Part 2:Antioxidation And Cytoprotection Of Acteoside And Its Derivatives: Comparison And Mechanistic Chemistry

Xican Li 1,2,*,† ID, Yulu Xie 1,2,†, Ke Li 3,4, Aizhi Wu 1,2,*, Hong Xie 1,2, Qian Guo 1,5 Penghui Xue 1, Yerkingul Maleshibek 1, Wei Zhao 6, Jiasong Guo 7 and Dongfeng Chen 3,

Contact: joanna.jia@wecistanche.com

1 School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China; xieyulu1900@163.com (Y.X.); xiehongxh1@163.com (H.X.); 15622178307@163.com (Q.G.); 15228738137@163.com (P.X.); pandiphd@163.com (Y.M.)

2 Innovative Research & Development Laboratory of TCM, Guangzhou University of Chinese Medicine,

Guangzhou 510006, China

3 School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China; ys1090992678@163.com (K.L.); chen888@gzucm.edu.cn (D.C.)

4 The Research Center of Basic Integrative Medicine, Guangzhou University of Chinese Medicine,

Guangzhou 510006, China

5 School of Basic Medical Science, Guangdong Pharmaceutical University, Guangzhou 510007, China

6 Zhongshan School of Medicine, Sun Yat-sen University, No. 74 Zhongshan Road. 2, Guangzhou 510080, China;

7 Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China;

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4.3. PTIO·-Scavenging Assay

The PTIO·-scavenging assays (at pH 4.5 or pH 7.4) were conducted based on our method [16]. In brief, the test sample solution (x = 0–20 βL, 1 mg/mL for pH 4.5 and 0.5 mg/mL for pH 7.4) was added to (20 . x) βL of 95% ethanol, followed by 80 βL of an aqueous PTIO· solution.The aqueous PTIO· solution was prepared using a phosphate-butter solution (0.1 mM, pH 4.5 or pH 7.4). The mixture was maintained at 37℃ for 2 h, and the absorbance was then measured at 560 nm using the aforementioned microplate reader. The PTIO· inhibition percentage was calculated as follows:

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

4.4. ABTS+·-Scavenging and DPPH·-Scavenging Assays

The ABTS·+-scavenging activity was evaluated according to the method [37]. The ABTS+· was produced by mixing 0.2 mL of ABTS diammonium salt (7.4 mmol/L) with 0.2 mL of potassium persulfate (2.6 mmol/L). The mixture was kept in the dark at room temperature for 12 h to allow completion of the radical generation before being diluted with distilled water (at a ratio of approximately 1:20) so that its absorbance at 734 nm was 0.35 o 0.01 using the aforementioned microplate reader. To determine the scavenging activity, the test sample (x = 4–20 βL, 0.05 mg/mL) was added to (20 . x) βL of distilled water followed by 80 βL of ABTS+· reagent, and the absorbance at 734 nm was measured 3 min after initial mixing, using distilled water as the blank.

DPPH· radical-scavenging activity was determined as previously described [18]. Briefly, 75 βL of DPPH· solution (0.1βM) was mixed with the indicated concentrations of the sample (0.025 mg/mL, 5–25 βL) dissolved in methanol. The mixture was maintained at room temperature for 30 min, and the absorbance was measured at 519 nm using the aforementioned microplate reader. The percentages of ABTS+·-scavenging activity and DPPH·-scavenging activity were calculated based on the formula presented in Section 4.3.

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4.5. UPLC.ESI.Q.TOF.MS/MS Analysis of DPPH· Reaction Products

This method was based on our previous study [25]. The methanol solution of acteoside 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-βm filter and analyzed using a UPLC system equipped with a C18 column (2.0 mm i.d. 土 100 mm, 1.6 βm, Phenomenex, Torrance, CA, USA). 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; 10–15 min, 100%A. The sample injection volume was set at 1 βL for the separation of the different components. ESI-Q-TOF-MS/MS analysis was performed using 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 parameters: ion spray voltage, .4500 V; ion source heater, 550℃; 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. The RAF products were quantified by extracting the corresponding molecular formula from the total ion chromatogram (Suppl. 2).

The aforementioned experiment was repeated using forsythoside B, poliumoside, and caffeic acid. The corresponding m/z peaks were extracted from the corresponding molecular formula from the total ion chromatogram (Suppl. 2).

4.6. UV-Vis-Spectra Analysis of Fe2+-Chelating Products

The Fe2+-chelating reaction products of acteoside-Fe2+ were evaluated using UV-Vis-spectroscopy [13]. For the experiment, 300 βL of a methanolic solution of acteoside (0.24 mM) was added to 700 βL of an aqueous solution of FeCl2×4H2O (168 mM). The solution was then mixed vigorously. Subsequently, the resulting mixture was scanned using a UV-Vis spectrophotometer after an hour (Unico 2600A, Shanghai, China) from 200–850 nm. The aforementioned experiment was repeated using forsythoside B, or poliumoside, instead of acteoside.

4.7. Pyrogallol Autooxidation Assay for Superoxide Anion (·O2.) Scavenging

Measurement of superoxide anion (·O2.) scavenging activity was based on our method [17]. Briefly, the sample was dissolved in ethanol at 1 mg/mL. The sample solution (x βL) was mixed with Tris-HCl buffer (980 . x βL, 0.05 M, pH 7.4) containing EDTA (1 mM). When 20 βL pyrogallols (60 mM in 1 mM HCl) were 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 a blank every 30 s for 5 min. The ·O2. scavenging ability was calculated as follows:

Here, △A325nm, control is the increment in A325nm of the mixture without the sample, and △A325nm, the sample is the increment in A325nm of the mixture with the sample; T = 5 min. The experimental temperature was 37 。C.

4.8. Cytoprotective Effect Towards Oxidatively Stressed bmMSCs (MTT Assay)

The bmMSCs were cultured according to our previous reports [38] with slight modifications. In brief, bone marrow was obtained from the femur and tibia of a rat. The marrow samples were diluted with DMEM (low glucose) containing 10% FBS. The bmMSCs were prepared by gradient centrifugation at 900 g for 30 min at 1.073 g/mL Percoll. The prepared cells were detached by treatment with 0.25% trypsin and passed into cultural flasks at 1 土 104/cm2. The bmMSCs at passage 3 were evaluated for cultured cell homogeneity using detection of CD44 using MTT assay [39].

The MTT assay was used to evaluate the cytoprotective effect of acteoside and its derivatives towards bmMSCs [40]. The injury model was established based on the previous study [41]. The experimental protocol is briefly illustrated in Figure 3.

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4.9. Statistical Analysis

Each experiment in Sections 4.2–4.4 and 4.7 were performed in triplicate, and the MTT assay experiment was performed in pentaplicate. Data were recorded as the mean o SD (standard deviation). The dose-response curves were plotted using Origin 6.0 professional software (OriginLab, Northampton, MA, USA). The IC50 value was defined as the final concentration of 50% radical inhibition (relative reducing power) [42]. Statistical comparisons were made by one-way ANOVA to detect significant differences using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) for Windows. p < 0.05 was considered to be statistically significant.

5. Conclusions

Three natural phenylpropanoid glycosides, namely, acteoside, forsythoside B, and poliumoside, can be involved in multiple pathways to exert antioxidant action, including ET, H+-transfer, and Fe2+-chelating, but not RAF. The ET and H+-transfer pathways may be hindered by rhamnosyl moiety or apiosyl moiety; however, the Fe2+-chelating pathway can be enhanced by sugar residues (especially rhamnosyl moiety). The general effect of rhamnosyl moiety or apiosyl moiety is to enhance multiple-pathway-involved ROS-scavenging ability. Thus, forsythoside B and poliumoside are superior to acteoside in cytoprotective effects.

Supplementary Materials: Supplementary materials are available online. 1. Dose-response curves; 2. HPLC-MS spectra; 3. Analysis certificates of acteoside, forsythoside B, and poliumoside.

Acknowledgments: This work was supported by the National Science Foundation of China (81573558, 81603269), Guangdong Science and Technology Project (2017A050506043), and Natural Science Foundation of Guangdong Province (2017A030312009, 2015A030310491).

Author Contributions: Xican Li and Dongfeng Chen conceived and designed the experiments; Aizhi Wu prepared poliumoside; Yulu Xie, Qian Guo, and Penghui Xue performed the antioxidant experiments; Ke Li and Wei Zhao performed the MTT experiments; Hong Xie analyzed the data; Jiasong Guo conducted the experiment of Figure 2D; Xican Li wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ABTS+· 2,2→-and-bis (3-ethylbenzene-thiazoline-6-sulfonic acid) radical bmMSCs bone marrow-derived mesenchymal stem cells

CUPRAC cupric reducing antioxidant capacity

dAMP 2’-deoxyadenosine-5’-monophosphate radical DMEM Dulbecco’s modified Eagle’s medium

dGMP 2’-deoxyguanosine-5’-monophosphate radical DPPH· 1,1-diphenyl-2-picryl-hydrazl radical

ET electron transfer; FBS: Fetal bovine serum

FRAP ferric ion reducing antioxidant power; HAT hydrogen atom transfer

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl PCET proton-coupled electron transfer

PTIO· 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical RAF radical adduct formation

ROS reactive oxygen species

SCNT somatic cell nuclear transfer

SEPT sequential electron-proton transfer

SPLET sequential proton loss single electron transfer TPTZ 2,4,6-tripyridyl triazine

Trolox (o)-6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic acid

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