bullet}OH$ radical ($IC_{50}$ $70.39{\pm}1.23{\mu}M$), ${\bullet}O_2{^-}$ radical ($IC_{50}$ $228.40{\pm}9.20{\mu}M$), $DPPH{\bullet}$radical ($IC_{50}$ $27.35{\pm}1.44{\mu}M$), and $ABTS{^+}{\bullet}$radical ($IC_{50}$ $2.53{\pm}0.070{\mu}M$), and reduce $Cu^{2+}$ ion ($IC_{50}$ $11.97{\pm}0.68{\mu}M$). In order to investigate the possible mechanism, the reaction product of [6]-gingerol and $DPPH{\bullet}$ radical was further measured using HPLC combined mass spectrometry. The product showed a molecular ion peak at m/z 316 $[M+Na]^+$, and diagnostic fragment loss (m/z 28) for quinone. On this basis, it can be concluded that: (i) [6]-gingerol can effectively protect against ${\bullet}OH$-induced DNA damage; (ii) a possible mechanism for [6]-gingerol to protect against oxidative damage is ${\bullet}OH$ radical scavenging; (iii) [6]-gingerol scavenges ${\bullet}OH$ radical through hydrogen atom ($H{\bullet}$) transfer (HAT) and sequential electron (e) proton transfer (SEPT) mechanisms; and (iv) both mechanisms make [6]-gingerol be oxidized to semi-quinone or quinone forms.

Introduction

As the most harmful reactive oxygen species (ROS), hydroxyl radical (•OH) can oxidatively damage DNA, lead to deleterious biological consequences, including genetic mutation,1 carcinogenesis,2 and cell death.2 Therefore, it is critical to search for potential therapeutic agents for oxida-tive DNA damage. Since ginger has been demonstrated to possess beneficial effects on cells,3 its relevant bioactive compounds had attracted considerable attention in recent years. As a major pungent principle of ginger, [6]-gingerol (Fig. 1) has therefore been intensively investigated for pharmacological and physiological activities.

Several studies suggested that [6]-gingerol can be respon-sible for the anti-inflammatory, anti-tumour and antioxidant activities of ginger. [6]-Gingerol can prevent against various cancers,4,5 especially prostate cancer,6 skin cancer,7 colon cancer.8 In addition, it has been observed to attenuate oxida-tive cell death9 and prevent genotoxicity.10

Figure 1.The chemical structure of [6]-gingerol.

These effects are thought to be related to its protective effects against oxidative DNA damage and antioxidant ability

Previously, Dugasani and colleagues have compared the antioxidant levels of four bioactive components in ginger (including [6]-gingerol).11 Unfortunately, some experimental data are not reliable, including superoxide radical scavenging and hydroxyl radical scavenging assays. In hydroxyl radical scaveng-ing assay, they used DMSO for sample solu-tion preparation, and the sample solution was directly used for hydroxyl radical-scavenging assay. As mentioned in our previous report,12 DMSO itself can scavenge •OH radical and bring about considerable interference. In fact, the reaction of DMSO with •OH had been recognized for over 30 years.13,14 The reaction rate was calculated as 9 × 109 L mol−1 s−1.15 The product of •OH reaction with DMSO has been demonstrated to be •CH3.16 In addition, in xanthine-luminal- xanthine oxidase assay, since alkaline DMSO can generate superoxide anion radicals,17,18 DMSO as the solv-ent undoubtedly cause interference with the determination of superoxide radical scavenging. In a word, it is necessary to reevaluate the antioxidant level of [6]-gingerol by a reliable method.

On the other hand, despite that there have been several computational studies for free radical-scavenging mech-anisms 19 and some hydrogen atom transfer kinetic studies for DPPH• scavenging,20 no mechanistic study based on oxidized product analysis has been reported. The present study, however, tried to use HPLC and MS to explain the mechanism. The structure elucidation of mass spectrum can be used to interpret the antioxidant mechanism of [6]-gin-gerol. Obviously, the present study will provide important insights into the mechanisms underlying the antioxidant of [6]-gingerol.

 

Experimental

Chemicals. [6]-Gingerol (CAS number: 519-34-6, 98%) was obtained from Weikeqi Biological Technology Co., Ltd (Chengdu, China). 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•), (±)-6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic acid (Trolox), and BHA (butylated hydroxy-anisole) were from Sigma-Aldrich Shanghai Trading Co. (Shanghai, China). Deoxyribose and 2,2′-azino-bis (3-ethyl-benzothiazoline- 6-sulfonic acid diammonium salt) (ABTS diammonium salt) were obtained from Amresco Inc. (Solon, OH, USA). DNA sodium salt (fish sperm, 98%) was pur-chased from Aladdin Chemistry Co. (Shanghai, China). Water and methanol were of HPLC grade. All other reagents were of analytical grade.

Protective Effect Against •OH-induced DNA Damage. The experiment was conducted using the method developed by our laboratory.21 Briefly, the sample was firstly dissolved in 95% ethanol at 2 mg/mL. The sample solution was then aliquoted into tubes. After evaporating the sample solutions in the tubes to dryness, 300 μL of phosphate buffer (0.2 M, pH 7.4) was added to the sample residue. Subsequently, 50 μL of DNA sodium (10 mg/mL), 75 μL of H2O2 (33.6 mM), 50 μL of FeCl3 (3.2 mM) and 100 μL of Na2EDTA (0.5 mM) solution were added. The reaction was initiated by adding 75 μL of ascorbic acid (1.2 mM). After incubation in a water bath at 55 °C for 20 min, the reaction was terminated by adding 250 μL of trichloroacetic acid (10%, w/w). The color was then developed by addition of 150 μL of TBA (5%, in 1.25% NaOH aqueous solution) and heating in an oven at 105 °C for 15 min. The mixture was cooled and the absorbance was measured at 530 nm against the buffer (as the blank). The inhibition percentage for •OH is expressed as follows:

Where A0 is the absorbance at 530 nm of the control without sample, and A is the absorbance at 530 nm of the reaction mixture with sample.

Hydroxyl Radical (•OH) Scavenging Assay. The experi-ment of •OH radical-scavenging was conducted according to our method.12 In brief, the sample ethanolic solution (1 mg/ mL) was separately added into tubes. After evaporating the sample solutions in the tubes to dryness, 550 μL of phos-phate buffer (0.2 M, pH 7.4) was added to the sample residue. Then, 50 μL glucose (2.8 mM), 50 μL Na2EDTA (1 mM), 50 μL FeCl3 (3.2 mM) and 50 μL H2O2 (2 mM) were added. The reaction was initiated by mixing 50 μL ascorbic acid (1.8 mM) and the total volume of the reaction mixture was adjusted to 800 μL with buffer. After incubation at 50 °C for 20 min, the reaction was terminated by 500 μL tri-chloroacetic acid (5%, w/w). The color was then developed by addition of 500 μL TBA (5%, in 1.25% NaOH aqueous solution) and heated in an oven at 105 °C for 15 min. The mixture was cooled and absorbance was measured at 532 nm (Unico 2100, spectrophotometer, Shanghai, China) against the buffer (as blank). The hydroxyl radical scavenging activity was expressed as:

Where A0 is the absorbance of the control without sample; and A is the absorbance of the reaction mixture with sample.

Superoxide Anion (•O2−) Radical-scavenging Assay. Measurement of superoxide anion (•O2−) scavenging activity was based on our method.22 Briefly, the sample was dis-solved in methanol at 2 mg/mL. The sample solution (x μL, where x = 0, 30, 60, 90, 120 and 150 μL) was mixed with 2960-x μL Tris-HCl buffer (0.05 mol/L, pH 7.4) containing Na2EDTA (1 mmol/L). When 40 μL pyrogallol (60 mmol/L in 1 mmol/L HCl) was added, the mixture was shaken at room temperature immediately. The absorbance at 325 nm of the mixture was measured (Unico 2100, Shanghai, China) against the Tris-HCl buffer as blank every 30 s for 5 min. The •O2− scavenging ability was calculated as:

Here, ΔA325nm,control/T is the increase in A325nm of the mix-ture without the sample and ΔA325nm,sample/T is that with the sample; T = 5 min. The experiment temperature was 37 °C.

DPPH• Radical-scavenging Assay. The DPPH• radical-scavenging activity was determined as described.23 Briefly, 500 μL of DPPH• solution (0.1 mM) was mixed with 250 μL sample 95% ethanol solution with various concentrations. The mixture was kept at room temperature for 30 min, and then the absorbance was measured at 519 nm against 95% ethanol (as blank). The DPPH• inhibition percentages of the samples were calculated:

Where A is the absorbance with samples; while A0 is the absorbance without samples. Trolox and BHA were used as the positive controls.

HPLC Analysis and Mass Spectrometry of Reaction Product of [6]-gingerol and DPPH• Radical. The product mixture of [6]-gingerol and DPPH• radical (10:3, mol/mol) was filtered using 0.45 μm filters then analyzed by a pre-parative HPLC system (Prominence LC-20A, Shimadzu, Japan), equipped with a Diamonsil C18 (250 mm × 4.6 mm, 5 μm) column (Dikma Co., Beijing, China). The mobile phase consisted of methanol-0.5% acetic acid (90:10, v: v) and the flow rate was 0.5 mL/min, injection volume was 27 μL, detection wavelength was 225 nm. [6]-Gingerol and DPPH• radical were also comparatively measured under the same chromatographic conditions.

The reaction product isolated by HPLC was further ana-lyzed by a microflex LT MALDI TOF-Q II (Bruker Daltonics, USA) mass spectrometer which was equipped with an electrospray ionisation (ESI) source and run in positive mode. The scan range was 50-3000 m/z. ESI parameters were optimised with direct infusion of dansylated amine mixture by an external syringe and set as follows: capillary, +4.5 kV; nebulizer pressure, 0.3 bar; dry gas flow, 4.0 L/ min; dry gas temperature, 180 °C. Argon was applied as the collision gas, and the collision energy was set to 10 eV to provide some structural information and to focus ion flux. High purity nitrogen was applied both as a nebulizer gas and a drying gas. The Q-TOF/MS parameters were optimised to the following: funnel 1 was 300.0 Vpp and 2 was 400.0 Vpp; hexapole Rf was 400.0 Vpp; quadrupole ion energy was 5.0 eV; collision Rf was 650.0 Vpp. The ion transfer time and prepulse storage time were set to 120 and 10 us, respectively.

ABTS+• Radical-scavenging Assay. The ABTS+• scaveng-ing activity was evaluated by the method.23 The ABTS+• was produced by mixing 350 μL ABTS diammonium salt (7.4 mM) with 350 μL K2S2O8 aqueous persulfate (2.6 mM). The mixture was kept in the dark at room temperature for 12 h to allow completion of radical generation, then diluted with 95% ethanol (about 1:50) so that its absorbance at 734 nm was 0.70 ± 0.02. Then, 0.6 mL diluted ABTS+• reagents were brought to 150 μL sample ethanolic solutions (0.01 mg/mL). After incubation for 6 min, the absorbance at 734 nm was read on a spectrophotometer (Unico 2100, Shanghai, China). The percentage inhibition was calculated as:

Where A0 is the absorbance of the control without any samples, A is the absorbance of the mixture with [6]-gingerol, Trolox, or BHA.

Cu2+-reducing Power Assay. The cupric ions (Cu2+) re-ducing power capacity was determined based on the method,23 with a slight modification. In brief, 100 μL CuSO4 aqueous solution (10 mM), 100 μL neocuproine ethanolic solution (7.5 mM) and 400 μL CH3COONH4 buffer solution (100 mM, pH 7.0) were brought to test tubes with different volumes of samples (0.1 mg/mL, 15-75 μL). Then, the total volume was adjusted to 800 μL with the buffer and mixed vigorously. Absorbance against a buffer blank was measured at 450 nm after 15 min. The relative reducing power of the sample as compared with the maximum absorbance, was calculated using the formula:

Here, Amax is the maximum absorbance in the test and Amin is the minimum absorbance in the test. A is the absorbance of sample.

Statistical Analysis. Each experiment was performed in triplicate and the data were recorded as mean ± SD (standard deviation). The IC50 value was defined as the final concen-tration of 50% radical inhibition (relative reducing power, or chelating effect). Statistical comparisons were made by one-way ANOVA to detect significant difference using SPSS 13.0 (SPSS Inc., Chicago, IL) for Windows. P < 0.05 was considered to be statistically significant.

 

Results and Discussion

In the present study, we used Fenton reaction (Equation 1) to produce •OH radicals for the investigation on the pro-tective effect of [6]-gingerol against oxidative DNA damage.

The data indicated that [6]-gingerol and the positive controls dose-dependently increased the protective effect against oxidative DNA damage at 0-200 μg/mL (Fig. 2). Based on the IC50 values (328.60 ± 24.41 and 690.76 ± 12.31 μM, respectively for [6]-gingerol and Trolox, Table 1), it can be inferred that [6]-gingerol presented 2.10 times higher pro-tective effect than the standard antioxidant Trolox in our model. This is consistent with the previous study, in which [6]-gingerol was found to prevent UVB-induced ROS pro-duction and oxidative DNA damage.24 Its protective effect against DNA oxidative damage may be primarily responsible for the pharmacological effects, including anti-inflammatory and anti-cancer effects.4-8 In fact, oxidative DNA damage has been observed to play a key role in inflammation-related carcinogenesis.25

Previous work has shown that there are two approaches for natural phenolic antioxidants to protect against oxidative DNA damage: one is to scavenge the •OH radical prior to DNA damage; and the other is to prevent the DNA radicals resulting from •OH radical attack.26

To further confirm whether the protective effect of [6]-gingerol against oxidative DNA damage is relevant to its radical-scavenging ability, we then determined the radical-scavenging abilities of [6]-gingerol on •OH and •O2−. In the •OH radical-scavenging assay (Suppl. Fig. S1), [6]-gingerol could effectively scavenge •OH radicals at 0-22.5 μg/mL (0-77.7 μm) and its IC50 value was calculated as 70.39 ± 1.23; In the •O2− radical-scavenging assay (Suppl. Fig. S2), [6]-gingerol along with the positive controls almost linearly increased the •O2− radical-scavenging percentages, and the IC50 value of [6]-gingerol was 228.40 ± 9.20 μM (Table 1). These data clearly suggest that ROS-scavenging (especially •OH-scavenging) is one possible mechanism for [6]-gin-gerol to protect against oxidative DNA damage.

Figure 2.The protective effect against •OH radical-induced DNA damage of [6]-gingerol and positive control Trolox (Each value is expressed as mean ± SD, n = 3).

Figure 3.Typical HPLC chromatogram of the reaction product of [6]-gingerol with DPPH• (1,1-Diphenyl-2-picrylhydrazyl radical).

To explore the possible mechanism for [6]-gingerol to scavenge ROS, we further explored the radical-scavenging effect on DPPH• and ABTS+• radicals.

The DPPH assay revealed that [6]-gingerol possessed a concentration-dependent effect at 0-10 μg/mL (0-34.01 μM, Suppl. Fig. S3), and the IC50 was calculated as 27.35 ± 1.44 μM (Table 1). It means that [6]-gingerol can effectively eliminate DPPH• radical.

In order to further explore the mechanism for [6]-gingerol to scavenge DPPH• radical, the reaction product of [6]-gingerol with DPPH• was measured using HPLC (Fig. 3). The HPLC profile clearly indicated a peak as the product at 11.98 min (retention time).

When the product was further analyzed by mass spectro-metry, it gave a molecular ion peak at m/z 316 [M+Na+] which was obviously one less than reactant [6]-gingerol at m/z 317 [M+Na+]. It indicated a hydrogen atom transfer of [6]-gingerol to product. In addition, fragment units of m/z 302.30, 274.27, and 246.24 in the product were also observed. Obviously, these fragment units showed a loss of C=O (m/z 28) which is regarded as the characteristic diagnostic frag-ment loss for quinone (Fig. 4(a)). On the other hand, the reactant [6]-gingerol, however, did not exhibit the charac-teristic losses at m/z 28 (Fig. 4(b)). In a word, the hydrogen atom has been shown to be transferred from [6]-gingerol to DPPH•, and [6]-gingerol has been oxidized to semi-quinone or quinone by DPPH•.

Based on the above data, and previous report which DPPH• may be scavenged through hydrogen atom (H•) transfer (HAT) to form DPPH-H molecule,27 the proposed reaction of [6]-gingerol with DPPH• can be illustrated in Figure 5. As shown in Figure 5, the reaction of [6]-gingerol with DPPH• was thought to yield a phenoxyl radical (I), which can be converted to semi-quinone (II). Semi-quinone (II), however, possessed various resonances formula, e.g. semi-quinone (III), semi-quinone (IV), and phenoxyl radical (I) which is some extent stable a stable species. Thus, step 1 for [6]-gingerol to transfer a hydrogen atom is easily to be initiated. Under the conditions of excessive DPPH•, however, semi-quinone (IV) may be further be extracted hydrogen atom to produce benzoquinone. However, since the ortho-position of –OH is –OCH3 not –OH, the hydrogen extraction isn’t so easy, therefore, the yield of benzoquinone is very less, and the peak at m/z 246.24 is very low in MS spectrometry (Fig. 4(a)).

Figure 4Mass spectra of the reaction product [6]-gingerol with DPPH• (a), and [6]-gingerol (b).

Figure 5.The proposed reaction of [6]-gingerol with DPPH• via HAT (hydrogen atom transfer) mechanism based on mass spectro-metry analysis.

Besides DPPH• scavenging, ABTS+• scavenging has also been used for investigation on the antioxidant mechanism of [6]-gingerol in the study. As seen in Suppl. Fig. S4, [6]-gingerol linearly (R=0.99925) increased its ABTS+• scaven-ging percentages at 0-1.33 μg/mL (0-4.55 μM) and its IC50 was 2.53 ± 0.070 μM (Table 1). As we know, ABTS+• scavenging is an electron (e) transfer process.28 In the pro-cess, e transfer is always accompanied by deprotonation, so it is termed a sequential electron proton transfer (SEPT) mechanism,29 or proton coupled electron-transfer (PCET) mechanism,30 sequential proton loss single electron transfer (SPLET).31 The SEPT mechanism for [6]-gingerol to scavenge ABTS+• was proposed as described in Figure 6. Through SEPT mechanism, [6]-gingerol might change to phenoxyl radical (I). The SEPT mechanism is also supported by Cu2+ assay, in which [6]-gingerol increased the reducing percent-age in a dose-dependent manner (Suppl. Fig. S5) with IC50 value being 11.97 ± 0.68 μM (Table 1). As we know, reduc-ing reactions are actually an electron (e) transfer process. Similarly, phenoxyl radical (I) was thought to be further converted into semi-quinones, even quinone under excess ABTS+• radicals or Cu2+ ions.

The fact that [6]-gingerol could effectively scavenge both ABTS•+ and DPPH• radicals, suggests the process of [6]-gingerol scavenging of ROS (especially •OH radicals) in cells would be mediated via HAT and SEPT mechanisms.

As shown in Eq. (1), the generation of •OH radicals may also bring about equal OH− ions in cells. Therefore, at high levels of •OH radicals, massive OH− ions could alkalize the cellular environment. Under alkaline conditions, however, the acidity may predominate over the chemical action of [6]-gingerol. As a phenol, [6]-gingerol presents a weak acidity (pKa ~10).32 Thus, [6]-gingerol might easily ionize to yield H+ ions, and [6]-gingerol− anion which could further donate an electron (e) to •OH radicals to form phenoxyl radical (I) (Fig. 7), even semi-quinones and benzoquinone. This is a possible mechanism for [6]-gingerol to directly scavenge •OH via the SEPT mechanism. The mechanism is similar to that of trans-resveratrol toward •OH radicals.33

Figure 6.The proposed reaction of [6]-gingerol with ABTS+• via SEPT (sequential electron proton transfer) mechanism.

Table 1.IC50 value is defined as the concentration of 50% effect percentage and calculated by linear regression analysis and expressed as mean ± SD (n = 3). The linear regression was analyzed by Origin 6.0 professional software. Means values with different superscripts in the same row are significantly different (p < 0.05), while with same superscripts are not significantly different (p < 0.05). *The positive control is Sodium citrate. Ratio = IC50,Trolox: IC50,[6]-gingerol. The dose response curves of [6]-gingerol in antioxidant assay were shown in Supplemental 1. N.D., not determined. BHA, butylated hydroxyanisole.

Figure 7.The proposed reaction for [6]-gingerol to scavenge •OH via SEPT (sequential electron proton transfer) mechanism.

Figure 8.The proposed reaction for [6]-gingerol to scavenge •OH via HAT (hydrogen atom transfer) mechanism.

However, at low levels of •OH radicals, the cellular environ-ment was almost neutral and the acidity could not predo-minate over the chemical action of [6]-gingerol. The rapid and direct attack of •OH radicals may cause homolysis of [6]-gingerol to generate phenoxyl radical (I) and hydrogen atom (•H). Hydrogen atom, however, would immediately be donated to •OH to form the stable H2O molecule (Fig. 8). Meanwhile, phenoxyl radical (I) changed into semi-quinones. This may be the HAT mechanism for [6]-gingerol to directly scavenge •OH. This mechanism agrees with the previous findings that the dopamine reaction towards •OH is mainly via HAT at physiological pH 7.4.28

To quantitatively evaluate the relative antioxidant level of [6]-gingerol, the ratio value was defined as IC50,Trolox/IC50,[6]-gingerol. As shown in Table 1, the ratio values of oxidative DNA damage, •OH-scavenging, ·O2 − scavenging, DPPH•scavenging, ABTS+• scavenging, and Cu2+-reducing were 2.10, 1.32, 0.99, 1.07, 2.91, and 3.08, respectively. The aver-age ratio value was calculated as 1.91 (Table 1). It implies that [6]-gingerol had 1.91 times higher the total antioxidant capacity than the standard antioxidant Trolox.

It must be emphasized that the IC50 values of [6]-gingerol in our ·O2− and •OH scavenging assays were respectively 228.40 ± 9.20 and 70.39 ± 1.23 μM, while they were calcu-lated as 4.05 and 4.62 μM respectively in the previous literature.11 Undoubtedly, there is a considerably difference. However, the IC50 values in DPPH scavenging assay are generally identical: As shown in Table 1, it was listed as 27.35 ± 1.44 μM in our study, while the previous literature shown as 26.3 μM.11 It clearly indicated that DMSO used as a solvent in the previous work indeed brought about considerable interference with the ·O2− and •OH scavenging assays.11

 

Conclusion

Based on the above discussion, it can be concluded that: (i) as the major bioactive constituent of ginger, [6]-gingerol can effectively protect against •OH-induced DNA damage; (ii) a possible mechanism for [6]-gingerol to protect against oxidative damage is •OH radical scavenging; (iii) [6]-gin-gerol scavenges •OH radicals possibly through hydrogen atom (H•) transfer (HAT) and sequential electron (e) proton transfer (SEPT) mechanisms; and (iv) radical scavenging makes [6]-gingerol be oxidized to semi-quinone or quinone forms.