Journal of Functional Foods 106 (2023) 105601

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Aporphine alkaloids identified from Xylopia aethiopica and their potential

hypoglycemic and hypolipidemic activities
Ye Liu a, Yawen Li a, b, Felix Wambua Muema a, b, Hui Zhang a, b, Armel Jackson Seukep a, c,
Mingquan Guo a, b, *, 1
a
CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Department of Biomedical Sciences, Faculty of Health Sciences, University of Buea, P.O Box 63, Buea, Cameroon

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

Keywords: Xylopia aethiopica has been widely utilized as food condiments in West African countries, as well as traditional
Xylopia aethiopica medicine for diabetes, but the responsible components remain unknown. In this work, the probable active
Aporphine alkaloids components in the contributing fraction were screened using bio-affinity ultrafiltration strategy targeting
Bio-affinity ultrafiltration
α-glucosidase and pancreatic lipase. Then, 11 alkaloids were identified, 9 of which were aporphines, including a
Hypoglycemic
new one. Further enzyme inhibitory validation of these compounds discovered that compounds 1, 2, 6 and 11
Hypolipidemic
showed better α-glucosidase inhibition activity than positive control, among which compounds 1, 2 and 6
simultaneously possessed potent inhibition against pancreatic lipase. The hypoglycemic and hypolipidemic ca­
pacities of these screened compounds were verified by in vitro glucose consumption and lipid accumulation
HepG2 cell models. This work provided solid evidences for the anti-diabetic benefit of X. aethiopica, and several
aporphine alkaloids could be the responsible compounds for its promising hypoglycemic and hypolipidemic
activities.

1. Introduction
Xylopia aethiopica, belonging to the family Annonaceae, mainly dis­
tributes in the Savanna region of Africa (Yin et al., 2019). The fruits of
X. aethiopica, known as Ethiopian pepper, are widely used as a food
condiment in many local dishes, and is served as a pepper substitute
(Adefegha et al., 2018). In addition to its application in garnishing the
taste of foods, X. aethiopica is also traditionally used as a medicinal agent
for the treatment of malaria, fungal infections, uterine fibroid, female
infertility, and so on, which also have been proven by modern phar­
macological study (Erhirhie and Moke, 2014); (Yin et al., 2019). Defi­
nitely, the hypoglycemic benefits of X. aethiopica fruits have been
verified traditionally by its application alone or within poly-herbal
formulation in the treatment of diabetes (Diallo et al., 2012); (Ogbon­
nia et al., 2010). Emerging pharmacological research also disclosed that
the X. aethiopica fruits extracts could inhibit the glucose synthesis
related enzymes (Adeyeoluwa et al., 2020), delay carbohydrate
digestion (Akolade et al., 2018), and ameliorate the oxidative stress in a
type 2 diabetes mellitus (T2DM) rat model (Mohammed and Islam,
2017). Meanwhile, it was found to be able to decrease the lipids level
and produce a favourable serum lipid profile to exert hypolipidemic
effect (Adefegha et al., 2018); (Nwozo et al., 2011). However, most anti-
diabetes research focuses on the X. aethiopica fruit extracts’ activity
screening, while the exact functional components remain unknown.
It is well known that T2DM, a chronic metabolic disease character­
ized by persistent hyperglycemia, remains a major public health prob­
lem due to its wide damage to various organ systems leading to severe
diabetes complications (Olokoba et al., 2012). The mechanism of T2DM
has been recognized as insulin secretion dysfunction and multiple organ
insulin resistance, which lead to a series of pathological manifestations,
such as hyperglycemia and dyslipidemia (Galicia-Garcia et al., 2020).
Currently, many oral medications have been approved to manage the
disease situation, but diet and exercise remain basic effective means for
diabetes control (Marin-Penalver et al., 2016). Hence, more and more

* Corresponding author at: Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences,
Ningbo 315201, China.
E-mail address: guomingquan@nimte.ac.cn (M. Guo).
1
Current address: Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo
315201, China.

https://doi.org/10.1016/j.jff.2023.105601
Received 20 March 2023; Received in revised form 17 May 2023; Accepted 28 May 2023
Available online 2 June 2023
1756-4646/© 2023 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Liu et al.
scientific researchers set their sights on the anti-diabetes natural herbs
or edible plants to search for natural effective agents, which could have
more possibility, and security using as drugs or dietary supplements (Xu
et al., 2018).
Family Annonaceae is one of the main resources of aporphine alka­
loids, characterized by a tetracyclic biphenyl structure, which belongs to
isoquinoline alkaloids (Xin et al., 2018). The evidences for aporphine
alkaloids’ important role in the improvement of diabetes have been
accumulated a lot in the past. For example, the typical aporphine al­
kaloids, nuciferine and pronuciferine derived from a functional food
Nelumbo nucifera, could ameliorate glucose and lipid metabolism (Ma
et al., 2014), promote insulin secretion (Nguyen et al., 2012), and inhibit
the activity of α-glucosidase hence preventing diabetes (H. (Zhang et al.,
2022). The phytochemical studies on X. aethiopica have revealed that
this plant is rich in terpenoids, especially the volatile class components
in the essential oil, which contributes to making food attractive and
palatable (Obiloma et al., 2019). Most research interests in this plant lay
in the functions and components of fruits’ essential oil, while those
intended to the anti-diabetes activity were limited to aqueous fruit or
defatted extract (Mohammed and Islam, 2017); (Okwari et al., 2010).
Combined with our primary screening results, the primary segmentation
and activity screening disclosed the active fraction with the most po­
tential hypoglycemic and hypolipidemic activities might be attributed to
the enrichment of aporphine alkaloids. Therefore, the biologically active
compounds in this functional food were subsequently subjected to ul­
trafiltration with α-glucosidase and pancreatic lipase followed by the
phytochemical isolation and identification. At last, and the biological
activities of these identified compounds were further verified by enzyme
inhibition, HepG2 cell glucose consumption and triglyceride (TG)
lowering tests to explore the most potent compounds with hypoglycemic
and hypolipidemic activities, which revealed the chemical bases un­
derlying its traditional use against diabetes.
2. Materials and methods
2.1. Plant material
The air-dried fruits of X. aethiopica were collected from Cameroon by
Doctor Armel Jackson Seukep. The specimens (voucher specimen No.
20200901) were deposited in Plant Chemical biology laboratory of
Wuhan Botanical Garden, Chinese Academy of Sciences, and authenti­
cated by a senior taxonomist Professor Guangwan Hu from the same
institution.
2.2. Chemicals and reagents
The α-glucosidase and its reaction substrate p-Nitrophenyl α-D-glu­
copyranoside (p-NPG) were purchased from Aladdin Biochemical
Technology Company (Shanghai, China), p-Nitrophenyl palmitate (p-
NPP) and orlistat were bought in Macklin Biochemical Company
(Shanghai, China), acarbose and porcine pancreatic lipase and were
bought from Yuanye Bio-Technology Company (Shanghai, China). The
organic reagents in analytical grade used in the isolation work, Na2CO3
and other inorganic chemicals used in buffer solution preparation were
obtained from Sinopharm Chemical Reagent Company (Shanghai,
China), while the solvents (methanol and acetonitrile) in HPLC grade
were bought from TEDIA Company (Fairfield, Ohio, USA). The water
used for HPLC was prepared by Millipore Ultrapure Water System
(Billerica, Massachusetts, USA). Various chromatographic fillers, such as
200–300 mesh of silica gel from Qingdao Marine Chemical Company
(Qingdao, Shandong, China), Sephadex LH-20 gel (GE Healthcare,
Sweden), 50 µm size of ODS gel from YMC Co., Ltd. (Kyoto, Japan), were
used for compounds separation and purification.
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Journal of Functional Foods 106 (2023) 105601
2.3. Extraction and segmentation
The dried X. aethiopica fruits (5.2 kg) were shredded, and extracted in
75% methanol (25 L per time) by the multifunctional extraction appa­
ratus (YC-050, Shanghai Pilotech instrument and equipment Co., Ltd.,
Shanghai, China) at 50 ◦ C (60 min per time) under reduced pressure.
After being suspended in water, the concentrated extract was sequen­
tially fractionated with petroleum ether, dichloromethane, and ethyl
acetate.
The dichloromethane fraction, with the proper polarity and amount,
was subjected to an atmospheric silica gel column eluting with a
gradient of petroleum ether and dichloromethane from 1:0 to 0:1 to
obtain eight fractions A − H. Fraction F was then sequentially parti­
tioned by the medium pressure liquid chromatography (MPLC, Shanghai
Tauto Biotech Co., Ltd., TBP 5010, ODS C18, 50 µm), eluted with a
gradient of MeOH − H2O from 40% to 100% to afford three sub-
fractions F1 − F3 (XA-F1 − XA-F3).
2.4. In vitro α-glucosidase inhibition assay
The α-glucosidase inhibition ability was evaluated according to a
previous method reported by Hamid and coworkers with minor modi­
fications (Hamid et al., 2015). Briefly, 40 µL phosphate buffer saline
(PBS) at pH 6.8, 25 µL α-glucosidase solution in PBS (0.2 U/mL) and 10
µL test samples were mixed in a 96-well plate and incubated for 15 min
at 37 ◦ C. Then, the addition of 25 µL p-NPG (0.5 mM) initiated the re­
action followed by further incubation (15 min) at 37 ◦ C. Finally, the
addition of 100 µL Na2CO3 solution (0.1 M) quenched the reaction, and
the absorbance was taken on a Tecan Infinite M200 PRO Microplate
Reader (Männedorf, Switzerland) at 405 nm. As a positive check, acar­
bose was utilized. Four groups were set as a blank control (without
enzyme and sample), sample control (with sample only), 100% enzyme
activity control (with enzyme only) and test sample groups (with sample
and enzyme) in all the tests. The following computational formula was
used for α-glucosidase inhibition ability evaluation: inhibition rate % =
(ΔAc − ΔAs)/ΔAc × 100%, where the ΔAc means the absorbance differ­
ence value between 100% enzyme activity control and blank control
groups and the ΔAs means the absorbance difference value between test
sample and sample control groups. All of the tests were conducted in
triplicate.
2.5. In vitro pancreatic lipase inhibition assay
The pancreatic lipase inhibition ability test was carried out referring
to a previously reported method (H. (Zhang et al., 2022). In brief, the
reaction mixture, which was composed of 90 µL PBS (pH 7.4), 60 µL
pancreatic lipase in PBS (1.0 mg/mL), and 10 µL test samples, was added
in a 96-well plate, afterward incubated for 10 min (37 ◦ C). Subsequently,
40 µL substrate p-NPP (2.5 mM) for the enzyme was added to start the
reaction at 37 ◦ C, and the absorbance was read on a microplate reader at
405 nm after 20 min incubation. Orlistat served as the positive control.
The groups setting and inhibition rate calculation methods were the
same as the α-glucosidase inhibition assay section and all the tests were
performed in triplicate.
2.6. Screening for potential ligands by ultrafiltration and HPLC-UV
analysis
The procedure for ultrafiltration (UF) screening referred to the
method in the literature with appropriate adjustment (Chen et al.,
2020). In short, 100 µL XA-F2 (F2 fraction of dichloromethane extract
from X. aethiopica fruits) solution (2.0 mg/mL in PBS) was pre-incubated
for 30 min with α-glucosidase (4 U in 100 µL pH 6.8 PBS) or pancreatic
lipase (0.2 mg in 100 µL pH 7.4 PBS) enzyme solution at 37 ◦ C,
respectively. In the meantime, the sample with inactivated α-glucosi­
dase or pancreatic lipase, which had been boiled in water for 15 min,
Y. Liu et al.
parallelly performed as the negative control. Then, the combinations
were moved to the 0.5 mL ultrafiltration tubes (Millipore) with 30 kD
membrane size (α-glucosidase) or 10 kD (pancreatic lipase) then
centrifugation for 10 min (10000 rpm). Afterward, the filtrate was dis­
carded, while the residuum on the membrane was washed and centri­
fuged by 200 µL PBS three times to eliminate the uncombined or
nonspecific binding compounds. Next, MeOH (200 µL) was added and
incubated for 10 min to release the binding ligands for these two en­
zymes. After three times repetitions and centrifugation at 10000 rpm
(10 min), these filtrates were dried and dissolved in 50 µL MeOH for the
HPLC-UV analysis.
The UF sample analysis was implemented on an Agilent 1220 HPLC
system fitted with a Waters column (C18, 3.5 µm, 150 × 4.6 mm, Mil­
ford, Massachusetts, USA) and 280 nm was chosen as the UV wave­
length. 10 µL of the prepared sample was injected and eluted by the H2O
(A) containing 0.1% (v/v) formic acid and ACN (B) as follows: 20%−
45% B in 0–15 min, 45%− 60% B in15 − 35 min, 60%− 85% B in 35–45
min with the flow rate of 0.8 mL/min.
2.7. Isolation and identification
Based on the previous enzyme inhibition screening results, the XA-F2
fraction was subjected to column chromatography (Sephadex LH-20)
washing by MeOH to divide into five sub-fractions F21 − F25 according
to the LC analysis under the UF-LC conditions as mentioned above. The
separation conditions of target compounds screened from UF-LC were
purposefully determined by comparing them with UF-LC chromatogram
under the same analytical conditions. Based on this tracking method, F21
was put through the semi-preparative column (PolyPak, 5 µm C18, 250
× 10 mm, Zhejiang Fuli Analytical Instruments Co., Ltd., Zhejiang,
China, flow rate: 3.0 mL/min) on a Qingbohua P2050 system (Beijing
Qingbohua Science and Technology company, China) eluting with 55%
MeOH − H2O to obtain compounds 1 (15.4 mg), 2 (9.2 mg) and 11 (1.4
mg), and collected impure peak 2 was further purified on an Agilent
1100 machine (Santa Clara, California, USA) with a YMC-Pack column
(C18, 250 × 10 mm, 5 µm, YMC, Tokyo, Japan), eluting with 30% ACN
− H2O supplied with 0.1% FA (2.0 mL/min) to obtain compound 8 (2.4
mg). The F22 sub-fraction was segmented by semi-preparative HPLC
(58% MeOH − H2O, 3.0 mL/min) and subsequently purified on the
Agilent 1100 system (25% ACN − H2O with 0.1% FA, 2.0 mL/min) to get
compounds 3 (3.9 mg) and 5 (2.4 mg). Then F23 was chromatographed
over the semi-preparative HPLC system with an elution of 45% MeOH −
H2O (3.0 mL/min) to get compound 4 (2.0 mg). Fraction F24 was
separated by the HPLC with elution of 60% MeOH − H2O (flow rate: 3.0
mL/min) to yield compounds 6 (4.5 mg) and 10 (3.5 mg). Subraction F25
was chromatographed successively on Qingbohua P2050 system (50%
MeOH − H2O, 3.0 mL/min) and Agilent 1100 system (30% ACN − H2O
with 0.1% FA, 2.0 mL/min) to yield compounds 7 (1.5 mg) and 9 (2.0
mg).
The chemical structures of the above isolated compounds were
identified by their 1D (1H NMR, 13C NMR, and DEPT 135 spectra) and
2D NMR (1H–1H COSY, HMBC, HSQC, and NOESY spectra), which were
analyzed by a 600 MHz NMR spectrometer (Bruker Avance, Karlsruhe,
Germany). The HR-ESI-MS data were acquired on an Aglient 1290 in­
finity II 6530C Q-TOF mass spectrometer (Santa Clara, CA, USA) using a
Waters ACQUITY C18 UPLC column (1.7 µm, Milford, MA, USA) under
the UF-LC chromatographic condition as mentioned before. The optical
rotations were carried out on a polarimeter and the UV spectra were
tested by a Lambda 35 UV spectrometer (PerkinElmer, Waltham, Mas­
sachusetts, USA).
2.8. Cytotoxicity assessment of selected compounds on HepG2 cells
Before the further cellular experiments, the cytotoxicity of these
compounds on HepG2 cells was assessed in advance. 100 µL cell sus­
pension (1 × 104 cells) were seeded in 96-well plate, 12 h later, the cell
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Journal of Functional Foods 106 (2023) 105601
supernatant was changed to culture medium (DMEM added with 10%
fetal bovine serum and 1% penicillin–streptomycin mixture solution,
Hyclone, UT, USA) with compound solutions in the final concentration
of 50 and 100 µM. 24 h later, the cell supernatant was discarded and
DMEM with 10% CCK8 reagent (Beyotime, Shanghai, China) was added,
and then the absorbance was measured at 450 nm after keeping out of
the light for 1 h.
2.9. The glucose consumption capacity test
The glucose consumption capacity was evaluated according to the
reported method (H. (Zhang et al., 2022). Briefly, 100 µL cell suspension
(8 × 103 cells) were seeded in 96-well plate for adherence for 12 h, then
the cell supernatant was replaced by DMEM without serum to starve the
cells for another 12 h. Afterwards, the cell supernatant was discarded
and low glucose DMEM medium with sample solution was added. After
incubation for 24 h, the supernatant was taken for centrifuging for 10
min at 1000 rpm, then the upper layer was used for glucose content test
by glucose determination kit (Nanjing Jiancheng Bioengineering Insti­
tute, China) at 505 nm. The metformin or medium without sample were
used as positive or negative control, respectively.
2.10. TG content determination in OA/PA induced HepG2 lipid
accumulation model
HepG2 cell suspension was seeded in 12-well plate (1 × 105cells/1
mL), and the culture medium with sample solution and 0.125 mM free
fatty acid composed of oleic acid (OA) and palmitic acid (PA) in a ratio
of 2:1 was added to replace the primary one 12 h later. After 24 h in­
cubation, the cells were digested by 0.25% trypsin (Hyclone, UT, USA)
and centrifuged for 10 min at 1000 rpm. Then the cell sediment was
lysed by radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime,
Shanghai, China) for 1 h and the supernatant was used for triglyceride
(TG) content determination using TG content assay kit (Nanjing Jian­
cheng Bioengineering Institute, China). The remaining supernatant was
centrifuged by 12000 rpm for 15 min at 4 ◦ C to obtain the upper layer for
protein content assay by bicinchoninic acid (BCA) method (Beyotime,
Shanghai, China). The TG content was expressed as the content of tri­
glyceride per gram of protein and orlistat was used as positive control.
2.11. Statistical analyses
All activities tests were carried out at least three times and were
manifested as means ± standard deviation (SD). The IC50 values were
calculated by the log (inhibitor) vs. nor-malized response–variable slope
method in GraphPad Prism 5 software. The statistically significant dif­
ference were analyzed by one-way ANOVA multiple comparisons of
Duncan test at the significance level of p < 0.05 (n = 3) in SPSS Statistics
26.0 software or one-way ANOVA Newman-Keuls multiple comparison
test by GraphPad Prism 5 software. In addition, the NMR data were
analyzed by MestReNova, the chemical structures were drawn by
ChemDraw 14.0, and the ultrafiltration-HPLC chromatograms were
drawn by OriginPro 9.0 software, respectively.
3. Results and discussion
3.1. The inhibition effects of XA-F2 fraction on α-glucosidase and
pancreatic lipase
α-Glucosidase is a carbohydrate hydrolytic enzyme presented in the
small intestine devoted to hydrolyzing poly- or oligosaccharides to
monosaccharides, which results in the increase of blood glucose con­
centration (Papoutsis et al., 2021). The inhibitors against α-glucosidase
could reduce the speed of digestion and absorption of carbohydrates to
maintain the postprandial blood glucose at a relatively lower level
(Abbas et al., 2017). Pancreatic lipase is one of the most important
Y. Liu et al.
enzymes in the lipolysis procedure of dietary lipids to release free fatty
acid, the excessive accumulation of which leads to dyslipidemia and
obesity, the pathologic abnormality and major risk factor of T2DM
(Rajan et al., 2020). Reducing lipid absorption by inhibiting lipases is an
efficient approach to the development of hypolipidemic drugs. Accord­
ing to the above understanding, α-glucosidase and pancreatic lipase
enzymes have been regarded as therapeutic targets for diabetes man­
agement, and the natural agents with inhibition effects on these two
enzymes also have attracted attention of researchers.
In this study, the aqueous methanol extract was successively defatted
through partitioned extraction by organic solvents with different po­
larities and normal phase silica gel column chromatography to remove a
large amount of oil. Therein, the sub-fraction F was further chromato­
graphed, and three fractions XA-F1 − XA-F3 were obtained. Considering
the crucial role of α-glucosidase and pancreatic lipase played in the
development of diabetes, the inhibiting activities of these fractions were
evaluated and compared.
As shown in Fig. 1, the XA-F2 fraction exhibited promising dose-
dependent inhibitory effects on α-glucosidase (IC50 141.5 ± 10.8 μg/
mL) (Fig. 1a), which was lower than acarbose (196.4 ± 27.6 μg/mL), a
glucosidase inhibitor severed as the positive control, while the other two
fractions did not show any activities. Meanwhile, this fraction also
showed best inhibition effect on pancreatic lipase with the IC50 value of
204.7 ± 21.0 μg/mL (Fig. 1b) compared with XA-F1 fraction (IC50 value:
362.8 ± 73.4 μg/mL) and XA-F3 fraction (the inhibition rate was 40.4%
at maximum detected concentration 500 μg/mL), along with the posi­
tive control possessing 0.048 ± 0.006 μg/mL. Similar screening has
done to compare the α-glucosidase inhibition activity of X. aethiopica
extracts obtained by different extraction methods (Adeyeoluwa et al.,
2020), while the best result was from the aqueous ethanol extract with
the IC50 value of 559.9 ± 44.9 μg/mL, which was much higher than that
of XA-F2. The above evidences indicated that the chromatographed
enrichment method removed the inactive or less active substances suc­
cessfully, and the XA-F2 fraction might be the target functional fraction
to find effective enzyme ligands for the α-glucosidase and pancreatic
lipase.
3.2. Screening for the potential α-glucosidase and pancreatic lipase
ligands from XA-F2 fraction by ultrafiltration
In consideration of the simultaneous inhibitory effects on α-gluco­
sidase and pancreatic lipase, the XA-F2 fraction was subjected to rapid
and preliminary screening for potential enzyme ligands by affinity
ultrafiltration-HPLC method. Affinity ultrafiltration is a membrane
technique with a certain molecular weight cut-off to separate target-
ligand complexes based on the interaction between small molecules
and biomacromolecules. And it is an efficient tool for high throughput
screening bioactive components from complex mixtures (Wei et al.,
2016). In this work, the XA-F2 fraction was incubated with the active
and denatured α-glucosidase or pancreatic lipase to generate enzyme-
Fig. 1. The inhibiting effects of XA-F2 fraction on α-glucosidase (a) or pancreatic lipase (b).
4
Journal of Functional Foods 106 (2023) 105601
compound complexes, which were subsequently intercepted by an ul­
trafiltration membrane with a certain pore size. The complexes were
unbound by incubating with methanol and the released ligands were
then analyzed by HPLC.
Accordingly, the XA-F2 original sample solution and compounds
released from the binding complex produced by ligands and active or
denatured enzymes were analyzed by HPLC, respectively. As shown in
Fig. 2, eleven peaks, speculated as alkaloids based on their molecular
weight preliminarily (Table 1), were listed as the main ingredients of
XA-F2, and seven of them were screened out in the α-glucosidase based
bio-affinity ultrafiltration, while six of them appeared in the pancreatic
lipase experiment. The relative affinity abilities between ligands
screened out and enzymes were described by the enrichment factor (EF)
value (Liu et al., 2021), which was defined as EF (%) = (As – Ac)/A0 ×
100%, wherein As, Ac and A0 represent the peak areas acquired from the
incubation of XA-F2 solution with activated, denatured and without
tested enzyme, respectively. Accordingly, the EF values of these seven
peaks screened out for α-glucosidase were calculated and listed in
Table 1, among which peak 11 possessed the highest EF value of 6.07%,
indicating its best binding affinity with α-glucosidase, followed by peak
2 (2.46%), peak 6 (2.39%) and peak 1 (2.20%). As for pancreatic lipase
(Table 1), six peaks were screened out and the best three ones possessed
close EF values displaying as 3.92% for peak 2, 3.63% for peak 1 and
3.40% for peak 6, followed by peak 3 (2.43%) and peak 8 (2.39%). To
some extent, the EF values reflected the affinity capacities of these
compounds with target enzymes, the differences between which might
be attributed to the competitive interactions of these active ingredients
with enzymes (Chen et al., 2019), and the experimental verifications
were quite essential to reveal the exact inhibitory abilities of these li­
gands against α-glucosidase and pancreatic lipase.
3.3. UF-HPLC guided isolation and structure elucidation of enzyme
ligands
Guided by the UF-HPLC results, the XA-F2 fraction was segmented
and purified by layers of progressive chromatography to obtain 11 al­
kaloids (1–11), corresponding to the UF-HPLC-UV results displayed in
Table 1 and Fig. 2. The structures of these alkaloids were mainly
determined by comprehensive 1D and 2D NMR analyses with the aid of
other spectral analytical methods. To the best of our knowledge, com­
pound 6 was an undescribed structure first found in this work, com­
pounds 2, 4, 5 and 7–9 were first isolated from Xylopia genus, and
compounds 10–11 were isolated from X. aethiopica for the first time,
which have been found in X. lemurica and X. ferruginea, respectively
(Nieto et al., 1975); Nik Abdullah (Zawawi and N. K., Ahmat, N.,
Ahmad, R., Jaafar, F. M., & Ab Ghani, N, 2012), while compounds 1 and
3 have already been reported in X. aethiopica (Harrigan et al., 1994). The
brief structure elucidation of compounds 6–8 was shown as follows.
Compound 6 was isolated as a yellowish-brown solid and its mo­
lecular formula was determined as C20H21NO5 according to the 13C NMR
Y. Liu et al. Journal of Functional Foods 106 (2023) 105601

Fig. 2. The ultrafiltration-HPLC chromatograms of XA-F2 fraction without ultrafiltration (black line), ultrafiltration with activated (red line), and inactivated (blue
line) α-glucosidase or pancreatic lipase, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

Table 1
Identification of potential α-glucosidase or pancreatic lipase ligands in the XA-F2 fraction.
Peak No.a Rt (min) EF (%)b for α-glucosidase EF (%)b for pancreatic lipase [M + H]+ (m/z) Compound namec

1 13.85 2.20 3.63 276.0669 Liriodenine

2 16.12 2.46 3.92 336.0881 Oxonantenine
3 17.56 0.91 2.43 292.0978 Lysicamine
4 17.80 – – 256.1357 N-benzoyl-L-phenylalaninol
5 18.20 – – 308.0940 Peruvianine
6 18.40 2.39 3.40 356.1487 N-formyllaurotetanine
7 18.80 – – 370.1671 N-Acetyllaurotetanine
8 18.99 1.65 2.39 354.1327 N-Acetylnordomesticine
9 19.38 1.14 0.76 268.1351 N-trans-cinnamoyltyramine
10 19.92 – – 306.0753 Lanuginosine
11 20.30 6.07 – 306.0771 Atherospermidine
a
The peak No. and Retention time was corresponding to LC results.
b
Enrichment factor.
c
Identified by comparing with the corresponding isolated pure compounds.

data and HR-ESI-MS peak at m/z 733.2733 [2 M + Na]+ (calcd for
733.2738). The signals in the 1H NMR spectrum (Table 2 and Fig. S2)
were attributed to an aldehyde hydrogen at δH 8.21, three aromatic
singlet hydrogens at δH 7.93 (1H, s, H-11), 6.80 (1H, s, H-3) and 6.70
(1H, s, H-8), one methine at δH 4.60 (1H, dd, J = 13.6, 4.3 Hz, H-6), three
methylene groups [δH 2.77 (2H, m, H-4); 3.91 (1H, 1H, dd, J = 12.8, 2.3
Hz, H-5) and 3.28 (1H, m, H-5); 2.73 (1H, dd, J = 13.6, 4.3 Hz, H-7) and
2.61 (1H, t, J = 13.6 Hz, H-7)], three methoxy groups at δH 3.81 (3H, s,
2-OCH3), 3.79 (3H, s, 10-OCH3), 3.60 (3H, s, 1-OCH3), with the assist of
HSQC signals (Fig. S5). The 13C NMR spectrum (Table 2 and Fig. S3),
with the aid of the DEPT 135 spectrum (Fig. S4), showed twenty carbons
consisting of a N-formyl carbon at δC 162.3, twelve aromatic carbons

5
Y. Liu et al. Journal of Functional Foods 106 (2023) 105601

Table 2
1
H NMR and 13C NMR spectroscopic data for the major isomers of compounds 6-8.a
6a 7a 8a

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

1 144.2, C 146.1, C 150.7, C

1a 127.0, C 128.9, C 128.4, C
2 151.9, C 153.6, C 145.2, C
3 6.80, s 111.2, CH 6.79, s 111.8, CH 6.66, s 115.7, CH
3a 129.3, C 130.9, C 131.2, C
4 2.77, m (overlap) 30.3, CH2 2.79, m 31.4, CH2 2.82, m (overlap) 31.1, CH2
2.74, m 2.69, m (overlap)
5 3.91, dd (12.8, 2.3) 41.0, CH2 4.13, dd (12.8, 2.3) 43.2, CH2 4.10, dd (12.9, 1.8) 43.3, CH2
3.28, m (overlap) 2.90, td (14.3, 12.8, 4.3) 3.28, dd (12.9, 2.4)
6 4.60, dd (13.6, 4.3) 48.8, CH 4.95, m (overlap) 52.3, CH 4.89, dd (13.6, 4.2) 52.2, CH
6a 123.8, C 126.3, C 125.3, C
7 2.73, dd (13.6, 4.3) 33.0, CH2 2.84, dd (13.5, 4.2) 34.3, CH2 2.88, dd (13.6, 4.2) 35.0, CH2
2.61, t (13.6) 2.68, t (13.5) 2.69, m (overlap)
7a 129.5, C 131.6, C 132.5, C
8 6.70, s 115.4, CH 6.73, s 116.2, CH 6.78, s 109.6, CH
9 146.3, C 147.6, C 148.2, C
10 146.1, C 147.4, C 148.2, C
11 7.93, s 112.4, CH 8.08, s 113.4, CH 7.93, s 109.2, CH
11a 122.1, C 124.3, C 126.4, C
N-CHO 8.21, s 162.3, C
N-COCH3 172.0, C 172.1, C
2.24, s 22.3, CH3 2.23, s 22.3, CH3
1-OCH3 3.60, s 59.3, CH3 3.66, s 60.2, CH3
2-OCH3 3.81, s 55.8, CH3 3.91, s 56.6, CH3 3.57, s 60.2, CH3
10-OCH3 3.79, s 55.7, CH3 3.89, s 56.4, CH3
9,10-OCH2O 5.98, s 102.4, CH2
a
Data (δ) were measured in DMSO‑d6 for 6, in CD3OD for 7 and 8 by 600 MHz for 1H NMR and 150 MHz for 13C NMR. The data listed here were belonged to the
major isomers 6a, 7a and 8a, and data for 6b, 7b and 8b could be found in Table S1 in supplementary materials.

contributing to two benzene rings (δC 111.2–151.9), one methine carbon
signal at δC 48.8, three methylene (δC 41.0, 33.0, 30.3) and three
methoxys (δC 59.3, 55.8, 55.7), corresponding to the proton signals. The
HMBC cross-peaks from H-3 to C-2 and C-4, H-4 to C-3a and C-6a, H-6 to
C-1a, C-6a and N-CHO, and H-7 to C-7a and C-11a (Fig. 3 and Fig. S7),
combined with the 1H− 1H COSY correlations of H-4/H-5 and H-6/H-7
(Fig. 3 and Fig. S6), indicated that compound 6 possessed an aporphine
alkaloid skeleton. The three methoxy substituents attached to the ben­
zene rings were determined by the HMBC correlations from OCH3 to C-1,
C-2 and C-10, respectively, while the remaining oxygenated aromatic
carbon at δC 146.3 substituted by a hydroxyl was deduced by the mo­
lecular weight of 6. The above characteristic data were quite similar to
those of the known compound laurotetanine (Costa et al., 2013) except
for the extra signals at δH 8.21 and δC 162.3, which were disclosed
belonging to the N-formyl group by further analysis of the HMBC
spectrum of 6, which showed the correlations from N-formyl to C-5 and
C-6. From the above findings, the planar structure of 6 should be N-
formyl substituted laurotetanine.
Interestingly, with a deep-going analysis of the NMR data, compound
6 exhibited two complete sets of signals with an approximate ratio of 2:1
calculated by the 1H NMR signal integrals. The major isomer was labeled
as 6a, while the minor one was marked as 6b (Fig. 3), and in consid­
eration of the similar profile of 6b (Table S1), the planar structure
elucidation above was based on the data of 6a. This phenomenon is
attributed to the partial double-bond property of the amide bond (Wang
et al., 2020). In the NOESY spectrum (Fig. S8), the aldehyde hydrogen of
6a (δH 8.21) correlated to H-5 (δH 3.91), while that of 6b (δH 8.34)
correlated to H-6 (δH 4.48), which indicated that the amide bond of 6a
was Z configuration and 6b was E configuration (Wang et al., 2020).
Meanwhile, the upfield moving of H-6 from δH 4.60 (6a) to δH 4.48 (6b)
and the downfield moving of CHO from δH 8.21 (6a) to δH 8.34 (6b)
were also consisted with the manners of reported isomers (Wang et al.,
2020). In addition, the absolute configuration of C-6 was determined as
S due to the positive optical rotation value of 6 (Wang et al., 2020).
Thus, compound 6 was named as N-formyllaurotetanine.
The 1D NMR data of compound 7 (Table 2 and Fig. S11–S12) was
quite similar to that of 6, and 7 was first and only mentioned in literature
reported in 1965 (Pachler et al., 1965) without any referable data, so
here was a supplement for its NMR data and isomer manner. The major
structure of 7 was also laurotetanine, along with the extra signals of N-
COCH3 at δH 2.24 (3H, s) and δC 172.0, 22.3, so the planar structure of 7
was determined as N-acetyl substituted laurotetanine. The 1D NMR of 7
displayed the same isomer manner as 6, with a pair of amide bond
isomers with the ratio of 3:1. The data of 7a and 7b was shown in Table 2
and Table S1, and compound 7 was accordingly named as N-
acetyllaurotetanine.
Compound 8 could be found in the Scifinder database, but without
any data and reference, so here gave the brief structure elucidation and
data supplement for it. The 1D NMR data of compound 8 was similar to
that of 7, except for the absence of two methoxy groups and the emer­
gence of a methylenedioxy group at δH 5.98 (2H, s) and δC 102.4 (Table 2
and Fig. S15–S16). The HMBC correlations (Fig. 3 and S18) of OCH3/C-2
and OCH2O/C-9, C-10 revealed that the remaining methoxy at δH 3.57
(3H, s) and δC 60.2 was attached to C-2 and the methylenedioxy group
was attached to C-9 and C-10. The substituent of hydroxyl at the
oxygenated C-1 (δC 150.7) was deduced by the molecular formula of 8,
which was determined by ion peak at m/z 729.2413 [2 M + Na]+ (calcd
for 729.2424) in HR-ESI-MS and 13C NMR. Based on the above analysis,
the planar structure of 8 was elucidated as N-acetyl substituted nordo­
mesticine (Indra et al., 2002). Similarly, the NMR data of 8 could be
separated into two sets with a ratio of 3:1 (Table 2 and Table S1), the
isomer manner of which was the same as that of 6. Consequently,
compound 8 was named as N-Acetylnordomesticine.
The other 8 known compounds yielded from XA-F2 were identified
as liriodenine (1) (Z. Z. (Zhang et al., 2002), Oxonantenine (2) (Teles
et al., 2015), lysicamine (3) (Teles et al., 2015), N-benzoyl-L-phenyl­
alaninol (4) (Barrow and Sun, 1994), peruvianine (5) (Menachery and
Cava, 1981), N-trans-cinnamoyltyramine (9) (Yang et al., 2002), lanu­
ginosine (10) (Nakano et al., 2013), atherospermidine (11) (Costa et al.,
2011), respectively, based on the careful analysis of their NMR data and
comparison with the reported spectroscopic data.
Data for N-formyllaurotetanine (6): tawny solid, [α]20 D + 325 (c 0.4,

6
Y. Liu et al. Journal of Functional Foods 106 (2023) 105601

Fig. 3. (a) Alkaloids 1–11 isolated from X. aethiopica (*new compound). (b) Key 1H− 1H COSY and HMBC correlations of compounds 6 and 8. (b) Isomers of
compounds 6–8.

MeOH); UV (MeOH), see Fig. S9, λmax (log ε) 215 (4.69), 274 (3.99), 302 ligands screened out by UF-HPLC, the in vitro inhibitory effects on
(3.67) nm; data for 1H NMR (600 MHz) and 13C NMR (150 MHz) in
DMSO‑d6 were listed in Table 2 and Table S1.
Data for N-Acetyllaurotetanine (7): white power; [α]20 D + 307 (c 0.1, Table 3
The α-glucosidase or pancreatic lipase inhibition activities of compounds 1–11.
MeOH); UV (MeOH), see Fig. S13, λmax (log ε) 214 (4.57), 282 (4.03),
305 (3.89) nm; data for 1H NMR (600 MHz) and 13C NMR (150 MHz) in Compound No. α-glucosidase pancreatic lipase
CD3OD were listed in Table 2 and Table S1. IC50 ± SD (µM) IC50 ± SD (µM)
Data for N-Acetylnordomesticine (8): white power; [α]20 D + 330 (c
1 148.8 ± 7.3b 306.2 ± 17.5b
0.2, MeOH); UV (MeOH), see Fig. S19, λmax (log ε) 217 (4.67), 284 2 193.3 ± 8.2c 358.2 ± 83.7bc
(4.01), 310 (4.23) nm; data for 1H NMR (600 MHz) and 13C NMR (150 3 – 421.2 ± 90.4c
MHz) in CD3OD were listed in Table 2 and Table S1. 6 115.6 ± 22.6ab 294.2 ± 49.0b
11 89.1 ± 3.7a –
Positive control Acarbose Orlistat
3.4. The inhibition effects validation of the isolated ligands on 304.2 ± 42.8d 0.096 ± 0.013a
α-glucosidase and pancreatic lipase a–d
Means the significant difference between these compounds and positive
control by one-way ANOVA Duncan’s multiple range test (DMRT) at the level of
To further validate the actual enzyme inhibition potential of the p < 0.05 (n = 3).

7
Y. Liu et al.
α-glucosidase and pancreatic lipase were tested for all the isolated
compounds (1–11). As the results shown in Table 3, compounds 1, 2, 6
and 11 possessed significant α-glucosidase inhibition activities, all the
IC50 values of which were lower than that of the oral hypoglycemic
α-glucosidase inhibitor acarbose, and showed statistical difference at the
level of p < 0.05, while other compounds didn’t exhibit more than 50%
inhibition rate at the maximum tested concentration. Among the iso­
lated compounds, 1, 3 and 9 have been reported for their α-glucosidase
inhibition activities, but only 1 showed potent inhibitory activity
(Hamid et al., 2015), which was identical to the screening results in this
work. For pancreatic lipase, compounds 1, 2, 3 and 6 were verified to
exhibit promising inhibition effects (Table 3), and all the compounds
were first reported for their lipase inhibitory abilities. Although the IC50
values were much higher than the positive control orlistat, that also
made sense for natural unmodified molecules and possible simultaneous
inhibition of multiple T2DM targets.
Comparing the results of the activity validation with those of UF-LC
screening, we found that the peaks screened out with higher EF values in
the ultrafiltration experiment did have better inhibitory activities. Peak
11, which possessed the distinguishing highest EF value of 6.07%,
showed the lowest IC50 value of 89.1 ± 3.7 against α-glucosidase, fol­
lowed by peaks 1, 2 and 6 with comparatively lower EF values and
higher IC50 values, while peaks missing in the UF experiment or with
lower EF values did not show any potential in the enzyme inhibition
assays. A roughly similar trend was also observed in the pancreatic
lipase verification assays, as expected, the best three compounds 1, 2
and 6 with relatively close EF values were revealed to have excellent
lipase inhibition capacities. The above results confirmed the enzyme
Fig. 4. (a) The cytotoxicity of compounds on HepG2 cells (***means p < 0.001). (b) The effects of compounds 2, 6, 11 (100 μM) and positive control metformin (1
mM) on glucose consumption of HepG2 cells (***means p < 0.001 compared with the blank group). (c) The effects of compounds 1, 2, 3, 6 (50 μM) and positive
control metformin (20 μM) on TG production in OA/PA induced HepG2 lipid accumulation cell model (###means p < 0.001 compared with the blank group,
***means p < 0.001, **means p < 0.01 compared with the model group).
8
Journal of Functional Foods 106 (2023) 105601
inhibitors screening efficiency of the ultrafiltration method from com­
plex naturally derived samples and provided several potential aporphine
alkaloids ligands for T2DM.
3.5. The active ligands promoted glucose consumption in HepG2 cells
The most direct manifestation of diabetes is hyperglycemia, which is
closely associated with the deficiency of glucose consumption. Anti-
diabetes agents take effects through different mechanisms of action,
but most of them could affect glucose consumption, including insulin
and metformin (Shi et al., 2019). In this study, HepG2 based glucose
consumption assay was used to verify the in vitro hypoglycemic effect of
α-glucosidase enzyme inhibitors screened out (compounds 1, 2, 6 and
11). Firstly, the cytotoxicity of these compounds were evaluated at the
concentration of 50 and 100 μM (Fig. 4a), as a result, the cell viability of
HepG2 treated with compounds 2, 6 and 11 were all over 90% in the two
tested concentrations, except that compound 1 showed some toxicity to
HepG2 cells at 100 μM (cell viability was lower than 80%). As shown in
Fig. 4b, the glucose content in the groups treated with 100 μM of com­
pounds 2, 6 and 11 significantly increased glucose consumption, and
compounds 6 and 11 showed greater efficacy than that of hypoglycemic
agent metformin with a much higher concentration of 1 mM. In
consideration of the toxicity of compound 1, same work was also per­
formed at 50 μM, but it turned out that only compound 11 exhibited an
effect of increasing glucose consumption (Fig. S20). In conclusion,
compounds 2, 6 and 11, which possessed better α-glucosidase inhibition
capacity than positive control, were confirmed for their great potenti­
ality in glucose-lowering effect.
Y. Liu et al.
3.6. The active ligands reduced TG production in OA/PA induced HepG2
lipid accumulation model
The elevation of free fatty acid level emerged in many metabolic
diseases, like obesity and diabetes, which will lead to insulin resistance
in insulin target organs, including liver. The mixture of OA and PA
induced lipid accumulation HepG2 cell model was used to evaluate the
lipid lowering effects of selected compounds. As shown in Fig. 4a,
compounds 1, 2, 3 and 6, lipase inhibitors screened out in XA-F2,
exhibited no significant cytotoxicity on HepG2 cells at 50 μM, which
was consequently chosen as the screening concentration. In Fig. 4c, OA/
PA induced a marked growing level of TG content in the model group,
which was significantly decreased by the treatment of compounds 1, 2,
3, 6 and positive control orlistat (20 μM). The above results confirmed
the lipid-lowering effects of these pancreatic lipase inhibitors screened
out.
4. Conclusion
Many studies have confirmed the effective role X. aethiopica played
in diabetes, but the responsible components remained unknown. Hence,
in this work, polarity-guided segmentation along with α-glucosidase and
pancreatic lipase-based enzyme inhibition screening ascertained the
active fraction XA-F2, which was further subjected to UF-LC, disclosing
the excellent bio-affinity of aporphine alkaloids with α-glucosidase and
pancreatic lipase. UF-LC guided phytochemical isolation led to the un­
equivocal identification of 11 alkaloids (1–11), including a new one (6),
among which compounds 1, 2, 6 and 11 exhibited potent α-glucosidase
inhibitory activities better than acarbose, while compounds 1, 2, 3 and 6
showed potential suppressing effects on pancreatic lipase. Meanwhile,
their glucose consumption and lipid-lowering capacity were validated,
and compounds 2 and 6 showed concurrently glucose consumption
increasing and TG production reducing capacity, which might be
promising anti-diabetes agents.
In conclusion, dietary food or condiments contains numerous sec­
ondary metabolites, which attributes to their health benefits beyond
their nutritional function, particularly in favor of those diseases related
to metabolism, such as obesity and diabetes. This work supplied new
evidences to support the anti-diabetes traditional use of X. aethiopica,
and disclosed its responsible components. In addition, two promising
candidate molecules, including an undescribed one, exhibited good
potential to be further developed as new natural therapeutics for the
treatment of diabetes and obesity through simultaneously inhibiting
α-glucosidase and pancreatic lipase, increasing glucose consumption
and reducing TG production, which might take cognizance of aporphine
alkaloids for their hypoglycemic and hypolipidemic activity except for
the frequently studied nuciferine and pronuciferine.
5. Ethics statement
No animal or human experiments are involved in this work.
CRediT authorship contribution statement
Ye Liu: Data curation, Funding acquisition, Methodology, Software,
Writing – original draft. Yawen Li: Methodology, Validation. Felix
Wambua Muema: Methodology, Software. Hui Zhang: Methodology,
Validation. Armel Jackson Seukep: Investigation, Resources. Min­
gquan Guo: Conceptualization, Funding acquisition, Project adminis­
tration, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
9
Journal of Functional Foods 106 (2023) 105601
Data availability
Data will be made available on request.
Acknowledgments
This work was partly supported by the National Natural Science
Foundation of China (Grant No. 32200324 to Y. Liu).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2023.105601.
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