The Horticulture Journal 89 (4): 384–393. 2020.
doi: 10.2503/hortj.UTD-163

The Japanese Society for
JSHS

Horticultural Science

http://www.jshs.jp/

Relationship Between the Limonoid Content in Different Parts of the Sour
Orange (Citrus aurantium L.) and the Ligand Activity of a Bile Acid Receptor,
TGR5
Fatema Tuz Zohra1**, Yuri Tominaga2**, Yomi Matsumoto3, Naoko Taguchi3, Ai Okubo3,
Susumu Mitsutake1,2,3 and Nobuhiro Kotoda1,2,3*
1

The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, JapanThe Graduate School of Agriculture, Saga University, Saga 840-8502, Japan
3
Faculty of Agriculture, Saga University, Saga 840-8502, Japan
2

Limonoids, a group of highly oxygenated triterpenoids mostly found in the Rutaceae and Meliaceae families,
have many biological and physiological activities, such as anti-cancer, anti-microbial, and insecticidal ones.
Recent studies suggest that some types of limonoids bind to a bile acid receptor, TGR5 (Takeda G protein–coupled receptor 5), and confer anti-obesity and anti-hyperglycemic effects. TGR5, also known as a G protein–coupled bile acid receptor 1 (GPBAR1), is a vital member of the membrane-bound G protein–coupled receptor(GPCR) family. In this study, we revealed the content of four types of limonoids (limonin, nomilin, obacunone,
and limonin glucoside) and TGR5 ligand activity in a sour orange extract. The total concentration of the four
limonoids was highest in the extract of ethyl acetate, followed by methanol and hexane in sour orange seeds.
On the other hand, a luciferase assay using CHO cells transfected with a TGR5 confirmed that TGR5 ligand
activity in the ethyl acetate extract of the seeds was as high as that in 50 μM nomilin, followed by that in themethanol extract of the seeds. The correlation coefficient between the limonoid content and the TGR5 ligand
activity showed the highest value (r = 0.867) for nomilin, which supported a previous report that the TGR5
ligand activity of nomilin is higher than that of limonin or obacunone. However, the activity of the extract
could not be explained by the nomilin content alone because the nomilin concentration in the extract used for
the TGR5 assay was 3.9 μM, much lower than that in the control (50 μM nomilin), suggesting unknown
compounds with higher TGR5 ligand activities in the seed extracts. In addition, the extract from cotyledons orgerminated seeds showed higher TGR5 activity. Taken together, these results indicate that the seeds of citrus,
such as the sour orange, may be a source of compounds that prevent obesity and metabolic disorders. In a
future study, it will be necessary to comprehensively investigate citrus seed extracts to identify unidentified
agonists for the TGR5 receptor.
Key Words: citrus, HPLC, luciferase assay, nomilin, seed.

Introduction
Received; November 4, 2019. Accepted; January 13, 2020.
First Published Online in J-STAGE on April 23, 2020.
This work was financially supported by a research fund from Karatsu
City, Saga Prefecture, Japan, and the Saga University President’s
discretionary funds.
Part of this study was presented at the Annual Meeting of The
Japanese Society for Horticultural Science, Kyushu branch, 2017 and
the Autumn Annual Meeting of The Japanese Society for
Horticultural Science, 2018.
* Corresponding author (E-mail: koto@cc.saga-u.ac.jp or nobu_
fruits@yahoo.co.jp).
** The first two authors have contributed equally to this work (cofirst authorship).

Limonoids represent a group of highly oxygenatedtriterpenoids that are mostly found in the Rutaceae andMeliaceae families (Hasegawa et al., 1999). Citrus planttissues and organs such as the stem, leaf, seed, and fruit(peel and juice sac) accumulate limonoids at a signifi‐
cant level as aglycones or glucosides (Gualdani et al.,2016; Herman et al., 1989; Li et al., 2014; Rouseff andNagy, 1982). The major neutral limonoids of citrus arelimonin, nomilin, obacunone, and deacetylnomilin.
Thirty-nine limonoid aglycones and 17 limonoid gluco‐
sides have been identified in the Citrus species and its© 2020 The Japanese Society for Horticultural Science (JSHS), All rights reserved.

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Hort. J. 89 (4): 384–393. 2020.

hybrids (Jayaprakasha et al., 2008; Patil et al., 2009).
Limonoid aglycones are found predominantly in citrus
seeds and peels, while limonoid glucosides are themajor components in the juice sac (Hasegawa et al.,1980, 1989; Russo et al., 2016; Vikram et al., 2007).
Limonin and nomilin are the two major limonoids incitrus and are responsible for the bitter taste in citrus
fruits (Dea et al., 2013; Endo et al., 2002; Hasegawa
et al., 1973; Higby, 1938). Many studies have demon‐
strated the significant biological and physiological im‐
pacts of limonoids related to human health, such as
anti-cancer (Jacob et al., 2000; Lam et al., 1994; Miller
et al., 2004) and anti-microbial (Chowdhury et al.,
2003; Govindachari et al., 2000) activities.
TGR5 (Takeda G protein–coupled receptor 5), also
known as novel G protein–coupled bile acid receptor 1(GPBAR1), is a vital member of the membrane-boundG protein–coupled receptor (GPCR) family (Kawamata
et al., 2003; Maruyama et al., 2002). TGR5 is expressedin various tissues and organs throughout the body, suchas the heart, liver, lung, spleen, kidney, placenta, stom‐ach, gallbladder, intestine, brown adipose tissue, andendocrine glands and is recognized and activated by thebinding of bile acids (BAs) as its endogenous ligands(Duboc et al., 2014; Kawamata et al., 2003; Maruyama
et al., 2002; Pols et al., 2011). Maruyama et al. (2006)
reported that TGR5/GPBAR1-disrupted mice fed a highfat diet showed significant fat accumulation with bodyweight gain as compared with wild-type mice, suggest‐
ing a potential role of the gene in energy homeostasis.
Notably, Watanabe et al. (2006) showed that the bind‐ing of bile acid with TGR5 induced energy expenditureby promoting thyroid hormone activation. Since then,
TGR5 has attracted the attention of researchers as animportant target for the potential treatment of several
metabolic disorders (Tiwari and Maiti, 2009; vanNierop et al., 2017). Recently, TGR5 was found to be
involved in glucose uptake into cells and in the remod‐
eling of energy-storing white fat into energy-expending
beige fat, suggesting its potential anti-obesity and antihyperglycemic effects (Lo et al., 2016; VelazquezVillegas et al., 2018).
As naturally occurring TGR5 agonists, oleanolic acidfrom the olive (Olea europaea L.) and limonoids, suchas the nomilin and obacunone abundant in citrus fruits,
have been discovered and investigated in detail (Genetet al., 2010; Horiba et al., 2015; Ono et al., 2011; Satoet al., 2007). These compounds derived from agricultur‐
al crops lowered serum glucose and/or insulin levels inmice fed with a high-fat diet (Horiba et al., 2015; Onoet al., 2011; Sato et al., 2007). Based on these reports,such TGR5 activators contained in agricultural crops
like olive and citrus could be expected to bind to the
TGR5 receptor expressed in the stomach and small in‐testine and also exert effects such as anti-obesity activi‐
ty after ingestion by humans. In recent years, obesity
has become a major problem in developed countries

due to nutritional bias in the diet and lack of exercise.
In the future, it will be important to make more use ofhealth-functional ingredients contained in foods andagricultural products that are locally available.
In the context described above, we preliminarily
screened the seed extracts of seven citrus plants for
TGR5 ligand activity and found the sour orange to havethe highest activity. Several previous studies reported
the limonoid content in sour orange fruit and seed ex‐
tracts (Bennett et al., 1991; Dandekar et al., 2008;
Matsumoto et al., 2008; Miyake et al., 1992; Ozaki
et al., 1991; Vikram et al., 2007). However, no studies
have considered the relationship between limonoid con‐tent and TGR5 ligand activity in citrus extracts. There‐
fore, in this study we aimed to clarify the relationshipbetween the content of four major limonoids and TGR5
ligand activity in sour orange fruit by using highperformance liquid chromatography (HPLC) and aluciferase assay system.

Materials and Methods
Plant materials
The sour orange (Citrus aurantium) fruit used in thisstudy was introduced from Italy and was collected in
February 2017 and April 2019 from the field of the Fac‐
ulty of Agriculture at Saga University (Fig. 1). The fruit
was separated into seeds, juice sac, and peel. Subse‐
quently, a single seed was divided into the seed coatand cotyledon. Sour orange seeds without seed coats
were germinated on a glass petri dish containing moist‐
ened blotting paper and grown under dark conditions at
25°C in a growth chamber. Ten-day-old germinatedseeds were transplanted from the petri dish to moist‐
ened potting soil composed of vermiculite and grown ina greenhouse under natural light conditions. In thisstudy, the peel, juice sac, seeds, seed coat, cotyledon,germinated seeds, and 2- and 4-week-old seedlings
were used as plant materials.
Preparation of plant extracts
Freeze-dried tissue samples were ground to a fine
powder with a multi-bead shocker (Yasui Kikai Corpo‐
ration., Osaka, Japan). The powder was subjected toFig. 1.

The sour orange fruit used in this study. Scale bar, 3 cm.

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F. T. Zohra, Y. Tominaga, Y. Matsumoto, N. Taguchi, A. Okubo, S. Mitsutake and N. Kotoda

solvent extraction prior to HPLC analysis.
Two grams of freeze-dried powder of the seed, peel,
and juice sac (sampled in February 2017) was placed ina Soxhlet extractor (TGK Co., Ltd., Tokyo, Japan) andextracted with 100 mL hexane at 60–70°C for 2 h.
Then, it was further extracted with 100 mL of ethyl ac‐
etate at the same temperature as above for 4 h. The
ethyl acetate extract was transferred to a flask, and25 mL of methanol was added to the residues. After
ultrasonication for 10 min, the extracts were kept atroom temperature for 1 h, followed by filtration. The
methanol extraction procedure was repeated twice. The
solvent was completely evaporated with a rotary evapo‐
rator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at
100 rpm and 40°C, then diluted with appropriate sol‐
vents to be used for HPLC analysis. For the intact seed,cotyledon, seed coat, germinated seed (10 days underdark conditions at 25°C after sowing), and 2- and 4week-old seedlings (derived from the seeds sampled in
April 2019), 1 g of freeze-dried powder was extractedwith 10 mL of 70% acetone three times. A total of
30 mL of the extract was evaporated with a rotary evap‐
orator (Tokyo Rikakikai) at 40°C under low pressure to
remove the acetone. The residual water was transferred
into a tube. After 10 mL of ethyl acetate was added, thetube was vortexed and centrifuged at 500 rpm for
10 min, and then the ethyl acetate layer was removed toa new tube (this step was repeated thrice). Sodium sul‐fate was added to the ethyl acetate layer, followed byfiltration. Then, the ethyl acetate was evaporated to dry‐ness with a rotary evaporator (Tokyo Rikakikai). Each
concentrated extract derived from 1 g of freeze-dried
powder was dissolved in 1 mL of dimethyl sulfoxide(DMSO) as a stock solution for HPLC analysis and aluciferase assay.
Quantitative and qualitative analysis of limonoids using
HPLC
Quantitative and qualitative analysis of limonoids
was performed using an HPLC system (JASCO Corp.,
Tokyo, Japan) with a UV detector (UV-1570; JASCO)
and an Inertsil ODS-3 column (3 μm, 4.6 × 100 mm;
GL Sciences Inc., Tokyo, Japan) at 40°C. The mobile
phases were 10% acetonitrile containing 3 mM phos‐
phoric acid (solvent A) and 50% acetonitrile containing
3 mM phosphoric acid (solvent B). The elution of a bi‐nary solvent was conducted in a gradient fashion, start‐
ing at 100/0 (A/B), changing to 15/85 (A/B) for 50 min,and maintained at 0/100 (A/B) for 5 min, with a flowrate of 1.2 mL·min−1. Five microliters of each sample
was injected, and an absorbance of 210 nm was mea‐
sured using the UV detector. Quantitative determina‐
tions were carried out based on the 25, 50, and 100 ppm
standards of limonin (Sigma-Aldrich, St. Louis, MO,
USA), nomilin (LKT Laboratories, Inc., St. Paul, MN,
USA), obacunone (provided by Dr. Hasegawa of the
Fruit and Vegetable Chemistry Laboratory, Agricultural

Research Service, United States Department of Agri‐culture), and limonin glucoside (LKT Laboratories)
using a chromatography data system (ChromNAV
1.0; JASCO). For qualitative analysis, four (limonin,
nomilin, obacunone, and limonin glucoside; used inquantitative analysis) and five (ichangin, deoxylimonin,deacetylnomilin, limonol, and limonin methoxime; pro‐vided by Dr. Hasegawa) standard limonoids were used.
Cell culture
CHO (Chinese hamster ovary) cells were cultured in
MEMα (alpha-modified minimum essential medium)(FUJIFILM Wako Pure Chemical Corp., Osaka, Japan)
supplemented with 1% penicillin/streptomycin (SigmaAldrich) and 10% fetal bovine serum (FBS, Gibco®;
Thermo Fisher Scientific, Waltham, MA, USA). The
cells were cultured in an incubator (MCO-5AC;
SANYO Electric Co., Ltd., Osaka, Japan) set at 37°C in
a humidified atmosphere of 5% CO2. Cells were sub‐cultured every three days.
Assay for TGR5 ligand activity
To measure TGR5 ligand activity, a luciferase assay
system was used in this study. p3 × FLAG-hTGR5 wasconstructed by cloning a polymerase chain reaction(PCR) fragment of human TGR5 cDNA derived from
IRAK064N14 (RIKEN Gene Bank, Tsukuba, Japan)
into a p3×FLAG-CMV vector (Sigma-Aldrich). First,
TGR5 genes (p3 × FLAG-hTGR5), CRE (cyclic AMP
response
element)
reportergenes[pGL4.29(luc2P/CRE/Hygro); Promega Corp., Madison, WI,
USA], and polyethylenimine max (Polysciences Inc.,
Warrington, PA, USA) were placed in a 96-well plate.
Second, CHO cells were placed at a density of 6 × 104
cells/well in 96-well plate for transfection. After a 4 h
incubation of the CHO cells, the medium was replacedwith MEMα containing 10% charcoal-stripped FBS.
Samples [stock solutions for each extract, positive con‐trols, and dimethyl sulfoxide (DMSO)] diluted with themedium (1:250) were added at a volume of 100 μL/well
24 h after transfection. Five hours after the samples
were added, a solution of Bright-GloTM (Promega
Corp.) was added to each well of the plate. Chenode‐
oxycholic acid (CDC, a bile acid; FUJIFILM Wako
Pure Chemical), limonin (Sigma-Aldrich), and nomilin(LKT Laboratories) were used as controls at a final
concentration of 50 μM. TGR5 ligand activity was mea‐
sured by detecting luciferase activity through lumines‐
cence with a luminometer (Luminoskan Ascent FL;
Thermo Fisher Scientific). In an assay of the extracts,
the stock solution (each sample derived from 1 g ofstarting materials was dissolved in 1 mL of DMSO) was
finally diluted 250 times in the medium as a 1/250 di‐luted sample (a sample derived from 4 mg of starting
materials per mL). The sample solution was further di‐luted in the medium and used for a dose-dependent
assay with 1/1250, 1/2500, and 1/5000 diluted samples.

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Hort. J. 89 (4): 384–393. 2020.

Results
Limonoid content in the sour orange
HPLC was used to determine the limonoid content in
the sour orange seed, peel, and juice sac sequentiallyextracted using three solvents (hexane, ethyl acetate,
and methanol). As shown in Figure 2, four limonoids(limonin, nomilin, obacunone, and limonin glucoside)
were found in the seed and peel, while only two
limonoids (nomilin and limonin glucoside) were foundin the juice sac. The total concentration of the four
limonoids was highest in the ethyl acetate extract of theseed (1201 μg·g−1 DW), followed by the methanol ex‐
tract of the seed (627 μg·g−1 DW) and the ethyl acetate
extract of the peel (230 μg·g−1 DW) (Table S1; Fig. 2).
In seeds, the total limonin, nomilin, obacunone, andlimonin glucoside contents were 948, 872, 40, and161 μg·g−1 DW, respectively (Table S1; Fig. 2).

assay system. The ethyl acetate extract of the seed
showed a level of TGR5 ligand activity (relative valueof luminescence = 3.3) similar to that (relative value ofluminescence = 3.1) of 50 μM CDC and nomilin(Fig. 3A). However, the final concentration of the totallimonoids (limonin, nomilin, and obacunone) in the ex‐
tract was 9.9 μM (5.6, 3.9, and 0.4 μM, respectively),
which was much lower than the 50 μM nomilin control(Table S1; Fig. 3A). The hexane and methanol extractof the seed and the hexane extract of the peel also
showed significantly higher activity than the blank(DMSO). In addition, a dose-dependence effect was ob‐
served in the assay in which the concentration of theseed extract was changed from 1/250 to 1/5000 of thefirst stock solution of the samples (Fig. 3B).
A positive correlation between TGR5 ligand activity
and limonoid content was demonstrated because theA

6.0
Relative luciferase activity(DMSO set to 1)

Statistical analysis
Data were analyzed using a one-way ANOVA andthe differences were contrasted using Tukey’s multiple
comparison test. The relationship between the limonoid
content and TGR5 ligand activity was investigatedusing Pearson’s correlation analysis. All statistical anal‐
yses were performed at a significance level of P < 0.05
using R-3.2.0 (The R Project for Statistical Computing,
http://www.R-project.org/, January 7, 2020).

5.0

d

4.0

bd

3.0
2.0

cd

bc

bc

b

a

1.0

a

a

a

a

a

0.0

Correlation of limonoid content and TGR5 ligand activ‐
ity
TGR5 ligand activity in the extract from the seed,
peel, and juice sac was measured using a luciferase

700

Limoninµg·g-1 DW

600

NomilinObacunone

500

Limonin glucoside
400

Peel

Juice sac

(1/250 dilution)B

6.0

Relative luciferase activity(DMSO set to 1)

800

Seed

5.0
4.0
3.0
2.0
1.0
0.0

300

Hexane

EtOAc

MeOH

Seed

200
100
0

Seed

Peel

Juice sac

Fig. 2. The concentrations of four limonoids (limonin, nomilin,
obacunone, and limonin glucoside) extracted from the seed,
peel, and juice sac of the sour orange using a Soxhlet extractor
with three different solvents (hexane, ethyl acetate, andmethanol) sequentially. All of the tissues were freeze-dried be‐fore extraction. Each extract derived from 1 g of the freezedried sample was dissolved in 1 mL of DMSO, and then a
1/250 dilution of the first prepared extract was used for theassay. The values are the mean of results from three biologicalreplicates. The error bars indicate the standard deviation (SD).

Fig. 3. TGR5 ligand activity assay. (A) Luciferase assay for TGR5
ligand activity in extracts from the seed, peel, and juice sac of
the sour orange. CDC (50 μM), nomilin (50 μM), and each ex‐
tract (using hexane, ethyl acetate, and methanol) of the sour or‐ange seed, peel, and juice sac (1/250 dilution of the firstprepared extract) were subjected to the assay. Different lettersat the top of each bar indicate significant differences among thesamples contrasted by Tukey’s multiple comparison test (P <0.05). (B) Luciferase assay for TGR5 ligand activity in differ‐
ent dilutions of the seed extract of sour oranges. Different dilu‐
tions (1/250, 1/1250, 1/2500, and 1/5000) of the first preparedextract (using hexane, ethyl acetate, and methanol) of sour or‐ange seeds were subjected to the assay. The values were ex‐pressed as a relative luciferase activity (DMSO used as a blank
was set to 1). Data are represented as the mean ± SD of fivetechnical replicates.

4

B

3
2
1
0

r = 0.787
0

200
400
600
800
Limonin content (µg・g-1 DW)

D

4
3
2
1
0

r = 0.444
0

10

20

30

40

50

Relative luciferase activity(DMSO set to 1)

Obacunone content (µg・g-1 DW)

ERelative luciferase activity(DMSO set to 1)

F. T. Zohra, Y. Tominaga, Y. Matsumoto, N. Taguchi, A. Okubo, S. Mitsutake and N. Kotoda

4

Relative luciferase activity(DMSO set to 1)

C

Relative luciferase activity(DMSO set to 1)

A

Relative luciferase activity(DMSO set to 1)

388

4
3
2
1
0

r = 0.867
0

200
400
600
Nomilin content (µg・g-1 DW)

4
3
2
1
0

r = 0.006
0

100

200

Limonin glucoside content (µg・g-1 DW)Hexane fraction
Ethyl acetate fraction
Methanol fraction

3
2
1
0

r = 0.808
0

250

500

extracts obtained from the intact seed, cotyledon andseed coat showed significantly higher luciferase activity
than the blank (DMSO). The cotyledon extract showed
the maximum luciferase activity, as compared to the in‐tact seed and seed coat, whereas the intact seed andseed coat extracts showed similar luciferase activities(Fig. 5B).

〇 seed extract□ peel extract△ juice sac extract

750 1000 1250

Total limonoid content (µg・g-1 DW)

Fig. 4. Scatter plots between the limonoid content (μg·g−1 DW) andthe values expressed as relative luciferase activity (DMSO was
set to 1). (A) Limonin, (B) nomilin, (C) obacunone, (D)limonin glucoside, and (E) total limonoids. Each panel shows
the plot of the values. The correlation coefficient was calculat‐
ed using Pearson’s correlation analysis.

order of the three extracts in the TGR5 ligand activity
was almost the same as that of the limonoid content de‐tected by HPLC (Fig. 4). Scatter plots of the limonoid
content (limonin, nomilin, obacunone, limonin gluco‐side, and total limonoids) and luciferase activity mea‐
sured for each extract showed a positive correlation
with a high correlation coefficient of 0.867 (Fig. 4B),
0.808 (Fig. 4E), and 0.787 (Fig. 4A) for nomilin, totallimonoids, and limonin, respectively.
Limonoid content and TGR5 ligand activity in cotyle‐dons and seed coats
Seeds were investigated in more detail by separatingthem into the cotyledon (seed without the seed coat)and the seed coat. The intact seed and cotyledon of thesour orange contained two major limonoids, limoninand nomilin, whereas the seed coat contained thoselimonoids a lower levels than the intact seed and cotyle‐don (Fig. 5A). The limonin and nomilin contents varied
depending on the tissues and showed a significant dif‐ference between them. The highest level of limonin wasdetermined in the cotyledon (1090 μg·g−1 DW), fol‐
lowed by the intact seed (720 μg·g−1 DW) and seed coat(300 μg·g−1 DW). The nomilin concentration was sig‐
nificantly higher in the cotyledon (770 μg·g−1 DW) thanin the intact seed (270 μg·g−1 DW) and seed coat (verysmall amount). The TGR5 ligand activity of the intact
seed-, cotyledon-, and seed coat–derived extract wasmeasured using a luciferase assay system (Fig. 5B). The

Limonoid content and TGR5 ligand activity in seedlings
at early developmental stages
Limonoid content was also determined via HPLC in
the germinated seeds (10 days under dark conditions at
25°C after sowing) and 2- and 4-week-old sour orange
seedlings (Fig. 5C). Limonin and nomilin were present
in significant amounts in all of the tissues. The limonincontent was found to be highest (1460 μg·g−1 DW) in
the germinated seeds, followed by 2-week-old(710 μg·g−1 DW) and 4-week-old (620 μg·g−1 DW)
seedlings. The nomilin concentrations were similar in
the germinated seeds (450 μg·g−1 DW) and 2-week-old(530 μg·g−1 DW) and 4-week-old (620 μg·g−1 DW)
seedlings. TGR5 ligand activity was also measured in
the extracts of the germinated seeds and the 2- and 4week-old seedlings using a luciferase assay system. All
extracts exhibited significant activity for TGR5
(Fig. 5D). As expected, the germinated seed extract ex‐hibited higher ligand activity, followed by the extract
derived from the 2- and 4-week-old seedlings.
TGR5 ligand activity in seven fractions of the ethyl ac‐
etate extract of intact seeds
An aliquot of the ethyl acetate extracts of intact seeds
were separated into seven fractions via HPLC
(Fig. 6A). After drying, each fraction was dissolved in
the same volume of DMSO as that injected for fraction‐
ation to adjust the concentration of each sample for the
TGR5 assay. The result showed that there were higher
activities in fractions 6 and 7, but lower or no activities
in the other fractions (Fig. 6B). To identify the peaks inthe chromatogram in Figure 6A, nine standard
limonoids often detected in citrus were analyzed togeth‐
er with an extract of intact seeds and a blank sample(50% MeOH) via HPLC with a C18 ODS column at
210 nm. Limonin glucoside was eluted first at a re‐tention time of 13.21 min, followed by ichangin,deoxylimonin, deacetylnomilin, limonol, limonin,
nomilin, obacunone, and limonin methoxime at reten‐tion times of 24.60, 24.75, 26.81, 27.06, 28.21, 31.21,
34.17, and 34.48, respectively (Fig. 7). Two broad
peaks eluted later than 36 min were derived from mo‐bile phases.

Discussion
Limonoid content in the sour orange
We confirmed the distribution of major limonoids(limonin, nomilin, obacunone, and limonin glucoside)in sour orange fruit by extracting these compounds

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Hort. J. 89 (4): 384–393. 2020.

(μg・g-1 DW)

1.0
1000
0.8
800

a

Limoninb

Nomilin

b

0.6
600
0.4
400

c

c

0.2
200

tr.

0.00

B

7.0

Relative luciferase activity(DMSO set to 1)

A 1200
1.2

a

6.0
5.0

1.0

b

bc

3.0
2.0

cd

d

0.0

50 μM
2.0
2000

Limonin
Nomilina(μg・g-1 DW)

1.6
1600
1.2
1200
0.8
800

b

b

b

b

b

0.4
400
0.00

D

1/250
a

4.0

Relative luciferase activity(DMSO set to 1)

C

b

b

4.0

3.5

ab

3.0
2.5
cd

2.0
1.5
1.0

e

bc

cd
de

0.5
0.0

50 μM

1/250

Fig. 5. Limonoid content and TGR5 ligand activity in seeds, germinated seeds, and seedlings. (A) The concentration of limonin and nomilin in
ethyl acetate extracts from the intact seed, cotyledon (seed without seed coat), and seed coat of the sour orange; tr. = trace amount. (B)Luciferase assay for TGR5 ligand activity in extracts from the intact seed, cotyledon, and seed coat of the sour orange. (C) The concentrationof limonin and nomilin in ethyl acetate extracts from the germinated seed (10 days under dark conditions at 25°C after germination) and 2and 4-week-old seedlings (germinated seeds were transplanted into pots containing vermiculite in a greenhouse) of the sour orange. (D)Luciferase assay for TGR5 ligand activity in extracts from the germinated seed and 2- and 4-week-old seedlings of the sour orange. CDC(50 μM), limonin (50 μM), nomilin (50 μM), and extracts (1/250 dilution of the first prepared extracts) from the intact seed, cotyledon, seed
coat, germinated seed, and 2- and 4-week-old seedlings of the sour orange were subjected to the assay. The ethyl acetate extracts were pre‐pared by extraction with ethyl acetate from the aqueous layer after concentration of 70% acetone extracts from each tissue. The values (B, D)
are expressed as a relative luciferase activity (DMSO was set to 1). Data are represented as the mean ± SD of three biological (A, C) and fivetechnical (B, D) replicates. Different letters on each bar indicate significant differences among the samples contrasted by Tukey’s multiple
comparison test (P < 0.05).

sequentially with three different solvents. Overall, thefour limonoids accumulated mainly in the seeds(2021 μg·g−1 DW), followed in order by the peel(443 μg·g−1 DW) and juice sac (83 μg·g−1 DW)(Table S1; Fig. 2). These results were consistent withthose of previous reports showing that the distributionof limonoids in citrus fruit is tissue-specific, as the con‐centration was different in each tissue, and that the seedis the source of abundant limonoids in fruit (Hasegawa
et al., 1980; Miyake et al., 1992; Ohta and Hasegawa,
1995; Rouseff and Nagy, 1982; Sun et al., 2005; Wanget al., 2016).
In the sour orange seeds used in this study, the con‐centrations of limonin, nomilin, obacunone, and limonin
glucoside were 948, 872, 40, and 161 μg·g−1 DW, re‐spectively (Table S1; Fig. 2). Rouseff and Nagy (1982)
reported the concentrations of limonin, nomilin, andobacunone to be 1256, 242, and 104 μg·g−1 DW, respec‐tively, in the common sour orange. Miyake et al. (1992)
reported a concentration of obacunone (32 μg·g−1 DW)
in sour orange seeds that was similar to that in ourstudy, although those of limonin, nomilin, and limonin
glucoside (2470, 178, and 610 μg·g−1 DW, respectively)

were different from those in our study. On the other
hand, Vikram et al. (2007) detected no nomilin in a sourorange seed extract. Considering reports that thelimonoid content depends on the maturation stage or
cultivar (Matsumoto et al., 2008; Sun et al., 2005; Wanget al., 2016), inconsistencies in the reported limonoid
concentration seem to be due to differences in the sea‐son of sampling, cultivar/strain of the sour orange, or
method of sample preparation.
Correlation of limonoid content and TGR5 ligand activ‐
ity
The ligand activity of TGR5 was significantly higher
in the extract from seeds, which contained a higher con‐centration of limonoids, than in extracts from otherorgans, except for the hexane extract from the peels(Fig. 3A). All of the samples that showed a value sig‐
nificantly higher than the blank (DMSO) contained
nomilin to some extent (Figs. 2 and 3A). The effect ofdose dependence on ligand activity indicated that theextract definitely contained compounds that highly acti‐
vated TGR5 (Fig. 3B). It is noteworthy that the correla‐tion coefficient between TGR5 ligand activity and

390

A

F-7

F-3

F-4
F-5
F-6

F-2

F-1

F. T. Zohra, Y. Tominaga, Y. Matsumoto, N. Taguchi, A. Okubo, S. Mitsutake and N. Kotoda(F=Fraction)

Solvent (EtOAc)
derivedIntensity (µV)

75000

50000
D

Mobile phase
derived

L

N

25000

0

0.0

B

10.0

20.0

30.0

Retention Time (min)

40.0

3

Relative luciferase activity(DMSO set to 1)

2.5
2
1.5
1
0.5
0

Fractions of intact seed extracts

Fig. 6. TGR5 ligand activity in fractions of the ethyl acetate extractof the sour orange seed. (A) Representative HPLC profile ofthe ethyl acetate extract of the sour orange seed at 210 nm. The
ethyl acetate extract was prepared in the same way as shown in
Figure 5. LG, limonin glucoside; L, limonin; D, deacetyl‐
nomilin; N, nomilin. Gray boxes show the fractions with higher
ligand activity for TGR5. (B) TGR5 ligand activity in the frac‐
tions of seed extract separated via high performance liquid
chromatography as shown in (A). CDC (50 μM) was used as apositive control. The values in (B) are expressed as a relative
luciferase activity (DMSO was set to 1). Data are represented
as the mean ± SD of five technical replicates.

nomilin content was highest among the four limonoids
analyzed in this study (Fig. 4B). On the other hand,
there was no correlation (r = 0.006) between TGR5 li‐gand activity and limonin glucoside content, suggestingthat limonin glucoside did not contribute to TGR5 re‐ceptor activation (Fig. 4D). Our results revealed that
nomilin in the sour orange extracts effectively activated
the TGR5 receptor. These results are consistent with thereport of Ono et al. (2011), in which the TGR5 ligand
activity of nomilin was higher than that of limonin or
obacunone. However, the ethyl acetate extract of theseeds showed activity equivalent to that of a positive
control of 50 μM nomilin despite the fact that the final
limonoid concentration (limonin: 5.6 μM; nomilin:
3.9 μM; obacunone: 0.4 μM) derived from the ethyl ac‐
etate extract in the assay was much lower than 50 μM(Figs. 2 and 3). This result suggests that some com‐pounds may have higher TGR5 ligand activity than
nomilin, which was expected to be a major compound
for activating TGR5 in the extracts.

Limonoid content and TGR5 ligand activity in seeds
and seedlings
Because higher TGR5 ligand activity was found in
the seed extract, we investigated both the limonoid con‐tent and the TGR5 ligand activity in more detail in theseeds and seedlings. It was found that limonin andnomilin accumulated more in the cotyledons than in theseed coats (Fig. 5A). As expected, the TGR5 ligand ac‐
tivity of the cotyledons was higher than that of the seedcoats. However, the TGR5 ligand activity of the seedcoats showed a level similar to that of the intact seeds,
suggesting that some seed coat compounds other than
nomilin activate the TGR5 receptor because only a
trace amount of nomilin was detected in the seed coats(Fig. 5).
Germinated seeds (10 days under dark conditions
after sowing) had a higher limonin content (1460 μg·g−1
DW) than cotyledons (seeds without seed coats)(1097 μg·g−1 DW), but a lower nomilin content(459 μg·g−1 DW) than cotyledons (771 μg·g−1 DW)(Fig. 5C). The total limonoid content (limonin +nomilin) before (1868 μg·g−1 DW in cotyledon) andafter (1919 μg·g−1 DW in the germinated seed) germi‐
nation was not greatly changed. This could be due tothe metabolism of nomilin to limonin through ichanginin the sour orange biosynthetic pathway (Miyake et al.,1992). In contrast, Ariza et al. (2015) reported that ahigher amount of limonoids, such as limonin, nomilin,
and ichangin, was present in germinating seeds than indormant seeds of Citrus aurantium. After germination(two and four weeks after transplanting to pots in agreenhouse), the limonin content decreased gradually,
whereas the nomilin content did not vary significantly
from germination to four weeks after transplanting(Fig. 5C). As expected, the TGR5 ligand activity was
higher in the germination stage, followed by that in 2and 4-week-old seedlings (Fig. 5D). The differences inlimonin content reflect those in TGR5 activity because
the nomilin content was almost the same among thethree samples.
In the seeds of citruses, including the sour orange, alarge amount of limonoid glucosides is accumulatedwith concentrations of 0.31 to 0.87% of the dry weight(Miyake et al., 1992; Ozaki et al., 1991), whereas
young seedlings contain practically no limonoid gluco‐
sides, suggesting the presence of β-glucosidase activity
in seeds (Ronneberg et al., 1995). On the other hand,
nomilin is synthesized in stem and root tissues, not inleaves, fruits, or seeds, in Citrus limon (Hasegawa
et al., 1986). In light of these facts, limonoid aglycones,
such as limonin and nomilin, in germinated seeds andseedlings may be derived partly from these glucosides
and partly from de novo synthesis in the stem and roots.
Because the concentration of nomilin did not greatlychange until at least four weeks after germination, thetotal amount of nomilin per plant increases during the
growth of the seedlings. Although the enzymatic libera‐

391

Hort. J. 89 (4): 384–393. 2020.
Solvent (DMSO) derived

34.17

Solvent (EtOAc)
derived

34.48

24.75
31.21

60000

28.21

Intensity (µV)

27.06
24.60

40000

26.81

13.21

20000

0

DL

LG
0.0

10.0

20.0

Mobile phase
derived peak

50% MeOH
Limonin methoxime
Obacunone
Nomilin
Limonin
Limonol
Deacetylnomilin
Deoxylimonin
Ichangin
Limonin glucoside
Intact seed

N

30.0

40.0

Retention Time (min)Fig. 7. Chromatograms of nine standard limonoids and the ethyl acetate extract of intact seeds separated via high performance liquid chromatog‐
raphy with a C18 ODS column detected at 210 nm. The ethyl acetate extract was prepared in the same way as shown in Figure 5. Methanol(MeOH, 50%) was used as a blank. Nine standard limonoids (limonin glucoside, ichangin, deoxylimonin, deacetylnomilin, limonol, limonin,
nomilin, obacunone, and limonin methoxime) were subjected to analysis. The value on each peak represents the retention time (min) of eachof the nine compounds. LG, limonin glucoside; D, deacetylnomilin; L, limonin; N, nomilin.

tion of limonin and nomilin from their glucosides could
be a problem in the juice industry because of their bit‐terness, it may contribute to the creation of a source of
bioactive limonoid aglycones.
Substances with TGR5 ligand activity in citrus seed ex‐
tracts
Our study suggested that the citrus extracts may also
contain compound(s) other than nomilin with TGR5
ligand activity. To investigate these unidentified active
substances, we separated the ethyl acetate extracts ofthe seeds into seven fractions via HPLC (Fig. 6A),
since the aqueous phase after extraction with ethyl ac‐
etate showed no activity in the TGR5 ligand assay (datanot shown). This result revealed that the two fractions
contained higher activity, as shown in Figure 6B (F-6
and F-7, shaded gray). As expected, fraction 6 (F-6),
which contained nomilin (N), showed high activity sim‐
ilar to that of 50 μM CDC. In addition, fraction 7 (F-7)
showed activity at the same level as 50 μM CDC (posi‐tive control). To search for known limonoids that could
be eluted around F-7, nine standard limonoids wereseparated via HPLC together with the ethyl acetate ex‐
tract and methanol as a blank. According to the chro‐
matogram shown in Figure 7, limonin glucoside (LG),ichangin and deoxylimonin, deacetylnomilin (D) andlimonol, and limonin (L) should be contained in F-2,
F-3, F-4, and F-5, respectively, which had no, or muchless, activity as compared to the positive control. On theother hand, obacunone and limonin methoxime should
be contained in F-7, but those limonoids were eluted

after nomilin, where there were only small or broad
peaks detected at 210 nm for the seed extracts (Figs. 6A
and 7).
Bennett and Hasegawa (1980) and Miyake et al.
(1992) reported that sour orange seeds contained
ichangin, isolimonic acid, and several kinds of limonoid
glucosides, in addition to limonoid aglycones including
the four examined in this study. TGR5 binds certain
limonoid aglycones, such as nomilin and obacunone,
but does not seem to bind their glucosides effectively.
However, water-soluble limonoid glucosides could be
utilized as TGR5 agonists after the liberation of their
aglycones by hydrolysis in humans, considering a re‐port that limonin glucoside is absorbed and metabolized
to produce limonin in humans (Manners et al., 2003).
Recently, Sasaki et al. (2017) constructed an hTGR5–
nomilin binding model and demonstrated that four hy‐
drophilic hydrogen-bonding interactions occurred be‐
tween four oxygen atoms of nomilin and three aminoacid residues of hTGR5. The binding mechanism of
nomilin to TGR5 is similar to that of obacunone, butdifferent from that of taurolithocholic acid (TLCA), akind of bile acid (Sasaki et al., 2017). Based on their re‐sults and ours, novel agonists with higher activity for
TGR5 could be found in citrus fruits, such as the sourorange, by considering compounds with some het‐
eroatoms which could form hydrogen bonds with spe‐cific amino acid residues of TGR5, like nomilin andobacunone.

392

F. T. Zohra, Y. Tominaga, Y. Matsumoto, N. Taguchi, A. Okubo, S. Mitsutake and N. Kotoda

Conclusion
In this study, we revealed the content of four kinds oflimonoids and theTGR5 ligand activity in extracts ob‐
tained via sequential extraction using three different
solvents from the seed, peel, and juice sac of the sourorange. In addition, we confirmed a high correlation be‐
tween nomilin content and TGR5 ligand activity, al‐though the activity could not be explained by the
nomilin content in the seed extract alone. Therefore,
sour orange seeds may contain components other than
nomilin with TGR5 ligand activity. The seed or germi‐
nated seed extracts of citruses such as the sour orange,
may be a source of compounds that prevent obesity andmetabolic disorders. To the best of our knowledge, thisis the first study to report on the TGR5 activity in crude
extracts from citrus tissues. In a future study, it will benecessary to comprehensively investigate citrus seedextracts for unidentified agonists for the TGR5 receptor.

Acknowledgements
The authors thank Dr. S. Hasegawa of the Fruit andVegetable Chemistry Laboratory, Agricultural ResearchService, United States Department of Agriculture, forkindly providing us with standard limonoids.

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