Effect and mechanism of wedelolactone as antioxidant-coumestan on 






•


OH




-treated mesenchymal stem cells

Xican Li; Tingting Wang; Jingjing Liu; Yulong Liu; Jun Zhang; Jian Lin; Zhongxiang Zhao; Dongfeng Chen

doi:10.1016/j.arabjc.2017.03.008

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Original article

13 (

); 184-192

doi:

10.1016/j.arabjc.2017.03.008

Effect and mechanism of wedelolactone as antioxidant-coumestan on $^{•} OH$ -treated mesenchymal stem cells

Xican Li^{^a,^b,⁎}, Tingting Wang^{^a,^b}, Jingjing Liu^{^a,^b}, Yulong Liu^{^a}, Jun Zhang^{^a}, Jian Lin^{^c}, Zhongxiang Zhao^{^a,^b}, Dongfeng Chen^{^c,^d,⁎}

a School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

b Innovative Research & Development Laboratory of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

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

d The Research Centre of Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

⁎Corresponding authors at: School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Waihuang East Road No. 232, Guangzhou Higher Education Mega Center, 510006 Guangzhou, China. Fax: +86 20 38892690. http://www.researchgate.net/profile/Xican_Li (Xican Li), lixican@126.com (Xican Li), CDF27212@21cn.com (Dongfeng Chen)

Received: 2016-6-10, Accepted: 2017-3-19, Published: 2017-01-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

The antioxidant properties of coumestans have been investigated previously using inappropriate methods, which has resulted in misleading and inaccurate mechanisms being reported to account for their antioxidant behavior. In this study, the phenolic coumestan wedelolactone increased the viability of $^{•} OH$ -treated mesenchymal stem cells (MSCs) in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl assay. Mechanistic analysis in vitro suggested that wedelolactone could scavenge various radicals, including $^{•} OH$ , $^{•} O_{2}^{-}$ , 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl ( ${DPPH}^{•}$ ) radicals, as well as reducing Cu²⁺ and chelating Fe²⁺. Configuration analysis based on a ball and stick model indicated that the 3′,4′-catechol moiety of wedelolactone was acting as an Fe²⁺-chelating site. The main product of the reaction between wedelolactone and ${DPPH}^{•}$ was analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry, which gave a mass ion with an m/z value of 709. This peak yielded several fragments with m/z values of 663, 543, 501 and 483. Quantitative evaluation revealed that the antioxidant activity of wedelolactone was 1.62 times higher than that of Trolox. Based on these results, we concluded that (i) wedelolactone can efficiently protect MSCs against $^{•} OH$ -induced damage and that this protective effect provides preliminary evidence for the application of wedelolactone in transplantation of MSCs (especially for osteoporosis); (ii) the main antioxidant mechanism of wedelolactone involves direct radical-scavenging via a single electron transfer (SET) → radical adduct formation (RAF) pathway, whereas the Fe²⁺-chelating activity of wedelolactone represents a minor pathway; and (iii) the direct radical-scavenging and Fe²⁺-chelating pathways can both be attributed to the catechol moiety rather than the coumestan skeleton.

Keywords

Wedelolactone

Phenolic coumestan

Mesenchymal stem cell

Antioxidant mechanism

SET → RAF pathway

•OH-induced damage

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1 Introduction

Some of the naturally occurring coumestans found in edible and medicinal plants have recently been reported as phytoestrogen or potent estrogen receptor antagonists, such as coumestrol and psoralidin (Wang et al., 2014; Liu et al., 2014a). Given that low levels of estrogen can result in osteoporosis (Strong et al., 2013), it could be possible to use coumestans for the treatment of this condition. Indeed, a coumestan wedelolactone (Fig. 1) has been shown to prevent bone destruction in osteoclastogenesis (Hsieh et al., 2015), as well as acting as a potential alternative therapy for the treatment of osteoporosis (Liu et al., 2014b; Shirwaikar et al., 2006). However, from the perspective of stem cell biology, the activity of this compound could be attributed to its beneficial effect on the proliferation of MSCs or its inhibitory activity toward the adipogenesis differentiation potential of MSCs (especially adipose tissue-derived MSCs) (Seyoung et al., 2012). During the proliferation and differentiation of MSCs, reactive oxygen species (ROS) have been demonstrated to play important roles. Excessive ROS can lead to cellular oxidative stress, which can hinder cell adhesion, induce cell detachment and even lead to cell death; in addition, excessive ROS can promote the differentiation of MSCs (especially bone marrow-derived MSCs) into adipose tissue (not bone). These processes can therefore limit the clinical application of MSC transplantation therapy (especially for osteoporosis) (Song et al., 2010; Chang et al., 2013).

As a coumestan, wedelolactone however contains a catechol moiety (i.e., its 3′,4′-dihydroxylbenzene group, Fig. 1) which has been reported to be effective scavengers of ROS. The phenolic characteristics of wedelolactone could therefore enable this compound to play a variety of different roles in the transplantation of MSCs for the treatment of osteoporosis, including (i) the repair of any ROS-induced damage to the MSCs; (ii) enhancing the viability of MSCs; and (iii) promoting the osteogenic differentiation of MSCs. However, there is currently a lack of evidence regarding the protective effects of wedelolactone against the ROS-induced damage of MSCs or wedelolactone’s mechanism of action.

In this study, we firstly used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay to explore the effects of wedelolactone on the MSCs induced by ^•OH radicals, which are an important form of ROS, and then used ultra-performance liquid chromatography electrospray ionization quadrupole-time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) to determine the antioxidant mechanism of wedelolactone in conjunction with several other chemical methods (including ^•OH radical-scavenging assay). It is noteworthy that Xi and Liu recently investigated the ^•OH radical-scavenging ability and mechanisms of action of seven synthetic coumestans. According to their experimental procedure, dimethyl sulfoxide (DMSO) solutions of the synthetic coumestans were measured directly without any pre-treatment process. DMSO however can rapidly scavenge ^•OH radicals at a rate of 9 × 10⁹ L mol⁻¹ s⁻¹ (Nakai et al., 2006), with ^•CH₃ radicals being produced as one of the major products (Chan et al., 1989). In an 800-µL reaction system, approximately 5.76 µL of DMSO would be able to scavenge 50% of the ^•OH radicals (Li, 2013). It is therefore clear that the use of DMSO in these experiments could result in considerable interference and that this solvent should be replaced with a suitable alternative or completely evaporated prior to performing any measurements (Li, 2013). The experimental protocol described by Xi and Liu is therefore unsuitable for the analysis of these compounds. Furthermore, the assertion that coumestans without any hydroxyl groups can act as antioxidants is incorrect (Xi and Liu, 2014). Therefore, it is necessary to reevaluate wedelolactone’s ^•OH radical-scavenging ability using an appropriate method, and then to explore the possible mechanisms.

Obviously, the study on phenolic coumestan wedelolactone will be of great significance. (i) Owing to the structural features and functional diversity of wedelolactone, the study will provide a novel candidate for the clinical application of MSC transplantation therapy (especially for osteoporosis); (ii) it will also give a satisfactory explanation of wedelolactone’s ^•OH radical-scavenging effect and correct the previous conclusion; (iii) in addition, it will provide available bioactive and mechanistic information of coumestans (especially phenolic coumestan) which have been poorly documented; and (iv) in our previous study, the Chinese herbal medicine Herba Ecliptae (i.e., the aerial part of Eclipta prostrata Linn. (Syn. Eclipta alba Linn.)) was observed to protect against ^•OH-induced damages to MSCs and wedelolactone was identified as its main active constituent by HPLC analysis (Han et al., 2014). However, there actually was no experimental evidence of wedelolactone itself. Therefore, the present study will be helpful for understanding the mechanisms responsible for the beneficial effects of H. Ecliptae.

2 Materials and methods

2.1

2.1 Mice test subjects and other chemicals

Four-week-old Sprague-Dawley (SD) rats (weighing 200–250 g) were obtained from the Animal Centre at the Guangzhou University of Chinese Medicine for the animal experiment. The protocol of this experiment was performed under the supervision of the Institutional Animal Ethics Committee in Guangzhou University of Chinese Medicine. Wedelolactone (C₁₆H₁₀O₇, M.W. 314, CAS: 519-34-6, purification >98%, Suppl. 1) was obtained from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT), (±)-6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic acid (Trolox), butylated hydroxyanisole (BHA), 2,9-dimethyl-1,10-phenanthroline hemihydrate (neocuproine), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•) and 3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-1,2,4-triazine disodium salt (Ferrozine) were obtained from Sigma-Aldrich (Shanghai, China). Deoxyribose and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS diammonium salt) were obtained from Amresco Inc. (Solon, OH, USA). 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). Methanol and water were purchased as the HPLC grade from Merck Ltd. (Darmstadt, Germany). FeCl₂·4H₂O, CH₃COONH₄, K₂S₂0₈ and all of the other reagents used in this study were purchased as the analytical grade from Guangzhou Chemical Reagent Factory (Guangzhou, China).

2.2

2.2 Protective effect of wedelolactone toward the ^•OH-induced damage of MSCs (MTT assay)

The MSCs were cultured according to a slightly modified version of the methods described in our previous report (Chen et al., 2007). Briefly, bone marrow samples were obtained from the femurs and tibias of rats, and the resulting samples were diluted with DMEM (LG: low glucose) containing 10% FBS. The MSCs were obtained by gradient centrifugation at 900g for 30 min on a 1.073 g/mL Percoll system. The cells were then detached by treatment with 0.25% trypsin and passaged into culture flasks at a density of 1 × 10⁴ cells/cm². The homogeneity of the MSCs was evaluated at passage 3 based on their CD44 expression by flow cytometry. These cells were then used for the following experiments.

These MSCs were seeded into 96-well plates (4 × 10³ cells/well). After adherence for 24 h, the cells were divided into three groups, including control, model and sample (wedelolactone) groups. The MSCs in the control group were incubated for 24 h in DMEM. The MSCs in the model group were injured for 1 h using FeCl₂ (100 μM) followed by H₂O₂ (50 μM). The resulting mixture of FeCl₂ and H₂O₂ was removed and the MSCs were incubated for 24 h in DMEM. The MSCs in the sample group were injured and incubated for 24 h in DMEM in the presence of various concentrations of wedelolactone (3.18, 31.8, 95.5, 159.1 and 318.2 μM). After being incubated, the cells were treated with 20 μL of MTT (5 mg/mL in PBS), and the resulting mixtures were incubated for 4 h. The culture medium was subsequently discarded and replaced with 150 μL of DMSO. The absorbance of each well was then measured at 490 nm using a Bio-Kinetics plate reader (PE-1420; Bio-Kinetics Corporation, Sioux Center, IA, USA). The serum medium was used for the control group and each sample test was repeated in five independent wells.

2.3

2.3 Antioxidant assays in vitro

2.3.1

2.3.1 Hydroxyl (^•OH) radical-scavenging assay of wedelolactone

The ^•OH radical-scavenging assay was performed according to the improved deoxyribose degradation method reported in our previous study (Li, 2013). Briefly, each sample was dissolved in ethanol at an appropriate concentration. Aliquots of the sample solutions were collected and placed in mini tubes and evaporated to dryness, before being treated with 10 μL of deoxyribose (50 mM), 20 μL of Na₂EDTA (1 mM), 10 μL of FeCl₃ (3.2 mM), 15 μL of H₂O₂ (50 mM) and 15 μL of ascorbic acid (1.8 mM). The total volume of each reaction mixture was adjusted to 130 μL with buffer and mixed thoroughly. After incubation at 50 °C for 20 min, each reaction was terminated by the addition of 50 μL of trichloroacetic acid (10%, w/w). The color was then developed by the addition of 30 μL of 2-thiobarbituric acid (5% in a 1.25% NaOH aqueous solution) and heated in an oven at 115 °C for 15 min. The mixture was cooled and its absorbance was measured at 530 nm using a Unico 2100 spectrophotometer (Shanghai, China) against the buffer (as a blank). The ^•OH radical scavenging activity was expressed as follows: $Inhibition % = \frac{A_{0} - A}{A_{0}} \times 100 % .$ where A₀ and A are the absorbance values of the blank and test samples, respectively.

2.3.2

2.3.2 Superoxide anion (^•O₂⁻) radical-scavenging ability of wedelolactone (pyrogallol autoxidation method)

The superoxide anion (^•O₂⁻)-scavenging activity was determined using a method previously developed in our laboratory (Li, 2012). Briefly, a 60–300 μL of sample solution (1 mg/mL) was added to Tris-HCl buffer (0.05 M, pH 7.4) containing Na₂EDTA (1 mM) and the total volume was adjusted to 980 μL using buffer. Twenty microliters of pyrogallol solution (60 mM in 1 mM HCl) was added to the sample, and the resulting mixture was vigorously agitated and read at 325 nm every 30 s for 5 min. The ^•O₂⁻ radical-scavenging ability was calculated as follows: $Inhibition % = \frac{(\frac{Δ A_{325 nm,control}}{T}) - (\frac{(Δ A_{325 nm,sample}}{T})}{(\frac{(Δ A_{325 nm,control}}{T})} \times 100 % .$ where ΔA_{325nm,control} is the increase in the A_325nm value of the mixture without the sample, ΔA_325nm,sample is the increase in the A_325nm value of the mixture with the sample and T is the determining time (5 min).

2.3.3

2.3.3 Colorimetry determination and ultraviolet (UV) spectra determination of Fe²⁺-chelating of wedelolactone

The Fe²⁺-chelating activity of wedelolactone was estimated using a colorimetric method (Gülçin, 2012). Briefly, a 0–100-μL aliquot of sample solution (1 mg/mL in methanol) was added to 100 μL of an aqueous FeCl₂·4H₂O solution (250 μM), and the resulting mixture was treated with 150 μL of aqueous ferrozine (1 mM) to initiate the reaction. The total volume of the system was then adjusted to 1000 μL with methanol, and the resulting mixture was vigorously shaken for 5 min before being allowed to stand at room temperature for 10 min. The absorbance of the solution was then recorded at 562 nm (Jinhua 754 PC, Shanghai, China). The percentage chelation activity was calculated using the formula described above in Section 2.3.

The wedelolactone-Fe²⁺ complex was evaluated by UV spectroscopy. For these experiments, 200 μL of a methanolic solution of wedelolactone (1 mg/mL) was added to 300 μL of an aqueous solution of FeCl₂·4H₂O (5 mg/mL). The resulting mixture was subjected to ultrasonic radiation before being centrifuged at 6500g for 10 min. The supernatant was collected and analyzed on an UV/Vis spectrophotometer (Jinhua 754 PC, Shanghai, China).

2.3.4

2.3.4 ABTS^•+ radical scavenging assay of wedelolactone

The ABTS^•+ scavenging activity was evaluated using a modified version of a previously reported method (Re et al., 1999). ABTS^•+ was produced by mixing 200 μL of ABTS diammonium salt (7.4 mM) with 200 μL of K₂S₂O₈ (2.6 mM). After being incubated in the dark for 12 h, the mixture was diluted with methanol (about 1:50, v/v) until its absorbance at 734 nm was 0.70 ± 0.02. The diluted ABTS^+• solution (800 μL) was then treated with 200 μL of ethanol containing different concentrations of wedelolactone and thoroughly mixed. The reaction mixture was then left to stand for 6 min and its absorbance was read at 734 nm on a spectrophotometer. The percentage inhibition was calculated using the formula described above in Section 2.3.1.

2.3.5

2.3.5 Cupric reducing antioxidant capacity (CUPRAC) assay of wedelolactone

A CUPRAC assay was carried out according to a previously reported method (Apak et al., 2004). Briefly, 125 μL of aqueous CuSO₄ (10 mM) was mixed with 125 μL of neocuproine solution (7.5 mM in CH₃OH), followed by solutions containing different concentrations of wedelolactone. The total volume of each mixture was then adjusted to 1000 μL with CH₃COONH₄ buffer and mixed vigorously. The absorbance of each solution was then measured against a buffer blank at 450 nm after 30 min. The relative Cu²⁺-reducing power was calculated using the following formula: $Relative reducing power % = \frac{A - A_{\min}}{A_{\max} - A_{\min}} \times 100 % .$ where A_max and A_min are respectively the maximum and minimum absorbance values in each test and A is the absorbance of the test sample.

2.3.6

2.3.6 DPPH^• radical scavenging assay of wedelolactone

The DPPH^• radical-scavenging ability was assessed according to the method (Sanchez et al., 1998). Briefly, 500 μL of a methanolic solution (at least five different concentrations were prepared) was mixed with 1000 μL of DPPH^• solution (100 μM in methanol, prepared daily). The mixture was shaken vigorously and left to stand for 30 min in the dark. The absorbance of the mixture was then measured at 519 nm against a blank. The DPPH^• radical-scavenging activity of each solution was calculated as a percentage inhibition, according to the equation described above in Section 2.3.1.

2.4

2.4 UPLC-ESI-Q-TOF-MS/MS analysis of reaction mixture between wedelolactone and DPPH^•

The analytical methods used in the current study were similar to those used in our previous study (Li et al., 2014). A solution of wedelolactone in methanol was mixed with a solution of DPPH^• radicals in methanol at a molar ratio of 1:2, and the resulting mixture was incubated for 24 h at room temperature. The product mixture was then filtered through a 0.22-μm filter and analyzed using a UPLC-ESI-Q-TOF-MS/MS system equipped with a C₁₈ column (2.0 mm i.d. × 100 mm, 2.2 μm, Shimadzu Co., Kyoto, Japan). The mobile phase was used for the elution of the system and consisted of a mixture of methanol (phase A) and water (phase B). The column was eluted at a flow rate of 0.3 mL/min with the following gradient elution program: 0–10 min, 60–100% A; 10–20 min, 100%A. The sample injection volume was set at 1 μL for the separation of the different components. Q-TOF-MS/MS analysis was performed on a Triple TOF 5600^plus Mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with an ESI source, which was run in the negative ionization mode. The scan range was set at 100–2000 Da. The system was run with the following parameter: ion spray voltage, −4500 V; ion source heater, 550 °C; curtain gas (CUR, N₂), 30 psi; nebulizing gas (GS1, Air), 50 psi; Tis gas (GS2, Air), 50 psi. The declustering potential (DP) was set at −100 V, whereas the collision energy (CE) was set at −40 V with a collision energy spread (CES) of 20 V.

2.5

2.5 Statistical analysis

All of the experiments described in the current study were performed in triplicate and the data reported as the mean values ± SD (standard deviation). The IC₅₀ value was defined as the concentration required for 50% radical inhibition (relative reducing power or chelating effect). Statistical comparisons were made by one-way ANOVA to detect significant differences using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) for windows. P values of <0.05 were considered to be statistically significant.

3 Results and discussion

Although the transplantation of MSCs has shown great promise for the treatment of various bone-related diseases (Gülçin et al., 2015), two major issues have prevented the clinical application of this technique in osteoporosis, including low osteogenic differentiation potential and poor viability. Both of these issues have been shown to be related to excessive ROS, which can not only oxidatively damage cells, but can also inhibit osteogenic differentiation (Song et al., 2010; Kim et al., 2010). Among the many forms of ROS reported in the literature, hydroxyl radicals (^•OH) are the most harmful. With this in mind, we have focused on the protective effects of wedelolactone against ^•OH-induced damage of MSCs. The results presented in Fig. 2 show that wedelolactone increased the viability of MSCs treated with Fenton reagent in a dose-dependent manner for concentrations in the range of 3.18–318.2 μM. Here the Fenton reagent (FeCl₂ plus H₂O₂) was used to generate ^•OH radicals. This result therefore indicates that wedelolactone could efficiently protect MSCs from ^•OH radical-mediated oxidative damage, and can partly explain the similar effects of H. Ecliptae toward MSCs in our previous study (Han et al., 2014).

The protective effect of wedelolactone against the Fenton reagent-induced damage to MSCs (mesenchymal stem cells) using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay. The Fenton reagent (FeCl2 plus H2O2) was used to generate • OH radicals. These data represent the mean ± SD (n = 3). *P < 0.05 vs model.

It is noteworthy that high levels of ROS-induced oxidative damage to cells (especially MSCs) can lead to various diseases, including cancer. The anticancer effects of wedelolactone are therefore believed to be linked to ability to protect MSCs from oxidative damage (Sarveswaran et al., 2012; Xu et al., 2014). Furthermore, we previously reported that chlorogenic acid exhibits similar effects to wedelolactone toward MSCs, although the effects of wedelolactone were much stronger than those of chlorogenic acid. This difference in the size of the effect could be partly attributed to the coumestan skeleton of wedelolactone, which was reported to promote stem cell proliferation (Nehybova et al., 2014). Taken together with its ability to promote osteogenic differentiation (Liu et al., 2014b; Hsieh et al., 2015), the protective effect of wedelolactone toward MSCs makes it a novel and effective therapeutic agent for the transplantation of MSCs for bone-related diseases (especially osteoporosis).

The ^•OH radical-scavenging ability of wedelolactone was also evaluated in terms of the level of thiobarbituric acid reactive substance (TBARS) to provide insight into its mechanism of action. The results of this analysis are shown in Fig. 3A and Table 1, and indicated that wedelolactone exhibited slightly lower ^•OH radical-scavenging activity compared with Trolox. This result was on the contrary to that of a previously reported study, which found that coumestans exhibited higher ^•OH radical-scavenging ability than Trolox (Xi and Liu, 2014). As mentioned above, however, the results of this particular study should be treated with caution because the samples were not pre-treated prior to their analysis and DMSO was used as a solvent. In fact, the results of this previous study contradict those of several other antioxidant assays. For example, the results of the ^•OH radical-scavenging assay reported by Xi and Liu suggested that the coumestans exhibited higher ^•OH radical-scavenging abilities than Trolox. Paradoxically, the same authors also reported that the coumestans exhibited much lower radical-scavenging abilities than Trolox in an ABTS^•⁺ assay. Furthermore, two coumestans with phenolic —OH groups exhibited much higher rate constants than those without phenolic —OH groups in the ABTS^•⁺, DPPH^• and galvinoxyl^• scavenging assays (Xi and Liu, 2014). Taken together, these results support our hypothesis that phenolic —OH groups play a key role in the antioxidant activity of phenolic coumestans. Our data may be helpful to explain the antioxidant effect of other coumestan in animal model (Chen et al., 2008), and predict the antioxidant levels of newly discovered natural coumestans or synthetic coumestans (Li et al., 2011; Tang et al., 2011).

Response curves for the ROS scavenging assays involving wedelolactone. (A) • OH OH scavenging assay; (B) • O 2 - scavenging assay. BHA, butylated hydroxyanisole. Each value represents the mean ± SD (n = 3).

Table 1 The IC₅₀ values of wedelolactone and the positive controls in various assays.

	Wedelolactone μg/mL (μM)	Positive controls		Ratio value
	Wedelolactone μg/mL (μM)	Trolox, μg/mL (μM)	BHA, μg/mL (μM)	Ratio value
$^{•} OH$ scavenging	399.4 ± 33.0(1270.9 ± 105.0^a)	289.8 ± 32.2(1157.9 ± 128.7^a)	355.1 ± 22.4(1969.9 ± 124.49^b)	0.91
$^{•} O_{2}^{-}$ scavenging	195.1 ± 0.6(620.9 ± 1.8^a)	511.1 ± 37.3(2041.7 ± 149.1^b)	450.9 ± 43.3(2501.4 ± 240.1^c)	3.29
Fe²⁺-chelating	47.3 ± 3.3 (150.4 ± 10.5^b)	2.6 ± 0.3(10.1 ± 1.1^a)	–	–
${ABTS}^{• +}$ scavenging	3.2 ± 0.1(10.1 ± 0.3^a)	2.6 ± 0.1(10.2 ± 0.2^a)	1.78 ± 0.06(9.9 ± 0.3^a)	1.02
Cu²⁺-reducing	8.7 ± 0.3(27.8 ± 1.0^a)	10.6 ± 0.2(42.2 ± 0.6^b)	4.4 ± 0.6(24.3 ± 3.3^a)	1.52
${DPPH}^{•}$ -scavenging	7.2 ± 0.2(22.9 ± 0.7^a)	7.8 ± 0.1(31.0 ± 0.4^b)	14.9 ± 0.1(82.6 ± 0.7^c)	1.35

Average				1.62

Note: IC₅₀ value is defined as the concentration of 50% effect percentage and calculated by linear regression analysis and expressed as Mean ± SD (n = 3). 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). BHA, butylated hydroxyanisole.

* Sodium citrate instead of Trolox. Ratio value is defined as IC_{50, Trolox}/IC_{50, wedelolatone}, based on μM unit.

The ^•O₂⁻ radical-scavenging ability of wedelolactone was also measured to evaluate its ability to scavenge other ROS. As shown in Fig. 3B, wedelolactone led to a dose-dependent increase in the level of ^•O₂⁻ radical scavenging. Notably, the IC₅₀ values determined in this experiment suggested that wedelolactone was a more effective ^•O₂⁻ radical scavenger than Trolox (Table 1). These results therefore clearly demonstrated that wedelolactone can be used as an efficient scavenger of ROS (including ^•OH and ^•O₂⁻), and that this ROS-scavenging (especially ^•OH-scavenging) activity could be one of the main mechanisms for its protective effects against the oxidative damage of MSCs.

The Fe²⁺-chelating ability of wedelolactone was determined to gain further insights into the mechanism responsible for its ROS-scavenging activity. Iron plays a key role in the intracellular generation of ROS (especially ^•OH and ^•O₂⁻ radicals) (Eqs. (1) and (2)), and iron overload has been reported to have an adverse impact on the microenvironment of bone marrow, including the quantity and quality of MSCs (Zhang et al., 2015).

(1)

Fenton reaction : {Fe}^{2 +} + H_{2} O_{2} \to {Fe}^{3 +} +^{•} OH + {OH}^{-}

(2)

Haber-Weiss reaction :^{•} O_{2}^{-} + H_{2} O_{2} \overset{Fe}{\to}^{•} OH + {OH}^{-} + O_{2}

As shown in Fig. 4A, wedelolactone chelated Fe²⁺ ions in a dose-dependent manner. The results of the UV analysis revealed that the green colored wedelolactone-Fe²⁺ complex gave two peaks at 470 and 800 nm, whereas colorless wedelolactone alone gave a single peak at 366 nm (Fig. 4B) (Suppl. 2). Based on these data and previous reports from the literature, where the chelation of a metal usually occurs from the ortho-hydroxyl groups (Torreggiani et al., 2005), we proposed that the Fe²⁺ ions were being chelated between the phenolic hydroxy groups of the catechol moiety on wedelolactone (Fig. 5A). As shown in the ball and stick model (Fig. 5B) and the related animation (Suppl. 3), the chelation of an Fe²⁺ ion to the catechol moiety of wedelolactone would result in the formation of a stable pentacyclic complex, which would expand the degree of aromatic conjugation, leading to considerable bathochromic shifts, i.e., λ_max 366 nm → 800 nm. However, the IC₅₀ values shown in Table 1 suggest that the Fe²⁺-chelating ability of wedelolactone is much lower than that of sodium citrate (a strong chelator), because it only has one chelating site (i.e., one 3′,4′-catechol moiety). It would not be possible for the site between the 4,5-positions of wedelolactone to form an Fe-complex, because of the rigidity of the furan ring. In a word, these results suggest that Fe²⁺-chelation could be a minor pathway in the ROS-scavenging activity of wedelolactone.

Dose response curve for the Fe2+-chelating activity of wedelolactone (A), and UV spectra for the chelation of Fe2+ by wedelolactone (B).

Proposed chemical structure (A) and ball and stick model (B) of the wedelolactone-Fe2+ complex.

Direct radical-scavenging in the absence of a metal catalyst can also take place at the stage. To test this possibility, we evaluated the radical-scavenging activity of wedelolactone toward ABTS^+•, which can be readily generated in the absence of a metal catalyst. As shown in Fig. 6A and Table 1, wedelolactone exhibited similar radical-scavenging activity to Trolox toward ABTS^+•. This result was in consistent with those of a previous study, where several coumestans exhibited ABTS^+• radical-scavenging activity that was proportional to the number of hydroxyl groups attached to the aromatic ring (Xi and Liu, 2014). This result also supports the aforementioned view that phenolic hydroxyl groups play a critical role in the antioxidant activity of coumestans. Our data also suggest that direct radical scavenging plays an important role in the ROS-scavenging activity of wedelolactone. It has been suggested that ABTS^+• scavenging proceeds via a single electron (e) transfer (SET) mechanism (Gülçin, 2012), thereby suggesting that the direct radical-scavenging activity of wedelolactone also proceeds via a SET pathway. This assumption was supported by the results of our Cu²⁺-reducing power assay, where wedelolactone exhibited similar reducing power to Trolox (Fig. 6B). Cu-reducing represents an actual electron transfer process. It is therefore highly likely that a SET pathway is involved in the ^•O₂⁻ radical-scavenging activity of wedelolactone (Slobodan et al., 1995).

The dose response curves for wedelolactone in the ABTS + • scavenging assay (A), Cu-reducing power assay (B) and DPPH • scavenging assay (C). BHA, butylated hydroxyanisole. Each value is expressed as mean ± SD (n = 3).

We also explored the DPPH^• radical-scavenging activity of wedelolactone using a colorimetric method and UPLC-ESI-Q-TOF-MS/MS analysis. As shown in Fig. 6C, wedelolactone acted in a dose-dependent manner in the DPPH^• radical-scavenging assay at concentration in the range of 2–10 μg/mL, giving an IC₅₀ value similar to that of Trolox (Table 1). This result therefore demonstrated that wedelolactone could scavenge DPPH^• radicals with high efficacy. The main product of the reaction between wedelolactone and the DPPH^• radical was separated and evaluated by UPLC-ESI-Q-TOF-MS/MS analysis. The total ion chromatograms of the product mixture are shown in Suppl. 4. The MS/MS spectrum of the major product is shown in Fig. 7. These data were further analyzed based on previously published results (Fig. 8A) (Ji and Wang, 1996; Yang et al., 2013).

MS/MS spectra of main product resulting from the reaction of wedelolactone with DPPH • .

Mass spectrometry analysis of the main product (A) and the proposed mechanism (B) for the reaction between wedelolactone and the DPPH • radicals. (Wedelolactone is oxidized to wedelolactone • radical by DPPH • radical through a SET pathway, and then bonds DPPH • radical via a RAF pathway to form a wedelolactone-DPPH adduct with m/z 709, 663, 543, 501 and 483 in mass spectrometry. It is called SET → RAF pathway. SET, single electron transfer; RAF, radical adduct formation).

Based on these results and previous results from the literature (Li et al., 2014; Gülçin, 2012; Galano and Alvarez, 2009; Iuga et al., 2011 ), we have proposed a plausible mechanism for the DPPH^• radical-scavenging activity of wedelolactone, which is shown in Fig. 8B.

We hypothesized that wedelolactone would be oxidized by DPPH^• during this process to afford a wedelolactone^• radical via a SET pathway. The wedelolactone^• radical would then proceed down a radical adduct formation (RAF) pathway, combining with DPPH^• to produce a wed-DPPH adduct, which would give rise to a peak with an m/z value of 709 (basic peak) by mass spectrometry. This pathway would therefore represent a SET → RAF pathway. It is noteworthy that (i) a SET pathway was proposed in this case rather than a HAT pathway because the MS data only gave an m/z value of 709 (no m/z 707); (ii) several other peaks were observed together with the basic ion peak (m/z 709) (Suppl. 4), which suggested that several other pathways were also available for wedelolactone to scavenge DPPH^•; (iii) fragments were observed with m/z values of 501 (89%) and 543 (10%), which indicated that the DPPH^• radial was bonding to the catechol or ortho-quinone moiety (i.e., the oxidized catechol moiety) rather than the coumestan skeleton; and (iv) the assumption that the benzofuran moiety could exhibit antioxidant activity in combination with the coumarin skeleton without any hydroxyl groups (Xi and Liu, 2014) is incorrect.

Finally, the ratio of the IC₅₀ values of Trolox and wedelolactone (IC_50,Trolox/IC_{50,wedelolactone}) was used to quantitatively evaluate the relative antioxidant level of wedelolactone. As shown in Table 1, the ratios for the ^•OH-scavenging^•O₂⁻ scavenging, ABTS^•+-scavenging, Cu²⁺-reducing and DPPH^•-scavenging activities were 0.91, 3.29, 1.02, 1.52 and 1.35, respectively. The average ratio was determined to be 1.62 (Table 1), which indicated that the antioxidant activity of wedelolactone is 1.62 times higher than that of the standard antioxidant Trolox. The results of our previous study showed that the IC₅₀ values of H. Ecliptae were generally much higher than those of the standard antioxidant Trolox, which indicated that H. Ecliptae was a weaker antioxidant than Trolox. The difference observed in the relative antioxidant levels between H. Ecliptae and wedelolactone therefore provides further evidence that wedelolactone is the main bioactive constituent in H. Ecliptae (Han et al., 2014).

4 Conclusions

Wedelolactone is a powerful coumestan antioxidant that can effectively protect MSCs against ^•OH-induced damage. The main antioxidant mechanism of wedelolactone appears to be direct radical-scavenging via a SET → RAF pathway, whereas Fe²⁺-chelation represents a minor pathway. Both of these pathways can be attributed to the catechol moiety of wedelolactone rather than its coumestan skeleton. Taken together with its phytoestrogen effect, the antioxidant protective effect of wedelolactone makes it a novel and effective therapeutic agent for the transplantation of MSCs for bone-related diseases, especially osteoporosis.

Acknowledgments

This work was supported by the National Nature Science Foundation of China (81573558, 81673770) and Guangdong Science and Technology Project 2016A050503039.

References

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Appendix A

Supplementary material

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2017.03.008.

Appendix A

Supplementary material

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[1] Apak R., Güçlü K., Özyürek M., Karademir S.E., . Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem.. 2004;52:7970-7981.
[Google Scholar]

[2] Chan J., Fujiwara T., Brennan P., McNeil M., Turco S.J., Sibille J.C., Snapper M., Aisen P., Bloom B.R., . Microbial glycolipids: possible virulence factors that scavenge oxygen radicals. Proc. Natl. Acad. Sci. U.S.A.. 1989;86:2453-2457.
[Google Scholar]

[3] Chang W., Song B.W., Moon J.Y., Cha M.J., Ham O., Lee S.Y., Choi E., Choi E., Hwang K.C., . Anti-death strategies against oxidative stress in grafted mesenchymal stem cells. Histol. Histopathol.. 2013;28:1529-1536.
[Google Scholar]

[4] Chen D.F., Zeng H.P., Du S.H., Li H., Zhou J.H., Li Y.W., Wang T.T., Hua X.C., . Extracts from plastrum testudinis promote proliferation of rat bone-marrow-derived mesenchymal stem cells. Cell Prolif.. 2007;40:196-212.
[Google Scholar]

[5] Chen Y., Wei X., Xie H., Deng H., . Antioxidant 2-phenylbenzofurans and a coumestan from Lespedeza virgata. J. Nat. Prod.. 2008;71:929-932.
[Google Scholar]

[6] Galano A., Alvarez J.R., . Guanosine + OH radical reaction in aqueous solution: a reinterpretation of the UV-vis data based on thermodynamic and kinetic calculations. Org. Lett.. 2009;11:5114-5117.
[Google Scholar]

[7] Gülçin G., Sema Y., Nejat G., Zehra S., Tijen U., . The therapeutic effects of resveratrol on MSCs cell-based strategies on degenerative bone disease. J. Biotechnol.. 2015;208:S35.
[Google Scholar]

[8] Gülçin I., . Antioxidant activity of food constituents: an overview. Arch. Toxicol.. 2012;86:345-391.
[Google Scholar]

[9] Han L., Sun C.J., Lin J., Li F., Li X.C., Wu Z.S., Liu Q.P., Bai Y., Mai W.Q., Chen D.F., . Herba Ecliptae protects against hydroxyl radical-induced damages to DNA and mesenchymal stem cells via antioxidant mechanism. J. Chin. Chem. Soc.. 2014;61:1161-1167.
[Google Scholar]

[10] Hsieh C.J., Kuo P.L., Hou M.F., Hung J.Y., Chang F.R., Hsu Y.C., Huang Y.F., Tsai E.M., Hsu Y.L., . Wedelolactone inhibits breast cancer-induced osteoclastogenesis by decreasing Akt/mTOR signaling. Int. J. Oncol.. 2015;46:555-562.
[Google Scholar]

[11] Iuga C., Alvarez-Idaboy J.R., Vivier-Bunge A., . ROS initiated oxidation of dopamine under oxidative stress conditions in aqueous and lipidic environments. J. Phys. Chem. B. 2011;115:12234-12246.
[Google Scholar]

[12] Ji X.S., Wang X.F., . Study on mass spectrum of coumarin derivatives. J. Chin. Mass Spectrom. Soc.. 1996;17:45-48.
[Google Scholar]

[13] Kim W.K., Meliton V., Bourquard N., Hahn T.J., Parhami F., . Hedgehog signaling and osteogenic differentiation in multipotent bone marrow stromal cells are inhibited by oxidative stress. J. Cell. Biochem.. 2010;111:1199-1209.
[Google Scholar]

[14] Li L., Deng X., Zhang L., Shu P., Qin M., . A new coumestan with immunosuppressive activities from Flemingia philippinensis. Fitoterapia. 2011;82:615-619.
[Google Scholar]

[15] Li X.C., . Improved pyrogallol autoxidation method: a reliable and cheap superoxide-scavenging assay suitable for all antioxidants. J. Agric. Food Chem.. 2012;60:6418-6424.
[Google Scholar]

[16] Li X.C., . Solvent effects and improvements in the deoxyribose degradation assay for hydroxyl radical-scavenging. Food Chem.. 2013;141:2083-2088.
[Google Scholar]

[17] Li X.C., Gao Y., Li F., Liang A., Xu Z., Bai Y., Mai W., Han L., Chen D., . Maclurin protects against hydroxyl radical-induced damages to mesenchymal stem cells: antioxidant evaluation and mechanistic insight. Chem. Biol. Interact.. 2014;219:221-228.
[Google Scholar]

[18] Liu X., Nam J.W., Song Y.S., Viswanath A.N., Pae A.N., Kil Y.S., Kim H.D., Park J.H., Seo E.K., Chang M.P., . Soralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia. Bioorg. Med. Chem. Lett.. 2014;24:1403-1406.
[Google Scholar]

[19] Liu Y.Q., Zhan L.B., Liu T., Cheng M.C., Liu X.Y., Xiao H.B., . Inhibitory effect of Ecliptae herba extract and its component wedelolactone on pre-osteoclastic proliferation and differentiation. J. Ethnopharmacol.. 2014;157:206-211.
[Google Scholar]

[20] Nakai K., Kadiiska M.B., Jiang J.J., Stadler K., Mason R.P., . Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin. Proc. Natl. Acad. Sci. U.S.A.. 2006;103:4616-4621.
[Google Scholar]

[21] Nehybova T., Smarda J., Benes P., . Plant coumestans: recent advances and future perspectives in cancer therapy. Anticancer Agents Med. Chem.. 2014;14:1351-1362.
[Google Scholar]

[22] Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C., . Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med.. 1999;26:1231-1237.
[Google Scholar]

[23] Sanchez M.C., Larrauri J.A., Saura C.F., . A procedure to measure the antiradical efficiency of polyphenols. J. Sci. Food Agric.. 1998;76:270-276.
[Google Scholar]

[24] Sarveswaran S., Gautam S.C., Ghosh J., . Wedelolactone, a medicinal plant-derived coumestan, induces caspase-dependent apoptosis in prostate cancer cells via downregulation of PKCε without inhibiting Akt. Int. J. Oncol.. 2012;41:2191-2199.
[Google Scholar]

[25] Seyoung L., Hyun J.J., Eun H.P., Jung K.K., Jung M.K., Eung K.K., Kyungmoo Y., Yun H.K., Whaseon L.K., Sung H.R., Pann G.S., . Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells. J. Cell. Biochem.. 2012;113:3436-3445.
[Google Scholar]

[26] Shirwaikar A., Prabhu R.G., Malini S., . Activity of Wedelia calendulacea less. In postmenopausal osteoporosis. Phytomedicine. 2006;13:43-48.
[Google Scholar]

[27] Slobodan V.J., Yukihiko H., Steen S., Michael G.S., . Antioxidant potential of gallocatechins. A pulse radiolysis and laser photolysis study. J. Am. Chem. SOC. 1995;117:9881-9888.
[Google Scholar]

[28] Song H., Cha M.J., Song B.W., Kim I.K., Chang W., Lim S., Choi E.J., Ham O., Lee S.Y., Hwang K.C., . Reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex. Stem Cells. 2010;28:555-563.
[Google Scholar]

[29] Strong A.L., Jiang Q., Zhang Q., Zheng S., Boue S.M., Elliott S., Burow M.E., Bunnell B.A., Wang G., . Design, synthesis, and osteogenic activity of Daidzein analogs on human mesenchymal stem cells. ACS Med. Chem. Lett.. 2013;5:143-148.
[Google Scholar]

[30] Tang L., Pang Y., Yan Q., Shi L., Huang J., Du Y., Zhao K., . Synthesis of coumestan derivatives via FeCl₃-mediated oxidative ring closure of 4-hydroxy coumarins. J. Org. Chem.. 2011;76:2744-2752.
[Google Scholar]

[31] Torreggiani A., Tamba M., Trinchero A., Bonora A., . Copper (II) – quercetin complexes in aqueous solutions: spectroscopic and kinetic properties. J. Mol. Struct.. 2005;744:759-766.
[Google Scholar]

[32] Wang C., Li C., Zhou H., Huang J., . High-throughput screening assays for estrogen receptor by using coumestrol, a natural fluorescence compound. J. Biomol. Screen. 2014;19:253-258.
[Google Scholar]

[33] Xi G.L., Liu Z.Q., . Coumestan inhibits radical-induced oxidation of DNA: is hydroxyl a necessary functional group? J. Agric. Food. Chem.. 2014;62:5636-5642.
[Google Scholar]

[34] Xu D., Lin T.H., Yeh C.R., Cheng M.A., Chen L.M., Chang C., Yeh S., . The wedelolactone derivative inhibits estrogen receptor-mediated breast, endometrial, and ovarian cancer cells growth. BioMed. Res. Int.. 2014;2014:713263.
[Google Scholar]

[35] Yang N., Ye X.L., Fan G.R., . Analysis of wedelolactone by ion trap mass spectrometry. Pharm. Care Res.. 2013;13:21-24.
[Google Scholar]

[36] Zhang Y.C., Zhai W.J., Zhao M.F., Li D.J., Chai X., Cao X.L., Meng J.X., Chen J., Xiao X., Li Q., Shen J.C., Meng A., . Effects of iron overload on the bone marrow microenvironment in mice. PLoS One. 2015;10:e0120219.
[Google Scholar]

Effect and mechanism of wedelolactone as antioxidant-coumestan on $^{•} OH$ -treated mesenchymal stem cells

Abstract