Bioactivities of 7'‑ethoxy‑trans‑feruloyltyramine from Portulaca oleracea L. and its metabolism in rats using ultra‑high‑performance liquid chromatography electrospray coupled with quadrupole time‑of‑flight mass spectrometry.

This research aims to study the antioxidation and anticholinesterase activities of 7'-ethoxy-trans-feruloyltyramine (ETFT), which was an alkaloid isolated from Portulaca oleracea for the first time. Furthermore, its main metabolites and metabolic pathways in rats were also explored. The antioxidation and anticholinesterase effects of ETFT were, respectively, examined using 1,1-diphenyl-2-picrylhydrazyl assay and modified Ellman's method. The results showed that ETFT exhibited both the good antioxidant and anticholinesterase effects. Its main metabolites in rats were implemented, and nine metabolites were finally found in the rat's plasma and urine, including the oxidation, reduction, hydrolysis, glucuronidation, sulfation, and glutathionylation process. Copyright:

Introduction

Portulaca oleracea L. is distributed in the temperate and tropical regions of the world, which has not only high nutritional value but also resistive effect of inflammatory,[1] neuroprotective,[2] antitumor,[3] and antioxidation.[4] This is mainly due to the fact that it contains many bioactive substances such as alkaloids[5678] and flavonoids,[9] in which the alkaloids presented remarkable anti-inflammatory.[101112]

In the study, the 7'-ethoxy-trans -feruloyltyramine (ETFT) [Figure 1] was obtained from purslane for the first time. Its structure was identified by comparing with literature.[13] The related spectrums are shown in Supplementary Figures 7-15, and its detailed 1H and 13C NMR data is shown in Supplementary Table 1.

Figure 1

Structure of 7'-ethoxy-trans-feruloyltyramine

Supplementary Table 1

1H NMR (500 MHz) and 13C NMR (125 MHz) data of 7’-ethoxy-trans-feruloyltyramine in MeOD

Position	δC	Type	δH (J in Hz)
1	128.32	C
2	111.58	CH	7.13 (1H, d, 1.85)
3	149.33	C
4	149.91	C
5	116.49	CH	6.79 (1H, m)
6	123.32	CH	7.03 (1H, dd, 1.90; 8.25)
7	142.21	CH	7.44 (1H, d, 15.7)
8	118.71	CH	6.47 (1H, d, 15.75)
9	169.24	C
3-OCH3	56.42	CH3	3.88 (3H, s)
1´	132.20	C
2´/6´	129.08	CH	7.18 (1H, m)
3´/5´	116.29	CH	6.79 (1H, m)
4´	158.41	C
7´	81.35	CH	4.37 (1H, q, 6.48)
8´	47.20	CH2	Ha=3.40; Hb=3.49 (2H, m)
1´´	65.13	CH2	3.37 (2H, m)
2´´	15.57	CH3	1.16 (3H, t, 7.05)

The antioxidation and anticholinesterase effects of ETFT were studied; in addition, the metabolism of ETFT in the rat's plasma, urine, as well as feces after intravenously administrated was also investigated by the ultra-high-performance liquid chromatography electrospray coupled with quadrupole time-of-flight mass spectrometry.

Experimental Method

Antioxidation assay

Different concentrations (0.25, 0.20, 0.15, 0.10, and 0.05 mg/mL) of ETFT and the 50 μg/mL 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution were prepared. Vitamin C and butylated hydroxyl anisole were used as positive controls, and the methanol was used as the blank control. The absorbance was detected by a U-3010 spectrophotometer at 517 nm.

Anticholinesterase assay

The concentrations of ETFT (0.30, 0.25, 0.20, 0.15, and 0.10 mg/mL) and 0.2 U/mL AChE, 15 mmol/L ATCI, and 15 mmol/L DTNB were prepared. For the positive control group, eserine was selected, and for the blank group, methanol was applied. The absorbance was detected at 405 nm using a 96-well microplate reader (HBS-1096A).

Instrumentation and condition

Agilent 1290 UHPLC (Agilent, Waldbronn, Germany) with ODS column (3.0 mm × 150 mm, particle size, 1.8 μm) was applied with the mobile phase of 0.1% formic acid (A) and acetonitrile (B). The program system was 15%–70% (B) at 0–10 min, with 0.3 mL/min, and the oven temperature was at 60°C. An Agilent 6520 Q-TOF/MS (Agilent, Waldbronn, Germany) was coupled to the UHPLC system through an ESI interface. The MS parameters in the positive mode: drying gas (nitrogen);10 L/min at 330°C; nebulizer pressure (45 psi); fragmentation voltage 150 V; ESI Vcap of 3500 V; and collision energy (30 eV). Full-scan information acquisition in the positive ionization mode was m/z 100–800.

Animal experiments

Male Sprague–Dawley rats (200 ± 20 g) were used for the animal assay, and this experiment was approved by the Committee of Ethics of Animal Experimentation of Liaoning University of Traditional Chinese Medicine with the approval number 2019YS(DW)-009-01.

After intravenous administration of ETFT (4 mg/kg), 300 μL blood sample was gathered from the orbital venous at 3, 10, and 30 min. Meanwhile, the urine as well as feces samples were respectively collected within 0–24 h.

Sample preparation

100 μL sample and 500 μL methanol were added into EP tubes, vortex mixing for 60 s, centrifuging for 15 min (3000 rpm) to remove the protein. The residue was reconstituted in a 100 μL initial mobile phase, and then centrifugating it for 3 min (10,000 rpm). Finally, a 5 μL aliquot was injected for determination.

Results

DPPH scavenging rate of ETFT is shown in Supplementary Figure 1, indicating that the oxidation effect increased along with the increase of concentration, with IC50 to be 0.058 mg/mL.

The result of the anticholinesterase assay is shown in Supplementary Figure 2, suggesting that the ETFT exerted a dose-dependent inhibitory effect against the AChE with IC50 values of 0.106 mg/mL.

Metabolism study

Analysis of plasma, urine, and feces samples

The chromatograms of the plasma, urine, and feces samples in positive ion mode are shown in Supplementary Figures 3 and 4. All of the fragment ions are shown in Supplementary Figures 5 and 6. The MS data of nine metabolic products are listed in Supplementary Table 2.

Supplementary Table 2

Ultra-high-performance liquid chromatography electrospray coupled with quadrupole time-of-flight mass spectrometry data of nine metabolites of 7’-ethoxy-trans-feruloyltyramine in rats

No.	Retention time (min)	Experimental (m/z)	Theoretical (m/z)	Formula	Metabolic process	Specimen
M-1	4.4	199.1240	199.1189	C8H6O4S	Oxidation, reduction, dehydration, sulfation	Plasma
M-2	5.0	309.1808	309.1301	C15H16O7	Hydrolysis, glucuronidation	Plasma
M-3	5.2	367.1857	367.1769	C18H22O8	Glucuronidation, dehydration, decarbonation	Plasma
M-4	6.5	337.1750	337.1750	C9H5O10S2	Oxidation, sulfation	Plasma
M-5	9.4	346.3313	346.1933	C19H23NO5	Oxidation	Plasma
M-6	9.4	346.3313	346.1611	C18H19NO6	Oxidation	Plasma
M-7	7.4	271.0609	271.2046	C12H16NO4S	Glutathionylation	Urine
M-8	7.8	188.0708	188.1268	C7H7O4S	Sulfation	Urine
M-9	9.7	348.2179	348.1769	C18H21NO6	Reduction	Urine

Identification of the metabolites

All metabolites and the metabolic pathway are shown in Figure 2.

Figure 2

Proposed major metabolic pathways of 7'-ethoxy-trans-feruloyltyramine

Oxidation

M-5 (m/z 346.3313) was in the same value with M-6. M-5 was from ETFT's olefine reduced and methoxy oxidization. M-6 was from ETFT in which both N and ethoxyl were oxidized to hydroxyl.

Glucuronide conjugates

M-2 (m/z 309.1808) was formed by ferulic acid, the hydrolysate of M-6, via the serial process of glucuronidation (370.1362 Da) and losing a H2O and a CO2. M-3 (m/z 367.1857) was formed from the fragmentation ion (138.0778), which experienced glucuronidation (491.2049 Da) and then losing two H2O and two CO2.

Sulfate conjugates

M-1 (m/z 199.1240) was formed by -SO3 (80.0642 Da) combining with the fragmentation ion (120.0626 Da) that was obtained when M-5 happened oxidation and reduction reaction, then losing H2O. M-4 (m/z 337.1750) was formed by two -SO3 (80.0642 Da) combining with the fragmentation ion (176.0523 Da) of M-6. M-8 (m/z 188.1268) was the combination of -SO3 (80.0642 Da) and the fragmentation ion (107.0547 Da).

Glutathione conjugates

M-7 (m/z 271.0609) was the combination of C9H11O2 (151.0857) and + C3H5NO2S (119.111 Da).

Reduction

M-9, the fragment ion at m/z 348.2179, was the reduction of M-6 in olefins.

Discussion

The bioactive assays indicated that the ETFT has a remarkably antioxidation effect compared with that of BHA, although its antioxidation effect has no more than that of Vitamin C. Moreover, it also exhibited an anticholinesterase effect with dose-dependent.

In the metabolism experiment, no ETFT was detected after intravenous administration, but a peak consisting of two molecules (M-5 and M-6) was found at 3 min and gradually enhanced at 10 and 30 min, which means that ETFT has converted into M-5 and M-6. And furthermore, other metabolites were formed from M-5 and M-6. Among nine metabolites, six metabolites (M-1 to M-6) were found in the plasma, and three metabolites (M-7 to M-9) were found in the urine samples. Any metabolites were not found in the feces, suggesting that ETFT was not metabolized via bile.

Financial support and sponsorship

The National Natural Science Foundation of China (No. 81573546).

Conflicts of interest

There are no conflicts of interest

Antioxidation effect of 7'-ethoxy-trans-feruloyltyramine

Anticholinesterase effect of 7'-ethoxy-trans-feruloyltyramine

The extracted ion chromatograms of metabolites from 7'-ethoxy-trans-feruloyltyramine in the rat plasma. (a) Blank rat plasma; rat plasma collected at (b) 3 min, (c) 10 min, and (d) 30 min after intravenous administration

The extracted ion chromatograms of metabolites from 7'-ethoxy-trans-feruloyltyramine in the rat urine and feces. (a) blank rat urine; (b) rat urine after intravenous administration of 7'-ethoxy-trans-feruloyltyramine; (c) blank rat feces; (d) rat feces collected after intravenous administration of 7'-ethoxy-trans-feruloyltyramine

MS/MS spectra of M-5 and M-6 in rat plasma collected at 3, 10, and 30 min after intravenous administration of 7'-ethoxy-trans-feruloyltyramine

MS/MS spectra that phase I and phase II metabolism in rat urine and plasma collected at 30 min after intravenous administration of 7'-ethoxy-trans-feruloyltyramine

Ultraviolet spectrum of 7'-ethoxy-trans-feruloyltyramine in methanol

Infrared spectrum of 7'-ethoxy-trans-feruloyltyramine

1H NMR (500 MHz) spectrum of 7'-ethoxy-trans-feruloyltyramine

13C NMR (125 MHz) spectrum of 7'-ethoxy-trans-feruloyltyramine

DEPT spectrum of 7'-ethoxy-trans-feruloyltyramine

1H-1H COSY spectrum of 7'-ethoxy-trans-feruloyltyramine

HMBC spectrum of 7'-ethoxy-trans-feruloyltyramine

HSQC spectrum of 7'-ethoxy-trans-feruloyltyramine

NOESY spectrum of 7'-ethoxy-trans-feruloyltyramin