New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid from the pericarp of Citrus grandis (L.) Osbeck

Article abstract of DOI:10.1016/j.bioorg.2018.06.024

Two new 3-hydroxy-3-methylglutaryl (HMG) flavanone 7-O-diglycosides, cigranosides A and B (1 and 2), the known naringenin 7-(2′′-α-rhamnosyl-6′′-(3′′′′-hydroxy-3′′′′-methylglutaryl)-glucoside (melitidin, 3), their common biosynthetic precursor flavanone 7-O-diglycoside (naringin, 4), and one known flavone 7-O-diglycoside (rhoifolin, 5) were isolated from the pericarp of Citrus grandis (L.) Osbeck. The structures of these compounds were elucidated by spectroscopic and chemical techniques. The relative ratios and absolute configurations of the C-2 diastereomers of compounds 1, 2 and 4 were determined by online normal-phase HPLC-CD using a Chiralcel column. The absolute configuration of the HMG fragment in compounds 1–3 was assigned to be S through spectroscopic analysis of the mevalonamide obtained by amidation and reduction of the HMG moiety. The NO inhibitory activities of compounds 1–5 were evaluated using lipopolysaccharide-induced RAW264.7 cells. Compounds 1–5 were not cytotoxic to RAW264.7 cells at 10 μM.

Full text of DOI:10.1016/j.bioorg.2018.06.024

Accepted Manuscript
New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-
methylglutaric acid from the pericarp of Citrus grandis (L.) Osbeck
Shuang-Gang Ma, Ru-Bing Wang, Wen-Rui Li, Yun-Bao Liu, Jing Qu, Yong
Li, Xiao-Jing Wang, Song Xu, Shi-Shan Yu
PII:
S0045-2068(18)30467-X
https://doi.org/10.1016/j.bioorg.2018.06.024
YBIOO 2404
DOI:
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 May 2018
16 June 2018
18 June 2018
Please cite this article as: S-G. Ma, R-B. Wang, W-R. Li, Y-B. Liu, J. Qu, Y. Li, X-J. Wang, S. Xu, S-S. Yu, New
C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid from the pericarp of
Citrus grandis (L.) Osbeck, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.024
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New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid
from the pericarp of Citrus grandis (L.) Osbeck
†
†
Shuang-Gang Ma , Ru-Bing Wang , Wen-Rui Li, Yun-Bao Liu, Jing Qu, Yong Li, Xiao-Jing Wang, Song Xu, Shi-Shan Yu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
ABSTRACT
Two new 3-hydroxy-3-methylglutaryl (HMG) flavanone 7-O-diglycosides, cigranosides A and B (1 and 2), the known
naringenin 7-(2′′-α-rhamnosyl-6′′-(3′′′′-hydroxy-3′′′′-methylglutaryl)-glucoside (melitidin, 3), their common biosynthetic
precursor flavanone 7-O-diglycoside (naringin, 4), and one known flavone 7-O-diglycoside (rhoifolin, 5) were isolated from
the pericarp of Citrus grandis (L.) Osbeck. The structures of these compounds were elucidated by spectroscopic and chemical
techniques. The relative ratios and absolute configurations of the C-2 diastereomers of compounds 1, 2 and 4 were determined
by online normal-phase HPLC-CD using a Chiralcel column. The absolute configuration of the HMG fragment in
compounds 1−3 was assigned to be S through spectroscopic analysis of the mevalonamide obtained by amidation and
reduction of the HMG moiety. The NO inhibitory activities of compounds 1−5 were evaluated using
lipopolysaccharide-induced RAW264.7 cells. Compounds 1-5 were not cytotoxic to RAW264.7 cells at 10 μM.
Key words: Citrus grandis; flavanone glycosides; diastereomers; HMG, cigranosides; NO inhibition

Corresponding author. Tel.: +86-10-63165326; fax: +86-10-63017757; e-mail: yushishan@imm.ac.cn
These authors contributed equally to this work.
†
1

1
. Introduction
Flavanone glycosides are found in large amounts in Citrus, and their distribution across various tissues has been widely
investigated [1]. More than 200 cultivars of Citrus grandis (L.) Osbeck, one of the three original citrus species, have been
planted in southern China [2]. Citri grandis exocarpium (huajuhong), the pericarp of the title plant, has been used as a
traditional Chinese medicine for the treatment of cough with asthma, abdominal pain and stomach ache [3]. The pericarp of C.
grandis is known to possess a wide variety of biological and pharmacological activities, including anti-inflammatory,
antioxidative, antibiotic and hypoglycemic activities [4-6]. Previous phytochemical investigations on the pericarp of C. grandis
led to the identification of flavonoids [7-8], coumarins [5, 9-11], limonins [12], and triterpenoids [13].
As part of our study seeking bioactive compounds from traditional Chinese medicinal resources, the pericarp of C. grandis
was investigated. In this study, we report the isolation and elucidation of two new flavanone 7-O-diglycosides in conjugation
with 3-hydroxy-3-methylglutaric acid from the pericarp of C. grandis, and the compounds were named cigranoside A (1,
naringenin-7-O-α- L -rhamnopyranosyl-(1→2)-[4″-O-(S)-(3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside) and cigranoside
B (2, naringenin-7-O-α-L-rhamnopyranosyl-(1→2)-[3″-O-(S)-(3-hydroxy-3- methylglutaryl)]-β-D-glucopyranoside). The
known compounds were identified from spectroscopic data as (3′′′′S)-melitidin (3, naringenin-7-O-α-L-rhamnopyranosyl-
(
1→2)-[6″-O-(S)-3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside) [14], naringin (4) [15], and rhoifolin (5) [16]. Their
anti-inflammatory activities based on their ability to inhibit NO production in lipopolysaccharide (LPS)-induced RAW264.7
cells and cytotoxicities toward RAW264.7 cells are also described.
2
. Materials and Methods
2
.1. General experimental procedures
Optical rotations were measured with a JASCO P-2000 polarimeter. The UV spectra were measured with a JASCO V-650
spectrometer. The IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer by the microscope transmission method.
The NMR spectra were acquired on BRUKER AV-500-III and BRUKER AV-600-III spectrometers. Chemical shifts are given
6 3
in δ (ppm) and are referenced to solvent peaks (DMSO-d or CDCl ). The ESIMS spectra were acquired on an Agilent 1100
Series LC/MSD ion-trap mass spectrometer. The HRESIMS data were recorded using a Micromass AutoSpec Ultima TOF
spectrometer. Preparative HPLC separations were performed on a Shimadzu LC-6AD instrument with an SPD-10A detector
using a YMC-Pack ODS-A column (250 × 20 mm, 5 µm). A YMC-Pack ODS-A column (250 × 4.6 mm, 5 µm) was used for
HPLC analyses. Online coupling HPLC/CD was realized using a Jasco LC-2000 Plus HPLC System (Jasco PU-2089 Plus
delivery pump, Jasco AS-2055 Plus autosampler, Jasco co-2060 Plus column oven, Jasco MD-2010 Plus DAD detector, and
Jasco CD-2095 Plus) with DAICEL CHIRALPAK OD-H and AD-H columns (5 μm × 250 mm) at a flow rate of 1.0 mL/min
and temperature of 30 °C. Polyamide (30-60 mesh, Jiangsu Linjiang Chemical Reagents Factory, Linjiang, People's Republic
of China), macroporous resin (D101 type, Chemical Plant of Nankai University, Naikai, People's Republic of China),
Sephadex LH-20 (GE Chemical Corporation), and ODS (50 µm, Merck, Germany) were used for column chromatography.
TLC analyses were carried out with glass plates precoated with silica gel GF254. The spots were visualized under UV light or
by spraying with 10% H
2
SO
4
in 95% EtOH followed by heating.
2

2
.2. Plant material
Citri grandis exocarpium (Huajuhong) were collected in Zhejiang Province, China, in July 2010 and were identified by Prof.
Lin Ma of the Institute of Materia Medica, Chinese Academy of Medical Sciences. A voucher specimen (ID-S-2859) was
deposited in the herbarium at the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical
Sciences.
2
.3. Extraction and isolation
Pieces of Huajuhong (20 kg) for decoction were extracted three times (2 h each) with 95% EtOH (180 L) under reflux
conditions. The EtOH extract was concentrated under reduced pressure. After the removal of almost all the EtOH solvent, 9.5
L of concentrated extract remained, and the concentrate was then separated on a D101 macroporous resin column (120 × 20 cm)
2
eluted with H O and 95% EtOH. The 95% EtOH fraction was further purified on a polyamide column (120 × 20 cm, 30-60
mesh, 3800 g) eluted sequentially with H
compound 4 (116 g) was crystallized from the concentrate. The 50% EtOH fraction (40 g) was subjected to Sephadex LH-20
column (120 × 8 cm) chromatography eluted with MeOH to yield fractions A –A17 based on HPLC analysis. Fraction A (11.5
2
O, 15% EtOH, and 50% EtOH. Then, the 15% EtOH fraction was concentrated, and
1
9
g) was subjected to ODS column (60 × 6 cm) chromatography eluted with MeOH-H
2
O (30:70→38:62) to give eight fractions,
O (30:70, v/v, 0.2% TFA, 6 mL/min) as the mobile
= 16.7 min), and 2 (45 mg, t = 17.3 min). Fraction A (320 mg) was
2 R
O (30:70, v/v, 0.2% TFA, 6 mL/min) as the mobile phase to afford 3 (120 mg, t = 21.5
A
9
B
1
–A
9
B
8
. Fraction A
9
B
2
(370 mg) was purified by HPLC with MeCN-H
2
phase to yield 5 (50 mg, t
R
= 12.3 min), 1 (62 mg, t
R
R
9 4
B
separated by HPLC using MeCN-H
min). The purity of each isolated compound was greater than 95% based on HPLC.
2
.4. Cigranoside A (1)
A pale, yellow powder, UV (MeOH) max 283, 324 (sh) nm; IR νmax 3412, 2978, 2936 1727, 1642, 1579, 1519, 1450, 1376,

1
1
13
1
348, 1297, 1269, 1173, 1088, 837 cm ; H NMR (500 MHz, DMSO-d
6
) data, see Table 1; C NMR (125 MHz, DMSO-d
6
)
+
+
data, see Table 1; HRESIMS (positive) m/z 725.2292 [M + H] (calcd for C33
H
41
O
18, 725.2287), 747.2099 [M + Na] (calcd
for C33
H
40NaO18, 747.2107).
.5. Cigranoside B (2)
A pale, yellow powder, UV (MeOH) max 283, 326 (sh) nm; IR νmax 3460, 2976, 2941 1728, 1701, 1632, 1572, 1520, 1449,
2

1
1
13
1
401, 1308, 1285, 1191, 1172, 1135, 1090, 1062, 837 cm ; H NMR (500 MHz, DMSO-d
6
) data, see Table 1; C NMR (125
+
MHz, DMSO-d
6
) data, see Table 1; HRESIMS (positive) m/z HRESIMS (positive) m/z 725.2281 [M + H] (calcd for
+
C
33
H
41
O
18, 725.2287), 747.2107 [M + Na] (calcd for C33H40NaO18, 747.2107).
2
.6. Acid hydrolysis of compounds 1-3 and determination of the absolute configuration of their sugar moieties [17-18]
Compounds 1, 2 and 3 (each 2 mg) were separately dissolved in 1 N HCl and refluxed for 6 h. After cooling, the yellow
precipitate was isolated by filtration and proved to be naringenin by NMR analysis.
Naringenin
3

1
Yellow solid; H NMR (500 MHz, DMSO-d
6
) δ 7.30 (2H, d, J = 8.5 Hz, H-2', -6'), 6.78 (2H, d, J = 8.5 Hz, H-3', -5'), 5.87
(
(
(
(
2H, s, H-6, -8), 5.42 (1H, dd, J = 13.0, 3.0, H-2), 3.25 (1H, dd, J = 17.0, 13.0, H-3a), 2.67 (1H, dd, J = 17.0, 3.0, H-3b); 12.14
1
3
6
1H, s, HO-5), 10.76 (1H, s, HO-7), 9.60 (1H, s, HO-4'); C NMR (125 MHz, DMSO-d ) δ 196.4 (C-4), 166.6 (C-7), 163.5
C-9), 162.9 (C-5), 157.7 (C-4'), 128.8 (C-1'), 128.3 (C-2', -6'), 115.1 (C-3', -5'), 101.7 (C-10), 95.8 (C-6), 94.9 (C-8), 78.4
+
C-2), 42.0 (C-3); ESIMS (positive) m/z 273 [M +H] .
2
After removal of the HCl from the combined filtrate by evaporation, the H O fraction was again concentrated and dried in
vacuo to afford a residue that contained the monosaccharide. The residue was dissolved in pyridine (1 mL), and 2 mg
of L-cysteine methyl ester hydrochloride was added to the solution. The mixture was kept at 60 °C for 2 h, and then the solvent
was evaporated under a stream of N
2
, and the residue was dried in vacuo. The residue was then trimethylsilylated by reacting
with N-(trimethylsilyl)imidazole (0.2 mL) for 2 h. The mixture was partitioned between n-hexane and H
2
O (2 mL each), and
the n-hexane extract was analyzed by GC under the following conditions: capillary column, SE-54 (30 m × 0.25 mm, i.d.);
detection, FID; detector temperature, 280 °C; injection temperature, 250 °C; column temperature, 270 °C; and carrier gas,
2
N at 250 kPa. The presence of L-rhamnose and D-glucose in the acid hydrolysates of 1, 2, and 3 was verified by comparison of
the retention times of their derivatives to those of the corresponding standard samples derivatized by the same procedure. The
retention times of L-rhamnose and D-glucose were 18.20 and 24.77 min, respectively.
2
.7. Determination of the absolute configuration of HMG in compounds 1−3 [19-24]
S)-1-Phenylethylamine (16 μL, 124.5 μmol), Et N (34 μL, 229 μmol), and DMAP (1.0 mg, 8.3 μmol) were added to a
(
3
solution of compound 1 (54 mg, 75 μmol) in 1.0 mL of DMF while cooling with ice. After 10 min, BOP (73 mg, 166 μmol)
was added to the mixture, and the reaction was stirred at room temperature for 6 h. The reaction was quenched with dilute
aqueous HCl and then dried under vacuum to afford a yellow residue, which was purified by preparative HPLC eluting with
3
2
5% MeCN-H O (35:65, v/v, 0.2% TFA) to give amide 1a (40.0 mg)
LiBH (30.5 mg, 1.4 mmol) was added to a solution of 1a (40.0 mg, 48 μmol) in THF (2.0 mL) under ice-cooling. After
4
stirring for 3 h at room temperature, the reaction was quenched with dilute aqueous HCl while cooling with ice, and the
resulting material was extracted with EtOAc. The organic layer was washed with saturated NaCl solution and concentrated
2
under reduced pressure to afford a residue. The residue was then acetylated with Ac O (14 μL, 160 μmol) and DMAP (4.8 mg,
4
0 μmol) in DCM (0.5 ml). After stirring for 24 h at room temperature, the reaction was quenched with water, and the mixture
was extracted with EtOAc. The organic layer was washed with saturated NaCl solution and concentrated to afford a yellow
1
residue, which was purified using preparative HPLC to afford 1b (1.5 mg) as a colorless oil. Its H NMR spectrum was
consistent with that of (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide rather than the (3S) isomer reported in the literature
[
20, 22]. The absolute configuration of the HMG moiety in compounds 2 and 3 was determined using the same method as was
described for 1, and all the final derivatization products were proved to be (3R)-5-O-acetyl-1-[(S)-phenylethyl] mevalonamide.
(3R)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide (1b)
4

1
Colorless oil; H NMR (CDCl
3
, 600 MHz) δ 7.27−7.37 (5H, m, C
6
H
5
-1′), 6.05 (1H, d, J = 7.2 Hz, NH), 5.14 (1H, quintet, J
COO), 1.84,
=
7.2 Hz, H-1′), 4.23 (2H, td, J = 6.6, 3.0 Hz, H-5), 2.41, 2.28 (each 1H, d, J = 14.4 Hz, H-2), 2.03, (3H, s, CH
3
1
.88 (each 1H, dd, J = 14.4, 7.2, H-4), 1.50 (3H, d, J = 7.2 Hz, H-2′), 1.23 (3H, s, H-6); HRESIMS m/z (%) 316.15192 [M +
+
Na] (calcd for C16
H
23
O
4
NNa, 316.15193).
2
.8. Assay of inhibitory ability toward LPS-induced NO production and cytotoxicity testing
RAW264.7 cells (immortalized mouse microglia) were cultured in DMEM supplemented with 10% fetal bovine serum, 100
U/mL penicillin, and 100 μg/mL streptomycin. After preincubation for 24 h in a 96-well plate, the cells were treated with the
test compounds and then stimulated with LPS (300 ng/mL) for 24 h. The production of NO was determined by measuring the
concentration of nitrite in the supernatant using the Griess assay. The absorbance was measured at 550 nm, and dexamethasone
was used as a positive control. Cell viability was measured by the MTT assay. The absorbance at 540 nm was read using a
microplate reader.
3
. Results and discussion
40
Compound 1 was isolated as a pale, yellow powder and its molecular formula was determined to be C33H O18 from the
+
+
HRESIMS signals at m/z 725.2292 [M + H] (calcd 722.2287), 747.2099 [M + Na] (calcd 747.2107) and its NMR data. Its IR
−1
−1
−1
spectrum showed typical absorption bands for OH (3412 cm ), carbonyl (1727 cm ), conjugated carbonyl (1642 cm ), and
−1
benzyl groups (1659 and 1519 cm ). Its UV spectrum showed absorption bands at 283 and 324 (sh) nm, indicating a flavanone
1
13
skeleton [25]. Careful analysis of the H and C NMR spectra of 1 showed two groups of downfield, highly similar signals
corresponding to the flavanone skeleton, which indicated compound 1 was a mixture of C-2 diastereomers. Additionally, the
ratio of the heights of the carbonyl signals at δ
C
197.3 and 197.2 (Supplementary data, Fig. S4) suggested that the ratio of the
1
two C-2 isomers was 1:1.5. For the main isomer of 1, the H NMR spectrum showed a pair of meta-coupled aromatic protons
at δ
H
6.10 (1H, d, J = 2.0 Hz) and 6.14 (1H, d, J = 2.0 Hz) and two protons in an AB spin system at δ
H
6.78 (2H, d, J = 8.0 Hz)
1
and 7.31 (2H, d, J = 8.0 Hz). The H NMR spectrum also exhibited a β-glucosyl anomeric proton at δ
H
5.36 (1H, d, J = 8.0 Hz)
and a group of proton signals characteristic of an α-rhamnosyl fragment including an anomeric proton at δ
the corresponding methyl protons at δ 1.12 (3H, d, J = 6.0 Hz). Similarly, the corresponding NMR data for the minor isomer
of 1 were assigned as shown in Table 1.
H
4.71(1H, brs) and
H
Acid hydrolysis of 1 afforded the aglycone naringenin (5,7,4'-trihydroxyflavanone), which was determined by comparison
of its NMR data with literature data [26], and two monosaccharides. The monosaccharides were identified by gas-liquid
chromatography as D-glucose and L-rhamnose through a comparison of the retention times of the derivatives of the sugar
residues to those of standard sugars. The linkages between the sugar and naringenin moieties were further determined from the
HMBC correlations from δ
anomeric proton of glucose) to δ 164.7/164.5 (C-7, naringenin). These results indicated compound 1 was a derivative
of flavanone 7-O-diglycoside (naringin, 4). In addition to the signals corresponding to the naringin moiety, the remaining H
H H
4.71 (the anomeric proton of rhamnose) to δ 75.7 (C-2″, glucose) and from δ 5.36/5.38 (the
1
1
3
and C NMR data showed the presence of a 3-hydroxy-3-methylglutaryl (HMG) moiety (Tables 1 and 2). This fragment was
confirmed by the HMBC correlations between δ
H
C H 2
2.76 (H-2a′′′′)/2.57 (H-2b′′′′) and δ 170.0 (C-1′′′′), between δ 2.53 (H -4′′′′)
5

and δ
C
172.4 (C-5′′′′), and between δ
H
1.32 (H
3
-6′′′′) and δ
C
69.2 (C-3′′′′), 46.0 (C-2′′′′), and 45.6 (C-4′′′′). Additionally, in the
HMBC spectrum, HMBC correlations from δ
H
5.07 (H″-4, Glu) to δ 170.0 (C-1′′′′, HMG), 75.7 (C-2″, glucose), and 67.6 (C-5″,
glucose) were observed. These data indicated that the HMG moiety was attached at C-4 of the internal glucose. The absolute
configuration of the chiral carbon in the HMG fragment was determined to be S according to a previously described method
(Scheme 1) [19−24]. Compound 1a was obtained by the amidation of 1 with (S)-1-phenylethylamine. Reduction of 1a with
1
LiBH
4
followed by acetylation gave 5-O-acetyl-1-[(S)-phenylethyl]mevalonamide (1b), and the H NMR data of 1b were
identical to those of (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide instead of the (3S) isomer [20, 22].
The absolute configurations of the C-2 diastereomers of 1 were determined from their online HPLC-electronic circular
dichroism (ECD) spectra using a Chiralcel OD-H column. As shown in Fig. 3, the ECD spectrum of the first peak,
corresponding to the minor C-2 diastereomer, revealed a positive Cotton effect for the π → π* electronic transition at 290 nm
and a negative Cotton effect for the n → π* electronic transition at 335 nm, which confirmed an R absolute configuration of
C-2, and in contrast, the ECD spectrum of the second peak, corresponding to the major C-2 diastereomer, revealed a positive
Cotton effect at 280 nm and a negative Cotton effect at 335 nm, which confirmed an S absolute configuration of C-2 [27,
2
8]. Thus, the structure of 1 was assigned as naringenin-7-O-α-L-rhamnopyranosyl-(1→2)-[4″-O-(S)-(3-hydroxy-3
methylglutaryl)]-β-D-glucopyranoside, and it was named cigranoside A.
Fig. 1. Chemical structures of compounds 1–5.
6

Fig. 2. Key HMBC correlations of compounds 1 and 2.
a
Scheme 1. Determination of the absolute configuration of the HMG moiety in compound 1
a
3 4 2
Reaction condition and reagent: (a) (S)-1-phenylethylamine, DMF, Et N, BOP, DMAP, r.t.; (b) LiBH , THF, 0℃; (c) Ac O,
DMAP, DCM, r.t..
7

Fig. 3. Online HPLC-CD experiments of compounds 1 (left) and 2 (right). Left upper: CD spectra of (2R)- and (2S)-1 obtained
from the first and second online HPLC eluted peaks (15.6 and 23.5 min) of 1, respectively. Right upper: CD spectra of (2R)-
and (2S)-2 obtained from the first and second online HPLC eluted peaks (32.5 and 37.1 min) of 2. Left middle and lower traces:
CD and UV signal at 280 nm from the continuous flow of (2R)-1 (peak 1) and (2S)-1 (peak 2). Right middle and lower traces:
CD and UV signal at 280 nm from the continuous flow of (2R)-1 (peak 1) and (2S)-1 (peak 2). Conditions: Chiralcel OD-H,
mobile phase of n-hexane/ethanol (70/30) doped with 0.1% of TFA at a flow rate of 1.0 ml/min for 1; and Chiralcel AD-H,
n-hexane/2-propanol (75/25) doped with 0.1% of TFA at a flow rate of 1.0 ml/min for 2.
Compound 2 was isolated as a pale, yellow powder and had the same molecular formula as 1, C33
H
40
O18, as determined
1
13
from its HRESIMS data. The UV, IR, H NMR, and C NMR data of 2 were similar to those of compound 1. Similarly, in
1
13
the H and C NMR spectra of 2, two groups of downfield, highly similar signals corresponding to the flavanone skeleton
were observed, indicating that compound 2 was a mixture of C-2 diastereomers. Additionally, the ratio of the heights of the
C
carbonyl signals at δ 197.3 and 197.2 (Supplementary data, Fig. S12) suggested that the ratio of the two C-2 isomers was
approximately 1:2.3. Acid hydrolysis also afforded D-Glu, L-Rha, and naringenin. The 1D and 2D NMR spectra allowed the
assignment of all signals, and the only structural difference between compounds 1 and 2 was the location of the HMG moiety,
and it was revealed that the HMG substituent of compound 2 was at C-3″ of the glucose instead of C-4″ as it is in 1, which was
supported by HMBC correlations from δ 4.65 (H-3″, Glu) to δ 169.7 (C-1′′′′, HMG), 74.4 (C-4″, Glu), and 60.2 (C-6″, Glu).
The NMR data of the C-2 diastereomers of 2 were assigned as shown in Table 1.
8

The absolute configurations of the C-2 diastereomers of 2 were determined by an online HPLC-ECD spectrum method.
Notably, the C-2 diastereomers cannot be resolved by Chiralcel OD-H, OZ-H, OJ-H, or AS-H chiral stationary phases
using n-hexane/ethanol or n-hexane/2-propanol doped with 0.1% of TFA as the mobile phase. Fortunately, partial resolution of
the isomers was achieved by a Chiralcel AD-H column with n-hexane/2-propanol doped with 0.1% of TFA as the mobile
phase.
As shown in Fig. 3, the ECD spectrum of the first peak, corresponding to the minor C-2 diastereomer, revealed a positive
Cotton effect at 290 nm and a negative Cotton effect at 335 nm, which confirmed an R absolute configuration of C-2, and in
contrast, the ECD spectrum of the second peak, corresponding to the major C-2 diastereomer, revealed a negative Cotton effect
at 280 nm and a positive Cotton effect at 335 nm, which confirmed an S absolute configuration of C-2 [27, 28]. Consequently,
compound 2 was assigned as naringenin-7-O-α-L-rhamnopyranosyl-(1→2)-[3″-O-(S)-(3-hydroxy-3-methylglutaryl)]-β-D-
glucopyranoside, and it was named cigranoside B.
Table 1
a
6
NMR spectroscopic data for 1 and 2 (500 MHz, DMSO-d ).
b
c
1
2
2
R
2S
2R
(J in Hz)
2S
no.
type
CH
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
δ
C
H
δ (J in Hz)
2
3
78.8
42.2
5.49, dd (13.5, 3.0)
3.33, m
78.7
42.2
5.52, dd (12.5, 3.0)
3.33, m
78.8
42.0
5.48, m
3.33, m
2.72, m
78.6
42.1
5.51, m
3.33, m
2.72, m
CH
2
2
.71, m
2.71, m
4
5
6
7
8
9
qC
qC
CH
qC
CH
qC
qC
qC
CH
CH
qC
197.3
162.9
96.5
197.2
162.8
96.5
197.4
162.8
96.3
197.2
162.7
96.3
6.10, d (2.0)
6.15, d (2.0)
6.10, d (2.0)
6.14, d (2.0)
6.10, d (2.0)
6.13, d (2.0)
6.09, d (2.0)
6.12, d (2.0)
164.5
95.4
164.7
95.3
164.5
95.2
164.7
95.1
163.0
103.6
128.7
128.4
115.4
157.9
163.0
103.6
128.8
128.3
115.4
157.9
162.9
103.4
128.5
128.5
115.2
157.9
162.9
103.4
128.6
128.4
115.2
157.8
1
1
2
3
4
5
4
0
'
', 6'
', 5'
'
7.32, d (8.0)
6.78, d (8.0)
7.31, d (8.0)
6.78, d (8.0)
7.32, d (8.0)
6.79, d (8.0)
7.32, d (8.0)
6.79, d (8.0
–OH
'–OH
12.04, s
9.60, s
12.03, s
9.60, s
12.04, s
9.60, s
12.03 (s)
9.60, s
Glc
1
2
3
4
5
6
''
''
''
''
''
''
CH
CH
CH
CH
CH
96.5
75.7
76.6
77.3
67.6
60.3
5.38, d (8.0)
3.60 m
96.5
75.7
76.6
77.3
67.6
60.3
5.36, d (8.0)
97.0
76.4
70.7
74.4
69.1,
60.2
5.27, d (8.0)
3.56, appt
97.1
76.3
70.7
74.3
69.1
60.2
5.25, d (8.0)
3.56, appt
3.60
3.58
3.58 m
4.65, dd (9.5, 9.5)
3.69
4.65, dd (9.5, 9.5)
3.69
5.07, dd (9.5, 9.5)
3.38
5.07, dd (9.5, 9.5)
3.38
3.66
3.66
CH
2
3.62
3.62
3.47, brd (12.5)
3.47, brd (12.5)
3
.45
3.45
3.29
3.29
Rha
1
2
3
4
'''
'''
'''
'''
CH
CH
CH
CH
100.9
70.7
70.5
71.9
4.71, brs
3.47
100.9
70.7
70.5
71.9
4.71, brs
3.47
100.7
70.4
70.3
71.8
5.04, brs
3.66
100.6
70.4
70.3
71.8
5.05, brs
3.66
3.25, dd (9.5, 3.0)
3.16, dd (9.5, 9.5)
3.25, dd (9.5, 3.0)
3.16, dd (9.5, 9.5)
3.30
3.30
3.18, dd (9.5, 9.5)
3.18, dd (9.5, 9.5)
9

5
6
'''
'''
CH
68.8
18.1
3.57
68.8
18.1
3.57
68.4
18.0
3.65
68.4
18.0
3.65
CH
3
1.12, d (6.0)
1.12, d (6.0)
1.15, d (6.0)
1.15, d (6.0)
d
HMG
1
2
''''
''''
qC
170.0
46.0
170.0
46.0
169.7
45.9
169.7
45.9
CH
2
2.76, d (14.0)
2.76, d (14.0)
2.57, d (14.0)
2.66, d (14.0)
2.59, d (14.0)
2.66, d (14.0)
2.59, d (14.0)
2
.57, d (14.0)
2.53, brs
1.32, s
3
4
5
''''
''''
''''
''''
qC
69.2
45.6
69.2
45.6
69.1
45.6
69.1
45.6
CH
2
2.53, brs
1.32, s
2.51, brs
1.29, s
2.51, brs
1.29, s
qC
172.4
27.1
172.4
27.1
172.4
27.1
172.4
27.1
6
CH
3
a
Overlapped signals are reported without designated multiplicities.
b
c
The diastereomeric ratio (2S/2R) of 1 is about 4/6.
The diastereomeric ratio (2S/2R) of 2 is about 3/7.
d
HMG = (S)-3-hydroxy-3-methylglutaric acid.
1
13
During the structural determination of the isolates, the H and C NMR data of the flavonone glycosides (3 and 4) were also
fully assigned (Supplementary data, Table S1). The structure of 3 isolated from C. bergamia was previously reported [14], but
the absolute configurations of the HMG moiety and C-2 were not assigned. These were investigated using the method
described above, and it was confirmed that the HMG moiety in compound 3 was also in the (S)-configuration, which was the
same as what was found for new compounds 1 and 2, and the relative ratio of the 2R- and 2S-diastereomers in 3 was 2:3.
Additionally, the relative ratio of the 2R- and 2S-diastereomers in naringin (4) was 54:46 (Supplementary data, Fig. S19 and
S22).
Compounds 1-5 were evaluated for their ability to inhibit LPS-induced NO production in RAW264.7 macrophages and their
cytotoxicity toward RAW264.7 cells, but none of the compounds were active; their inhibition rates were lower than 7.7% at 10
μM. As a positive control, dexamethasone inhibited NO production by 78.8% at a concentration of 1.0 μM. Compounds 1–5
were not cytotoxic to RAW264.7 cells at 10 μM.
In conclusion, two new flavanone 7-O-diglycosides conjugated with 3-hydroxy-3-methylglutaric acid, cigranosides A-B
(
(
1-2), were isolated from the pericarp of C. grandis. Known compounds were identified from their spectroscopic data as
3′′′′S)-melitidin (3, naringenin-7-O-α-L-rhamnopyranosyl-(1→2)-[6″-O-(S)-3-hydroxy-3-methylglutaryl)]-β-
D-glucopyranoside), naringin (4), and rhoifolin (5). As reported previously, flavanone glycosides from mature fruits of
some Citrus are often isolated as mixtures of diastereomers (epimers) [1, 29-31]. The relative ratios of the C-2 diastereomers of
naringin (4, the major flavanone diglycoside in Citri grandis exocarpium) and its HMG derivatives (1-3) were determined by
an online HPLC-ECD spectroscopy method. According to a previous report [32], just over 50 examples of flavonoid
glycosides acylated with 3-hydroxy-3-methylglutaric acid (HMG-glycosides) have been reported to date, and only six
flavanone HMG-glycosides (O-linked) have been reported from the families of Rutaceae [33], Dryopteridaceae [34], and
Woodsiaceae [35], where the HMG moieties are all connected at C-6 of the glucose. Our study is the first report of the
simultaneous isolation of three flavanone 7-O-glycoside isomers (1−3) with their HMG moieties located at C-4, C-3, and C-6,
respectively, of glucose. The absolute configuration of HMG in compounds 1−3 was determined to be S through spectroscopic
10

analysis of the mevalonamide obtained by amidation and reduction of the HMG moiety. The anti-inflammatory activities of
compounds 1-5 based on their ability to inhibit NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells and their
cytotoxicities toward RAW264.7 cells are also described.
Acknowledgments
The authors are grateful to the Department of Instrumental Analysis of our institute for the acquisition of the IR, NMR, and
MS spectra. This work was supported by grants from CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1-010 and
No. 2016-I2M-3-011), from the Natural Science Foundation of China (No. 21732008), and from the National Mega-project for
Innovative Drugs (No. 2018ZX09711001-008).
Conflict of interest
The authors declare that there are no conflicts of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at https://doi.org/10.1016/j.bioorg. 2018.
0
0.000.
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Graphical abstract
Highlights
Two new flavanone 7-O-diglycosides conjugated with 3-hydroxy-3-methylglutaric acid, cigranosides A-B (1-2) were
isolated from the pericarp of C. grandis. The relative ratios of the C-2 diastereomers of naringin (4, the
major flavanone diglycoside in Citri grandis exocarpium) and its HMG derivatives (1-3) were determined by an online
HPLC-ECD spectroscopy method. Our study is the first report of the simultaneous isolation of three flavanone 7-O-glycoside
isomers (1−3) with their HMG moieties located at C-4, C-3, and C-6, respectively, of glucose. The absolute configuration of
HMG in compounds 1−3 was determined to be S through spectroscopic analysis of the mevalonamide obtained by amidation
and reduction of the HMG moiety. Their anti-inflammatory activities based on their ability to inhibit NO production in
lipopolysaccharide (LPS)-induced RAW264.7 cells and their cytotoxicities toward RAW264.7 cells are also described.
12