A new phenolic glycoside and two new monoterpenoid furocoumarins from Aurantii Fructus Immaturus

Article abstract of DOI:10.1080/14786419.2015.1118634

A new phenolic glycoside, citrauranoside A (1), and two new monoterpenoid furocoumarins, citraurancoumarin A (2) and citraurancoumarin B (3), along with four known compounds (4–7) were isolated from the young fruit of Citrus aurantium L. The structures were elucidated by their comprehensive analysis including 1D, 2DNMR, IR and mass spectra.

Full text of DOI:10.1080/14786419.2015.1118634

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new phenolic glycoside and two new
monoterpenoid furocoumarins from Aurantii
Fructus Immaturus
Ying Xiong, Meiyan Chang, Kezhong Deng & Yongming Luo
To cite this article: Ying Xiong, Meiyan Chang, Kezhong Deng & Yongming Luo (2016): A
new phenolic glycoside and two new monoterpenoid furocoumarins from Aurantii Fructus
Immaturus, Natural Product Research, DOI: 10.1080/14786419.2015.1118634
To link to this article: http://dx.doi.org/10.1080/14786419.2015.1118634
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Date: 05 February 2016, At: 14:41

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2015.1118634
A new phenolic glycoside and two new monoterpenoid
furocoumarins from Aurantii Fructus Immaturus
Ying Xiong, Meiyan Chang, Kezhong Deng and Yongming Luo
college of Pharmacy, Jiangxi university of traditional chinese Medicine, Nanchang, china
ARTICLE HISTORY
ABSTRACT
received 13 august 2015
accepted 10 october 2015
A new phenolic glycoside, citrauranoside A (1), and two new
monoterpenoid furocoumarins, citraurancoumarin
A (2) and
citraurancoumarin B (3), along with four known compounds (4–7)
were isolated from the young fruit of Citrus aurantium L. The structures
were elucidated by their comprehensive analysis including 1D,
2DNMR, IR and mass spectra.
KEYWORDS
aurantii Fructus Immaturus;
coumarin; phenolic glycoside
1. Introduction
Aurantii Fructus Immaturus is derived from the young fruit of Citrus aurantium L. and its
cultivated varieties or Citrus sinensis Osbeck (Rutaceae) which is mainly produced in the
province of Jiangxi, Hunan and Sichuan. It has been used for treatment of food retention
syndromes, chest impediment and epigastric stuffiness in Traditional Chinese Medicine.
Previous phytochemical investigations have reported flavonoids, coumarins (Zhang et al.
2005; Feng et al. 2012; Wen et al. 2014), limonoids, alkaloids (Zhang et al. 2015), chromone
(Jiang et al. 2015) and essential oils in this plant. This article deals with the isolation and struc-
tural determination of seven compounds from the young fruit of C. aurantium L., including
a novel phenolic glycoside (1) and two new monoterpenoid furancoumarins (2,3) together
with two known coumarins (4,5) and two known benzene derivatives (6,7).
2. Results and discussion
The ethyl acetate soluble fraction of ethanol extract of the young fruit of C. aurantium L. was
purified by column chromatography (silica gel, ODS, Sephadex LH-20 and HPLC) to afford four
coumarins and three benzene derivatives, including three new compounds (1–3) (Figure 1).
CONTACT Kezhong deng
dengkezhong@jxutcm.edu.cn
the supplementary material for this paper is available online at http://dx.doi.10.1080/14786419.2015.1118634.
© 2016 taylor & Francis

2
Y. XIONg eT AL.
Figure 1. the structures of compounds 1–3.
Compound 1, a white amorphous powder, had the molecular formula of C21H30O10
deduced from the positive HReSIMS for the peak at m/z 460.2186, [M + NH4]+, Calcd 460.2183.
The IR spectrum exhibited absorption bands at 3436, 1709, 1609 and 1562 cm−1 for hydroxyl
1
group, carbonyl group and benzene ring system, respectively. The H NMR spectrum of
1 exhibited signals characteristic for an isopropyl group[δH 2.82 (1H, m), δH 1.15 (3H, d,
J = 7.2 Hz), δH 1.14 (3H, d, J = 7.2 Hz)], a methylene [δH 4.00 (1H, d, J = 17.6 Hz), 4.18 (1H,
d, J = 17.6 Hz)], a methoxyl [δH 3.72 (3H, s)] and a 1,2,3,4-tetrasubstituted aromatic ring[δH
7.21 (1H, d, J = 8.4 Hz), 6.72 (1H, d, J = 8.4 Hz)]. The 1H and 13C NMR spectra at δH 4.56 (1H, d,
J = 7.6 Hz), 3.46 (1H, t, J = 8.0 Hz), 3.37 (2H, m), 3.11 (1H, m), 3.77 (1H, dd, J = 12.0, 2.5 Hz), 3.63
(1H, dd, J = 12.0, 5.2 Hz) and δC 105.1, 76.8, 76.7, 74.1, 70.0 and 61.2 suggested the presence
of one beta glucose moiety, In addition, the sugar was also determined as D-glucose by
the acid hydrolysis and then comparison with authentic sample on TLC. The glucopyranose
moiety was determined to have a β-configuration, supported by the coupling constant of
the glucose anomeric proton H-1‴(J = 7.6 Hz). Correlations from H-1‴ (δH 4.56) to C-2′ (δc
154.1) in the HMBC spectrum indicated that the β-glucopyranose group was located at C-2′.
The isopropyl group was linked with benzyl group through a carbonyl, while the methoxyl
was connected to C-4′, as evidence by the HMBC correlations from H-3″, H-4″ and H-5″ to
C-2″, from H-1″ to C-2′, C-3′, C-4′, C-2″ and from methoxy protons to C-4′. Furthermore, a
propionyloxy (δC 177.5, 35.7, 25.3) was linked to C-1′ confirmed by HMBC [between H-2,H-3
and C-1′]. The structure of compound 1 was therefore determined as 3-[4-methoxy-3-(3-
methyl-2-oxobutyl)-2-O-β-d-glucopyranosyl-phenyl]propanoic acid, named citrauranoside A.
Compound 2 was obtained as a faint yellow powder. Its molecular formula, C21H24O6, was
deduced from its positive HReSIMS (m/z 373.1643, [M + H]+ Calcd 373.1646) and NMR data
analysis. The IR spectrum showed absorption bands at 3434, 1700 and 1617 cm−1, indicating
the presence of hydroxyl group, carbonyl group and aromatic ring system, respectively.
1
In the H NMR and 13C NMR spectra of 2, two pairs of one-proton doublets [δH 6.18 (1H,d,
J = 9.6 Hz), 8.34 (1H, d, J = 9.6 Hz); δH 7.68 (1H, d, J = 2.0 Hz),7.04 (1H, d, J = 2.0 Hz)] in con-
junction with δC 162.3, 157.1, 150.7, 147.0, 143.8, 140.8, 113.7, 109.9, 109.5, 105.0, 103.3 were
indicative of a 3,4-unsubstituted furanocoumarin. The remaining 10 carbon signals together

NATuRAL PRODuCT ReSeARCH
3
with HSQC experiments suggested the presence of three methyl singlets [δH 1.83 (3H, s), δC
26.2; δH 1.03 (3H, s), δC 24.3; δH 0.98 (3H, s), δC 23.8], two methylenes [δH 2.64 (1H, m), 2.02
(1H, m), δC 38.2; δH 1.31 (1H, m), 1.13 (1H, m), δC 26.9], two quaternary carbons (δC 44.5 and
72.5), one oxymethine [δH 3.23 (1H, m), δC 79.2] together with one double bond [δH 6.47
(1H, dd, J = 10.8, 17.6 Hz),5.00 (1H, br d, J = 17.6 Hz), 4.93 (1H, br d, J = 10.8 Hz) and δC 147.7,
109.3]. A myrcenyl moiety, 6,7-dihydroxy-3,7-dimethyloct-1-en-3-yl, in the molecule was
deduced from the 1H-1H COSY [between H-4″ and H-5″] and HMBC [between H-1″,2″,4″,10″
and C-3″ (δC 44.5); the correlation of H-5″,8″,9″ with both C-6″ (δC 79.2) and C-7″ (δC 72.5) ].
Furthermore, the HMBC correlations between H-2″,4″,10″ and C-8 (δC 109.9) displayed that
the substituted group and furocoumarin backbone were linked via C-3″-C8. Thus, based on
the above evidence, compound 1 was established as 8-(6,7-dihydroxy-3,7-dimethyloct-1-
en-3-yl)-5-hydroxy-6,7-furocoumarin, named citraurancoumarin A.
Compound 3, isolated as white amorphous powder, was determined to have a molecular
formula of C21H24O7 by its HReSIMS (m/z 389.1590, [M + H]+ Calcd 389.1595). The IR spectrum
suggested the presence of hydroxyl (3431 cm−1), carbonyl (1721 cm−1) and phenyl groups
(1625, 1578 cm−1). Analysis of the 1H and 13C NMR spectra indicated compounds 3 and 2 had
the same furocoumarin skeleton, but the different side chain. In the NMR spectrum for the
side chain, instead of three olefinic proton signals at δH 6.47, 5.00 and 4.93 and one methyl
singlets at δH 1.83 in 2, five signals due to oxymethine, oxymethylene and olefinic methylene
appeared at δH 4.57 (1H, m), δH 4.41 (2H, m) and δH 5.26 (1H,br d, J = 11.2 Hz), 5.07 (1H,br s)
respectively, in the 1H NMR spectrum of 3. The HMBC correlations of H-4 (δH 8.15), 1″ (δH 4.41)
with C-5 (δC 148.3) supported this unit connected to C-5. The NMR data of 3 was also very
similar to those of praealtin D (Wilzer et al. 1989) except for one more furan ring [δH 7.55
(1H, d, J = 2.0 Hz), 6.95 (1H, d, J = 2.0 Hz), δC 145.0, 104.5]. Thus, compound 3 was assigned as
5-(2,6,7-trihydroxy-7-methyl-3-octaenyloxy)-6,7-furocoumarin, named citraurancoumarin B.
The stereochemistry of the two new furocoumarin remains to be determined. According
to the previous literature, the tentative absolute configuration of C-6″ was determined as R
in praealtin D on biogenetic grounds (Wilzer et al. 1989).
Two known coumarins, Praealtin D (4) (Wilzer et al. 1989), bergapten (5) (guan et al. 2007)
and two known benzene derivatives, 3,5-dihydroxyphenyl-β- -glucopyranoside (6) (Sakar et
d
al. 1993), benzoic acid (7) (Yu et al. 2015), were identified by the comparison of their physical
and spectroscopic data with literature values.
3. Experimental
3.1. General experimental procedures
IR spectra (KBr) were obtained on a Bio-Rad FTS 6000 infrared spectrometer. HReSIMS spec-
tra were performed on an Ionspec 7.0T FTICR MS. 1D and 2D NMR spectra were recorded
on a Bruker AVANCe-400 NMR spectrometer using TMS as internal standard. Preparative
HPLC was performed on a CXTH LC-3000 liquid chromatography with an ODS column (YMC-
pack ODS-A, 5 μm, 250 × 20 mm). Silica gel (200–300 mesh, 300–400 mesh, Qingdao Ocean
Chemical group Co. of China), Sephadex LH-20 (Merck Co.) and HW-40F (TOSOH) for column
chromatography as well as silica gel gF254 (Qingdao Ocean Chemical group Co. of China)
for TLC were used.

4
Y. XIONg eT AL.
3.2. Plant material
The young fruit of C. aurantium L. were purchased from Zhangshu, Jiangxi province, China,
in July of 2013 and was identified by Dr. Ke-Zhong Deng. A voucher specimen (No. ZS13008)
is deposited in the College of Pharmacy, Jiangxi university of Traditional Chinese Medicine,
China.
3.3. Extraction and isolation
The young fruit of C. aurantium L. (50 kg) were powdered and successively extracted with
95 and 75% etOH by diacolation and filtered. After removal of the solvent under reduced
pressure, the residue (12 kg) was suspended in water and partitioned with petroleum ether,
ethyl acetate and n-BuOH successively. The etOAc extract (1 kg) was subjected to silica gel
column chromatography (CC, 18 × 150 cm, 200–300 mesh, 5 kg), eluted with CH2Cl2/CH3OH
(1:0, 98:2, 97:3, 95:5, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) to give 29 fractions. Fraction 4（49 g）was
chromatographed on silica gel CC (8 × 120 cm, 200–300 mesh, 900 g) eluted with a gradient
solvent of petroleum ether/etOAc (1:0 → 0:1) to obtain nineteen fractions (Fr.4–1-Fr.4–19).
Compound 6 (240 mg) and 7 (178 mg) were crystallised from Fr.4–3 and Fr.4–10, respectively.
Fraction 15（20 g）was subjected to silica gel CC (6 × 100 cm, 300–400 mesh, 500 g) eluted
with a gradient solvent of CH2Cl2/CH3OH (1:0 → 8:2) to obtain 20 fractions (Fr.15–1-Fr.15–20).
Fr.15–8 and Fr.15–10 applied to Sephadex LH-20 CC eluted with CH2Cl2/CH3OH (1:1) to obtain
compounds 3 (26 mg) and 4 (35 mg), respectively. The further separation of Fr.15–9 by
Sephadex LH-20 CC eluted with CH2Cl2/CH3OH (1:1) and preparative TLC (silica gel, thickness
1 mm) with CH3Cl/CH3OH (15:1) to give compound 2 (45 mg). Fraction 25 (12 g) was loaded
on an open ODS column and eluted with H2O/MeOH (3:7 → 0:1,v/v) to give 9 subfractions
Fr.25–1-Fr.25–9. Fr.25–4 was applied to pre-HPLC (YMC-pack ODS-A, 250 mm × 20 mm,
MeOH/H2O 4:6, 5 ml/min, 210 nm) and purified by Sephadex LH-20 column eluted with
MeOH to yield compound 1 (28 mg) and 5 (34 mg).
Citrauranoside A (1), C21H30O10, white amorphous powder; IR (KBr): 3436, 2969, 2929,
1709, 1709, 1609, 1562, 1489, 1466, 1403, 1385, 1316, 1273, 1201, 1098, 1074, 1046, 896, 808,
576 cm−1; 1H NMR (400 MHz, CD3OD): δ 2.57 (2H, t, J = 8.0, H-2), 3.05 (2H, t, J = 8.0, H-3), 6.72
(1H, d, J = 8.4 Hz, H-5′), 7.12 (1H, d, J = 8.4 Hz, H-6′), 4.00 (1H, d, J = 17.6 Hz, H-1″a), 4.18 (1H,
d, J = 17.6 Hz, H-1″b), 2.82 (1H, m, H-3″), 1.15 (3H, d, J = 7.2 Hz, H-4″), 1.14 (3H, d, J = 7.2 Hz,
H-5″), 4.56 (1H, d, J = 7.6 Hz, H-1‴), 3.46 (1H, t, J = 8.0 Hz, H-2‴),3.37 (2H, m, H-3‴, 4‴), 3.11
(1H, m, H-5‴), 3.77 (1H, dd, J = 12.0, 2.5 Hz, H-6‴a), 3.63 (1H, dd, J = 12.0, 5.2 Hz, H-6‴b), 3.72
(3H, s, OCH3); 13C NMR (100 MHz, CD3OD): δ 177.5 (C-1), 35.7 (C-2), 25.3 (C-3), 127.1 (C-1′),
154.1 (C-2′), 118.3 (C-3′), 156.9 (C-4′), 106.9 (C-5′), 128.2 (C-6′), 36.9 (C-1″), 215.4 (C-2″), 40.4
(C-3″), 17.5 (C-4″), 17.4 (C-5″), 105.1 (C-1‴), 74.1 (C-2‴), 76.7 (C-3‴), 70.0 (C-4‴), 76.8 (C-5‴),
61.2 (C-6‴), 54.7 (OCH3). HReSIMS m/z 460.2186 [M + NH4]+ (Calcd for C21H34O10 N, 460.2183).
Citraurancoumarin A (2), C21H24O6, faint yellow powder; IR (KBr): 3434, 2965, 2926, 1700,
1617, 1467, 1384, 1351, 1266, 1207, 1146, 1078, 827, 753, 728, 574 cm−1; 1H NMR (400 MHz,
CD3OD): δ 6.18 (1H, d, J = 9.6, H-3), 8.34 (1H, d, J = 9.6, H-4), 7.68 (1H, d, J = 2.0 Hz, H-2′), 7.04
(1H, d, J = 2.0 Hz, H-3′), 5.00 (1H, br d, J = 17.6 Hz, H-1″a), 4.93 (1H, br d, J = 10.8 Hz, H-1″b),
6.47 (1H, dd, J = 10.8, 17.6 Hz, H-2″), 2.64 (1H, m, H-4″a), 2.02 (1H, m, H-4″b), 1.31 (1H, m,
H-5″a), 1.13 (1H, m, H-5″b), 3.23 (1H, m, H-6″), 1.03 (3H, s, H-8″), 0.98 (3H, s, H-9″), 1.83 (3H, s,
H-10″); 13C NMR (100 MHz, CD3OD): δ 162.3 (C-2), 109.5 (C-3), 140.8 (C-4), 150.7 (C-5), 113.7

NATuRAL PRODuCT ReSeARCH
5
(C-6), 157.1 (C-7), 109.9 (C-8), 147.0 (C-9), 105.0 (C-10), 143.8 (C-2′), 103.3 (C-3′), 109.3 (C-1″),
147.8 (C-2″), 44.5 (C-3″), 38.2 (C-4″), 26.9 (C-5″), 79.2 (C-6″), 72.5 (C-7″), 24.3 (C-8″), 23.8 (C-9″),
26.2 (C-10″). HReSIMS m/z 373.1643 [M + H]+ (Calcd for C21H25O6, 373.1646).
Citraurancoumarin B (3), C21H24O7, white amorphous powder; IR (KBr): 3431, 2971, 2931,
1721, 1625, 1579, 1458, 1384, 1347, 1284, 1206, 1156, 1134, 1079, 941, 900, 826, 750 cm−1;
1H NMR (400 MHz, CDCl3): δ 6.20 (1H, d, J = 9.6, H-3), 8.15 (1H, d, J = 9.6, H-4), 7.07 (1H, s, H-8),
7.55 (1H, d, J = 2.0 Hz, H-2′), 6.95 (1H, d, J = 2.0 Hz, H-3′), 4.41 (2H, m, H-1″), 4.57 (1H, m, H-2″),
2.32 (1H, m, H-4″a), 2.17 (1H, m, H-4″b), 1.70 (1H, m, H-5″a), 1.55 (1H, m, H-5″b), 3.43 (1H, m,
H-6″), 1.20 (3H, s, H-8″), 1.15 (3H, s, H-9″), 5.26 (1H, br d, J = 11.2 Hz, H-10″a), 5.07 (1H, br s,
H-10″b); 13C NMR (100 MHz, CDCl3): δ 161.3 (C-2), 112.4 (C-3), 139.4 (C-4), 148.3 (C-5), 113.7
(C-6), 158.0 (C-7), 94.2 (C-8), 152.1 (C-9), 106.9 (C-10), 145.0 (C-2′), 104.5 (C-3′), 75.6 (C-1″),
73.8 (C-2″), 147.3 (C-3″), 28.2 (C-4″), 29.9 (C-5″), 78.0 (C-6″), 73.0 (C-7″), 26.3 (C-8″), 23.1 (C-9″),
113.0 (C-10″). HReSIMS m/z [M + H]+ 389.1590 (Calcd for C21H25O7, 389.1595).
3.4. Acidic hydrolysis of 1
Compound 1 (4 mg) was refluxed with 5% HCl (5 ml) in 50% methanol for 2 h. The reac-
tion mixture was neutralised with 2% KOH/H2O and extracted with ethyl acetate. The Rf
value checked by TLC (etOAc/MeOH/H2O/acetic acid, 13:4:4:3) and optical activity of the
monosaccharide in the residue were identical with those (Rf :5.1; optical activity: positive)
of authentic d-glucose.
4. Conclusion
In our study, a novel phenolic glycoside, citrauranoside A (1), and two new monoterpenoid
furocoumarins, citraurancoumarin A (2) and citraurancoumarin B (3), as well as four known
compounds, praealtin D (4), bergapten(5), 3,5-dihydroxyphenyl-β- -glucopyranoside (6) and
d
benzoic acid (7) were isolated from Aurantii Fructus Immaturus. This is the first report of cou-
marin possessing a 6,7-dihydroxy-3,7-dimethyloct-1-en-3-yl group from Citrus L. Compounds
4–7 were isolated from this plant for the first time.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant number
81260621] and the Innovation Fund Designated for graduate Students of Jiangxi Province [grant
number YC2015-S347].
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