Flavonoids and a Limonoid from the Fruits of Citrus unshiu and Their Biological Activity

Article abstract of DOI:10.1021/acs.jafc.6b03465

The fruits of Citrus unshiu are one of the most popular and most enjoyed fruits in Korea. As we continue to seek for bioactive metabolites from Korean natural resources, our study on the chemical constituents of the fruits of C. unshiu resulted in the isolation of a new flavonoid glycoside, limocitrunshin 1, along with seven other flavonoids 2-8 and a limonoid 9. All structures were identified by spectroscopic methods, namely 1D and 2D NMR, including HSQC, HMBC, and ¹H-¹H COSY experiments, HRMS, and other chemical methods. Compounds 3, 5, and 9 are reported to be isolated from this fruit for the first time. The isolated compounds were applied to activity tests to verify their inhibitory effects on inflammation and nephrotoxicity. Compounds 6 and 9 showed the most potent inhibitory activity on renal cell damage and nitric oxide production, respectively. Thus, the fruits of C. unshiu could serve as a valuable natural source of bioactive components with health benefits for potential application in functional foods.

Full text of DOI:10.1021/acs.jafc.6b03465

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Article
Flavonoids and a Limonoid from the Fruits
of Citrus unshiu and their Biological Activity
Hee Jeong Eom, Dahae Lee, Seulah Lee, Hyung Jun Noh, Jae
Wook Hyun, Pyoung Ho Yi, Ki Sung Kang, and Ki Hyun Kim
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03465 • Publication Date (Web): 08 Sep 2016
Downloaded from http://pubs.acs.org on September 9, 2016
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Flavonoids and a Limonoid from the Fruits of Citrus unshiu and
their Biological Activity
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Hee Jeong Eom,† Dahae Lee,†,‡ Seulah Lee,† Hyung Jun Noh,§ Jae Wook Hyun,#
Pyoung Ho Yi,# Ki Sung Kang,‡,* Ki Hyun Kim†,*
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†School of Pharmacy, Sungkyunkwan University, Suwon 440ꢀ746, Republic of Korea
‡College of Korean Medicine, Gachon University, Seongnam 461ꢀ701, Republic of Korea
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§Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science,
RDA, Eumseoung 369ꢀ873, Republic of Korea
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#Citrus Research Station, National Institute of Horticultural & Herbal Science, Rural
Development Administration, Jeju 697ꢀ943, Republic of Korea
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*Corresponding author (Tel: +82ꢀ31ꢀ290ꢀ7700; Fax: +82ꢀ31ꢀ290ꢀ7730; Eꢀmail:
khkim83@skku.edu)
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ABSTRACT: The fruits of Citrus unshiu are one of the most popular and mostly enjoyed
fruits in Korea. As we continue to seek for bioactive metabolites from Korean natural
resources, our study on chemical constituents of the fruits of C. unshiu resulted in the
isolation of a new flavonoid glycoside, limocitrunshin 1, along with seven other flavonoids
2–8 and a limonoid 9. All structures were identified by spectroscopic methods, namely 1D
and 2D NMR, including HSQC, HMBC and 1Hꢀ1H COSY experiments, HRMS, and chemical
methods. Compounds 3, 5, and 9 are reported to be isolated from this fruit for the first time.
The isolated compounds were applied to activity tests to verify their inhibitory effects on
inflammation and nephrotoxicity. Compounds 5 and 9 showed the most potent inhibitory
activity on renal cell damage and nitric oxide production, respectively. Thus, the fruits of C.
unshiu could serve as a valuable natural source of bioactive components with health benefits
for potential application in functional foods.
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KEYWORDS: Citrus unshiu; Rutaceae; flavonoid; nitric oxide; nephrotoxicity
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INTRODUCTION
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Citrus unshiu Markovich (Rutaceae), also known as the Satsuma mandarin or Satsuma
tangerine, is cultivated in subtropical countries with moderate climate, such as Korea, Japan,
China and Russia. As the fruits of C. unshiu are seedless and are easily peeled, they are one
of the mostly enjoyed fruits in Korea. Citrus fruit is a major product of Jeju Island in Korea,
and many varieties are cultivated there. Peels of mature fruits of C. unshiu have been used as
a Chinese traditional medicine, as well as in Korea.1ꢀ3 The peels of citrus fruits contain
phenolic compounds, such as flavanones and hydroxyl cinnamic acids; hesperidin and
narirutin are two main constituents of the flavanones in C. unshiu fruit.4ꢀ6 The peels of C.
unshiu are known to possess a wide variety of biological and pharmacological activity by in
vitro and in vivo studies. For example, the peels showed antioxidant activity7 and inhibition
of the growth of tumor in murine renal cell carcinoma in mice through immuneꢀmediated
pathways.2 The peels also exhibited the inhibitory activities of hydroperoxide production,8
certain virus such as hepatitis C,9 as well as growth of certain bacteria.10 From a recent study,
the peels of C. unshiu were reported to have the inhibitory effects on the proꢀinflammatory
cytokines in lipopolysaccharide (LPS)ꢀactivated RAW 264.7 cells.1
In spite of intensive research on the pharmacological features of C. unshiu peels, the flesh of
the whole fruit has not drawn much attention in biological research although it is the portion
that people actually consume. There are few reports dealing with chemical constituents of the
whole fruit.11 The whole fruit has also been used in natural Korean medicine to improve skin
elasticity, relieve cough and fatigue, and prevent bronchitis, flu, and cancers.12 In our
continuation to seek for bioactive metabolites from Korean natural resources, the fresh whole
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parts of C. unshiu fruit were investigated to explore the antiꢀinflammatory properties of C.
unshiu fruits. From our recent study, we reported the isolation of a novel cyclic peptide,
citrusin XI, and its antiꢀinflammatory effects in LPSꢀstimulated RAW 264.7 cells.11 As part
of our study to seek for novel bioactive constituents from C. unshiu fruit, phytochemical
investigation of the whole fruit was carried out and nine compounds including a new
flavonoid glycoside, seven flavonoids and a limonoid, were isolated. The structure of the new
compound was identified using spectroscopic techniques including 1D and 2D NMR, HRMS,
and other chemical methods. The isolates were further assessed for the inhibition on nitric
oxide (NO) production and renal cell damage.
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MATERIALS AND METHODS
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General Experimental Procedures. Infrared (IR) spectra were measured on an IFSꢀ66/s
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FTꢀIR spectrometer (Bruker, Karlsruhe, Germany). Ultraviolet (UV) spectra were acquired on
an Agilent 8453 UVꢀvisible spectrophotometer (Agilent Technologies, Santa Clara, CA).
Optical rotations were recorded on a Jasco Pꢀ1020 polarimeter (Jasco, Easton, MD). Highꢀ
resolution (HR)ꢀelectrospray ionization (ESI) mass spectra were obtained on an UPLCꢀQTOF
Xevo G2ꢀS mass spectrometer (Waters Corporation, Milford, CT). Nuclear magnetic
resonance (NMR) spectra were obtained from a Bruker AVANCE III 700 NMR spectrometer
operating at 700 MHz (1H) and 175 MHz (13C) (Bruker). Agilent 1200 Series HPLC system
(Agilent Technologies) equipped with a photo diode array (PDA) detector was used for
preparative highꢀperformance liquid chromatography (HPLC) using a 250 mm × 20 mm i.d.,
10 ꢁm, YMCꢀPack ODSꢀAM C18(2) column (YMC America, Inc., Allentown, PA). Agilent
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1200 Series HPLC system equipped with a diode array detector and a 6130 Series ESI mass
spectrometer was used for LCꢀMS analysis using an analytical Kinetex C18 100 Å column
(100 mm × 2.1 mm i.d., 5 ꢁm) (Phenomenex, Torrance, CA). Column chromatography used
silica gel 60, 230ꢀ400 mesh, and RPꢀC18 silica gel, 230ꢀ400 mesh (Merck, Darmstadt,
Germany). Sephadex LHꢀ20 (Pharmacia, Uppsala, Sweden) was used for molecular sieve
column chromatography. Thinꢀlayer chromatography (TLC) analysis was conducted by using
precoated silica gel F254 plates and reverseꢀphase (RP)ꢀ18 F254s plates (Merck). Spots on TLC
were detected using UV and by heating after dipping in solvent of anisaldehydeꢀsulfuric acid.
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Plant Material. Fully ripe fruits of the ‘Miyagawaꢀwase’ variety of Satsuma mandarin (C.
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unshiu Marc.) were cultivated from the National Institutes of Horticultural and Herbal
Science, Jeju, Korea, in August 2012. The materials were identified by one of the authors (J.
W. Hyun). A voucher specimen (SKKUꢀCU 2012ꢀ8) has been stored in the herbarium of the
School of Pharmacy, Sungkyunkwan University, Suwon, Korea.
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Extraction and Isolation. The whole parts of fresh C. unshiu fruits (1.0 kg) were partially
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chopped, then extracted with 100% EtOH for 2 d twice at room temperature, accompanied
with slight shaking or swirling. The resultant extracts were then filtered and the filtrate was
concentrated under vacuum pressure. After suspension of the crude extract (89.0 g) in
distilled water (10 L), it was subjected to solvent partition using hexane, EtOAc, and nꢀBuOH,
which yielded residues of 230 mg, 3.0 g, and 15.0 g, respectively.
The EtOAcꢀsoluble fraction (3.0 g) was fractionated on silica gel column chromatography
(300 g, 3 × 55 cm) with a gradient solvent system of nꢀhexaneꢀEtOAc [1:1 (0.3 L)], CHCl3ꢀ
MeOH [50:1 (0.2 L), 10:1 (0.2 L), 5:1 (0.3 L), 2:1 (0.3 L), and 1:1 (0.3 L)], and 100% MeOH
(0.5 L) to give thirty fractions (E1 – E30), based on TLC analysis. Fraction E7 (260 mg) was
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subjected to an RPꢀC18 silica gel column chromatography (20 g, 1 × 10 cm) using MeOHꢀ
H2O [2:1 (0.3 L)] as a solvent to yield 10 subꢀfractions (E7a – E7j). Fraction E7b (52 mg)
was further purified by using semiꢀpreparative reverseꢀphase HPLC (250 mm × 10 mm i.d.,
10 ꢁm, Phenomenex Luna C18(2) column, flow rate; 1.5 mL/min) with MeOHꢀH2O (2:1) to
afford compounds 2 (12.0 mg), 4 (6.4 mg), and 9 (8.0 mg).
The nꢀBuOHꢀsoluble fraction (15.0 g) was fractionated on HPꢀ20 column chromatography
(500 g, 5 × 55 cm) with the use of a gradient solvent system of MeOHꢀH2O (from 0:1 to 1:0)
to give six fractions (B1 – B6) according to TLC analysis. Fraction B2 (4.5 g) was subjected
to a column chromatography using silica gel (300 g, 3 × 55 cm) with CHCl3ꢀMeOHꢀH2O
[7:3:1 (2.0 L)] as a solvent system to obtain 32 subꢀfractions (B2(1) – B2(32)). Fraction
B2(11) (122 mg) was further purified with preparative reverse phase HPLC (250 mm × 20
mm i.d., 10 ꢁm, YMCꢀPack ODSꢀAM C18(2) column, flow rate; 8.0 mL/min) by using 40%
MeOH to obtain compound 8 (38.0 mg). Fraction B2(31) (277 mg) was also separated by
preparative HPLC using the same column (flow rate; 10.0 mL/min) with 40% MeOH to yield
compound 5 (9.0 mg), along with subfraction B2(31)A (87.0 mg), which was then purified by
semiꢀpreparative reverse phase HPLC (250 mm × 10 mm i.d., 10 ꢁm, Phenomenex Luna
C18(2) column, flow rate; 2.0 mL/min) with 30% MeOH to furnish compound 7 (30.0 mg).
Fraction B4 (2.8 g) was applied to a silica gel column (300 g, 3 × 55 cm) using CHCl3ꢀ
MeOHꢀH2O [8:3:1 (0.8 L), 6:3:1 (0.8 L), and 6:4:1 (0.5 L)] and 100% MeOH (1.0 L) as a
gradient solvent system to give 29 subꢀfractions (B4(1) – B4(29)). Fraction B4(9) (44 mg)
was further purified using semiꢀpreparative RP HPLC (250 mm × 10 mm i.d., 10 ꢁm,
Phenomenex Luna C18(2) column, flow rate; 2.0 mL/min) with 40% MeOH to afford
compound 6 (5.2 mg). Fraction B4(15) (168 mg) was also separated by semiꢀpreparative
HPLC using the same column system with 40% MeOH to yield compound 1 (21.5 mg).
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Finally, fraction B6 (3.2 g) was applied to column chromatography with silica gel (300 g, 3 ×
55 cm) using a gradient solvent system of CHCl3ꢀMeOHꢀH2O [8:3:1 (1.0 L)] and 100%
MeOH (1.0 L) to give 20 subꢀfractions (B6(1) – B6(20)). Fraction B6(10) (383 mg) was
further purified using semiꢀpreparative HPLC (250 mm × 10 mm i.d., 10 ꢁm, Phenomenex
Luna C18(2) column, flow rate; 3.0 mL/min) with 40% MeOH to obtain compound 3 (6.0
mg).
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Limocitrunshin (1). Amorphous yellow powder. [α]2D5 +13.57 (c 0.14, MeOH); UV
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(MeOH) λmax (log ε): 204 (4.5), 260 (3.2), 272 (3.1) 358 (2.3) nm; IR (KBr) νmax: 3326, 2942,
2826, 1714, 1672, 1590, 1455, 1352, 1110, 1030 cmꢀ1; 1H NMR (CD3OD, 700 MHz): δ 7.97
(1H, br s, Hꢀ2'), 7.73 (1H, br d, J = 8.0 Hz, Hꢀ6'), 6.94 (1H, d, J = 8.0 Hz, Hꢀ5'), 6.29 (1H, br
s, Hꢀ6), 5.36 (1H, d, J = 7.5 Hz, Hꢀ1''), 4.25 (1H, dd, J = 11.0, 1.0 Hz, Hꢀ6''a), 4.14 (1H, dd, J
= 11.0, 3.0 Hz, Hꢀ6''b), 3.98 (3H, s, 3'ꢀOCH3), 3.94 (3H, s, 8ꢀOCH3), 3.53 (1H, m, Hꢀ2''), 3.50
(1H, m, Hꢀ5''), 3.39 (1H, m, Hꢀ3''), 3.33 (1H, m, Hꢀ4''), 2.52 (4H, m, Hꢀ2''', Hꢀ4'''), 1.23 (3H, s,
13
Hꢀ6'''); C NMR (CD3OD, 700 MHz): δ 178.5 (Cꢀ4), 171.5 (Cꢀ5'''), 171.3 (Cꢀ1'''), 157.9 (Cꢀ
7), 157.6 (Cꢀ2), 157.1 (Cꢀ5), 150.1 (Cꢀ4'), 149.4 (Cꢀ9), 147.5 (Cꢀ3'), 134.4 (Cꢀ3), 128.2 (Cꢀ8),
123.1 (Cꢀ6'), 122.1 (Cꢀ1'), 115.2 (Cꢀ5'), 113.3 (Cꢀ2'), 104.7 (Cꢀ10), 103.0 (Cꢀ1''), 99.3 (Cꢀ6),
77.0 (Cꢀ3''), 75.0 (Cꢀ5''), 74.9 (Cꢀ2''), 70.8 (Cꢀ4''), 69.7 (Cꢀ3'''), 63.6 (Cꢀ6''), 61.2 (8ꢀOCH3),
55.8 (3'ꢀOCH3), 45.2 (Cꢀ2'''), 45.1 (Cꢀ4'''), 26.9 (Cꢀ6'''); Highꢀresolution (HR)ꢀESIMS
(negativeꢀion mode) m/z: 651.1547 [M – H]ꢀ (calcd for C29H31O17, 651.1561).
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Acid Hydrolysis of Compound 1. Compound 1 (1.0 mg) was hydrolyzed with 1 N HCl
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(1.0 mL) for 6 h at 100 °C. Then the hydrolysate was cooled and filtered, and a yellowish
precipitate was obtained, which was identified by NMR to be the aglycone portion,
limocitrin.13 The filtrate was then neutralized by passage through an Amberlite IRAꢀ67 ionꢀ
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exchange resin column (Rohm and Haas, Philadelphia, PA). The H2O eluent was repeatedly
evaporated until the liquid was completely removed, then was analyzed using TLC over silica
gel (CHCl3/MeOH/H2O, 8:5:1), loaded with authentic sugar [TLC Rf (glucose) = 0.30] for the
comparison. The sugar residue and Lꢀcysteine methyl ester hydrochloride (1.0 mg) were
dissolved in 0.1 mL of anhydrous pyridine, and the resultant mixture was gently stirred at
60 °C for 2 h. The mixture was then evaporated under vacuum, and the reaction mixture was
trimethylsilylated using 0.3 mL of hexamethyldisilazane (HMDS, SigmaꢀAldrich, St. Louis,
MO)/trimethychlorosilane (TMCS, SigmaꢀAldrich)/pyridine (3:1:9) at 60 °C for another 1.5
h. It was then concentrated and was solventꢀpartitioned using nꢀhexane, to yield nꢀhexaneꢀ
soluble and H2Oꢀsoluble layers. The nꢀhexane layer was examined using gas chromatography
(GC).14,15 The tR value of the standard
Dꢀglucose (DꢀGlc) derivative prepared in the same way
was 18.58 min. ꢀGlc was detected from compound 1 by coꢀinjection of hydrolysate with
D
standard silylated sample, giving a single peak at 18.59 min.
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Determination of the Absolute Configuration for Compound 1. (S)ꢀ1ꢀ
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Phenylethylamine (1.9 ꢁL, 15.0 ꢁmol), Et3N (3.2 ꢁL, 22.5 ꢁmol), (Benzotriazolꢀ1ꢀ
yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, SigmaꢀAldrich) (5.8 mg,
11.5 ꢁmol), and hydroxybenzotriazole (HOBt, SigmaꢀAldrich) (2.0 mg, 15.0 ꢁmol) were
added to the solution containing compound 1 (4.9 mg, 7.5 ꢁmol) and 0.3 mL of DMF under
iceꢀcooling, and the resultant mixture was stirred at 25 °C for 9 h.16 The reaction was
quenched with dilute aqueous HCl, and a yellowish residue was obtained after drying under
vacuum. The residue was separated on Sephadex LHꢀ20 column chromatography using 50%
MeOH to furnish amide 1a (4.2 mg). Compound 1a was identified by LCꢀMS analysis, where
a molecular ion peak at m/z 756.2 [M + H]+ was observed. LiBH4 (2.6 mg, 79.5 ꢁmol) was
added to the solution containing 1a (4.0 mg, 5.3 ꢁmol) and THF (0.3 mL) under iceꢀcooling.
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The solution was stirred for 24 h at 25 °C, then the reaction was quenched with dilute
aqueous HCl and the resultant mixture was extracted with EtOAc. The resulting extract was
separated using a silica gel Waters SepꢀPak Vac 6 cc (CHCl3ꢀMeOH, 3:1) and a colorless oil
was obtained, which was then acetylated with Ac2O (2.5 ꢁL, 26.5 ꢁmol) in pyridine (30 ꢁL).
The reaction mixture was stirred for 24 h at 25 °C, diluted with H2O, extracted with EtOAc,
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1
and concentrated to yield 1b (4.5 mg) as a colorless oil. The H NMR spectrum of 1b was
found to be consistent with that of (3R)ꢀ5ꢀOꢀacetylꢀ1ꢀ[(S)ꢀphenylethyl]ꢀmevalonamide when
compared, rather than the (3S) isomer previously reported.17,18
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(3R)-5-O-Acetyl-1-[(S
)-phenylethyl]-mevalonamide (1b). Colorless oil. 1H NMR
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(CDCl3, 700 MHz): δ 7.27–7.37 (5H, m, Ph), 6.11 (1H, br s, NH), 5.14 (1H, m, Hꢀ1'), 4.23
(2H, t, J = 6.5 Hz, Hꢀ5), 2.41, 2.28 (each 1H, d, J = 14.5 Hz, Hꢀ2), 2.04, (3H, s, Ac), 1.85ꢀ
1.83 (2H, m, Hꢀ4), 1.50 (3H, d, J = 6.5 Hz, Hꢀ2'), 1.23 (3H, s, Hꢀ6); ESIMS m/z 294.1 [M +
H]+.
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Chemicals and Reagents. Cisplatin and LPS were obtained from SigmaꢀAldrich (Seoul,
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South Korea). The cell viability assay kit (EzꢀCytox) was obtained from Dail Lab Service Co.
(Seoul, Korea). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium
(DMEM) were obtained from Invitrogen Co. (Grand Island, NY).
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Inhibitory Activity towards NO Production by LPS-induced Macrophages. RAW
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264.7 cells were purchased from the American Type Culture Collection (Rockville, MD) and
cultured in DMEM (Cellgro, Manassas, VA) supplemented with 10% FBS, 1% penicillin and
streptomycin (Invitrogen Co.) and 4 mM Lꢀglutamine in an atmosphere of 5% CO2 at 37 °C.
When the cells were approximately 80% confluent, they were seeded in 96ꢀwell culture plates
at 1×105 cells per well and incubated for 24 h for adhesion. The cells were then treated with
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control (0.5% DMSO) or with the indicated concentrations of the isolates 1-9 or 1 µg/mL of
LPS. After incubation for 24 h, 80 µL of cell culture medium was mixed with 80 µL of Griess
reagent and the mixture was incubated for 10 min. The absorbance was measured at 540 nm
using a microplate reader. The quantity of nitrite was determined from a sodium nitrite
standard curve. After the nitric oxide assay, cell viability was determined. When the cells
were approximately 80% confluent, they were seeded in 96ꢀwell culture plates at 5×105 cells
per well and incubated for 24 h for adhesion. The cells were treated with control (0.5%
DMSO), with the indicated concentrations of isolates 1-9, or with 1 µg/mL of LPS. After
incubation for 24 h, 10 ꢁL of EzꢀCytox reagent was added to each well, and the cells were
incubated for 2 h. Quercetin was used as a positive control. Cell viability was measured by
absorbance at 450 nm using a microplate reader.
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Protective Effect against Cisplatin-induced Damage in LLC-PK1 Renal Cells. Pig
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kidney epithelium LLCꢀPK1 cells were purchased from the American Type Culture
Collection (Rockville) and cultured in DMEM (Cellgro), supplemented with 10% FBS, 1%
penicillin and streptomycin (Invitrogen Co.), and 4 mM Lꢀglutamine in an atmosphere of 5%
CO2 at 37 °C. Cell viability was determined using the EzꢀCytox cell viability detection kit.
Pig kidney epithelium LLCꢀPK1 cells were used to evaluate renoprotective activity against
cisplatinꢀinduced cytotoxicity. When the cells were approximately 80% confluent, they were
seeded in 96ꢀwell culture plates at 1×104 cells per well and incubated for 24 h for adhesion.
Then cells were treated with control (0.5% DMSO) or the indicated concentrations of isolates
1-9. After incubation for 2 h, 30 ꢁM of cisplatin was added to each well, and incubated for
another 24 h. After incubation, 10 ꢁL of EzꢀCytox reagent was added to each well, and the
cells were incubated for 2 h. Nꢀacetyl cysteine (NAC) was used as a positive control. Cell
viability was measured by absorbance at 450 nm using a microplate reader.
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Western Blotting Analysis. LLCꢀPK1 cells cultured in 6ꢀwell plates were treated with
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250 ꢁM of 5 and 8 for 24 h, and cells were lysed with radioimmunoprecipitation assay
(RIPA) buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) immediately
before use. Concentration of protein was determined using the Protein Assay Kit (Thermo
Fisher Scientific, Waltham, MA). Equal amounts (20 ꢁg/lane) of protein (wholeꢀcell extracts)
were separated by electrophoresis and transferred onto PVDF transfer membranes. Specific
proteins were analyzed using epitopeꢀspecific primary antibodies to phosphoꢀJNK, p53,
cleaved caspaseꢀ3, glyceraldehyde 3ꢀphosphate dehydrogenase (GAPDH) and horseradish
peroxidase (HRP) conjugated antiꢀrabbit antibodies (Cell Signaling Technology). Bound
antibodies were detected using ECL Advance Western Blotting Detection Reagents (GE
Healthcare, Little Chalfont, UK) and visualized with a FUSION Solo Chemiluminescence
System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).
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Statistical Analysis. Oneꢀway analysis of variance (ANOVA) followed by a multiple
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comparison test with Bonferroni adjustment was used for statistical analysis using SPSS ver.
19.0 (SPSS Inc., Chicago, IL). P values of less than 0.05 were considered statistically
significant.
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RESULTS AND DISCUSSION
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Isolation and Structural Elucidation of Compounds. Whole C. unshiu fruits were
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extracted with 100% EtOH to give crude EtOH extracts (89.0 g) which were solventꢀ
partitioned using hexane, EtOAc, and nꢀBuOH. Chemical investigation of the two fractions,
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EtOAcꢀsoluble and nꢀBuOHꢀsoluble fractions, using repeated column chromatography and
HPLC purification resulted in isolation and identification of a novel flavonoid glycoside,
limocitrunshin 1, along with seven other flavonoids 2–8 and a limonoid 9 (Figure 1).
Compound 1 was isolated as an amorphous, yellowish powder; its molecular formula,
C29H32O17 was established by HRꢀESIMS in negative ion mode at m/z 651.1547 [M – H]ꢀ
(calcd for C29H31O17, 651.1561). The absorption bands for OH (3326 cmꢀ1), carbonyl (1714
cmꢀ1), conjugated carbonyl (1672 cmꢀ1), and benzyl groups (1590 and 1455 cmꢀ1) were
observed in the IR spectrum. In the UV spectrum, absorption bands that appeared at 260, 272,
13
and 358 nm suggested that compound 1 possesses a flavonol skeleton. The 1H and C NMR
data consisted of signals that are similar to that of 5,7,8,3',4'ꢀpentasubstituted flavonol
glycoside19 with two methoxy groups [δH 3.98 (s); δC 55.8 and δH 3.94 (s); δC 61.2] and one
sugar unit as shown by signals of an anomeric proton at δH 5.36 (d, J = 7.5 Hz, Hꢀ1'') and its
corresponding anomeric carbon at δC 103.0 (Cꢀ1''). The heteronuclear multiple bond
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correlations (HMBC) between δH 3.98 (OCH3) and δC 147.5 (Cꢀ3') and between δH 3.94
(OCH3) and δC 128.2 (Cꢀ8) was observed and thus confirmed the locations of methoxy
groups at Cꢀ3' and Cꢀ8 of the aglycone, respectively (Figure 2). Acid hydrolysis of 1 yielded
a sugar and limocitrin, 3,4',5,7ꢀtetrahydroxyꢀ3',8ꢀdimethoxyflavone, which was verified
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through comparison with reported NMR data.13 The sugar was identified as
Dꢀglucose using
GC analysis, where the retention time of the derivative of the sugar residue and the standard
sugar were compared. The position of glycosidic linkage was determined by the correlation
between δH 5.36 (Hꢀ1'') and δC 134.4 (Cꢀ3) in HMBC experiment, indicating the glucose unit
to be located at Cꢀ3 of the aglycone. The remaining signals in the 1H and 13C NMR spectra of
1 were identified as a 3ꢀhydroxyꢀ3ꢀmethylglutaryl (HMG)ꢀmoiety by the HMBC correlations
of Hꢀ2''' (δH 2.52)/Cꢀ1''' (δC 171.3), Hꢀ4''' (δH 2.52)/Cꢀ5''' (δC 171.5), and Hꢀ6''' (δH 1.23)/Cꢀ2'''
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(δC 45.2), Cꢀ3''' (δC 69.7), and Cꢀ4''' (δC 45.1). On the basis of the HMBC correlations
between δH 4.25 and 4.14 (Hꢀ6'') and δC 171.3 (Cꢀ1'''), the HMG substituent was established
to be located at Cꢀ6 of the glucose moiety (Figure 2). The absolute configuration of the HMG
unit was determined by the refined method using the steps of amination and reduction.16
Amination with (S)ꢀ1ꢀphenylethylamine gave compound 1a (Figure 3). Reduction of 1a with
LiBH4 followed by acetylation with Ac2O yielded 5ꢀOꢀacetylꢀ1ꢀ[(S)ꢀphenylethyl]ꢀ
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1
mevalonamide (1b): the H NMR data of 1b were identical to those of (3R)ꢀ5ꢀOꢀacetylꢀ1ꢀ
[(S)ꢀphenylethyl]ꢀmevalonamide, instead of (3S) isomer.17,18 Thus, with the above evidences,
compound
1
was unambiguously identified as limocitrinꢀ3ꢀOꢀ[(S)ꢀ3ꢀhydroxyꢀ3ꢀ
ꢀglucopyranoside, and was named limocitrunshin. Previously, the
methylglutarylꢀ(1→6)]ꢀβꢀ
D
gross structure of 1 was identified from C. unshiu without verifying the absolute
configuration,20,21 but the complete structure of 1 including the identification of the (3S)ꢀ
HMGꢀmoiety were established for the first time in this study. The previously reported NMR
data in the literature were very similar to those of compound 1, but they had apparent
13
differences in the C NMR data of the HMG unit [particularly, δC 69.7 (Cꢀ3''') in 1 and δC
68.8 (Cꢀ3''') in the reported data],21 which suggested that compound 1 is not identical to the
previously reported one. Compound 1 is the first example of the limocitrin 3ꢀβꢀDꢀ
glucopyranoside possessing the (3S)ꢀHMGꢀmoiety.
The naturally occurring products conjugated with HMG group are seldom present in various
types of natural products including sesquiterpenoids,18,22 diterpenoids,23 triterpenoids,24ꢀ27
steroids,28 and flavonoids.29ꢀ33 The majority of compounds with the HMG moiety belong to
flavonoid glycosides, and the HMG moiety tends to be attached at Cꢀ6 of sugar such as
glucose or galactose.29ꢀ33 Citrus fruits have been reported to contain the flavonoid glycosides
carrying the HMG unit,20,21,30,31 but this study is the first report of the verification of 3Sꢀ
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configuration for the HMG group using the refined method in the flavonoid glycosides of C.
unshiu. An extensive literature survey showed that the HMG group does not seem to have
significance in biological activities. Several HMGꢀconjugated flavonoid glycosides did not
have inhibitory effects on NO production in lipopolysaccharide (LPS)ꢀinduced RAW 264.7
cells33 nor antimicrobial activity against methicillinꢀresistant Staphylococcus aureus (MRSA)
and Helicobacter pylori (H. pylori), although the other related flavonoids without the HMG
group showed the antimicrobial activity.30 In addition, the presence of the HMG group in
triterpenoids did not affect cytotoxic activities against several cancer cell lines25,26 nor
inhibitory effects on LPSꢀinduced NO production in murine microglia BVꢀ2 cells.26 However,
there was an interesting report for the HMG group that the linkage of the HMG group at Cꢀ3
in triterpenoids markedly increased the selective inhibition of COXꢀ1 activity when
compared to the related triterpenoids without the HMG group, which were selective
inhibitors of COXꢀ2.27
The known compounds were identified as nobiletin (3',4',5,6,7,8ꢀhexamethoxyflavone) (2),34
kaempferol 3ꢀOꢀrutinoside (3),35 limocitrin 3ꢀglucoside (4),19 kaempferol 3ꢀ(2Gꢀ
rhamnosylrutinoside) (5),35 didymin (4'ꢀmethoxyl naringenin 7ꢀOꢀrutinoside) (6),36 (2S)ꢀ
narirutin 4'ꢀOꢀglucoside (4'ꢀβꢀD
ꢀglucosyl naringenin 7ꢀOꢀrutinoside) (7),37 naringenin 7ꢀOꢀ
rutinoside (8),38 and methyl nomilinate (9)39 respectively, by comparing their spectroscopic
and physical data with those in the literature as well as by measurement of their specific
rotations. Compounds 3, 5, and 9 were reported from C. unshiu for the first time.
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Evaluation of Biological Activity of Compounds 1–9. Compounds 1–9 were evaluated for
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inhibitory effects on inflammation and nephrotoxicity to verify their potential health benefits.
Murine macrophage RAW 264.7 cells were used for evaluating inhibitory activity towards
NO production by LPSꢀactivated macrophages.40,41 Treatment of RAW 264.7 cells with up to
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200 ꢁM of compounds 1-9 did not show any cytotoxic effects (Figure 4A). Conversely, all of
the compounds significantly inhibited NO production (Figure 4B), which is involved in
inflammatory processes.42 Particularly, compound 9 (IC50 = 65 ꢁM) was the strongest
inhibitor as compared to the positive control used in this study [IC50 (quercetin): 150
ꢁM].43,44 The other compounds also showed inhibitory activity on NO with IC50 values in the
range 70ꢀ110 ꢁM [IC50 (1): 75 ꢁM, IC50 (2): 95 ꢁM, IC50 (3): 110 ꢁM, IC50 (4): 75 ꢁM, IC50
(5): 70 ꢁM, IC50 (6): 85 ꢁM, IC50 (7): 70 ꢁM, and IC50 (8): 70 ꢁM]. The most potent inhibitor,
methyl nomilinate (9) is a class of limonoids which are highly oxygenated nortriterpenoids
with a prototypical structure and a βꢀsubstituted furan ring. Its occurrence is abundant in
citrus fruits and other plants of the families Rutaceae and Meliaceae. There has been only
limited work that focused on the antiꢀinflammatory effects of limonoids, but recently many
limonoids were reported to inhibit NO production in RAW 264.7 macrophage cells induced
by LPS,45ꢀ48 which suggests the potential of limonoids for the development of antiꢀ
inflammatory agents. The other active compounds were all flavonoids and their inhibitory
effects on NO production have been extensively studied.49 In the structureꢀactivity
relationships, it has been reported that a Cꢀ2,3 double bond in the flavonoid skeleton is
essential for the activity and that the efficacy of activity was dependent upon the substitution
patterns within the flavonoids.49
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Next, the kidney protective effects of compounds 1-9 were assessed in LLCꢀPK1 cells using
a WST assay.50 The kidney protection effects of isolates 1-9 are shown in Figure 5A.
Pretreatment of LLCꢀPK1 cells with compounds 1, 2, 5, 8 and 9 at concentrations of 125 and
250 ꢁM significantly abrogated cisplatinꢀinduced nephrotoxicity (Figure 5A). Of these,
compounds 5 and 8 were selected for further mechanistic studies because of their
ameliorating effects on cell viability damage, leading to recovery of more than 90% at the
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250 ꢁM concentration (Figure 5A).
Western blotting was performed in order to investigate the protective mechanism of
compounds 5 and 8 on the expression of proteins involved in the apoptotic response. Results
from Western blot analysis are shown in Figure 5B. We determined that cisplatin promoted
high levels of phosphorylated JNK (phosphoꢀJNK), p53 and cleavage of caspaseꢀ3, which
triggered apoptosis of LLCꢀPK1 cells, while pretreatment with compounds 5 and 8 decreased
levels of phosphoꢀJNK, p53, and cleaved caspaseꢀ3 protein (Figure 5B). Therefore, the
kidney cell protective effects of compounds 5 and 8 are shown to involve the inhibition of
pathways of apoptosis through the JNKꢀp53ꢀcaspase apoptotic cascade.
Recently, it was reported that several flavonoids isolated from peat moss Sphagnum palustre
showed the protective effects against kidney damage induced by cisplatin51 and that
flavonoids in a multiꢀherbal decoction, known as Chungsimyeonjaꢀtang, possessed protective
effects against cisplatinꢀinduced nephrotoxicity.52 Unfortunately, it was difficult to find any
significant relevance between structure and activity of the flavonoid molecules in this kidney
protection assay.
In conclusion, chemical investigation of the EtOH extract of C. unshiu fruit led to the
isolation and identification of a total of 9 components including a new flavonoid glycoside,
which may be at least partially responsible for the health benefits of C. unshiu fruit. Among
the isolates, compound 9 inhibited potent NO production in LPSꢀstimulated macrophages,
and compounds 5 and 8 showed the most potent inhibition of renal cell damage. Thus, this
study revealed the possible application of the fruits of C. unshiu as a beneficial natural source
of bioactive metabolites with health benefits in functional foods.
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ASSOCIATED CONTENT
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*Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
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■
AUTHOR INFORMATION
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Corresponding Authors
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*(Ki Sung Kang) (Tel: +82ꢀ31ꢀ750ꢀ5402; Fax: +82ꢀ31ꢀ750ꢀ5416; Eꢀmail:
kkang@gachon.ac.kr)
*(Ki Hyun Kim) (Tel: +82ꢀ31ꢀ290ꢀ7700; Fax: +82ꢀ31ꢀ290ꢀ7730; Eꢀmail:
khkim83@skku.edu)
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Author Contributions
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H.J.E., D.L., S.L., and H.J.N. performed most of the experimental work. H.J.N. conceived the
project and designed the experiments. J.W.H. and P.H.Y. provided the needed materials. H.J.E.
and K.H.K. designed and implemented the separation and purification protocols. D.L. and
K.S.K. designed and implemented the biological test protocols. K.S.K., S.L., and K.H.K.
drafted and revised the manuscript. All authors read and approved the final manuscript.
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Funding
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This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future
Planning (2015R1C1A1A02037383).
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Note
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The authors declare there are no conflicts of interest.
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Figure captions
Figure 1. Chemical structures of compounds 1-9.
563
564
565
566
567
568
569
570
571
572
573
574
575
576
Figure 2. Key 1Hꢀ1H COSY (
) and HMBC (
) correlations of 1.
Figure 3. Determination of the absolute configuration of HMG group of compound 1.
Figure 4. (A) Comparison of the cell viability of compounds 1-9 in RAW 264.7 cells. (B)
Nitric oxide inhibition of compounds 1-9 in LPSꢀactivated macrophage RAW 264.7 cells.
*p<0.05 means compared to the LPSꢀtreated value.
Figure 5. (A) Comparison of the protective effects of compounds 1-9 against cisplatinꢀ
induced nephrotoxicity in LLCꢀPK1 cells. (B) Effects of compounds 5 and 8 on phosphoꢀ
JNK, JNK, phosphoꢀp53, p53, and cleaved caspaseꢀ3 expression on cisplatinꢀinduced
nephrotoxicity in LLCꢀPK1 cells. *p<0.05 means compared to the cisplatinꢀtreated value.
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577
OH
OCH3
3'
4' OH
OCH3
HO
O
2'
OCH3
OCH3
8
HO
O 2
7
9
5'
OCH3
1'
O
6'
3
H3CO
H3CO
O
6
OH
O
10
O
5
4
6'''
O
O
O
OH
O
OH
OH
O
O
6''
5''
OCH3O
3'''
1'''
5'''
HO
O
OH
2'''
4'''
O
4'' OH
OH
OH
OH
2
1''
OR
2''
OH
OH3''
3 R = H
5 R = rhamnosyl
OCH3
OH
1
OCH3
HO
O
OR
O
O
O
O
O
OH
O
O
O
OH
HO
O
O
OAc
O
OH
OH
OH
OH
OH
H3CO
O
OH
O
O
O
H
OH
OH
HO
OH
6 R = CH3
7 R = glucosyl
9
8
R = H
4
Figure 1.
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OCH3
OH
OCH3
HO
O
O
O
O
OH
O
OH
HO
O
O
OH
OH
OH
Figure 2.
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OCH3
OH
OCH3
OH
OCH3
OCH3
HO
O
HO
O
O
O
O
O
OH
O
O
O
a
OH
O
OH
OH
O
OH
b
N
H
N
HO
OH
O
O
H
O
O
OH
OH
c
OH
OH
OH
OH
1
1a
6
O
OH
5
1'
1
N
OAc
2'
H
1b
(a) (S)-1-phenylethylamine, DMF, Et3N, PyBOP, HOBt; (b) LiBH4, THF; (c) Ac2O, pyridine.
Figure 3.
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