PART 1 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, Aichi 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 3,4

1 School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China;

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;

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;

Received: 24 January 2018; Accepted: 20 February 2018; Published: 23 February 2018

Abstract: The study tried to explore the role of sugar-residues and mechanisms of phenolic phenylpropanoid antioxidants. Acteoside, along with its opposite forsythoside B and rhamnoside podium side, were comparatively investigated using various antioxidant assays. In three electrontransfer (ET)-based assays (FRAP, CUPRAC, PTIO*-scavenging at pH 4.5), the relative antioxidant levels roughly ruled as acteoside >forsythoside B > poliumoside. Such order was also observed in H⁺-transfer-involved PTIO*-scavenging assay at pH 7.4, and in three multiple-pathway-involved radical-scavenging assays, i.e., ABTS+ •-scavenging, DPPH• -scavenging, and • O2 - -scavenging. In UV-vis spectra, each of them displayed a red-shift at 335—364 nm and two weak peaks (480 and 719 nm), when mixed with Fe²⁺; however, acteoside gave the weakest absorption. In Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC—ESI—Q-TOF—MS/MS) analysis, no radical adduct-formation (RAF) peak was found. MTT assay revealed that poliumoside exhibited the highest viability of oxidative-stressed bone marrow-derived mesenchymal stem cells. In conclusion, acteoside, forsythoside B, and poliumoside may be involved in multiple pathways to exert the antioxidant action, including ET, H⁺-transfer, or Fe²⁺-chelating, but not RAF. The ET and H+ transfer may be hindered by rhamnosyl and apiosyl moieties; however, the Fe²⁺-chelating potential can be enhanced by two sugar-residues (especially rhamnosyl moiety). The general effect of rhamnosyl and apiosyl moieties is to improve the antioxidant or cytoprotective effects.

Keywords: acteoside; apiosyl; forsythoside B; phenylpropanoid glycosides; poliumoside; rhamnosyl

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1. Introduction

Recently, acteoside (verbascoside, Figure1, a phenolic phenylpropanoid glycoside occurring in vervain and other plants, was found to improve the efficiency of canine somatic cell nuclear transfer (SCNT) during the dog-cloning process . In addition, acteoside could protect human neuroblastoma SH-SY5Y cells against p-amyloid- induced damage and human skin fibroblasts against X-ray-induced damage. Its apposite forsythoside B (Figure 1) is also distributed in plants and has been observed to exert potent neuroprotective effects with a favorable therapeutic time window. These beneficial effects on cells and tissues are thought to be associated with the protection of some biomolecules, such as lipids and DNA. In fact, acteoside and its analog czstanoside F have been demonstrated to inhibit mitochondriel lipid peroxidation in rats. Lipid peroxidation is known to be a result of cellular oxidative stress and ultimately from reactive oxygen species (ROS).

From a DNA protection aspect, acteoside and its derivatives can quickly repair DNA radicals, such as 2'-deoxyadenosine-5'-monophosphate (dAMP) and 2'-deoxyguanosine-5'-monophosphate (dGMP) [8]. These secondary deoxynucleotide radicals can further oxidatively damage cells leading to mutagenesis and carcinogenesis [9]. The biophysical study indicated that acteoside and its derivatives could dock into DNA minor grooves and quickly repair such oxidative DNA damage [10]. The preferential-conformation-based ball-stick model proposes (Figure1, right) that these molecules are prolate and can easily reach the loop of DNA, and thus, they are able to quickly repair the damage of DNA radicals [8].

However, from the viewpoint of biochemistry, the repair is essentially fulfilled via a ROS-scavenging (i.e., antioxidant) pathway. As previously mentioned, the generation of deoxynucleotide radical nations are ultimately from the attack of ROS, including free radicals (e.g.,・OH radical 11) and oxidant molecules (eg, H2O2 [12]). ROS-scavenging is thought to efficiently lower cellular oxidative status [13,14] and improve the quality of stem cells [12,15]. Therefore, it is necessary to study their antioxidant ability and further discuss possible mechanisms.

In this study, three phenolic phenylpropanoid glycosides (acteoside, forsythoside B, and poliumoside) were comparatively investigated using various antioxidant assays, including the 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO・)scavenging assay, ferric ion reducing antioxidant power (FRAP) assay, cupric reducing antioxidant capacity (CUPRAC) assay, 2,2‘-casino-bis(3-ethylbenzene-thiazoline-6-sulfonic acid radical (ABTS+ •) scavenging assay, 1,1-diphenyl-2-picryl-hydrazine (DPPH・)assay, and ・。2 --scavenging assay as well as Fe²⁺-chelating UV-spectra analysis. Subsequently, their reaction products with DPPH・ were determined using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC—ESI—Q-TOF—MS/MS) analysis. Finally, their cytoprotective effect towards bone marrow-derived mesenchymal stem cells (bmMSCs) was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. As seen in Figure 1, the difference among the three phenolic phenylpropanoid glycosides (i.e., acteoside, forsythoside B, and poliumoside) is the glycoside type: forsythoside B is an apposite of acteoside, while poliumoside is a rhamnoside of acteoside. Thus, this comparative study also helps in understanding the roles of apiosyl and rhamnosyl in antioxidant effects.

2. Results

2.1. Metal-Reducing Assays (FRAP & CUPRAC)

In the study, we carried out two metal-reducing assays, i.e., FRAP assay and CUPRAC assay. As illustrated in Suppl. 1, acteoside and its derivatives increased the relative FRAP (or Cu²⁺-reducing power) percentages at 0-10 昭/L in a dose-dependent manner. Their order of relative metal-reducing levels roughly decreased in the order of acteoside > forsythoside B > poliumoside (Table 1).

2.2. PTIO・-ScavengingAssay

The PTIO*-scavenging assay was recently developed by our laboratory [16]. It can be used to evaluate the antioxidant levels and to explore the antioxidant pathways. As shown in Suppl. 1, acteoside and its derivatives could successfully scavenge PTIO* at pH 4.5 and pH 7.4. However, according to the IC50 values in Table 1, their relative PTIO*-scavenging levels at the same pH value were different from each other. Also, each acteoside and its derivatives exhibited different PTIO• -scavenging levels between pH 4.5 and pH 7.4. In general, the PTIO^-scavenging levels at 7.4 were higher than those at pH 4.5.

2.3. ABTS+9-Scavenging and DPPH・-Scavenging Assays

The ABTS⁺—scavenging, and DPPH—scavenging are now widely used for the antioxidant studies of pure compounds or extracts. As seen in Suppl. 1, acteoside and its derivatives concentration-dependently increased the ABTS+ —scavenging or DPPH・-scavenging percentages. However, in terms of the IC50 values in Table 1, their relative ABTS+ ^-scavenging levels were found in the order of acteoside > forsythoside B > poliumoside > Trolox. A similar order could also be observed in the DPPH—scavenging assay.

2.4. UPLC-E SI—Q-TOF—MS/MS Analysis of DPP H Reaction Products

The reaction product of DPPH・ with each of three phenylpropanoid glycosides was explored using UPLC—ESI—Q-TOF—MS/MS analysis. In the analysis (Suppl. 2), none of the three phenylpropanoid glycosides produced the RAF product peak. By comparison, caffeic acid was found to yield a dimer product (m/z 359-360, Suppl. 2).

2.5. UV-Vis-Spectra Analysis ofFe²⁺-Chelating Products

Since UV-vis-spectra can characterize metal complexes, the study thus used UV-vis-spectra to analyze the possible Fe²⁺-chelating reaction with three phenylpropanoid glycosides. As shown in Figure 2, each of them could produce a bathochromic shift (335 nmT364 nm) in the UV spectra. Meanwhile, each of them could provide two weak vis-spectra peaks in the visible spectra (480 nm and 719 nm) and lead to a light green appearance. However, the peak intensity was in descending order of poliumoside > forsythoside B > acteoside.

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2.6. Pyrogallol Autooxidation Assay for Superoxide Anion (•O2~) Scavenging

The pyrogallol autooxidation assay was improved by our laboratory in 2012 [17]. It was used to estimate the ・O2--scavenging potential in the present study. As seen in Suppl. 1, all of acteoside and its derivatives could dose-dependently increase the --scavenging percentages. However, the relative bioactivity decreased in the order of poliumoside > forsythoside B > acteoside, according to the IC50 values in Table1.

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2.7. Cytoprotective Effect towards Oxidatively Stressed bmMSCs (MTT Assay)

To assess the cytoprotective effects of acteoside and its derivatives, we performed an MTT assay. In the assay, bmMSCs were damaged by oxidative reagent H₂O₂, the damaged bmMSCs were then treated by acteoside and its derivatives. The A_490nm values were used to assess the relative cytoprotective effects. As seen in Table 2, each acteoside and its derivatives dose-dependently increased the A_490nm values. However, at the same concentration, poliumoside gave the highest A490nm values.