Investigation of the in vivo metabolism of harpagoside and
distribution of its metabolites in rats by HPLC‐IT‐TOF‐MS
Division of Pharmacognosy, School of
Pharmaceutical Sciences, Peking University,
Ming‐Ying Shang, Division of Pharmacognosy,
School of Pharmaceutical Sciences, Peking
University, Beijing, China.
National Natural Science Foundation of China,
Grant/Award Number: 81274074
Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese
medicine Scrophulariae Radix. High‐performance liquid chromatography with a diode array
detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrom-
) was used to profile and identify the metabolites of harpagoside in
rats in vivo and to study the distribution of these metabolites in rats for the first time. A total
of 45 metabolites were identified, 37 of which were postulated to be new compounds. The num-
ber of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine
was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside
should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats
are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitu-
tion, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites
were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These
findings are useful for better comprehension of the effective forms, target organs and pharmaco-
logical effects of harpagoside. Moreover, these findings provide a reference for studying the
metabolism and distribution of iridoid compounds.
bioactivities prediction, distribution, harpagoside, HPLC‐IT‐TOF‐MS
Harpagoside is an iridoid glycoside. It is the main ingredient in
Scrophulariae Radix (SR) and has been determined to be one of the
index components for quality control of SR in the 2015 edition of the
Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2015).
Harpagoside exhibits a variety of pharmacological actions including
anti‐inflammatory (Lanhers, Fleurentin, Mortier, Vinche, & Younos,
1992; Ortiz, Martin, Fernandez, San, & Cubillo, 1994), abirritation
(Lanhers et al., 1992), antioxidation (Jeong, Lee, Kim, Sung, & Kim,
2008; Zhang et al., 2011), immunologic enhancement (Garg et al.,
1994), cardiovascular effects (Circosta et al., 1984; Costa et al., 1985)
and neuroprotection (Sun et al., 2012). Scrophulariae Radix, which
comes from the dried roots of Scrophularia ningpoensis Hemsl.
(Scrophulariaceae), is widely used in China. It was first recorded in
Shen Nong's herbal classic. Its traditional efficacy is cooling blood
and nourishing Yin, purging fire and detoxification (Chinese Pharmaco-
poeia Commission, 2015). The primary constituents of SR are iridoid
glycosides, phenylpropanoid glycosides, phenylethanol glycosides, ter-
penoids, fatty acids, sterols and polysaccharose (Xu, Xu, & Chen, 2013).
The pharmacopoeia stipulates that the total content of harpagoside
and harpagide should not be <0.45% (Chinese Pharmacopoeia Com-
mission, 2015). Research suggests that harpagoside might be hydro-
lyzed to harpagide and cinnamic acid during processing using
traditional methods (Bai et al., 2011; Wang, Xie, Liu, Wang, & Cai,
2000). Preliminary in vivo studies on the metabolism of Scrophulariae
Radix have shown that most metabolites of Scrophulariae Radix come
from harpagoside. Therefore, metabolic research of harpagoside can
not only provide evidence for the material basis of its pharmacological
effect, but can also further understanding of harpagoside metabolism
To explore the metabolism and distribution of bioactive com-
pounds, high/ultra‐performance liquid chromatography tandem
Abbreviations: SR, Scrophulariae Radix.
Jing‐Zhe Wang and Yi‐Fan Zhang contributed equally to this work.
Received: 21 November 2017 Revised: 5 February 2018 Accepted: 9 February 2018
Biomedical Chromatography. 2018;32:e4218.
Copyright © 2018 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc 1of10