Fitoterapia 154 (2021) 105020

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Three new alkaloids from Portulaca oleracea L. and their bioactivities

Xinyu Cui a, Zheming Ying b, c, Xixiang Ying a, *, Lianqun Jia c, Guanlin Yang b, c, **
a
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, Liaoning, PR China
b
School of The First Clinic, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, PR China
c
Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, PR
China

A R T I C L E I N F O A B S T R A C T

Keywords: Three novel alkaloids, named oleracone L (1), portulacatone B (2), and portulacatal (3), were isolated from
Portulaca oleracea L. P. oleracea L.. The structures were determined using UV, IR, 1D and 2D NMR spectroscopy and UHPLC-ESI-
Alkaloid QTOF/MS. The three compounds in a dose-dependent manner significantly reduced the secretion of IL-1β in
Anticholinesterase activity
the lipopolysaccharide-stimulated macrophages RAW 264.7 cell culture supernatant, moreover, exhibited the
Anti-inflammatory activity
anticholinesterase activities.

1. Introduction terpenes [20] and so on.

Portulaca oleracea L. is an annual fleshy herbaceous plant belonging
to the Portulaca of the family Portulacaceae [1]. It has strong repro­
ductive capacity and adaptability and is widely distributed in temperate
and tropical regions of the world [2]. P. oleracea is rich in many nutri­
ents, such as ω-3 and ω-6 fatty acids, α-linolenic acid, β-carotene,
α-tocopherol, vitamin B, C, E, [3,4] and a variety of trace elements
required by the human body, such as Ca, P, Fe, and K, etc. [5]. Thus,
P. oleracea is commonly used as a vegetable, traditional herbal medicine,
and animal feed in many areas.
P. oleracea, as a plant with the same medicinal and edible source, is
often referred to as a “longevity vegetable”. A great deal of literature
report that P. oleracea had a variety of beneficial effects on the human
body, such as anti-oxidation [6], anti-inflammation [7,8], neuro­
protection [9], analgesic [10], and anticholinesterase activities [11].
The World Health Organization lists P. oleracea as one of the commonly
used medicinal plants and was awarded the title of “Global Panacea”
mainly owing to its many active ingredients including alkaloids
[12–14], flavonoids [15,16], lignans [17,18], organic acids [19],
Alzheimer's disease (AD), the major killers of human health, its
pathogenesis is mainly attributed to the lack of acetylcholine and the
release of inflammatory factors in the brain [21–23]. Recently the al­
kaloids were found to have significant anticholinesterase and anti-
inflammatory effects in our lab [8,13], thus three novel alkaloids iso­
lated from P. oleracea were investigated the anticholinesterase and anti-
inflammatory activities in this study. As P. oleracea had anti-
inflammation and anticholinesterase activities aforementioned and its
extract was used to treat UC [24] and IL-1β is the main inflammatory
factor induced UC, and therefore the inflammatory factor, IL-1β was
selected as the indicator in the anti-inflammation assay [25].
2. Experimental
2.1. Chemical and reagents
Methanol and acetonitrile were all HPLC grade produced in Kaixin
Chemical Reagent Plant (Tianjin, China) and the water was Wahaha
purified water (Shenyang, China). Other reagents were analytical grade

Abbreviations: IR, infrared; UV, ultraviolet; NMR, nuclear magnetic resonance; UHPLC-ESI-QTOF-MS, ultra-high-performance liquid chromatography coupled
with electrospray ionization quadrupole time-of-flight mass spectrometry; AChE, acetylcholinesterase; ATCI, acetylthiocholine iodide; DMEM, Dulbecco's modified
eagle medium; IL-1β, interleukins-1β; LPS, lipopolysaccharide; Dex, dexamethasone; DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; FBS, fetal bovine
serum; AD, Alzheimer's disease; UC, ulcerative colitis.
* Correspondence to: X. Ying, School of Pharmacy, Liaoning University of Traditional Chinese Medicine, No.77, Shengming 1 Road, DD Port, Dalian 116600, PR
China.
** Correspondence to: G. Yang, School of The First Clinic and Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications, Liaoning
University of Traditional Chinese Medicine, No. 79, Chongshan Eastern Road, Shenyang, Liaoning 110847, PR China.
E-mail addresses: yingxixiang@163.com (X. Ying), yang_guanlin@163.com (G. Yang).

https://doi.org/10.1016/j.fitote.2021.105020
Received 6 July 2021; Received in revised form 12 August 2021; Accepted 13 August 2021
Available online 19 August 2021
0367-326X/© 2021 Elsevier B.V. All rights reserved.
X. Cui et al. Fitoterapia 154 (2021) 105020

Table 1
1 13
H NMR (600 Hz) and C NMR (150 Hz) Spectral Data of Compound 1–3 in MeOD-d4.
1 2 3

position δC type δH mult (J in Hz) δC type δH mult (J in Hz) δC type δH mult (J in Hz)

1 119.4 CH 7.14 d (4.3) 132.4 CH 8.13 m 110.7 CH 6.08 d (4.0)

2 163.1 C 119.7 CH 7.06 dd (4.0, 2.4) 126.9 CH 6.91 d (4.0)
C2-OMe 52.3 CH3 3.83 s
3 118.1 CH 6.93 d (4.3) 140.9 CH 8.03 m 133.6 C
C3-CHO 180.4 CHO 9.30 s
4 146.9 C N N
4a 140.5 C
5 NH 57.2 CH2 5.12 t (5.0) 48.7 CH2 4.63 m
6 48.6 CH2 4.87 m 45.0 CH2 3.95 t (5.0) 32.6 CH2 3.10 m
6a 141.1 C 130.5 C
7 36.1 CH2 3.17 dd (4.4, 6.0) 129.5 CH 7.42 d (8.0) 117.0 CH 6.62 s
7a 136.4 C
8 117.2 CH 6.67 s 129.6 CH 7.73 d (8.0) 144.9 C
9 145.9 C 155.2 C 145.4 C
10 152.4 C 163.2 C 117.6 CH 6.63 s
10a 128.1 C 128.9 C
11 118.8 CH 7.54 s 194.2 C 33.6 CH2 3.99 s
11a 129.2 C 141.9 C 145.1 C
12 182.9 C
12a 127.5 C

Fig. 1. Structures of the compounds isolated from Portulaca oleracea L.. 1: Oleracone L, 2: Portulacatone B, 3: Portulacatal.

Fig. 2. HMBC correlations of the compounds 1–3.

Fig. 3. 1H–1H COSY and ROESY correlations of the compounds 1–3.

from Kaixin Chemical Co. (Tianjin, China). The RAW 264.7 macrophage purchased was from Boster Biological Technology Co., Ltd. (CA, USA).
cells (ATCC TIB-71) were purchased from American Type Culture Penicillin-streptomycin solution and fetal bovine serum (FBS) were
Collection (ATCC, Manassas, VA, USA). Cell counting kit-8 (CCK-8) was purchased from Gibco (Grand Island, NY, USA). Dimethyl sulfoxide

2
X. Cui et al. Fitoterapia 154 (2021) 105020

Table 2 other chemicals were provided by Jinfeng Chemical Reagent Co., Ltd.
IC50 (μM) for anticholinesterase activity of compounds 1–3. (Tianjin, China).
Compounds and standard Inhibitors IC50 (μM)

1 46.00 ± 0.22 2.2. Plant material

2 50.49 ± 0.17
3 58.37 ± 0.30 The whole herbs of P. oleracea were purchased in Shijiazhuang
Eserine 38.31 ± 0.13
(Hebei, China) in June 2017, and identified by Prof. Xixiang Ying. The
Eserine as positive control, and values are expressed as the means ± SD for voucher specimen (No. 20171001) was displayed at Liaoning University
n = 5. of Traditional Chinese Medicine.

(DMSO) was from Sigma-Aldrich (Santa Clara, CA, USA). Trypsin-EDTA

2.3. General experimental procedures
solution was from Beyotime Biotechnology (Shanghai, China). Dulbec­
co's modified Eagle medium (DMEM), LPS (E. coli strain 055:B5), and
In the separation process, column chromatography (CC) included
interleukin 1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits
silica-gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Co.,
were obtained from Solarbio Technology Co., Ltd. (Beijing, China). All
Qingdao, China), polyamide resin (80–100 mesh, Taizhou Luqiao Sijia

Fig. 4. Cell viability of the LPS-induced macrophage RAW 264.7 cells pretreated with the compounds 1–3. The data represent the mean ± SD of three independent
experiments. (#) p < 0.05 vs. LPS group.

Fig. 5. Inhibitory effect on IL-1β production in the culture media of LPS-induced RAW 264.7 cells pre-incubated with compounds 1–3. Dexamethasone as positive
control. The data represent the mean ± SD of three independent experiments. (*) p < 0.05 vs. con group; #p < 0.05 vs. LPS group.

3
X. Cui et al.
Biochemical Plastic Factory, Zhejiang, China), ODS (20–40 μm, GE
Healthcare, Marlborough, MA) and Sephadex LH-20 (GE Healthcare,
Marlborough, MA). The TLC was prepared with silica gel GF254 (Qing­
dao Marine Chemical Co., Qingdao, China). The dual fluorescence cy­
tometer was Nexcelom ceilometer (Nexcelom Bioscience., Boston, US).
2.4. Equipment
The drying P. oleracea was extracted and concentrated using a
multifunctional extraction and concentration machine (Shanghai Shu­
nyi Tech. Co. Ltd., Shanghai). The NMR spectra were recorded by an
AVANCE 600 MHz instrument (Bruker Corporation, Switzerland) while
the compounds were dissolved in MeOD-d4. Relative molecular mass
was recorded using a 6520 quadrupole-time-of-flight mass spectrometer
(Agilent, Palo Alto, CA). The samples were prepared on a Nexera X2
UHPLC LC-30A system (Shimadzu, Kyoto, Japan). The Kromasil C18
column (150 mm × 4.6 mm, 5 μm, Dalian Jiangshen Separation Science
and Technology Co.) was used for separation and analysis. The ultra­
violet (UV) spectrum was obtained from a HITACHI U-3010 spectro­
photometer (Hitachi Ltd., Tokyo, Japan), the infrared (IR) spectrum,
and data was obtained from IR 200 spectrophotometer (Thermo Elec­
tron Corporation, Waltham, MA). The absorbance was measured with a
microplate reader (Molecular Devices Corporation, Silicon Valley, US).
The clean bench and CO2 incubator were from Esco Micro Pte. Ltd.
(Singapore).
2.5. Extraction and isolation
The sample of the drying P. oleracea (150 kg) was extracted twice
with 10 volumes of 50% ethanol for 2 h each time to obtain an extract
(approximately 21 kg). The extract was concentrated and further sub­
jected to a 100–200 mesh silica-gel column chromatography (120 × 80
cm, approximately 150 kg) with a 2 volumes of ethyl acetate 3 times as
the isocratic elution, the fraction was concentrated under reduced
pressure (1.5 kg), and then was added to an 80–100 mesh polyamide
resin column chromatography (120 × 8 cm, approximately, 2.5 kg),
eluted with water and 95% ethanol to obtain 2 fractions (4 L each). The
fraction of 95% ethanol extract was condensed by distillation to obtain
the extract, which was subjected to a 200–300 mesh silica-gel column
chromatography (120 × 8 cm, approximately, 2.5 kg) using ethyl ace­
tate, ethyl acetate and methanol (5:1, 2:1, v/v) as the gradient eluent,
obtaining 3 fractions (4 L each). Then the fractions were spotted on thin-
layer chromatography (TLC) plate and sprayed with ferric chloride re­
agent. The part of ethyl acetate turned cyan and then it was eluted with a
gradient of methanol (60%, 70%, 80%, 90%, respectively) using 20–40
μm ODS under moderate pressure (500 mL each). The 70% methanol
fraction was combined and condensed according to the results of thin-
layer color development to obtain 5 parts (A1-A5, 100 mL each). A1
was purified on a Sephadex LH-20 column chromatography (100 g, φ2
× 150 cm) using methanol solvent (50 mL each), according to the results
of thin-layer color development to give fractions B1-B6. And then B5 was
isolated by the HPLC using acetonitrile-0.1% formic acid (35:65, v/v)
with the flow rate of 2 mL/min and temperature of 40 ◦ C to obtain C1-
C10. C-3 was further purified with the UHPLC using acetonitrile-0.1%
formic acid (45:55, v/v) as the mobile phase to obtain compound 1 (3
mg, purity of >98% with UHPLC, tR 3.112 min). C-5 was further purified
with the UHPLC using acetonitrile-0.1% formic acid (35:65, v/v) as the
mobile phase to obtain compound 2 (5 mg, purity of >98% with UHPLC,
tR 8.694 min). C-7 was further purified with the UHPLC using acetoni­
trile-0.1% formic acid (25:75, v/v) as the mobile phase to obtain com­
pound 3 (3 mg, purity of >98% with UHPLC, tR 8.231 min). The
structures of the above compounds were fully proved by UHPLC-ESI-
QTOF/MS, 1H NMR, and 13C NMR spectral data.
2.5.1. Oleracone L (1)
Yellowish-brown powder, IR (KBr) υmax 3340, 1707, 1568, 1508,
4
Fitoterapia 154 (2021) 105020
1440, 1356, 1268, 929, and 892 cm− 1; UV (MeOH) λmax 348, 313 and
243 nm; 1H and 13C NMR spectroscopic data (MeOD-d4), see Table 1;
UHPLC-ESI-QTOF/MS, m/z 300.0878 [M − H]− (calcd: C16H14NO5− ,
300.0877).
2.5.2. Portulacatone B (2)
Yellow powder, IR (KBr) υmax 3438, 1610, 1554, 1521, 1437, 1404,
1047, 1260, and 808 cm− 1; UV (MeOH) λmax 346 and 280 nm; 1H and
13
C NMR spectroscopic data (MeOD-d4), see Table 1; UHPLC-ESI-QTOF/
MS, m/z 230.0812 [M + H]+ (calcd: C13H12NO3+, 230.0812).
2.5.3. Portulacatal (3)
Reddish-brown powder, IR (KBr) υmax 3367, 2922, 2852, 1711,
1632, 1569, 1244, 893, and 856 cm− 1; UV (MeOH) λmax 452 and 297
nm; 1H and 13C NMR spectroscopic data (MeOD-d4), see Table 1;
UHPLC-ESI-QTOF/MS, m/z 244.0969 [M + H]+ (calcd: C14H14NO3+,
244.0969).
2.6. Assay of anti-inflammatory activity
2.6.1. Cell culture
The macrophage cell line RAW 264.7 was cultured in DMEM con­
taining 10% heat-inactivated fetal bovine serum (FBS) and antibiotics
(100 units/mL penicillin and 100 μg/mL streptomycin) in a humidified
incubator at 37 ◦ C with 5% CO2.
2.6.2. Cell viability
The WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium) assay was used to evaluate the viability
of the three compounds [26]. RAW 264.7 cells were seeded in 96-well
plates at an initial density of 1 × 104 cells/well in 100 μL and incu­
bated for 24 h. The cells were then pretreated for 1 h with the com­
pounds at various concentrations (0, 5, 10, 25, and 50 μM), and 1 μg/mL
LPS was added for pretreatment for 24 h. Finally, 10 μL cell counting kit-
8 (CCK-8) was added to each well and incubation at 37 ◦ C for 3 h. The
absorbance of each well was measure at 450 nm and 650 nm by a
microplate reader. The LPS group was considered to be 100% feasible.
2.6.3. Determination of IL-1β production
The supernatant of RAW 264.7 cells induced by LPS pre-cultured
with or without compounds of the different concentrations (1, 5, 10,
and 20 μM) were collected and determined the production of IL-1β using
an ELISA kit. 10 μM dexamethasone (Dex) was used as the positive
control for IL-1β release inhibitor.
2.7. Assay of anticholinesterase activity
According to the improved method of Ellman [27], the anticholin­
esterase activity of the compounds was tested in 96-well plates. The 20
μL samples solutions in five different concentrations, 15 μL AChE solu­
tion (0.2 U/mL), 140 μL PBS (pH 8.0, containing 0.1 mol/L Na2HPO4
and NaH2PO4) were mixed in the 96-well microplate and incubated for
10 min at 37 ◦ C. Then 10 μL DTNB (5,5′ -dithiobis-(2-nitrobenzoic acid)
(15 mmol/L) and 10 μL acetylthiocholine iodide (ATCI) (15 mmol/L)
were added and initiated the reaction for 20 min at 20 ◦ C. The absor­
bance was monitored spectrophotometrically at 405 nm. The eserine
was prepared into the same concentrations as the samples as the positive
control. Methanol was added instead of the sample in the blank group.
Each sample was measured three times in parallel, and the percentage of
inhibition of AChE was calculated using the following formula [28]:
( )/
Anticholinesterase (%) = Ablank − Asample Ablank × 100%
In which, Asample was the absorbance of the test compound, and
Ablank was the absorbance of the blank group. The anticholinesterase
activities were evaluated by the value of IC50, and values were expressed
X. Cui et al.
as the means ± SD for n = 5.
2.8. Statistics analysis
The data results were expressed in the form of mean ± standard
deviation (SD). Use Excel and SPSS 25.0 to analyze and process the
obtained data, and calculate the IC50 value. All data were evaluated and
compared using analysis of variance (ANOVA) and Dunnett's t-test.
3. Results and discussion
3.1. Structure elucidation
The structures (Fig. 1) of compounds 1–3 were confirmed by analyses
of comprehensive 1D and 2D NMR. According to the analysis data, there
is no report about compounds 1–3 before.
Compound 1 was obtained as a yellowish-brown powder, which
turned orange with Dragendorff's reagent. The obvious absorption bands
of the NH group (3340 and 1567 cm− 1) and the carbonyl group (1707
cm− 1) were observed in the IR spectrum. The UV (MeOH) spectrum
showed an absorption maximum at 348, 313, and 243 nm. Its molecular
formula was C16H15NO5, as deduced from the UHPLC-ESI-QTOF/MS
protonated molecularion at m/z 300.0878 [M − H]− (calcd. For
C16H14NO5− 300.0877), requiring 10 degrees of unsaturation. The 13C
NMR and heteronuclear single quantum correlation (HSQC) spectra and
distortionless enhancement by polarization transfer (DEPT) 135 dis­
played signals of 16 C-atoms, including one methoxy group, one
carbonyl carbon, two CH2 groups, four aromatic methines, four O-
bearing C-atoms, and other four quaternary C-atoms. In the 1H NMR
spectrum of compound 1, signals at δH 7.54 (1H, s, H-11) and δH 6.67
(1H, s, H-8) indicated the presence of a 1,2,4,5-tetrasubstituted phenyl
ring; signals at δH 7.14 (1H, d, J = 4.3 Hz, H-1) and δH 6.93 (1H, d, J =
4.3 Hz, H-2) indicated the presence of a 1,2,3,5-tetrasubstituted phenyl
ring. In addition to the unsaturated H-atoms, the chemical shift at δH
3.83 (3H, s) was a typical methoxy signal. According to the hetero­
nuclear multiple bond correlation (HMBC) spectrum, the signal of H-6 at
δH 4.87 coincides with the water peak signal. And the H-6 linked to an N-
atom was observed at δH 4.87 (2H, H-6) and δH 3.17 (2H, dd, J = 4.4/
6.0 Hz, H-7). The data of 1H NMR (600 MHz, MeOD-d4) and 13C NMR
(150 MHz, MeOD-d4) were listed in Table 1. The HMBC correlations
(Fig. 2) from H-8 to C-9, C-10, C-11a, and H-11 to C-7a, C-9, C-10, C-11a
together with their respective carbons at δC 117.2 and δC 118.8
demonstrated that two hydroxyl groups located at C-9 and C-10 of a
benzene ring. On the other side of the molecule, the assignment of the
methoxy group was based on the Rotating Frame Overhauser Effect
Spectroscopy (ROESY) (Fig. 3) correlations of MeO-C2 with H-1 and H-
3. Therefore, it was possible to establish the presence of a 2-methoxy-4-
hydroxyphenyl according to the HBMC correlations from H-1 to C-3, C-
4a, 12a, and H-3 to C-1, C-4a, C-12a. Besides, C-7 close to C-8 and C-12
close to C-11 were confirmed by the HMBC correlations of H-7 with C-
7a, C-8, and C-11a and of H-11 with C-7a, C-11a, and C-12. The carbon
chemical shift in position 12 (δC 182.9) demonstrated that C-12 was a
carbonyl carbon. The N-CH2CH2 bond can be established based on the
proton and carbon chemical shifts in position 6 (δH 4.87/δC 48.6) and
the Correlation Spectroscopy (COSY) (Fig. 3) of H-6 with H-7. Therefore,
the 6, 7-dihydroazocin-12-one subunit was established based on their
HMBC correlations. Other correlations in COSY and ROESY spectra
further corroborate this structure. From the above analysis, the structure
of compound 1 was elucidated as 4,9,10-trihydroxy-2-methoxy-6,7-
dihydrodibenzo[b,e]azocin-12(5H)-one, and named oleracone L (1).
The biogenesis pathway of 1 was shown in Fig. S32. According to
speculation, compound 1, as a new skeleton alkaloid, may be originated
from the shikimate pathway to generate dopamine, and the dehydration
and acylation of dopamine and 2,3-dihydroxy-5-methoxybenzoic acid
structural element gave compound 1.
Compound 2 was obtained as a yellow powder, which turned orange
5
Fitoterapia 154 (2021) 105020
with Dragendorff reagent. The obvious absorption bands of the carbonyl
group (1610 cm− 1) and hydroxyl group (3438 cm− 1) were observed in
the IR spectrum. And the UV (MeOH) spectrum showed an absorption
maximum at 346 nm and 280 nm. Its molecular formula was C13H11NO3,
as deduced from the UHPLC-ESI-QTOF/MS protonated molecularion at
m/z 230.0812 [M + H]+ (calcd. For C13H12NO3+ 230.0812), requiring 9
degrees of unsaturation. The 13C NMR, HSQC, and DEPT 135 spectra
displayed the signals of 13 C-atoms, including one carbonyl carbon, two
CH2 groups, two aromatic methines, three olefinic methines, two O-
bearing C-atoms, and other three quaternary C-atoms. In the 1H NMR
spectrum of compound 2, signals at δH 7.42 (d, J = 8.0 Hz, H-7) and δH
7.73 (d, J = 8.0 Hz, H-8) indicated the presence of a 1,2,3,4-substituted
phenyl ring; the other three were cis-coupled olefinic H-atoms at δH 8.03
(m, H-1), δH 7.06 (dd, J = 4.0/2.4 Hz, H-2) and δH 8,13 (m, H-3). In
addition to the unsaturated H-atoms, the signals of a moiety linked to an
N-atom were observed at δH 5.12 (m, H-5) and δH 3.95 (m, H-6). The
data of 1H NMR (600 MHz, MeOD-d4) and 13C NMR (150 MHz, MeOD-
d4) were listed in Table 1. The HMBC (Fig. 2) correlations from H-7 to C-
8, C-9, C-10a, and H-8 to C-6a, C-9, C-10a demonstrated that two hy­
droxyl groups located at C-9 and C-10 of a benzene ring. On the other
side of the molecule, the COSY (Fig. 3) spectrum defined a proton
sequence H-1/H-2/H-3, and the proton and carbon chemical shifts in
position 3 (δH 8.03/δC 140.9) demonstrated that C-3 was linked to an N-
atom. Therefore, it was possible to establish a pyrrole subunit based on
their HMBC correlations from H-1, H-2, and H-3 with non-protonated
carbon of the ring. Besides, C-6 close to C-7, C-5 close to C-3, and C-
11 close to C-1 and C-10 were confirmed by the HMBC correlations of H-
6 with C-6a, C-7, and C-10a; of H-5 with C-3 and C-11a and of H-1 with
C-11, C-11a, and H-7 with C-11. The N-CH2CH2 bond can be established
based on the proton and carbon chemical shifts in position 5 (δH 5.12/δC
59.2) and the COSY of H-5 with H-6. Therefore, the 5, 6-dihydroazepin-
11-one subunit was established based on their HMBC correlations. Other
correlations in COSY and ROESY spectra (Fig. 3) further corroborated
this structure. From the above analysis, the structure of compound 2 was
elucidated as 9,10-dihydroxy-5,6-dihydro-11H-benzo[d]pyrrolo[1,2-a]
azepin-11-one and named portulacatone B (2).
It is speculated that compound 2 has two biosynthetic pathways in
Fig. S32 and Fig. S33. Compound 2 might be originated from the
deamination of dopamine produced by the shikimate pathway with 1H-
pyrrole-2-carboxylic acid structural element, followed by acylation. In
addition, compound 2 can be considered as an acylationproduct of
portulacaldehyde [29] after oxidation to the corresponding carboxylic
acid.
Compound 3 was obtained as a reddish-brown powder, which turned
orange with Dragendorff reagent. The obvious double peak absorption
bands of the aldehyde group (1711, 2922, and 2852 cm− 1) and hydroxyl
group (3349 cm− 1) were observed in the IR spectrum. And the UV
(MeOH) spectrum showed an absorption maximum at 452 nm and 297
nm. Its molecular formula was C14H13NO3, as deduced from the UHPLC-
ESI-QTOF/MS protonated molecularion at m/z 244.0969 [M + H]+
(calcd. For C14H14NO3+ 244.0969), requiring 9 degrees of unsaturation.
The 13C NMR and HSQC and DEPT 135 spectra displayed signals of 14 C-
atoms, including one aldehyde carbon, three aliphatic methylenes, two
aromatic methines, two olefinic methines, two O-bearing C-atoms, and
other four quaternary C-atoms. In the 1H NMR spectrum of compound 3,
signals at δH 6.62 (1H, s, H-7) and δH 6.63 (1H, s, H-10) indicated the
presence of a 1,2,4,5-substituted phenyl ring; the other two were cis-
coupled olefinic H-atoms at δH 6.08 (d, J = 4.0 Hz, H-1) and δH 6.91 (d, J
= 4.0 Hz, H-2). In addition to the unsaturated H-atoms, the signal of a
CH2 was observed at δH 3.99 (s, H-11), and the signals of two CH2 linked
to an N-atom were observed at δH 4.63(m, H-5) and δH 3.10 (m, H-6).
The data of 1H NMR (600 MHz, MeOD-d4) and 13C NMR (150 MHz,
MeOD-d4) were listed in Table 1. The HMBC (Fig. 2) correlations from H-
7 to C-9, C-10a, and H-10 to C-6a and C-8 together with their respective
carbons at δC 117.0 and δC 117.6 demonstrated that two hydroxyl
groups were located at C-8 and C-9 of a benzene ring. On the other side
X. Cui et al.
of the molecule, the assignment of the aldehyde group was based on the
ROESY (Fig. 3) correlations of CHO-C3 with H-2 and H-5. And the COSY
(Fig. 3) spectrum defined a proton sequence H-1/H-2; the HMBC cor­
relations of aldehyde hydrogen with C-3 and the proton and carbon
chemical shifts in position 3 (δC 133.6) demonstrated that C-3 was
linked an N-atom. Therefore, it was possible to establish a pyrrolo-3-
carbaldehyde subunit according to their HMBC correlations from H-1
to C-3 and C-11a; H-2 to C-3 and C-11a. Besides, C-5 close to C-3, C-6
close to C-7, and C-11 close to C-1 and C-10 were confirmed by the
HMBC correlations of H-5 with C-6a, C-11a, H-6 with C-6a, C-7, C-10a,
and H-11 with C-1, C-6a, C-10, C-10a, C-11a. The N-CH2CH2 bond could
be established based on the proton and carbon chemical shifts in posi­
tion 5 (δH 4.63/δC 48.7) and the COSY of H-5 with H-6. Therefore, the 5,
6, 11-trihydroazepine subunit was established based on their HMBC
correlations. Other correlations in COSY and ROESY spectra (Fig. 3)
further corroborated this structure. From the above analysis, the struc­
ture of compound 3 was elucidated as 8,9-dihydroxy-6,11-dihydro-5H-
benzo[d]pyrrolo[1,2-a]azepine-3-carbaldehyde and named portulacatal
(3).
Fig. S32 and Fig. S33 present two reasonable biogenesis pathways
for 3, respectively. The biogenesis of 3 might be divided from the
dehydration of dopamine produced by the shikimate pathway and 5-
(hydroxymethyl)-1H-pyrrole-2-carbaldehyde structural element.
Another biogenesis pathway precursor may be derived from portulaca­
tone A [14], which is produced by hydrolysis and reduction reaction to
finally obtain compound 3.
AD, a degenerative disease of the central nervous system that often
occurs in the elderly, is mainly manifested as a progressive decline in
memory and cognitive function, abnormal behavior, and social disor­
ders. Its main pathological feature is that extracellular β-amyloid (Aβ)
plaque deposition, intracellular neurofibrillary tangles, development,
and neuronal death [30,31]. The pathogenesis of AD is complex, and one
of the important pathogenesis was the loss of central cholinergic neu­
rotransmitters, which leads to memory decline in patients [32]. Studies
have shown that AChE is the key enzyme in the formation of AD; AChE
inhibitors are the main drugs for the treatment of AD, which can in­
crease the level of acetylcholine neurotransmitters in the brain and
improve the symptoms of patients. It has been reported that in addition
to direct neurotoxicity [33], there was a widespread inflammatory
response in the brains of AD patients. The main reason was that the
deposition of Aβ can further trigger the inflammatory response mediated
by microglia and astrocytes [34–36], secrete a large number of inflam­
matory factors, such as IL-1β, to promote neuronal apoptosis, and ulti­
mately lead to the nervous system disorders [37–39]. In addition, the IL-
1β was increased after NLRP3 inflammatory signaling pathway was
activated, also the activation of caspase-1 by the NLRP3 inflammasome
will cause pro-IL-1β to cleavage and release IL-1β, which in turn induces
UC that was also listed as one of the most difficult to cure diseases by the
World Health Organization as it is hard to heal, and easy to relapse with
the characteristics of a long course of the disease [25,40]. Therefore, the
anti-inflammatory activities of the three alkaloids isolated from
P. oleracea were investigated as the production of IL-1β is one of the
important causes of UC using RAW 264.7 cells induced by LPS as an
inflammatory cell model [41].
In this study, the result of the anticholinesterase activity assay sug­
gested that the three compounds exerted different levels of dose-
dependent inhibitory activity on AChE (Fig. S31, Supporting Informa­
tion). According to Table 2, the IC50 values of oleracone L (1), portu­
lacatone B (2), and portulacatal (3) were 46.00 μM, 50.49 μM, and
58.37 μM, meaning the anticholinesterase activity of oleracone L (1) was
the highest and was close to that of eserine at the same concentration.
After detection by CCK-8 assay (Fig. 4), it was found that the cell
viability of the three sample groups were not statistically different from
the LPS group, indicating that LPS induced RAW 264.7 cells within the
selected concentration range (5, 10, 25, and 50 μM) did not affect cell
viability and was not cytotoxic. Therefore, the three compounds at
6
Fitoterapia 154 (2021) 105020
concentrations of 1, 5, 10, and 20 μM were selected for a subsequent
experiment. Fig. 5 shows the result of the three compounds against the
production of IL-1β in LPS-induced RAW 264.7 cells. The three com­
pounds inhibited the secretion of inflammatory mediators in a dose-
dependent manner. Compared with the LPS group, oleracone L (1)
and portulacatone B (2) showed significant differences at 5 μM for IL-1β,
at the same time oleracone L (1) found no difference compared with the
control group indicating that it showed the highest activity of anti-
inflammative. In addition, portulacatal (3), which had similar effects
to the DEX-treated cells, showed significant differences compared with
the LPS group at 10 μM for IL-1β.
4. Conclusions
In the study, three novel alkaloids were obtained from P. oleracea.,
named oleracone L (1), portulacatone B (2), and portulacatal (3), whose
results of the bioactivities assay suggested that the three compounds all
exerted inhibitory activity on the production of AChE and IL-1β at the
different levels of dose-dependent, among which oleracone L (1) showed
the highest anti-inflammative bioactivity.
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was funded by the Project of the National Natural Science
Foundation of China (Grant No. 82073990).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fitote.2021.105020.
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