J. Yang, et al.
FoodChemistry315(2020)126270
we known, an attempt to use the technology to obtain information
about potential oxidative transformation and metabolic pathway of
natural products has rarely been reported so far.
obtained by a Millipore Milli-Q purification system (Billerica, MA).
2.2. Instrumentation
Danshen (the root of Salvia miltiorrhiza Bunge), a well-known med-
icinal plant and functional food, has been widely used in the treatment
of cardiovascular, cerebrovascular, liver and chronic renal failure dis-
eases, especially angina pectoris, myocardial ischemia, coronary heart
2002). The main active compounds of Danshen were classified into two
groups: hydrophilic phenolics and lipophilic diterpenoid quinines (Li,
from the caffeic acid construction unit via various kinds of condensa-
tion reactions, including caffeic acid, danshensu, rosmarinic acid (RA),
salvianolic acid C (Sal C), lithospermic acid (LA), salvianolic acid B (Sal
were considered as the phytochemical markers in the Chinese Phar-
macopoeia 2015 due to various biological effects. The active water-
soluble constituents of phenolic acids in Danshen are very complicated.
Therefore, it is essential to illuminate the whole metabolic pathways of
phenolic acids for a comprehensive understanding about the active
constituents of Danshen. So far, most metabolic reports on phenolic
acids have been carried out under different modes in vivo, including gut
microflora, rat plasma, bile, urine, feces, WZS-pig urine (Bel-Rhlid
However, these investigations only focused on a single analyte or a few
characteristic compounds of phenolic acids, which were insufficient to
demonstrate the metabolic features of Danshen. In addition, the com-
plex in vivo process after administration increased the difficulty in de-
tection. Thus, a simple, fast and comprehensive EC/MS method is vital,
which is proposed for a holistically metabolic study of multiple active
constituents in Danshen.
In this work, electrochemistry coupled to online quadrupole time-
of-flight mass spectrometry (EC/Q-TOF/MS) was presented as a simple,
rapid method to investigate the oxidative transformation of character-
istic phenolic acids (RA, protocatechuic aldehyde (PA), Sal C, LA, Sal B)
in Danshen. Oxidation reactions were carried out in a three-electrode
controlled-potential electrochemical reactor employing a glassy carbon
electrode as the working electrode. High-resolution Q-TOF/MS was
used to predict and elucidate the structure of intermediates and oxi-
dation products. The generation and reactivity of metabolites and the
possibility to simulate its biomolecule (glutathione) were also dis-
cussed. The obtained results were compared with a conventional in vitro
microsomal approach with the use of rat liver microsomes (RLM), and
in vivo assays, which indicated that it was a powerful tool in the in-
vestigation of the metabolic reactions of natural products.
The electrochemical oxidation of phenolic acids was performed in
the ROXY™ system (Antec, Zoeterwoude, The Netherlands), which was
committed to investigating the oxidative metabolism of drugs. EC was
conducted in an electrochemical thin-layer cell (ReactorCell, Antec
Leyden), which composed by a three-electrode arrangement, containing
a glassy carbon working electrode, a Pd counter electrode and a HyREF
(Pd/H2) reference electrode. The glassy carbon electrode and the aux-
iliary electrode inlet module were separated by a 50 µm spacer, and the
effective volume was 0.7 µL. The active surface area (wetted area) of
the working electrode was approximately 14 mm2, which was de-
termined by the spacer. Potentials were ramped between 0 and
3000 mV using a dedicated ROXY potentiostat (Antec Leyden). Buffer
solution was passed through the electrochemical cell by a syringe pump
at a constant flow rate of 10 μL/min. The EC cell temperature was kept
at 35 °C.
Mass analyses of the electrochemical products, were performed by a
Q-TOF/MS from Agilent Technologies (Santa Clara, CA, USA) equipped
with an Dual AJS electrospray ionization (ESI) source. Mass spectra
were recorded in the negative ion mode under the following para-
meters: capillary voltage (V), 3500; drying gas temperature (°C), 350;
nebulizer pressure (psig), 45; drying gas (N2) flow rate (L/min), 12;
fragmentor pressure (V), 175; OCT/RF (V), 750; skimmer voltage (V),
65. The mass-to-charge (m/z) ratio range was set from 100 to 1500. All
the data were obtained and analyzed by Mass Hunter software (version
B 08.00, Qualitative Analysis).
For all analyses of rat microsomal incubation, UHPLC/Q-TOF-MS
separation was carried out on
a
ZORBAX SB C18 column
(4.6 mm × 150 mm i.d., 5 μm, Agilent). During the separation process,
the column constant temperature was kept at 35 °C and the flow rate of
was set as 0.4 mL/min. The injection volume was 2 μL. The mobile
phases were 0.1% formic acid in water and acetonitrile, and the cor-
responding gradient profile was shown below: 8%–20% B, 0–2 min;
20%–40% B, 2–4 min; 40%–80% B, 4–6 min; 80%–100% B, 7–8 min.
The corresponding conditions for the Q-TOF/MS measurements were
same as before.
2.3. Generation of metabolites by electrochemical oxidation.
20 mM ammonium formate buffer was prepared with water/acet-
onitrile (50/50, v/v) and used throughout the electrochemical experi-
ments. For the electrochemical conversion, a 100 μM sample of target
compound in a 20 mM buffer solution was used. The pH was adjusted to
7.4 with 20–22% ammonium hydroxide solution. A setup for on-line
oxidation of metabolites by EC is shown in Fig. S1(a) (see
Supplementary material). The samples were injected into the electro-
chemical flow-through cell at a constant flow rate of 10 μL/min with a
syringe pump (1 mL glass syringe). The potentiostat was set to perform
a potential scan from 0 to 3 V at a scan rate of 100 mV/s. Potential and
time applied by the electrochemical cell are shown in Table S1. After
electrochemical conversion of target analytes, the products were flowed
to MS for real-time monitoring.
2. Experimental
2.1. Chemicals
RA, PA, Sal C, LA, Sal B, were supplied by Shanghai Winherb
Medical Technology Co., Ltd. (Shanghai, China). Chromatographic
grade acetonitrile was obtained from Tedia Company Inc. (Fairfield,
US). 20–22% ammonia was LC/MS grade supported by ANPEL
Laboratory Technologies (Shanghai) Inc. (Shanghai, China).
Glutathione (GSH) was obtained from Fluka (Buchs, Switzerland).
Ammonium formate, sodium dihydrgen phosphate, disodium hydro-
genorthophosphate and β-Nicotinamide adenine dinucleotide phos-
phate (β-NADPH) were all purchased from Sigma-Aldrich (Schnelldorf,
Germany). Microsomes in vitro derived from male SD rat liver cells at
the concentration of 10 mg/vial were obtained from Research Institute
for Liver Diseases (Shanghai) Co. Ltd. (Shanghai, China). The micro-
somes were placed at −70 °C before experiments. Pure water was
2.4. Adduct formation using GSH
For the investigation of target analyte trapping experiments with
GSH, a slightly modification of EC/MS set-up was applied, as shown in
Fig. S1(b). The electrochemical oxidation process was conducted under
the same conditions mentioned above. After the electrochemical oxi-
dation, 20 mM ammonium formate containing 300 μM GSH was added
to the oxidized effluent of target analyte via a T-piece immediately. The
GSH aqueous solution was constantly added at a flow rate of 10 μL/min
via a syringe pump, resulting in the final flow of 20 μL/min to Q-TOF/
2