Scholarly article on topic 'Flos Chrysanthemi Indici protects against hydroxyl-induced damages to DNA and MSCs via antioxidant mechanism'

Flos Chrysanthemi Indici protects against hydroxyl-induced damages to DNA and MSCs via antioxidant mechanism Academic research paper on "Chemical sciences"

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{"Flos Chrysanthemi Indici " / "Hydroxyl-induced damage" / "Antioxidant Mechanism" / "Hydrogen atom transfer HAT" / "Sequential electron proton transfer SEPT" / "Chlorogenic acid" / "Total phenolics"}

Abstract of research paper on Chemical sciences, author of scientific article — Xican Li, Qiuping Hu, Shuxia Jiang, Fei Li, Jian Lin, et al.

Abstract Flos Chrysanthemi Indici (FCI) is a Chinese herbal medicine used in China for over 2000years. In this study, its ethanol extract (EFCL) was found to protect against hydroxyl radical ( OH) induced oxidative damages to mesenchymal stem cells (MSCs) and DNA. To explore the mechanism, EFCL was further determined by chemical and antioxidant assays. Folin–Ciocalteu colorimetric assay suggested that content of total phenolics was 93.85±3.56mg catechin/g and HPLC analysis indicated a content of 10.05±1.44mg/g as chlorogenic acid in EFCI. Antioxidant assays revealed that EFCI could also scavenge O2 − radical (IC50 58.74±1.30μg/mL), DPPH radical (IC50 46.46±1.35μg/mL) and ABTS+ radical (IC50 20.59±0.52μg/mL), bind Fe2+ (IC50 364.27±19.57μg/mL) and Cu2+ (IC50 734.77±34.69μg/mL), reduce Fe3+ (IC50 147.11±11.09μg/mL) and Cu2+ (IC50 35.69±3.14μg/mL). On the basis of mechanistic analysis, we concluded that FCI can effectively protect against hydroxyl-induced damages to DNA and MSCs, therefore FCI shows promise as a possible therapeutic reagent for many diseases. The effect may be mainly attributed to phenolics especially chlorogenic acid, which exerts the antioxidant action possibly through metal-chelating, and radical-scavenging which is via hydrogen atom transfer (HAT) and/or sequential electron proton transfer (SEPT) mechanisms.

Academic research paper on topic "Flos Chrysanthemi Indici protects against hydroxyl-induced damages to DNA and MSCs via antioxidant mechanism"

Journal of Saudi Chemical Society (2015) 19, 454-460

King Saud University Journal of Saudi Chemical Society

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Flos Chrysanthemi Indici protects against cMa*

hydroxyl-induced damages to DNA and MSCs via antioxidant mechanism

Xican Lia *1, Qiuping Hua1, Shuxia Jiang a,b, Fei Lic, Jian Linc, Lu Hana, Yuling Hong a, Wenbiao Lu a, Yaoxiang Gao a, Dongfeng Chenc

a School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China b School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, China

c School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

Received 11 February 2014; revised 18 June 2014; accepted 24 June 2014 Available online 4 July 2014

KEYWORDS

Flos Chrysanthemi Indici; Hydroxyl-induced damage; Antioxidant Mechanism; Hydrogen atom transfer HAT;

Sequential electron proton transfer SEPT; Chlorogenic acid; Total phenolics

Abstract Flos Chrysanthemi Indici (FCI) is a Chinese herbal medicine used in China for over 2000 years. In this study, its ethanol extract (EFCL) was found to protect against hydroxyl radical ("OH) induced oxidative damages to mesenchymal stem cells (MSCs) and DNA. To explore the mechanism, EFCL was further determined by chemical and antioxidant assays. Folin-Ciocalteu colorimetric assay suggested that content of total phenolics was 93.85 ± 3.56 mg catechin/g and HPLC analysis indicated a content of 10.05 ± 1.44 mg/g as chlorogenic acid in EFCI. Antioxidant assays revealed that EFCI could also scavenge "O2 radical (IC50 58.74 ± 1.30 ig/mL), DPPH" radical (IC50 46.46 ± 1.35 ig/mL) and ABTS + " radical (IC50 20.59 ± 0.52 ig/mL), bind Fe2+ (IC50 364.27 ± 19.57 ig/mL) and Cu2 + (IC50 734.77 ± 34.69 ig/mL), reduce Fe3 + (IC50 147.11 ± 11.09 ig/mL) and Cu2 + (IC50 3 5.69 ± 3.14 ig/mL). On the basis of mechanistic analysis, we concluded that FCI can effectively protect against hydroxyl-induced damages to DNA and MSCs, therefore FCI shows promise as a possible therapeutic reagent for many diseases. The effect may be mainly attributed to phenolics especially chlorogenic acid, which exerts the antioxidant action possibly through metal-chelating, and radical-scavenging which is via hydrogen atom transfer (HAT) and/or sequential electron proton transfer (SEPT) mechanisms.

© 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

Corresponding author. Address: School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Waihuan East Road No.232, Guangzhou Higher Education Mega Center, 510006 Guangzhou, China. Tel.: +86 20 39358076; fax: +86 20 38892690. E-mail address: lixican@126.com (X. Li).

1 Both authors are equally contributed to the work. Peer review under responsibility of King Saud University.

1. Introduction

It is well known that mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into all mesodermal cell types and neuro-ectodermal cells. These abilities make MSCs an excellent seed cell for cell transplantation, tissue engineering, and gene therapy [1]. However, these clinical applications are usually limited by the oxidative damages induced by reactive oxygen species (ROS) especially hydroxyl radical ("OH)

http://dx.doi.Org/10.1016/j.jscs.2014.06.004

1319-6103 © 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

[2]. The hydroxyl radical ('OH) is well-known as the most powerful reactive oxygen species (ROS) and can damage DNA to bring about various oxidative lesions (Supplemental file 1). These oxidative lesions, however, can result in genomic instability and lead to cell death [3,4].

Therefore, it is critical to search for potential therapeutic agents for oxidative damage. In recent years, medicinal plants especially Chinese medicinal herbals have attracted much attention.

As a typical Chinese herbal medicine, Flos Chrysanthemi Indici (FCI, ^Mffi in Chinese, Supplemental file 2) has been used in traditional Chinese medicine (TCM) for over 2000 years [5,6]. From the perspective of TCM, FCI can clear away heat and toxicity [5]. Therefore, FCI is widely consumed as a healthcare tea which is generally called Chrysanthemum Tea (Mffi^ in Chinese) in China. The so-called heat and toxi-city in TCM, however can be regarded as oxidative stress in free radical biology and medicine, and excessive ROS can be a resource of heat and toxicity [7].

Phytochemical analyses have demonstrated that there are total flavones, chlorogenic acid, and some volatile compounds (such as 1,7,7-trimethyl-bicyclo[2,2,1]heptan-2-one and isobor-neol) [8-10]. Obviously, flavones and chlorogenic acid belong to the family of total phenolics.

In the present study, we employed a Soxhlet extractor to prepare its ethanolic extract (EFCI). The extract (EFCI) was then evaluated for the protective effect against oxidative damages to MSCs and DNA, and the relevant chemical contents especially total phenolics and chlorogenic acid. On this basis, the antioxidant mechanism of CFI was further discussed using an in vitro model. Undoubtedly, it will be helpful to understand the pharmacological effects or functions in TCM of CFI, and to discover a new therapeutic agent against oxidative damage in MSC transplantation or tissue engineering.

2. Materials and methods

2.1. Plant material and animals

Flos Chrysanthemi Indici was collected at an elevation of 500 m in the region of Mount Huangshan (Latitudes 30° 1' N and longitudes 118°1' E, Anhui, China). It was authenticated by Professor Shuhui Fan and sun-dried. A voucher specimen has been deposited in our laboratory. Sprague-Dawley (SD) rats of 4 weeks of age were obtained from the animal center of Guangzhou University of Chinese Medicine.

2.2. Chemicals

DPPH' (1,1-diphenyl-2-picryl-hydrazl radical), pyrogallol, murexide (5,5'-nitrilodibarbituric acid monoammonium salt), ferrozine [3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine], neocuproine (2,9-dimethyl-1,10-phenanthroline), cat-echin, and Folin-Ciocalteu reagent were purchased from Sigma Co. (Sigma-Aldrich Shanghai Trading Co., China); ABTS [2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt)] was obtained from Amresco Co. (Solon, OH, USA); Chlorogenic acid and caffeic acid were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were

purchased from Gibco (Grand Island, NY, USA); CD44 was purchased from Wuhan Boster Co., Ltd. (Wuhan, China). All other reagents were of analytical grade.

2.3. Preparation and HPLC characterization of ethanolic extract from Flos Chrysanthemi Indici (EFCL)

Dried Flos Chrysanthemi Indici (10 g) was powdered then extracted by 150 mL absolute ethanol using a Soxhlet extractor for 6 h. The extract was filtered using a Buckner funnel and Whatman No. 1 filter paper. The filtrate was then concentrated to dryness under reduced pressure to yield ethanolic extract of Flos Chrysanthemi Indici (EFCI, 2.36 g). It was stored at 4 0C for analysis.

The HPLC characterization of EFCI was performed on a Syltech P510 system (Los Angeles, California, USA), equipped with a Diamonsil C18 (250 mm x 4.6 mm, 5 im) column (Dik-ma Co., Beijing, China). All samples were dissolved in methanol at 10 mg/mL and filtered using 0.45 im filters. The mobile phase consisted of methanol-0.1% phosphoric acid (85:15, v:v) and the flow rate was 0.5 mL/min, injection volume was 15 iL, detection wavelength was 254 nm. In the study, chlorogenic acid in EFCI was firstly identified using an external standard method based on the retention time (23.18 min), it was then further identified by an internal standard method. The content of chlorogenic acid was calculated based on the linear equation (y = 715502.78x - 975704.97, R = 0.9993, Supplemental file 3).

2.4. Determination of total phenolics

The total phenolic content of EFCI was determined using Folin-Ciocalteu assay [11] with catechin as a standard. In brief, 0.5 mL sample methanolic solution (3 mg/mL) was mixed with 0.5 mL Folin-Ciocalteu reagent (0.25mol/L). After incubation for 3 min, 1.0 mL of Na2CO3 aqueous solution (15%, w/v) was added. After standing at room temperature for 30 min, the mixture was centrifuged at 3500r/min for 3 min. The absorbance of the supernatant was measured at 760 nm (Unico 2100, Shanghai, China). The total phenolic content of EFCI was calculated based on a linear regression equation of catechin (y = 72.322x + 0.1346, x for catechin content, y for absorbance at 760 nm, R = 0.994), and was expressed as catechin equivalents in milligrams per gram of extract.

2.5. Protective effect against 'OH-induced damage to MSCs (MTT assay)

MSC culture was carried out according to our previous report [12] with slight modifications. In brief, bone marrow was obtained from the femur and tibia of rat. The marrow samples were diluted with DMEM (LG: low glucose) containing 10% FBS. MSCs were prepared by gradient centrifu-gation at 900g for 30 min on 1.073 g/mL Percoll. The prepared cells were detached by treatment with 0.25% trypsin and passaged into cultural flasks at 1 x 104/cm2. MSCs at passage 3 were evaluated for cultured cell homogeneity using detection of CD44 by flow cytometry and were used for the investigation.

These MSCs were seeded at 1 x 104 cells per well in 96-well plates. After adherence for 24 h, these MSCs were then divided into normal, model, and sample (EFCI and chlorogenic acid) groups. In the normal group, MSCs were incubated for 24 hr in DMEM; In the model and sample groups, MSCs however were added by FeCl2 (100 iM) followed by H2O2 (50 iM). After incubation for 20 min, the mixture of FeCl2 and H2O2 was removed. MSCs in the model group were incubated in DMEM for 24 hr, while MSCs in the sample group were incubated in DMEM with 3 and 30 ig/mL EFCI (or 3 and 30 ig/ mL chlorogenic acid) for 24 hr. All groups had five independent wells. After incubation, 20 iL MTT (5 mg/mL) was added and then incubated for further 3 h. Culture medium was discarded and was replaced with 150 iL DMSO. Absor-bance at 490 nm was measured by a Bio-Kinetics reader (PE-1420; Bio-Kinetics Corporation, Sioux Center, IA, USA). In the experiment, culture with serum medium was used for the control group and each sample test was repeated in five independent wells.

2.6. Protective effect against hydroxyl-induced DNA damage

The protective effect against hydroxyl-induced DNA damage of EFCI was estimated by our method [13]. Briefly, sample was dissolved in methanol at 4 mg/mL. Various amounts (9-36 iL) of sample methanolic solutions were then separately taken into tubes. After evaporating the sample solutions in tubes to dryness, 300 iL of phosphate buffer (0.2 mol/L, pH 7.4) was added to the sample residue. Subsequently, 50 iL DNA sodium (10.0 mg/mL), 75 iL H2O2 (33.6 mmol/L), 50 iL FeCl3 (3.2mmol/L), and 100 iL Na2-EDTA (0.5 mmol/L) were added. The reaction was initiated by adding 75 iL of ascorbic acid (12 mmol/L). After incubation in a water bath at 50 0C for 20 min, the reaction was terminated by adding 250 iL of trichloroacetic acid (10 g/ 100 mL water). The color was then developed by addition of 150 iL of TBA (2-thiobarbituric acid) (0.4 mol/L, in 1.25% NaOH aqueous solution) and heated in an oven at 105 0C for 15 min. The mixture was cooled and absorbance was measured at 530 nm against the buffer (as blank). The percent of protection against DNA damage is expressed as follows:

Protective effect % =

Ap - A A0

x 100%

where A0 is the absorbance of the control without sample, and A is the absorbance of the reaction mixture with sample.

2.7. Superoxide anion ("O2) radical-scavenging assay

Measurement of superoxide anion ("O22) scavenging activity was based on our improved method [14]. Briefly, the sample was dissolved in methanol at 3 mg/mL. The sample solution (x iL, where x = 0, 5, 10, 15, 20, and 25 iL) was mixed with 2950-x iL Tris-HCl buffer (0.05 mol/L, pH 7.4) containing Na2EDTA (1 mmol/L). When 50 iL 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:

Inhibition % =

DA325 nm .control \ _ / DA325 nm,sample \

T \ T )

( AA325

x 100%

Here, DA325 nmi control is the increment in A325 nm of the mixture without the sample and DA325 nm, sample is that with the sample; T = 5 min. The experiment temperature was 37 0C.

2.8. Fe + -chelating assay

The Fe2+-chelating capacity was evaluated by the method as described by Li [11]. Briefly, x iL sample solutions (6 mg/ mL, x = 0, 20, 40, 60, 80, and 100) were added to 100 iL FeCl2 aqueous solutions (0.25 mmol/L). The reaction was initiated by the addition of 150 iL ferrozine aqueous solutions (0.50 mmol/L) and the total volume of the reaction mixture was adjusted to 1000 iL with methanol. Then, the mixture was shaken vigorously and stood at room temperature for 10 min. Absorbance of the solution was measured spectropho-tometrically at 562 nm (Unico 2100, Shanghai, China). The percentage of chelating effect on Fe2+ was calculated by the following formula:

Inhibition % =

A0 - A A0

x 100%

where A0 is the absorbance without sample, and A is the absorbance with sample.

2.9. Cu2 + -chelating assay

The Cu2+-chelating capacity was measured by a complexo-metric method [15]. Briefly, murexide solution (1.2 mL, 0.25 mmol/L) and CuSO4 aqueous solution (60 iL, 20 mmol/ L) were added to hexamine HCl buffer (pH 5.0, 30 mmol/L) containing 30 mmol/L KCl. After incubation for 1 min at room temperature, 30-240 iL sample solutions (3 mg/mL in methanol) were added. The final volume was adjusted to 1500 iL with methanol. Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 485 nm and 520 nm (Unico 2100, Shanghai, China). The absorbance ratio (A485 nm/A520 nm) reflected the free Cu2+ content. Therefore, the percentage of cupric chelating effect was calculated by the following formula:

Chelating effect % =

A485 1

A520 ,

A485 1 A520 1

(A485 -

\A520 >

(A485 -

\A520 >

■ x 100%

A485 -

A520 :

is the absorbance ratio of the sample, while

is the maximum absorbance ratio and

A4851 A520 1

is the minimum absorbance ratio in the test. 2.10. DPPH' radical-scavenging assay

DPPH' radical-scavenging activity was determined as previously described by Li [16]. Briefly, 1 mL DPPH' ethanolic solution (0.1 mmol/L) was mixed with 0.5 mL sample alcoholic solution (0.06-0.18 mg/mL). The mixture was kept at room temperature for 30 min, and then measured with a spectropho-

tometer (Unico 2100, Shanghai, China) at 519 nm. The DPPH' inhibition percentage was calculated as:

A0 - A

Inhibition % =:

■ x 100%

where A is the absorbance with samples; while A0 is the absor-bance without samples.

2.11. ABTS+' radical-scavenging assay

were analyzed using Origin 6.0 professional software (Origin-Lab Corporation, Northampton, MA, USA). Determination of significant differences between the mean IC50 values of the sample and positive controls was performed using one-way ANOVA the T-test. The analysis was performed using SPSS software 13.0 (SPSS Inc., Chicago, IL) for windows. P < 0.05 was considered to be statistically significant.

3. Results and discussion

The ABTS +'-scavenging activity was measured as described [11] with some modifications. The ABTS + ' was produced by mixing 0.2 mL ABTS diammonium salt (7.4 mmol/L) with 0.2 mL potassium persulfate (2.6 mmol/L). 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. To determine the radical-scavenging activity, 1.2 mL aliquot of diluted ABTS + ' reagent was mixed with 0.3 mL of sample ethanolic solution (0.05-0.25 mg/mL). After incubation for 6 min, the absorbance at 734 nm was read on a spec-trophotometer (Unico 2100, Shanghai, China). The percentage inhibition was calculated as:

Inhibition %

Ao - A Ao

Here, A0 is the absorbance of the mixture without sample, A is the absorbance of the mixture with sample.

2.12. Cu2 + -reducing power assay

Cupric ion (Cu2+) reducing capacity was determined by the method [17], with minor modifications. Briefly, 125 iL CuSO4 aqueous solution (0.01 mol/L), 125 iL neocuproine ethanolic solution (7.5 mmol/L) and (750 - x) iL CH3COONH4 buffer solution (0.1 mol/L, pH 7.5) were brought to test tubes with different volumes of samples (3 mg/mL, x = 20-100 iL). Then, the total volume was adjusted to 1000 iL with the buffer and mixed vigorously. Absorbance against a buffer blank was recorded at 450 nm after 30 min (Unico 2100, Shanghai, China). The relative reducing power of the sample as compared with the maximum absorbance, was calculated by the formula:

Relative reducing power% =

where, Amax is the maximum absorbance at 450 nm and Amin is the minimum absorbance in the test. A is the absorbance of sample.

2.13. Statistical analysis

Each experiment was performed for three times, and the results were presented as mean ± standard deviations (SD). Based on the data of dose-response curve, the regression equation for calculating IC50 value was established, and the IC50 value was defined as the final concentration of 50% radical inhibition (relative reducing power, or chelating effect) in the study. Based on the calibration curve, the regression equation for calculating total contents of phenolics was established, and the correlation coefficient R was quoted. All linear regressions

It is well known that ROS are various forms of activated oxygen including free radicals and non-free-radical species, especially hydroxyl radical ('OH) and superoxide anion ('O2-). For example, the most reactive ROS form, hydroxyl radical ('OH), once generated via Fenton reaction in vivo (Eq. (1)), can oxidatively damage DNA to yield malondialdehyde (MDA) and a number of oxidative lesions (Supplemental file 4). These oxidative lesions along with ROS lead to apoptosis of cells especially stem cell [3,4].

Fe2+ + H2O2 ! 'OH + OH- + Fe3+ (1)

In the study, however, ECFI was observed to be able to protect MSCs against oxidative damage (Fig. 1).

Undoubtedly, the protective effect is assumed to be related to its protection on DNA. Therefore, we used our method to estimate its protective effect against hydroxyl-induced DNA damage. The data indicated that EFCI increased dose-depen-dently the protection percentages (Supplemental file 4). It means that EFCI can effectively protect against hydroxyl-induced DNA damage, and the IC50 value was observed to be 405.72 ± 4.08 ig/mL (Table 1). Since cancer and inflammation have been demonstrated to be closely related with the oxi-dative stress, the protective effect against oxidative damages to DNA and MSCs may be therefore responsible for the anti-cancer and anti-inflammation effects of CFI [18,19]. In addition, the fact that FCI can reduce the oxidative stress and protect against damage induced by ROS in cells, can also account for the effects of clearing away heat and toxicity in TCM, and may show promise as a new therapeutic reagent against oxidative damage in MSC transplantation or tissue engineering.

Figure 1 The protective effect of extracts from Flos Chrysanthemi Indici (ECFI) and chlorogenic acid against 'OH-induced damage to MSCs (mesenchymal stem cells) using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay. Each value is expressed as mean ± SD (n = 3). *P < 0.05 vs model.

Table 1 The IC50 values of EFCI (ig/mL). and positive controls

Assays EFCI Positive controls Catechin

DNA protecting 'O- scavenging Fe2+-chelating Cu2+-chelating DPPH' scavenging ABTS + ' scavenging Cu2+-reducing 405.72 ± 4.08b 58.74 ± 1.30b 364.27 ± 19.57b 734.77 ± 34.69b 46.46 ± 1.35b 20.59 ± 0.52b 35.69 ± 3.14b 192.07 ± 13.74a 7.12 ± 0.49*,a 3.21 ± 0.14 **,a 191.58 ± 2.10 **,a 4.66 ± 0.070a 0.92 ± 0.010a 6.22 ± 0.060a

IC50 value is defined as the concentration of 50% effect percentage and expressed as mean ± SD (n = 3). Means values with different superscripts in the same row are significantly different (one-way ANOVA, p < 0.05). The positive control is caffeic acid. The positive control is sodium citrate. EFCI, ethanol extract of Flos Chrysanthemi Indici.

rr ^ Iron ion ^

'O2 + H2O2 —o ' OH + OH + O2

(or Cu2+)

A number of phytochemical researches suggest that the antioxidant ability in plants can be attributed to the existence of total phenolics. In the Folin-Ciocalteu assay, EFCI exhibited a high amount of total phenolics (93.85 ± 3.56 mg cate-chin/g). As a phenolic compound, chlorogenic acid has also been found to be of substantial amount (10.05 ± 1.44 mg/g) in EFCI (Fig. 2). The high ratio of chlorogenic acid versus total phenolics (10.05:93.85) suggests that chlorogenic acid is one of main phenolic compounds. On the other hand, chlorogenic acid itself has been known as a good antioxidant [20], and presented a protective effect against MSCs oxidative damage in the study (Fig. 1). Thereby, the antioxidant ability of ECFI can be mainly attributed to the existence of chlorogenic acid. The assumption is further supported by the previous studies, in which chlorogenic acid has been considered as the functional component in Chrysanthemum [21].

Earlier investigators have pointed out that there are two approaches for natural phenolic antioxidant to protect against oxidative DNA damage: one approach is to scavenge ROS then to reduce their attack; another approach is to repair the deoxynucleotide radicals which are damaged by free radicals [22]. In order to further confirm whether the protective effect of EFCI against oxidative DNA damage was related to its ROS scavenging, we determined its 'O- radical scavenging ability.

As we know, superoxide anion ('O2-) is also regarded as an important form of ROS in living cells. Although 'O2- is much weaker than 'OH, however, it is able to directly attack DNA and lipids too [23], or transform into 'OH via Haber-Weiss reaction (Eq. (2)) to damage biomolecules (e.g., DNA) [24].

To obtain more reliable values, the 'O2--scavenging ability of EFCI was determined at physiological pH 7.4 [14]. As shown in Supplemental file 4, EFCI increased dose-dependently the 'O2- radical-scavenging percentages, and the IC50 value was 58.74 ± 1.30 ig/mL (Table 1). The fact that EFCI can effectively scavenge 'O- radical suggests ROS scavenging as one possible approach for EFCI to protect against oxidative DNA damage.

As shown in Eqs. (1) and (2), transition metals (especially Fe and Cu) can catalyze the generation of ROS (especially 'OH and 'O2- radicals). The metal-chelating ability of EFCI was thus explored in the study. The dose-response curves in Supplemental file 4 demonstrated an effective metal-chelating capacity of EFCI, and the IC50 values were calculated as 364.27 ± 19.57 and 734.77 ± 34.69 ig/mL, respectively for Fe2 + -chelating and Cu2 + -chelating (Table 1). It was previously reported that the chelating ability might mainly result from the existence of the ortho- or adjacent hydroxyl group (-OH) and carbonyl group (C=O) [11]. For example, chlorogenic acid occurring in FCI [25], may bind metal ions based on the proposed reaction (Fig. 3).

Undoubtedly, metal-chelating was regarded as one mechanism for EFCI to scavenge ROS.

To verify whether EFCI can directly scavenge free radicals, we further measured its radical-scavenging on DPPH' and ABTS' +. The assays revealed that EFCI can efficiently eliminate both DPPH' and ABTS'+ radicals (Supplemental file 4). Its IC50 values were respectively 46.46 ± 1.35 and 20.59 ± 0.52 ig/mL (Table 1). As we know, both DPPH' and ABTS'+ radicals could be yielded without transition metal-catalysis. On this basis, it can be induced that another mechanism for EFCI to scavenge ROS is direct radical-scavenging.

Taken together, one approach for EFCI to protect against oxidative damages to DNA or MSCs may be ROS scavenging, which would be mediated via metal-chelating and direct radical-scavenging.

Furthermore, the previous works suggested that DPPH' scavenging has been demonstrated to be a hydrogen atom (H') transfer process (HAT). In the process, DPPH' was thought to convert to DPPH-H molecule, and the antioxidant was assumed to donate a H' atom to form semi-quinone even quinone form [26]. For instance, chlorogenic acid, an antioxi-dant occurring in FCI [22,25], may scavenge DPPH' via the following proposed mechanism [27,28] (Fig. 4).

Unlike DPPH' radical, ABTS + ' radical cation however needs an electron (e) to neutralize the positive charge, and ABTS +' scavenging is considered an electron (e) transfer reaction [29]. For example, the proposed reaction for chlorogenic acid to scavenge ABTS + ' can be briefly illustrated by Fig. 5,

Figure 2 A typical HPLC profile of ethanol extract of Flos Chrysanthemi Indici (ECFI).

Figure 3 The proposed reaction of chlorogenic acid binding metal ions.

Homolysis

COOHA^wO^dpph.

✓"к 1Л

DPPH-H

Ortho-dihydroxyl moiety

Chlorogenic acid

COOH О'

OH (Il)Semi-quinone О

OH OH (IV)Quinone

( I JChlorogenic acid* О

DPPH« DPPH-H OH

ли I . .

(III) Semi-quinone <

Orffto-benzoquinone form

Figure 4 The proposed reaction for chlorogenic acid to scavenge DPPH" (1,1-diphenyl-2-picrylhydrazyl radical).

соон^

—Гон

Chlorogenic acid

ABTSt■

соон^Л.уу1

- он OH ( I (Chlorogenic acid

ABTS + H+

Figure 5 The proposed reaction of chlorogenic acid and ABTS + " (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid radical anion).

in which (I) can be further converted into semi-quinone (II) or quinone (IV).

The fact that EFCI can effectively scavenge ABTS +" radical, suggests that donating electron (e) may be another approach for EFCI to directly scavenge radicals. The donating electron (e) mechanism is further supported by the Cu2+-reducing power assay. The results in Supplemental file 4 showed that EFCI could dose-dependently reduce Fe3+ and Cu2 + , and the IC50 values were 147.11 ± 11.09 and 35.69 ± 3.14 ig/mL, respectively (Table 1). Obviously, the data indicate an effective reducing power of FCI. As we know, the reducing reaction is actually an electron (e) transfer process. Since e transfer is always accompanied by deprotonation, it is called sequential electron proton transfer (SEPT) [30].

4. Conclusions

In conclusion, Flos Chrysanthemi Indici can effectively protect against hydroxyl-induced damages to DNA and MSCs, therefore FCI shows promise as a possible therapeutic reagent for many diseases. The effect may be mainly attributed to pheno-lics (including chlorogenic acid), which exert the antioxidant action possibly through metal-chelating, and radical-scavenging which is via hydrogen atom transfer (HAT) and/or sequential electron proton transfer (SEPT) mechanisms.

Acknowledgement

This work was supported by the National Nature Science Foundation of China (81273896).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at http://dx.doi.Org/10.1016/j.jscs. 2014.06.004.

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