DPPH-HPLC-MS assisted rapid identification of endothelial protective substances from Xiao-Ke-An

doi:10.1016/j.jep.2017.07.016

Journal of Ethnopharmacology

Volume 211, 30 January 2018, Pages 188-196

https://doi.org/10.1016/j.jep.2017.07.016 Get rights and content

Abstract

Ethnopharmacological relevance

Xiao-Ke-An formula (XKA) is a Chinese medicine widely used for treating diabetes and associated complications. Endothelial protection is thought to be one of its therapeutic mechanisms. However, the protective effect of XKA on endothelial cells remains unclear, especially in oxidative injury induced endothelial dysfunction.

Materials and Methods

A novel strategy to rapidly screen and identify potential endothelial protective substances from XKA was established by combining the 2,2′-diphenyl-1-picrylhydrazyl high performance liquid chromatography coupled with mass spectrometry (DPPH-HPLC-MS) approach with cell-based verification. Firstly, the DPPH-HPLC-MS approach was employed to identify the antioxidants in XKA. Then, the potent endothelial protective effect of XKA, and the potential active substances and mechanism of action were revealed in EA.hy926 cells injured by tert-butyl hydroperoxide (t-BHP).

Results

XKA exhibited potent antioxidant activity and endothelial protective effect. Phenolic acids derived from Salvia miltiorrhiza Bunge, root and rhizome, xanthones from Anemarrhena asphodeloides Bunge, rhizome, and acteoside from Rehmannia glutinosa (Gaertn.) DC., root, were identified as the major endothelial protective components in XKA.

Conclusions

An efficient method for rapid identifying endothelial protective substances from complex mixtures was developed and used to identify the major endothelial protective components in XKA. This method would help reveal the material base of herbal medicine with endothelial protective effect, and could also be applied to discover novel natural-origin endothelial protective substances.

Graphical abstract

Introduction

Vascular endothelium is a continuous monolayer of cells lining the luminal surface of blood vessels, which plays an important role in vascular homeostasis (Tang et al., 2014). Increasing evidence suggests that oxidant stress induced endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases, e.g., atherosclerosis, diabetes and its long-term complications (Fatehi-Hassanabad et al., 2010, Malakul et al., 2011). Microvascular and macrovascular complications, including retinopathy, nephropathy, neuropathy and atherosclerosis, are the principal causes of death and disability in patients with diabetes (Creager et al., 2003). Hyperglycemia, dyslipidemia and insulin resistance induce excessive generation of reactive oxygen species (ROS) within endothelial cells, which leads to oxidative stress and cellular injury (Minuz et al., 2006, Spitaler and Graier, 2002). Antioxidant therapy exhibits the endothelial protective effect by preventing oxidative stress (Rosa et al., 2010). Protection of endothelial cells against ROS is a beneficial strategy for intervention in endothelial dysfunction.

The conventional method to discover endothelial protective substances from complex mixtures is time-consuming and labor intensive, which usually includes fractionation guided by endothelial cell assay, purification, and structure identification. For example, Zhou et al. (Zhou et al., 2013) studied the endothelial protective substances from Platycladus orientalis (L.) Franco, branch and leaf, with a lipopolysaccharide induced human umbilical vein endothelial cell damage model, and found that ethyl acetate extract was the main effective fraction while quercitrin and myricitrin were the corresponding effective substances. To the best of our knowledge, there is no existing method for rapidly identifying endothelial protective substances from complex mixtures.

Xiao-Ke-An formula (XKA) is a Chinese medicine widely used for treating diabetes (Nan et al., 2005). It includes Rehmannia glutinosa (Gaertn.) DC., root, Anemarrhena asphodeloides Bunge, rhizome, Coptis chinensis Franch., rhizome, Lycium chinense Mill., root bark, Lycium barbarum L., fructus, Polygonatum odoratum (Mill.) Druce, rhizome, Panax ginseng C.A.Mey., root and rhizome, and Salvia miltiorrhiza Bunge, root and rhizome, with a weight ratio of 6:5:2:4:2:3:2:3. Our previous studies have indicated that XKA could regulate critical targets involved in atherosclerosis (Yang et al., 2015a) and lower the atherogenic index (Yang et al., 2015b). Therefore, XKA might have the potential to attenuate or delay the occurrence of diabetes complications. However, the protective effect of XKA on endothelial cells is unclear, especially in oxidative injury induced endothelial dysfunction.

In this study, a strategy to rapidly screen and identify potential endothelial protective substances from complex mixtures was established as illustrated in Fig. 1, and XKA was used as a case study to investigate its ingredients of endothelial protection from the antioxidant perspective. A 2,2′-diphenyl-1-picrylhydrazyl high performance liquid chromatography coupled with mass spectrometry (DPPH-HPLC-MS) approach (Zhang et al., 2012) was adopted for the rapid screening and identification of antioxidant compounds from XKA. The screening results were verified by the conventional DPPH radical scavenging assay. The antioxidants discovered, treated as candidates of endothelial protective substances, were further investigated for their protective effects on tert-butyl hydroperoxide (t-BHP)-induced cellular injury in human endothelium-derived EA.hy926 cells. In total, 8 compounds with antioxidant effect were discovered from XKA, among which phenolic acids derived from Salvia miltiorrhiza Bunge, root and rhizome, xanthones derived from Anemarrhena asphodeloides Bunge, rhizome, and acteoside derived from Rehmannia glutinosa (Gaertn.) DC., root were assumed as main endothelial protective substances in XKA. These compounds might be the major effective substances for prevention of diabetic vascular complications.

Section snippets

Materials

EA.hy926 cells were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Corning (USA). Fetal bovine serum (FBS), penicillin, streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco (NY, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), t-BHP and vitamin C were obtained from Sigma (St. Louis, MO, USA). Total superoxide dismutase (SOD) assay kit and BCA protein assay kit,

Antioxidant activity of XKA

To investigate the antioxidant activity of XKA, the reactivity toward the free radical DPPH was measured at 510 nm. XKA exhibited potent activity of scavenging DPPH radical with the IC₅₀ value of 124.5 ± 2.6 μg/mL. The dose-response curve of XKA extract against DPPH was shown in Fig. 2.

Identification of constituents in XKA

A practical method was adopted to characterize the constituents in XKA by the combinatorial analysis of HPLC-MSⁿ and HR-MS data (Li et al., 2015, Shi et al., 2011). HR-MS data provided the accurate molecular weight

Conclusions

In the present study, a rapidly screening strategy for potential endothelial protective substances from complex mixtures was established based on DPPH-HPLC-MS analysis sequenced by cell-based verification. The DPPH-HPLC-MS approach identified the antioxidants in XKA, which narrowed down the number of candidates for further research. Through the study with t-BHP induced injury in EA.hy926 cells, the potent endothelial protective effect of XKA, the potential effective substances and mechanism of

Author contributions

Zhenzhong Yang and Zheng Li conceived and designed the experiments. Zhenzhong Yang carried out the experiments and wrote the manuscript. Zhenzhong Yang and Zhenhao Li analyzed the data. Zheng Li reviewed the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by Zhejiang TCM Funds for Outstanding Young Talent (No. 2016ZQ024) and National Natural Science Foundation of China (No. 81603268). The authors sincerely thank Feng Zhang for her assistance with the cellular experiments.

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DPPH-HPLC-MS assisted rapid identification of endothelial protective substances from Xiao-Ke-An

Abstract

Ethnopharmacological relevance

Materials and Methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Materials

Antioxidant activity of XKA

Identification of constituents in XKA

Conclusions

Author contributions

Conflict of interest

Acknowledgements

Eur. J. Pharmacol.

Food Chem.

J. Chromatogr. A

J. Ethnopharmacol.

Anal. Chim. Acta

Food Chem.

Biochem. Pharmacol.

Diabetes and vascular disease pathophysiology, clinical consequences, and medical therapy: part I

Circulation

Oxidative stress, antioxidants, and vascular damage

Br. J. Clin. Pharmacol.