Biochemical Systematics and Ecology 37 (2009) 678682

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Biochemical Systematics and Ecology

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Triterpenes and avonoids from Ilex hainanensis Merr. (Aquifoliaceae)

Xiao-Qing Chen a, Ke Zan a, Hai Liu b, Jie Yang a, Mao-Xiang Lai c, Qiang Wang a, *
a

Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, 1 Shennong Road, Nanjing 210009,
Peoples Republic of China
Shanghai Mass Spectrometry Center, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, Peoples Republic of China
c
Guangxi Academy of Chinese Medicine and Pharmaceutical Science, Guangxi 530022, Peoples Republic of China
b

a r t i c l e i n f o
Article history:
Received 22 April 2009
Accepted 22 August 2009
Keywords:
Ilex hainanensis Merr.
Acetylilexsaponin A1
Ursane-type triterpene
Saponin
Flavonoids

1. Subject and source

Ilex hainanensis Merr. (Aquifoliaceae) is distributed mainly in the southern region of the Peoples Republic of China. Its
leaves are used as a traditional tea product, known as Shan-Lu-Cha. Leaves of I. hainanensis were collected in Guangxi
Province, Peoples Republic of China, in July 2006 and authenticated by Prof. Qiang Wang. A voucher specimen (IH-060716)
was deposited at department of Chinese Mwateria Medica Analysis, China Pharmaceutical University.
2. Previous work
Six triterpenes, six triterpenoid saponins, ve avonoids and caffeic acid were identied from I. hainanensis, and most of
the triterpenes and aglycones of saponins were pentacyclic triterpenes with a 24,28-dicarboxylic acid group (Min and Qin,
1984; Wen et al., 1999; Zhou et al., 2007a,b; Chen et al., 2009a,b).
3. Present study
The dried and powdered leaves of I. hainanensis Merr. (12 kg) were exhaustively extracted with 90% ethanol to yield
a crude extract after evaporation of the solvent in vacuo. The residue was suspended in H2O and partitioned with petroleum
ether, chloroform, ethyl acetate and n-butanol successively. The chloroform soluble fraction (200 g) was fractionated by
column chromatography on silica gel eluted with a gradient of CHCl3:MeOH (50:1, 19:1and 10:1each eluent). Repeated
column chromatography of the eluent (50:1) with CHCl3: MeOH (100:1) afforded ursolic acid (1, 500 mg) (Cheng et al., 2000).
Ilexgenin A (2, 10 g) (Hidaka and Ito, 1987) was gained by recrystallization with MeOH from the eluent (20:1) and the mother

* Corresponding author. Tel.: 86 (0)25 85391 1253; fax: 86 (0)25 8530 1528.
E-mail address: qwang49@163.com (Q. Wang).
0305-1978/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2009.08.006

X.-Q. Chen et al. / Biochemical Systematics and Ecology 37 (2009) 678682

679

Fig. 1. Structure and the key HMBC correlations of compound 8.

liquor was subjected to an ODS column eluted with (MeOHH:O, 40:60 / 95:5) to give 3b, 24-dihydroxyurs-12-en-28-oic
acid (3, 10 mg) (Fourneau et al., 1996) and 27-p-E-coumaroyloxyursolic acid (4, 20 mg) (Siddiqui et al., 1990). The n-butanol
extract (500 g) was subjected to HPD-400 porous polymer resin and eluted with 30% and 70% ethanol, successively to afford
fractions (I, 200 g) and (II, 60 g). Fraction II was fractionated by column chromatography on silica gel eluted with a gradient of
CHCl3: MeOH (19:1, 10:1 and 4:1) to get subfraction II-18. Subfraction II-1 on Sephadex LH-20 column eluted with MeOH
furnished 7-O-b- glucosyloxy-5-hydroxy-chromone (12, 50 mg) (Simon et al., 1994). Subfraction II-3 was subjected to an ODS
column with a stepwise gradient elution with H2O/MeOH to get ilexsaponin A1 (5, 5 g) (Hidaka et al., 1987a). Repeated ODS

Table 1
1
H and 13C NMR for compound 8.
qi

Position

d13C

d1H

Position

d13C

d1H

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

39.8
29.2
78.3
49.2
56.9
21.0
33.9
40.4
47.2
37.9
24.4
128.5
139.2
42.2
29.2
26.7
48.7
54.5
72.6

1.02 (m), 1.71 (m), 2H

1.94 (m), H
3.37 (dd, J 11.9, 4.4 Hz), H

20
21
22
23
24
25
26
27
28
29
30
10
20
30
40
50
60
OAc

42.1
26.1
37.7
24.3
180.9
13.9
17.3
24.7
176.9
27.0
16.6
95.6
73.9
78.6
71.1
76.0
64.5
170.7
20.73

1.32
1.24
1.98
1.69

1.05 (m), H
2.15 (m), H
1.59 (m), H
1.81 (8.9, J 8.9 Hz), H
2.07 (m), H
5.57 (t, J 3.4 Hz), H

1.31 (m), 2.45 (m), 2H

2.01 (m), 3.08 (m), 2H
2.93 (s), H

The experiments were carried out at 500 MHz for 1H and 125 MHz for

13

C in C5D5N.

(m), H
(m), 1.94 (m), 2H
(m), 2.07 (m), 2H
(s), 3H

1.18 (s), 3H
1.24 (s), 3H
1.69 (s), 3H
1.38
1.03
6.20
4.21
4.22
4.09
4.10
4.75

(s), 3H
(d, J 6.7 Hz), 3H
(d, J 7.9 Hz), H
(m), H
(m), H
(m), H
(m), H
(m), 4.84 (m), 2H

1.91 (s), 3H

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X.-Q. Chen et al. / Biochemical Systematics and Ecology 37 (2009) 678682

column of Subfraction II-7 yielded Ilexsaponin B1 (6, 50 mg), ilexsaponin B2 (7, 30 mg) (Hidaka et al., 1987b) and compound 8.
Repeated purication of Subfraction I on a Sephadex LH-20 column with a stepwise gradient elution with H2O/MeOH to give
kaempferol 3-O-a-rhamnopyranosyl-(1 / 6)-glucopyranoside (9, 10 mg), kaempferol 7-O-b-glucopyranoside (10, 20 mg)
(Markham et al., 1978), isorhamnetin 3-O-a-rhamnopyranosyl-(1 / 6)-glucopyranoside (11, 15 mg) (Fukunaga et al., 1988).
The structures of all these compounds obtained were established by comparing NMR (Bruker DRX-500 spectrometer:
500 MHz for 1H and 125 MHz for 13C) data with those reported in the literature.
Compound 8 was obtained as a white amorphous power, [a] 30 (c 0.11, MeOH). Positive results for both LiebermannBurchard and Molisch reactions indicated it to be a triterpenoid saponin. The HRTOFMS showed a quasimolecular ion peak [MH] at m/z 705.3798, corresponding to the molecular formula C38H57O12, supported by the 1H NMR and
13
C NMR analysis. The IR spectrum showed absorptions at nmax 3451 (OH), 1727 (C]O), 1637 (C]C) cm1. The 1H NMR
spectrum indicated typical features of a triterpene mono-glycoside, showing signals for six methyls at d 1.18, 1.24, 1.38,

Fig. 2. Structures of compounds 17, 912.

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681

1.69  2 (s, 3H, each) and 1.03 (d, J 6.7 Hz), an olenic proton at d 5.57 (t, J 3.4 Hz) and an anomeric proton at d 6.20 (d,
J 7.9 Hz). The 13C NMR spectrum contained resonances for a C]C bond (d 128.5, 139.2), and two C]O functions (d 180.8,
176.4), which suggested the aglycone was an ursane-type with two carboxyl groups. The correlation between a proton at
d 3.37 (dd, J 11.9, 4.4 Hz) and an oxygenated carbon (d 78.2) in HSQC spectrum indicated that the former was typical for an
axial H-3 atom, which supposed that the 3-OH group in 8 was b-equatorial in orientation (Ouyang et al., 1996). In the HMBC
spectrum (Fig. 1), correlation peaks were observed from d 3.37 (H-3) to d 180.8 (COOH), and from d 1.69 (Me) to d 180.8
(COOH), 78.2 (C-3), and 49.2 (C-4), which indicated that a COOH and an Me group were both attached to C-4. The Me(C-23)
group was in an a-equatorial position on the basis of its 13C NMR chemical shift (Table 1) (Mahato and Kundu, 1994). A
characteristic signal for H-18 at d 2.93 (s) indicated a 19-OH group in 8, and oxygenated C-19 was inferred from the low-eldshifted signal at d 72.6 showing long-range HMBC correlations with H-18 (Hidaka et al., 1987a). Comparison of the 1H and 13C
NMR data of 8 (Table 1) with those of ilexsaponin A1, indicated that 8 was a triterpene saponin with a 3b, 19a-dihydroxyurs12-ene-24, 28-dioic acid aglycone. The 13C NMR chemical shift of C-28 (d 176.9) which is normally expected to resonance
approximatively at d 180.2 suggested the presence of the O-glycosidic linkage at C-28. This result was further conrmed by the
HMBC correlation from anomeric proton at d 6.28 (d, J 8.1 Hz) to the C-28. The D-glucose was identied by GC after acid
hydrolysis experiment and the large J H1H2 coupling constant H-10 (7.9 Hz) suggested a b-conguration for the glucose unit
(Liu et al., 2007). The main difference between 8 and ilexsaponin A1 in sugar component was the appearance of carbon signals
at d 20.73 and d 170.7, which was a typical for an acetyl. The chemical shift of C-60 (d 64.5) is normally expected to resonance
approximatively at d 62.0 suggested the presence of an acetyl linkage at C-60 (Komatsu et al., 1998). This result was further
conrmed by the HMBC correlation between the 60 -CH2 protons of the Glc residue (4.84, 4.75 ppm) and the carbonyl carbon
resonance of the Ac residue at 170.7 ppm. Consequently, the structure of compound 8 was established as 28-O-(6-O-acetyl-Dglucopyranosyl) ester of 3b,19-dihydroxy-12-ursene-24,28-dioic acid, named acetylilexsaponin A1 (Fig. 1) and its 1H NMR and
13
C NMR data (Table 1) were assigned unambiguously by HSQC and HMBC experiments.
Sugar identication by hydrolysis and GC analysis: Compound 8 (4 mg) was heated in 2 ml of 10% HCldioxane (1:1) at
80  C for 4 h. After the dioxane was removed, the solution was extracted with EtOAc (2 ml). The aqueous fractions were
evaporated and the residues were prepared to their derivatives for GC analysis according to the methods described in the
literature (Tang et al., 2005). The D-glucose was conrmed by comparison of its retention time (49.48 min) with that of
authentic standards (D-glucose and L-glucose eluted at 49.50 and 49.61 min).
4. Chemotaxonomic signicance
The present study reports the isolation of four triterpenes(14), four triterpene saponins (58), three avonoid glycosides(911), and a chromone glycoside (12) (Fig. 2). Ursolic acid (1), ilexgenin A (2) and ilexsaponin A1 (5) have been isolated
previously from I. hainanensis (Wen et al., 1999). Compound 8 is isolated as a new acetylated saponin. Besides, six acetylated
saponins have been reported from Ilex crenata and Ilex amara (Miyase et al., 1990; Hata et al., 1992; Pezzuto de Andrade et al.,
2002; Taketa et al., 2004). This suggests that the presence of acylated saponins in the Ilex genus could be of taxonomic
importance. The occurrence of ilexgenin A (2), ilexsaponin A1 (5), ilexsaponin B1 (6), ilexsaponin B2 (7) in I. hainanensis is in
agreement with compounds previously reported from Ilex pubescens (Hidaka et al., 1987a, 1987b), especially 6, 7 had been
isolated only from I. pubescens before, which suggests a phylogenetical similarity between I. hainanensis and I. pubescens. This
supports Hus classication of the Ilex genus in so far as both of these two species are members of series Prinifoliae (Hu, 1950).
Ilexsaponin A1 (5) has also been reported from Ilex psammophila (Pires et al., 2002); 27-p-E-coumaroyloxyursolic acid (4) has
previously been isolated from Ilex kudincha (Nishimura et al., 1999) and Ilex aquifolium of this genus (Budzikiewicz and
Thomas, 1980). Therefore compounds 2, 47 might also be useful taxonomic markers for the genus. Although ursane-type
triterpenes appear widespread in the genus Ilex, this is the rst report of 3b, 24-dihydroxyurs-12-en-28-oic acid (3).
The isolation of compounds 9, 10, 11 agrees with distribution pattern of avonoids aglycones in Ilex genus. It was
considered that avonols with kaempferol, quercetin and isorhamnetin as aglycones were widely distributed in Ilex genus.
(Martnez et al., 1997). 7-b-D-glucosyloxy-5-hydroxy-chromone (12) is the rst chromone isolated from Ilex genus.
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