Suppression of cytokine production by newly isolated flavonoids from pepper

Article abstract of DOI:10.1016/j.fitote.2021.104903

New flavonoid glycoside, kaempferol 3-O-α-[(6-P-coumaroyl galactopyranosyl-O-β-(→4)-O-α-rhamnopyranosyl-(1→4)]-O-α-rhamnopyranoside 1, in addition to five known flavonoid glycosides (2–6) kaempferol 3-O-[α-rhamnopyranosyl-(1→4)-O-α-rhamnopyranosyl-(1→6)-O]-β-galactopyranoside (kaempferol 3-O-β-isorhamninoside) 2, quercetin 3-O-[(2,3,4-triacetyl-α-rhamnopyranosyl)-(1 → 6)-β-galactopyranoside 3, quercetin 3-O-[(2,4-diacetyl-α-rhamnopyranosyl)-(1 → 6)]-3,4-diacetyl-β-galactopyranoside 4, quercetin 3-O-[(2,4-diacetyl-α-rhamnopyranosyl)-(1→6)]-2,4-diacetyl-β-galactopyranoside 5, quercetin 3-O-[(2,3,4-triacetyl-α-rhamnopyranosyl)-(1 → 6)-3-acetyl-β-galactopyranoside 6 were isolated from bell pepper (Capsicum annum L.) fruits and tested for both anti-inflammatory activity through cytokine production (TNF-α and IL-1β) and antioxidant activity through scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Compounds 1–3 significantly suppressed production of TNF-α / IL-1β in cultured THP-1 cells previously co-stimulated by LPS in a dose-dependent manner (10.2/49.1, 28.1/55.7, and 35.2/57.5 μM respectively) whereas compounds 4–6 have relatively weaker inhibitory activity. (45.3/73.5, 48.2/65.6, and 42.2/67.4 μM respectively). All compounds 1–6 showed no cytotoxic activity against the growth of THP-1where the percentage of cell viability was (127.4, 108.5, 105.4, 103.9, 103.4, and 104.2 μM respectively). All isolated compounds exhibited higher radical scavenging activity than ascorbic acid in (DPPH) assay. These results indicated that bell pepper fruits could be an effective candidate for ameliorating inflammatory-associated complications.

Full text of DOI:10.1016/j.fitote.2021.104903

Fitoterapia 151 (2021) 104903
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Suppression of cytokine production by newly isolated flavonoids
from pepper
Ahmed E. Allam
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
New flavonoid glycoside, kaempferol 3-O-α-[(6-P-coumaroyl galactopyranosyl-O-β-(→4)-O-α-rhamnopyranosyl-
Anti- inflammatory
Antioxidant activity
Bell pepper
(1→4)]-O-α-rhamnopyranoside 1, in addition to five known flavonoid glycosides (2–6) kaempferol 3-O-
[
α
-rhamnopyranosyl-(1→4)-O-
rhamninoside) 2, quercetin 3-O-[(2,3,4-triacetyl-
3-O-[(2,4-diacetyl- -rhamnopyranosyl)-(1 → 6)]-3,4-diacetyl-β-galactopyranoside 4, quercetin 3-O-[(2,4-diace-
tyl- -rhamnopyranosyl)-(1→6)]-2,4-diacetyl-β-galactopyranoside 5, quercetin 3-O-[(2,3,4-triacetyl- -rhamno-
pyranosyl)-(1 → 6)-3-acetyl-β-galactopyranoside 6 were isolated from bell pepper (Capsicum annum L.) fruits and
tested for both anti-inflammatory activity through cytokine production (TNF- and IL-1β) and antioxidant ac-
tivity through scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Compounds 1–3 significantly
suppressed production of TNF- / IL-1β in cultured THP-1 cells previously co-stimulated by LPS in a dose-
dependent manner (10.2/49.1, 28.1/55.7, and 35.2/57.5 M respectively) whereas compounds 4–6 have rela-
tively weaker inhibitory activity. (45.3/73.5, 48.2/65.6, and 42.2/67.4 M respectively). All compounds 1–6
showed no cytotoxic activity against the growth of THP-1where the percentage of cell viability was (127.4,
α
-rhamnopyranosyl-(1→6)-O]-β-galactopyranoside
(kaempferol
3-O-β-iso-
α
-rhamnopyranosyl)-(1 → 6)-β-galactopyranoside 3, quercetin
Cytokine production
α
α
α
α
α
μ
μ
108.5, 105.4, 103.9, 103.4, and 104.2 μM respectively). All isolated compounds exhibited higher radical scav-
enging activity than ascorbic acid in (DPPH) assay. These results indicated that bell pepper fruits could be an
effective candidate for ameliorating inflammatory-associated complications.
1. Introduction
Alzheimer’s, and Parkinson’s disease [6,7] that evoked us to carry out
this study.
Because the discovery of new drugs requires a long time and expense,
searching for new compounds from natural sources known for their high
2. Materials and methods
safety and applicability will be
a good avenue to deal with
inflammatory-associated complications. From which, bell pepper
(Capsicum annum L.), a member of the family Solanaceae is the most
commonly consumed fruity vegetable well known for its nutritional and
antioxidant significance [1]. Bell pepper exhibits medicinal as well as
food value all over the world. Both warm and dry climates are suitable
for its cultivation [2]. It is a rich source of neutral phenolic compounds,
especially luteolin, quercetin, capsaicinoids, nordihydrocapsaicin,
homodihydrocapsaicin, homocapsaicin, norcapsaicin, and nornorcap-
saicin [3,4]. Carotenoids exhibit antioxidant properties and prevent
tissue damage by acting as singlet molecular oxygen; reactive oxygen
species (ROS); peroxyl radicals and reactive nitrogen species (RNS)
scavengers [5]. The utilization of bioactive compounds plays key health-
promoting functions such as protecting against oxidative cell damage,
cancer insurgence, diabetes prevalence, cardiovascular disorders,
2.1. General
The optical activity was determined with Horiba SEPA-3000 high-
sensitivity polarimeter (Horiba). UV analysis was determined on Shi-
madzu UV-1600 UV–visible spectrometer, IR analysis was operated on
Shimadzu FTIR-8400. NMR analysis was carried out on JEOL GSX-600
spectrometer in CD3OD. Fast atom bombardment (FABMS) and high-
resolution fast atom bombardment (HRFABMS) were carried out on
JEOL JMS SX-102 mass spectrometry. Reversed-phase high-perfor-
mance liquid chromatography (HPLC) was undertaken on an ODS col-
umn (particle size: 5
Diaion HP-20 (Mitsubishi) (Tokyo Japan), silica gel (63–210
Kagaku), and ODS (63–212 m; Wako Pure Chemical) (Tokyo Japan)
were used for open column chromatography. Thin-layer
μ
m, TOSO, 18 × 250 mm) RP-23 (5
μ
m; Waters).
μ
m; Kanto
μ
E-mail address: aallam81@yahoo.co.uk.
https://doi.org/10.1016/j.fitote.2021.104903
Received 26 January 2021; Received in revised form 30 March 2021; Accepted 30 March 2021
Available online 2 April 2021
0367-326X/© 2021 Elsevier B.V. All rights reserved.

A.E. Allam
Fitoterapia 151 (2021) 104903
chromatography (TLC) was carried out on silica gel (SiO2, 60–100 mesh;
Table 1
13C and 1H NMR assignments for compound 1 recorded in CD3OD.
Wako Pure Chemical) 60 F254 and RP-18 F254S (Merck).
Position
13C NMR
1H NMR
Position
13C NMR
1H NMR
2.1.1. Extraction and isolation
(δ,
[δ, mult, J
(δ,
[δ, mult, J
Air-dried bell pepper fruits (2 kg) were extracted thrice with MeOH
(5 l each) to yield methanol extract (310 g) which was partitioned be-
tween distilled water, chloroform, ethyl acetate, and n-butanol (1 l each)
to yield chloroform fraction (90 g), ethyl acetate fraction (60 g), the n-
butanol fraction (50 g) and the rest aqueous fraction (100 g). All frac-
tions were screened for the antioxidant and cytokine production in
cultured THP-1 cells activities where the ethyl acetate was the most
active fraction and hence, and hence it was fractionated by ODS column
using six mobile phase systems of CH3CN-H2O (10, 25, 40, 50, 70 and
90% v/v; elution volume: 500 ml of each) to give six corresponding
subfractions. Subfraction eluted with 40% CH3CN (3.8 g) was further
isolated by silica gel column chromatography with gradient elution by
CHCl3:MeOH (ratios of 9:1, 6:1, 4:1, 3:1 and 1:1, v/v, elution volume:
200 ml each) to give five corresponding subfractions. The subfraction
eluted by 6:1 CHCl3: MeOH was further chromatographed by prepara-
tive HPLC, ODS column equipped with a UV detector (210 nm) with
mobile phase 20% CH3CN in H2O which afforded compounds 3–6, (15,
18, 22, and 9 mg respectively). These preparative HPLC conditions were
also used after gradually increasing the mobile phase to 50% CH3CN in
H2O to isolate the same fraction to afford compounds 1 and 2, (13 and
24 mg respectively).
mult.)
(Hz)]
mult.)
(Hz)]
2
3
4
159.2,s
134.1,s
179.4,s
–
–
–
Galactopyranosyl
1′′′′
100.5,d
5.62,d,7.5
3.82,dd,
9.9,7.5
2′′′′
72.4,db
5
163.1,s
3′′′′
73.8,d
3.87,dd,
9.9,3.4
6
7
8
99.8,d
165.7,s
94.7,d
6.09,d, 2.06
–
6.29,d, 2.06
4′′′′
5′′′′
6′′′′
72.0,db
77.7,d
67.1,t
5.20,d,3.4
3.84,m
3.41,m,
3.13,m
9
158.3,s
105.9,s
123.1,s
–
–
–
P-coumaroyl
10
1′′′′
2′′′′
127.6,s
–
1′
116.0,d
6.66,2H,
d,8.0.9
2′
3′
4′
5′
132.2, d
115.9,d
161.3,s
115.9,d
7.97, 2H,d, 8.9
6.82, 2H,d, 8.9
–
3′′′′
4′′′′
5′′′′
6′′′′
134.0,d
160.0,s
134.0,d
116.0,d
7.65,2H,d,8.9
–
7.65,2H,d,8.9
6.66,2H,
d,8.0.9
6.82, 2H,d, 8.9
6′
132.2,d
7.97, 2H,d, 8.9
7
146.1,d
115.9,d
167.6,s
6.86,d,13.0
5.77,d,13.0
Rhamnopyranosyl
8
1′′
2′′
102.7,d
4.36, brs.
3.88,dd, 3.4,
1.7
C=O
72.1,da
3′′
4′′
72.3,da
74.0,d
3.39,dd, 9.6,
3.4
2.1.2. Acid hydrolysis
3.25,dd, 9.9,
9.6
Acid hydrolysis of the flavonoid glycosides was carried out by
refluxing 5 mg of the compound in 5 ml of 6% HCl in MeOH for 3 h. The
reaction mixture was partitioned against EtOAc (3 × 10 ml). The agly-
cones were obtained from the EtOAc layer and identified as kaempferol
and quercetin by co-chromatography on silica gel with reference sam-
ples (Sigma) (Tokyo, Japan). Identification of galactose and rhamnose
present in the sugar fraction was carried out by comparison with
authentic samples, galactose (Rf 0.41), glucose (Rf 0.46), and rhamnose
(Rf 0.66) (Sigma) (Tokyo, Japan) in TLC on silica gel (CHCl3-MeOH-H2O
8:5:1) using 5% H2SO4 in MeOH as spraying reagent followed by heating
the plates at 120◦C for 15–20 min.
5′′
6′′
69.9,d
17.4,q
4.0,dd,9.9,6.1
0.85,d, 6.1
Rhamnopyranosyl
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
102.6,d
71.9,d
73.7,d
74.0,d
69.9,d
17.88,q
5.10,brs.
3.71,m
3.74,m
3.12,m
3.29,m
0.96,d, 6.1
(180 μl) were seeded in 96-well plates at 1.0 × 105 cells per well with
tested samples (purity >93%) (20 μl in DMSO/ PBS) at various con-
2.1.3. Measurements of the optical rotation of D-galactopyranose and L-
rhamnopyranose tetrabenzoate derivatives
centrations. After 48-h cultivation, supernatants were removed, non
adherent cells (THP-1) incubated with 3-(4, 5-dimethylthiazol-2-yl)-2,5-
Benzoyl chloride (0.5 ml) was added to each ice-cooled solution of
either D–galactopyranose (6.0 mg) or L-rhamnopyranose (4.0 mg) in dry
pyridine (1.0 ml) and each mixture was stirred at room temperature for
15 h. MeOH (1.0 ml) was added dropwise to the reaction mixture, stirred
for 30 min, and then diluted with EtOAc and aqueous Na2CO3, and the
layers were separated. Each organic layer was washed with brine and
the combined aqueous layers for each was extracted with EtOAc. Each
combined organic extract was dried over MgSO4 and concentrated. The
corresponding residual dark brown oil fractions were individually pu-
rified by silica gel cc (eluted by hexane/EtOAc 5:1) to give either
31
diphenyltetrazolium bromide (MTT; 10 μl, 5 mg/mL in PBS) for 4 h, and
then solubilized with 10% (w/v) sodium dodecyl sulfate (SDS; in 60%
[v/v] dimethyl formamide) solution (100 l) for 18 h. The absorbance
μ
was measured at 570 nm using a microplate reader, and the cytotoxicity
was calculated by comparing absorbance with that of the non-treated
control culture. The cell growth curve was graphed using statistical
analysis software (Kaleida Graph version 4.00; Synergy Software), and
IC50 values calculated using simple linear regression. The cytotoxic
activity of all of isolates was determined by MTT colorimetric assay
(Segun, et al., 2019; Alley et al., 1988) [12,13].
D–galactopyranose tetrabenzoate [
α
]
+ 53.5 (c= 1.2, CHCl3) or L-
+ 75.0 (c =1.6, CHCl3) as a
D
2.1.6. DPPH radical scavenging activity
29.6
rhamnopyranose tetrabenzoate [
α]
DPPH assay was performed by a method previously reported by
D
(Kumar et al.; 2011) [14]. 100 μl of the tested samples at different
colorless oil, respectively [8,9].
concentrations in MeOH and 1.0× 10–4 M DPPH in MeOH (300 μl) were
added to the 96-well microtiter plate. The plate was shaken for 1 min on
a plate shaker and incubated for 30 min at room temperature in the dark.
After incubation, the absorbance was recorded at 517 nm. The tested
samples at different concentrations without DPPH solution were used as
a blank control to eliminate the influence of sample color. Ascorbic acid
was used as a positive control [15] and DPPH solution in MeOH served
as a negative control.
2.1.4. Evaluation of cytokine production in cultured THP-1 cells and
cytotoxic assay
To determine the effect of both fractions and the isolated compounds
(1–6) on the production of inflammatory cytokines in monocytes THP-1
cells (Dainippon Pharmaceutical Company), a method modified by
´
(Bornstein et al; 2004 and Nehme et al.; 2008) was used [10,11].
2.1.5. MTT in vitro assay
To determine the cytotoxic activity of the tested samples, THP-1 cells
2

A.E. Allam
Fitoterapia 151 (2021) 104903
Fig. 1. Structure of compounds 1–6.
Table 2
3. Results and discussion
Effects of methanol extract and fractions isolated from a particular extract on
TNF-α production in cultured THP-1.
3.1. Structure elucidation of compound 1
Extract/
fraction
Inhibitory activity
(IC50) ( g/ml)
DPPH radical
scavenging
activity
μ
Compound 1 (Table 1, Fig. 1) (13 mg) was obtained as yellow
30.1
amorphous powder, soluble in methanol, with [
α]
-54.5◦ (c =
D
TNF-
α
Dexamethasone;
positive control 5
Ascorbic acid
n
3
production
μM
positive control
0.333, MeOH). The structure of compound 1 was elucidated by UV, IR,
one- and two dimensional NMR spectroscopy including H, 13C NMR,
1
12 μM
Total
52 ± 8
32 ± 8
DEPT-135H-H COSY, HMQC, and HMBC experiments, as well as HRFAB
mass spectrometry. UV spectrum (in MeOH) exhibited absorption
maxima at 256 nm (band-II) and 352 nm (band-I) indicating a flavonol
type. IR spectrum of 1 (in CHCl3) indicated the presence of hydroxyl
(3445 cm-1), carbonyl (1782 cm-1) and phenyl (2980, 1640, 1533 cm-
methanol
extract
Chloroform
fraction
130 ± 6
19 ± 9
75 ± 3
45 ± 5
21 ± 8
65 ± 4
3
3
3
Ethyl acetate
fraction
n-butanol
fraction
3

A.E. Allam
Fitoterapia 151 (2021) 104903
two features characteristic of a flavonol with hydroxyl functionality at
positions 5, 7 and 4. 1HNMR spectrum showed also signals characteristic
to p-coumaroyl moiety, where there is a typical coupling pattern of 1,4-
disubstituted benzene ring [a pair of doublets each is equivalent to two
protons at δH 7.65 (H-2′′′′,H-6′′′′) and at δH 6.66 (H-3′′′′,H-5′′′′)] in addi-
tion to a trance olefinic protons at δH 6.86 and 5.77 (H-7,H-8) respec-
tively. 13C NMR agreed with 3-substituted kaempferol moiety.
Substitution of kaempferol in C-3 was evident from the chemical shift of
neighboring C-2 (δC 159.2 ppm) whereas in flavonols with unsubstituted
hydroxyl functionality at C-2 was detected around δC 147 ppm [16]. A
long-range correlation was observed in HMBC experiment between C-3
of kaempferol (δC 134.1 ppm) and the anomeric proton of rhamnose (δH
4.36 ppm) confirmed that this was the site of glycosylation and rham-
nose was the first sugar. Other long-range correlations observed in
HMBC experiment, between C-4 of the first rhamnose (δC 74 ppm) and
the anomeric proton of the middle one (δH 5.1 ppm) and between C-4 of
the middle rhamnose (δC 74 ppm) and the anomeric proton of the ter-
minal galactose (δH 5.62 ppm) confirmed that the attachment between
the middle rhamnose and first one is (1 → 4) and that between the
terminal galactose and the middle rhamnose is (1 → 4). Another HMBC
correlation was observed between H-6 protons of the terminal galactose
(δH 3.41 and 3.13 ppm) and the carbonyl group of P-coumaroyl moiety
(δC 167.6 ppm), confirming that the P-coumaroyl moiety is attached to
the terminal galactose. Also, 13C- downfield shift of both C-6 of
Table 3
Activities of compounds 1–6 on the production of inflammatory cytokines and
their cytotoxic activity in vitro.
Compound
Inhibitory activity (IC50) (
μ
M)
% of Cell viability
Cell lines THP-1
n
Cytokines (TNF-α/IL-1β)
1
10.2/49.1
28.1/55.7
35.2/57.5
45.3/73.5
48.2/65.6
42.2/67.4
42.1/40.5
127.4
108.5
105.4
103.9
103.4
104.2
ND
2
2
2
2
2
2
2
2
3
4
5
6
Dexamethasone
Values are mean values from two experiments (n = 8 in total) n: number of
experiments (one experiment: n = 4).
Dexamethasone (positive control for the activity of cytokine inhibition) ND: not
determined.
1). 1H and 13C NMR spectra of 1 indicated the presence of kaempferol
moiety, three sugar moieties (one hexose unite and two pentose unites)
in addition to the presence of P-coumaroyl moiety attached to a terminal
hexose. 1H NMR spectrum showed pair of doublets at δH 6.09 (H-6) and
δH 6.29 (H-8) and two-spin system with the typical coupling pattern of
1,4-disubstituted benzene ring [a pair of doublets each is equivalent to
two protons at δH 7.97 (H-2′, H-6) and δH 6.82 (H-3′, H-5)] which are
Fig. 2. Effects of flavonoid glycosides on the production of inflammatory cytokines in cultured THP-1 cells.
Cells were directly treated with various concentrations (1, 10 and100 M) of compounds 1–6 co-stimulated with 1 μM LPS. Culture supernatants were collected 24 h
μ
after LPS stimulation and cytokine levels such as TNF-α (A) and IL-1β (B) were measured using ELISA. Data are means ± SD of quadruplicate cultures. Correlation
coefficients for TNF-
α
production co-stimulated with LPS: r2 = 0.89629 (1), r2 = 0.96711 (2), r2 = 0.92035 (3), r2 = 0.95413 (4), r2 = 0.99115 (5), r2 = 0.87086 (6).
Correlation coefficients for IL-1β production co-stimulated with LPS: r2 = 0.99760 (1), r2 = 0.99454 (2), r2 = 0.99024 (3), r2 = 0.90043 (4), r2 = 0.99944 (5), r2
0.95169 (6).
=
Data are means ± SD of three independent experiments (n = 12 in total); n: numbers of experiment (one experiment: n = 4). a Original methanol extract. b Original
extract was loaded onto HP-20, and successive elution with water, methanol and acetone were carried out yielding three fractions.
4

A.E. Allam
Fitoterapia 151 (2021) 104903
immunosuppressive glucocorticoid agent dexamethasone was used as a
Table 4
DPPH radical scavenging activity of compounds 1–6.
positive control for inhibition of cytokine production at 5 μM, dexa-
Compound
IC50 (μ
M)a
methasone strongly decreased levels of TNF-
pernatants of THP-1 cells co-stimulated with LPS [21]. It was noticed
that compounds 1–3 strongly suppressed the production of TNF- / IL-1β
in THP-1 cells in a dose-dependent manner (10.2/49.1, 28.1/55.7, and
35.2/57.5 M respectively) whereas compounds 4–6 have relatively
α, and IL-1β in culture su-
1
1.37
1.42
3.62
3.29
1.87
7.21
12
α
2
3
4
μ
5
weaker inhibitory activity.(45.3/73.5, 48.2/65.6, and 42.2/67.4
μM
6
respectively), These represents results more or less more potent than the
positive control Dexamethasone.
Ascorbic acid
a
IC50 values were determined by regression anal-
ysis and expressed as the mean of four replicates.
A possible mechanism for flavonoids used to alleviate some
inflammation-related symptoms is to act by inhibiting cyclooxygenase
Fig. 3. DPPH radical scavenging activity of compounds 1–6.
galactose, C-4 of the middle rhamnose, and C-4 of the first rhamnose
confirmed the mentioned site of attachment [17]. The HRMS spectrum
showed a quasi-molecular ion peak at m/z 887.2629 [M + H] + calcu-
lated as 886.2532 per the molecular formula C42H46O21. Hence 1 could
activity or by blocking cytokine receptors [22,23].
As SAR study, it was noticed that the effect of kaempferol and
quercetin on cytokine-induced pro-inflammatory status of cultured
human endothelial cells was always more strongly inhibited in
kaempferol-treated than in quercetin-treated cells [24], Kaempferol can
modulate the Th1/Th2 balance and could be useful for the treatment of
cell-mediated immune diseases [25] which may explain the strong
suppression activity of compounds (1–2) (kaempferol derivative) over
compounds (4–6) (quercetin derivatives), for compound 3, it has a
relatively weaker activity.
unequivocally be identified as Kaempferol 3-O-
actopyranosyl-O-β-(1 → 4)-O-
nopyranoside which is a new compound.
In addition, five known falvonoid glycosides (2–6) Fig. 1, Kaemp-
ferol 3-O-[ –rhamnopyranosyl-(1 → 4)-O- -rhamnopyranosyl-(1 → 6)-
O]-β-galactopyranoside (kaempferol 3-O-β-isorhamninoside) 2, quer-
cetin 3-O-[(2,3,4-triacetyl- -rhamnopyranosyl)-(1 → 6)-β-galactopyr-
anoside 3, quercetin 3-O-[(2,4-diacetyl- -rhamnopyranosyl)-(1 → 6)]-
3,4-diacetyl-β-galactopyranoside 4, quercetin 3-O-[(2,4-diacetyl-
-rhamnopyranosyl)-(1 → 6)]-2,4-diacetyl-galactopyranoside 5, quer-
α
-[(6-P-coumaroyl gal-
α
-rhamnopyranosyl-(1 → 4)]-O- -rham-
α
α
α
α
α
3.3. DPPH radical scavenging activity
α
The isolated flavonoid compounds 1–6 exhibited free radical scav-
cetin 3-O-[(2,3,4-triacetyl-
α
-rhamnopyranosyl)-(1 → 6)-3-acetyl-β-gal-
enging activity with IC50 values of 1.37 and 1.42
μ
M for 1and 2
actopyranoside 6 were isolated and their structure were identical with
the reported data [18–20].
(kaempferol derivatives) and 3.62, 3.29, 1.8, and 7.21 μM for 3–6
(quercetin derivatives), (Table 4; Fig. 3). This may be attributed to the
differences in the sites of the acetyl groups in compounds 3–6.
3.2. Inhibition of production of inflammatory cytokines in THP-1 cells
4. Conclusion
Both fractions and isolated compounds (1–6) were subjected to 24-h
Based on the previous results, ball pepper fruit extracts may be safely
considered potential anti-inflammatory and antioxidant candidates for
inflammatory diseases.
stimulation by lipopolysaccharide (LPS) at 1μM (Tables 2, 3; Fig. 2)
where ethyl acetate fraction showed the strongest inhibitory activity.
This fraction inhibited TNF- production and DPPH radical scavenging
α
activity with IC50 values of 19.9 and 21 μg/ml respectively. Its inhibi-
Funding
tory activity was 2.5–1.5 times stronger than those of the original
methanol extract.
This work was financially by the Egyptian Ministry of Higher Edu-
cation and Scientific Research.
Ethyl acetate fraction was subjected to isolation by HPLC yielding
the previously mentioned six flavonoids 1–6 which in turn were sub-
jected to the same assay as the parent fraction. Putative
5

A.E. Allam
Fitoterapia 151 (2021) 104903
´
[11] A. Nehme, J. Edelman, Dexamethasone inhibits high glucose-, TNF-alpha- and IL-
1beta-induced secretion of inflammatory and angiogenic mediators from retinal
microvascular pericytes, Invest. Ophthalmol. Vis. Sci. 49 (5) (2008) 2030–2038.
[12] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D. Fine, B.
J. Abbott, J.R. Mayo, R.H. Shoemaker, M.R. Boyd, Feasibility of drug screening
with panels of human tumor cell lines using a microculture tetrazolium assay,
Cancer Res. 48 (1988) 589–601.
Conflicts of interests
No potential conflict of interest was reported by the authors.
Acknowledgments
[13] P.A. Segun, O.O. Ogbole, F.M.D. Ismail, L. Nahar, A.R. Evans, E.O. Ajaiyeoba, S.
D. Sarker, Resveratrol derivatives from Commiphora africana (A. Rich) Endl. display
cytotoxicity and selectivity against several human cancer cell lines, Phytother. Res.
33 (1) (2018) 159–166.
The author acknowledges Pharmacognosy and Chemistry of Natural
Products Department, School of Pharmaceutical Sciences, Kanazawa
University, Kanazawa, Japan, for carrying out NMR analysis.
[14] V. Kumar, M. Mohan, Physicochemical status and primary productivity of Ana
Sagar Lake, Ajmer (Rajasthan), India. Univ. J. of Envir. Res. Tech. 1 (3) (2011)
286–292.
Appendix A. Supplementary data
˜
[15] P. Valentao, E. Fernandes, F. Carvalho, P.B. Andrade, R.M. Seabra, M.I. Bastos,
Hydroxyl radical and hypochlorous acid scavenging activity of small Centaury
(Centaurium erythraea) infusion, A comparative study with green tea (Camellia
sinensis), Phytomedicine 10 (6–7) (2003) 517–522.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fitote.2021.104903.
[16] A.N. Panche, A.D. Diwan, S.R. Chandra, Flavonoids: an overview, J. Nutr. Sci. 5
(2016) 1–15.
[17] J.Y. Salib, H.N. Michael, E.F. Eskande, Anti-diabetic properties of flavonoid
compounds isolated from Hyphaene thebaica epicarp on alloxan induced diabetic
rats, Pharm. Res. 5 (1) (2013) 22–29.
References
[18] M. Halabalaki, A. Urbain, A. Paschali, S. Mitakou, F. Tillequin, A.L. Skaltsounis,
˘
[1] R. Soare, M. Dinu, C. Babeanu, M. Popescu, A. Popescu, Nutritional value and
Quercetin and Kaempferol3-O-[alpha-L-Rhamnopyranosyl-(1-→2)-α-L-
antioxidant activities in fruit of some cultivars of pepper (Capsicum annuum L.),
J. Agroaliment. Process. Technol. 23 (4) (2017) 217–222.
arabinopyranoside]-7-O-α-rhamnopyranosides from Anthyllis hermanniae: structure
determination and conformational studies, J. Nat. Prod. 74 (9) (2011) 1939–1945.
[2] G.E. Igbokwe, G.C. Aniakor, C.O. Anagonye, Determination of β-carotene &
vitamin C content of fresh green pepper (Capsicum annum), fresh red pepper
(Capsicum annum) and fresh tomatoes (Solanum lycopersicum) fruits, Bioscientist. 1
(1) (2013) 89–93.
´
˜
´
´
´
´
[19] J.M. Calderon-Montano, E. Burgos-Moron, C. Perez-Guerrero, M.A. Lopez-Lazaro,
Review on the dietary flavonoid kaempferol, Mini-Rev. Med. Chem. 11 (4) (2011)
298–344.
[20] A.E. De Sousa, A.C.A. Da Silva, A.J. Cavalheiro, J.H.G. Lago, M.H. Chaves, A new
flavonoid derivative from leaves of Oxandra sessiliflora R. E. Fries, J. Braz. Chem.
Soc. 25 (4) (2014) 704–708.
[3] G. Oboh, T.B.G. Rocha, Distribution and antioxidant activity of polyphenols in ripe
and unripe tree pepper (Capsicum pubescens), J. Food Biochem. 31 (2007) 456–473.
[4] N.B.C. Prasad, R. Shrivastava, G.A. Ravishankar, Capsaicin as multifaceted drug
from Capsicum spp, Evid. Based Intern. Med. 2 (2005) 147–166.
[21] W.H. Watson, Y. Zhao, R.K. Chawla, S-adenosylmethionine attenuates the
lipopolysaccharide-induced expression of the gene for tumour necrosis factor
alpha, Biochem. J. 342 (1999) 21–25.
[5] Y.J. Kim, Y.A.E. Kim, T. Yokozawa, Protection against oxidative stress,
inflammation, and apoptosis of high-glucoseexposed proximal tubular epithelial
cells by astaxanthin, J. Agric. Food Chem. 57 (19) (2009) 8793–8797.
[22] L. Nayely, P. Erick, G. Gutierrez, L.A. Dulce, H. Basilio, Flavonoids as cytokine
modulators: a possible therapy for inflammation-related diseases, Int. J. Mol. Sci.
17 (6) (2016) 921–936.
´
´
[6] G.M. Lalanne, A.J. Alvarez, M.J. Fernandez, E. Azuara, Oleoresins from Capsicum
spp.: extraction methods and bioactivity, Food Bioprocess Technol. 10 (2017)
51–76.
´
[23] A. Nehme, J. Edelman, Dexamethasone inhibits high glucose-TNF-alpha- and IL-
1beta-induced secretion of inflammatory and angiogenic mediators from retinal
[7] K. Sanatombi, S. Rajkumari, Effect of processing on quality of pepper: a review,
Food Rev. Int. 36 (6) (2020) 626–643.
microvascular pericytes, Invest. Ophthalmol. Vis. Sci. 49 (2008) 2030–2038.
´
´
˜´
[24] I. Crespo, M. García-Mediavilla, B. Gutierrez, S. Sanchez-Campos, M. Tunon,
[8] Y. Kasai, K. Komatsu, H. Shigemori, M. Tsuda, Y. Mikami, J. Kobayashi,
A. Cladinol, A polyketide glycoside from marine-derived fungus Gliocladium
species, J. Nat. Prod. 68 (2005) 777–779.
´
J. Gonzalez-Gallego, A comparison of the effects of kaempferol and quercetin on
cytokine-induced pro-inflammatory status of cultured human endothelial cells Br,
J. Nutr. 100 (5) (2008) 968–976.
[9] T. Hasegawa, F. Takano, T. Takata, M. Niiyama, T. Ohta, Bioactive monoterpene
glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus,
Phytochemistry. 69 (2008) 747–753.
[25] I. Okamoto, K. Iwaki, S. Koya-Miyata, T. Tanimoto, K. Kohno, M. Ikeda,
M. Kurimoto, The flavonoid Kaempferol suppresses the graft-versus-host reaction
by inhibiting type 1 cytokine production and CD8+ T cell engraftment, Clin.
Immunol. 103 (2) (2002) 132–144.
[10] S.R. Bornstein, H. Rutkowski, I. Vrezas, Cytokines and steroidogenesis, Mol. Cell.
Endocrinol. 215 (2004) 135–141.
6