Structure–activity relationships of flavanones, flavanone glycosides, and flavones in anti-degranulation activity in rat basophilic leukemia RBL-2H3 cells

Article abstract of DOI:10.1007/s11418-017-1169-3

The incidence of type I allergies, which are associated with mast cell degranulation and local inflammation, is increasing, and new treatments are needed. To date, structure–activity relationships of flavonoids in their degranulation-inhibiting activity have not been systematically characterized. In the current study, the degranulation-inhibiting activity of a series of flavonoids was evaluated. The following three observations were made: (1) the activity disappears when a sugar moiety is introduced into the A ring of the flavanone; (2) the activity depends on the number of hydroxyl groups on the B ring; (3) the activity is markedly enhanced when a double bond is introduced into the C ring. The information obtained in the current study may guide the development of a therapy for type I allergies.

Full text of DOI:10.1007/s11418-017-1169-3

Journal of Natural Medicines
https://doi.org/10.1007/s11418-017-1169-3
NOTE
Structure–activity relationships of favanones, favanone glycosides,
and favones in anti‑degranulation activity in rat basophilic leukemia
RBL‑2H3 cells
Toshiro Noshita1 · Kaori Miura2 · Kaoru Ikeda2 · Hidekazu Ouchi3 · Takuya Matsumoto4 · Akihiro Tai1
Received: 14 October 2017 / Accepted: 26 December 2017
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
The incidence of type I allergies, which are associated with mast cell degranulation and local infammation, is increasing, and
new treatments are needed. To date, structure–activity relationships of favonoids in their degranulation-inhibiting activity
have not been systematically characterized. In the current study, the degranulation-inhibiting activity of a series of favonoids
was evaluated. The following three observations were made: (1) the activity disappears when a sugar moiety is introduced
into the A ring of the favanone; (2) the activity depends on the number of hydroxyl groups on the B ring; (3) the activity
is markedly enhanced when a double bond is introduced into the C ring. The information obtained in the current study may
guide the development of a therapy for type I allergies.
Keywords Flavonoids · Anti-degranulation · Type I allergy · Structure–activity relationship · Rat basophilic leukemia
RBL-2H3 cell
Introduction
from various sources. Flavonoids have been shown to be
benefcial for human health [2] and to possess a wide range
of biological and pharmacological activities in vitro, such as
antimicrobial [3], antioxidative [4], anti-infammatory [5],
antihypertensive [6], anti-metastatic [7], anti-hypercholester-
olemic [8], hypolipidemic [9], AMP-activated protein kinase
(AMPK)-inhibiting [10], and antitumor [11] activities.
Japan is currently facing the societal problem of an
increasing number of cases with type I allergies. According
to one report, in 2006, ca. 30% of Japanese individuals in
the Tokyo metropolitan area had cedar pollinosis, one of
the typical allergic diseases [12]. Antigen-induced mast cell
degranulation leads to local infammation in type I allergies;
therefore, natural compounds that can attenuate this degran-
ulation have received particular attention as possible thera-
peutics. Recently, we showed that methyl or ethyl etherifca-
tion at the 7-OH of hesperetin, a typical favanone of Citrus
fruits, slightly enhanced the inhibition of antigen-induced
degranulation of rat basophilic leukemia cells (RBL-2H3)
[13]. The degranulation-inhibiting activity is an important
index of the anti-allergy action of a drug. However, no struc-
ture–activity relationship studies on the anti-degranulation
efects of favonoids have been published, with the excep-
tion of one study on the degranulation-inhibiting activities
of favanone glycosides and their aglycon derivatives [14].
Flavonoids are one of the most widespread and important
plant pigments, with over 9000 naturally occurring favo-
noids from various plants characterized to date [1]. New
favonoids, including crude drugs, are continuously isolated
* Toshiro Noshita
noshita@pu-hiroshima.ac.jp
* Akihiro Tai
atai@pu-hiroshima.ac.jp
1
Department of Life Sciences, Faculty of Life
and Environmental Sciences, Prefectural University
of Hiroshima, 5562 Nanatsuka, Shobara,
Hiroshima 727-0023, Japan
2
Program in Biological System Sciences, Graduate School
of Comprehensive Scientifc Research, Prefectural
University of Hiroshima, 5562 Nanatsuka, Shobara,
Hiroshima 727-0023, Japan
3
Department of Pharmacy, Faculty of Pharmacy, Kindai
University, 3-4-1 Kowakae, Higashiosaka 577-8502, Japan
4
Department of Environmental Sciences, Faculty
of Life and Environmental Sciences, Prefectural
University of Hiroshima, 5562 Nanatsuka, Shobara,
Hiroshima 727-0023, Japan
Vol.:(0123456789)
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Journal of Natural Medicines
In the current study, a series of favonoids were tested as
inhibitors of degranulation in the RBL-2H3 cell line (a mast
cell model). Combined with the results of our earlier study,
their structure–activity correlations have now been clarifed.
(benzene/ethyl acetate 12:1) yielded 16 (85 mg, 0.32 mmol,
63%). 7-O-Methylchrysin (16). Pale yellow crystal. M.p.
163°C (lit. [17] 164–166°C). 1H NMR (CDCl3, 500 MHz)
δ: 3.87 (3H, s, O-CH3), 6.36 (1H, d, J = 2.3 Hz, 6-H), 6.49
(1H, d, J = 2.3 Hz, 8-H), 6.65 (1H, s, 3-H), 7.49–7.56 (3H,
m, Ar–H), 7.86–7.89 (2H, m, Ar–H). 13C NMR (CDCl3,
125 MHz) δ: 55.6, 92.6, 98.1, 105.6, 105.7, 126.2, 129.0,
131.2, 131.8, 157.7, 162.1, 163.9, 165.5, 182.4. IR (diamond
ATR) cm−1: 1663, 1606, 1586, 1493, 1350, 1201, 1158,
1039, 848, 805, 768. ESIMS m/z: 269.0 [M + H]+.
Materials and methods
Chemicals
Eriodictyol (1), eriodictyol-7-O-glucoside (4), eriocitrin (5),
and narirutin (9) were purchased from Extrasynthese (Lyon,
France). Hesperetin (2), hesperidin (6), and luteolin (11)
were purchased from Cayman Chemical Co. (Ann Arbor,
MI, USA), Nacalai Tesque Inc. (Kyoto, Japan), and LKT
laboratories Inc. (St. Paul, MN, USA), respectively. Nar-
ingenin (3), neohesperidin (7), naringin (8), pinocembrin
(10), diosmetin (12), and quercetin (15) were obtained from
Sigma-Aldrich Co. (St. Louis, MO, USA). Apigenin (13)
and chrysin (14) were purchased from Wako Pure Chemi-
cal Industries (Osaka, Japan). 7-O-Methylchrysin (16) and
7-O-methyldiosmetin (17) were prepared according to modi-
fed methods of Lim et al. [15] and Yang et al. [16], respec-
tively, and their physical data were comparable. The 1H and
13C NMR spectra of compounds 16 and 17 were recorded
using an ECA-500 spectrometer (JEOL, Tokyo, Japan) with
tetramethylsilane as the internal standard. The IR spectra
were obtained using a Nicolet iS10 FT-IR spectrometer
(Thermo Fisher Scientifc, Waltham, MA, USA) with a dia-
mond horizontal attenuated refectance (ATR) accessory and
co-addition of 16 interferograms. Calibration models were
generated using OMNIC 9.2.98 software (Thermo Fisher
Scientifc). Mass spectra were recorded using an API-2000
mass spectrometer (AB Sciex LLC, Framingham, MA,
USA).
7‑O‑Methyldiosmetin (17)
A mixture of luteolin (143 mg, 0.5 mmol), K2CO3 (280 mg,
2.0 mmol), and dimethyl sulfate (150 mg, 1.2 mmol) in
10 mL of acetone was refuxed for 1 h. Then, the reaction
mixture was poured into water and extracted with CH2Cl2.
The extract was washed with water and brine, dried over
anhydrous CaCl2, and concentrated under pressure. Purifca-
tion of the residue by column chromatography on silica gel
and elution with CHCl3/MeOH (30:1) yielded 17 (110 mg,
0.37 mmol, 73%). 7-O-Methyldiosmetin (17). Pale yellow
powder. M.p. 234–235 °C, decomp. (lit. [18] 230–232 °C,
1
decomp.). H NMR (CDCl3:DMSO-d6 = 2:1, 60 °C,
500 MHz) δ: 3.89 (3H, s, O-CH3), 3.93 (3H, s, O-CH3),
6.31 (1H, d, J = 2.1 Hz, 6-H), 6.53 (1H, d, J = 2.1 Hz, 8-H),
6.54 (1H, s, 3-H), 6.99 (1H, d, J = 8.4 Hz, 5′-H), 7.41 (1H,
d, J = 2.1 Hz, 2′-H), 7.43 (1H, dd, J = 8.4, 2.1 Hz, 6′-H),
8.81 (1H, br-s, Ar-OH), 12.77 (1H, s, Ar-OH). 13C NMR
(CDCl3/DMSO-d6 = 2:1, 60 °C, 125 MHz) δ: 54.6, 54.8,
91.3, 96.9, 103.0, 104.2, 110.8, 112.2, 117.5, 122.6, 146.0,
150.1, 156.5, 160.8, 163.1, 164.3, 180.9. IR (diamond ATR)
cm−1: 3350, 1655, 1603, 1505, 1438, 1325, 1187, 1141,
1039, 813, 770. ESIMS m/z: 315.1 [M + H]+.
p-Nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside
and the penicillin–streptomycin solution were obtained from
Nacalai Tesque Inc. The monoclonal anti-dinitrophenyl
(DNP) antibody, cromolyn sodium salt (DSCG), and DNP-
labeled human serum albumin were purchased from Sigma-
Aldrich Co. Dulbecco’s modifed Eagle’s medium was pur-
chased from Corning (Corning, NY, USA). Fetal bovine
serum was acquired from HyClone (Logan, UT, USA).
Evaluation of degranulation‑inhibiting activity
The inhibitory activity of the compounds against the
release of β-hexosaminidase from RBL-2H3 cells was
evaluated using a modified method of Watanabe et al.
[19]. RBL-2H3 cells were purchased from the JCRB
Cell Bank (Osaka, Japan). Dulbecco’s modified Eagle’s
medium containing 10% heat-inactivated fetal bovine
serum, 100 U mL−1 penicillin, and 100 μg mL−1 strep-
tomycin was used as growth medium. The cells were
cultured in 96-well plates (5.0 × 104 cells/well) for 24 h
at 37 °C under a humidified 5% CO2 atmosphere. Then,
they were incubated in growth medium supplemented
with 50 ng mL−1 mouse monoclonal anti-DNP IgE for
2 h. The cells were washed with a modified Tyrode’s
(MT) buffer before the test compounds or wortmannin
(2.5 μM) was added. The test compounds and wortman-
nin were dissolved in DMSO and diluted in the MT
7‑O‑Methylchrysin (16)
A mixture of chrysin (127 mg, 0.5 mmol), K2CO3 (83 mg,
0.6 mmol), and dimethyl sulfate (76 mg, 0.6 mmol) in 5 mL
of acetone was refuxed for 1 h. Then, the reaction mix-
ture was poured into water and extracted with CH2Cl2. The
extract was washed with water and brine, dried over anhy-
drous CaCl2, and concentrated under pressure. Purifcation
of the residue by preparative thin-layer chromatography
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Journal of Natural Medicines
buffer at a final DMSO concentration of 0.25%. After
20 min of incubation, DNP-labeled human serum albumin
(50 ng mL−1 final concentration) was added to the cells,
and the culture was continued for 1 h. The supernatant
was then collected, and the cells were lysed with MT
buffer containing 0.1% Triton X-100. β-Hexosaminidase
activity in the supernatants and cell lysates was deter-
mined by the method of Demo et al. [20]. Briefly, the
supernatant or cell lysate (20 μL) was mixed with 3.3 mM
p-nitrophenyl-2-acetamide-2-deoxy-β-d-glucopyranoside
(40 μL) in 100 mM citrate buffer (pH 4.5), and the mix-
ture was incubated in a 96-well plate at 37 °C for 90 min.
The reaction was terminated by the addition of 2 M gly-
cine–NaOH buffer (pH 10.4, 40 μL), and the absorbance
at 405 nm was measured using a microplate reader.
Results and discussion
Flavonoids have the general structure of a C6–C3–C6
skeleton, consisting of two benzene rings (A and B) and a
heterocyclic C ring. In the current study, the anti-degran-
ulation efects of 17 favonoids were evaluated using the
β-hexosaminidase release assay [19]; the tested favonoids,
including favanones, favanone glycosides, and favones, are
shown in Fig. 1. These favonoids were chosen so as to inves-
tigate the efects of the sugar on the A ring; the substituent
on the B ring; and the double bond in the C ring.
We frst investigated the efect of the presence of a sugar
moiety in the favonoid A ring on the anti-degranulation
activity. As shown in Fig. 2, eriodictyol (1) and hespere-
tin (2) both exhibited degranulation-inhibiting activities,
although these were weak. Naringenin (3) tended to show
degranulation-inhibitory activity. In contrast, glycosides of
eriodictyol (1) [eriodictyol-7-O-β-d-glucopyranoside (4) and
eriocitrin (5), Fig. 2a], hesperetin (2) [hesperidin (6) and
neohesperidin (7), Fig. 2b], and naringenin (3) [narirutin
(9) and naringin (8), Fig. 2c] did not exhibit any observ-
able degranulation-inhibiting activity. On the basis of these
observations, it was apparent that the degranulation-inhib-
iting activity is lost when a sugar moiety (regardless of the
type and number of sugars) is connected to the favonoid
A ring.
Analysis of degranulation‑inhibition assay data
The results are expressed as the mean and SD from three
independent cultures. Multiple data comparisons were
performed by analysis of variance followed by Dunnett’s
test; p values below 5% were regarded statistically sig-
nificant. A single asterisk (*) indicates p < 0.05; two
asterisks (**) indicate p < 0.01. The differences between
two samples were analyzed using an unpaired Student’s t
test (#p < 0.05, ##p < 0.01).
Then, the efect of the structure of the B ring on the
degranulation-inhibiting activity was evaluated. Eriod-
ictyol (1), which has two hydroxyl groups in the B ring,
Fig. 1 Structures of favonoids used in the current study
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Journal of Natural Medicines
Fig. 3 Inhibitory efects of favanones with a hydroxyl substitution on
the B ring on antigen-induced degranulation of RBL-2H3 cells. Wort
wortmannin. All data represent the mean SD of three independent
cultures. *p < 0.05, **p < 0.01 (Dunnett’s test), compared to the con-
trol
Next, the efect of the structure of the C ring on the
degranulation-inhibiting activity was investigated. As shown
in Fig. 4, the degranulation-inhibiting activity markedly
increased when a double bond was present between posi-
tions C-2 and C-3 of the C ring. Luteolin (11), which has a
double bond, exhibited very strong degranulation-inhibiting
activity at a concentration of 10 μM, whereas eriodictyol
(1), which does not possess the double bond, showed little
activity. Similar diferences in activity were observed for
other favanones and their corresponding favones [2 vs. 12,
3 vs. apigenin (13), and 10 vs. chrysin (14)]. On the other
hand, the introduction of a hydroxyl group at the C-3 posi-
tion of the C ring did not afect the activity as there was
no apparent diference in activity between luteolin (11) and
Fig. 2 Inhibitory efects of favanones and their glycosides on anti-
gen-induced degranulation of RBL-2H3 cells. a Eriodictyol (1) and
its glycosides 4 and 5; b hesperetin (2) and its glycosides 6 and 7; c
naringenin (3) and its glycosides 8 and 9. Wort wortmannin. All data
represent the mean SD of three independent cultures. *p < 0.05,
**p < 0.01 (Dunnett’s test), compared to the control
clearly exhibited the strongest activity, as shown in Fig. 3.
Moreover, hesperetin (2) and naringenin (3), which have
one hydroxyl group, exerted weak activity; no activity
was observed for pinocembrin (10), which does not have
a hydroxyl group on the B ring. These observations indi-
cated that the degranulation-inhibiting activity of favanones
depends on the number of hydroxyl groups on the B ring.
Fig. 4 Inhibitory efects of favanones and favones on antigen-
induced degranulation of RBL-2H3 cells. Wort wortmannin. All data
represent the mean SD of three independent cultures. **p < 0.01
(Dunnett’s test), compared to the control. ##p < 0.01 (t test), fa-
vanones vs. favones
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Journal of Natural Medicines
quercetin (15) (data not shown). For the favones, the activ-
ity depended on the number of hydroxyl groups on the B
ring, which was also observed for the favanones (activity:
11 = 12 > 13 > 14). On the basis of these results, when the
structure of the B ring was the same, the favone showed a
considerably stronger degranulation-inhibiting activity than
the favanone (Fig. 4). In other words, the double bond of the
C ring of the favonoid is important for the degranulation-
inhibiting activity. In addition, the activity depends on the
number of hydroxyl groups of the B ring in both the fa-
vanone and the favone (Figs. 3, 4), and these two efects [the
efect of the double bond of the C ring and the efect of the
hydroxyl group(s) in the B ring] are additive.
In conclusion, the inhibiting activity of 17 favonoids
against antigen-induced degranulation of rat basophilic leu-
kemia cells (RBL-2H3) was examined, and several structural
requirements for the activity were clarifed. The experiments
revealed that the inhibitory activity was lost when a sugar
unit was coupled to the A ring of the favanone and that the
activity depended on the number of hydroxyl groups on the
B ring. The inhibiting activities of favones were remark-
ably more pronounced than those of favanones, indicating
the importance of the double bond in the C ring. The spatial
confguration of favones and favanones is considerably dif-
ferent, resulting in diferences in activity. The fndings of
this study might be helpful in designing lead compounds for
the development of therapeutics for type I allergies.
We recently reported that the methylation or ethylation
of the 7-O-hydroxyl group clearly increases the degran-
ulation-inhibiting activity of hesperetin [13]; thus, two
7-O-methylated derivatives, 7-O-methylchrysin (16) and
7-O-methyldiosmetin (17), were synthesized, and the efects
of the methylation on their activity were examined. Com-
parison of the degranulation-inhibiting activities of 14 and
7-O-methylchrysin (16) confrmed that methylation of the
C-7 hydroxyl group potentiated the activity (Fig. 5). In con-
trast, 7-O-methyldiosmetin (17) showed a weaker activity
than diosmetin (12), as shown in Fig. 5. In other words, the
efect of 7-O-methylation of the A ring of favones was dif-
ferent from that of the introduction of a sugar moiety on the
A ring of favanones. The degranulation-inhibiting activities
were lost following glycosidation of the A ring, as shown
in Fig. 2, but in the case of 7-O-methylation, the efect was
not as clear-cut (Fig. 5). Thus, an investigation of additional
compounds is necessary to reach a conclusion about the
efect of 7-O-methylation of the A ring of favonoids on the
degranulation-inhibiting activity.
Acknowledgements The authors would like to thank Editage (http://
www.editage.jp) for English language editing.
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