全 文 ：700 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn
A new homoisoflavone compound as a potent antibacterial agent from
Aspidistra typica Baill.
Xiaoxia Liang*, Lingxi Kong, Min He
Natural Medicine Research Center, College of veterinary medicine, Sichuan Agricultural University, Chengdu 611130 , China
Abstract: A novel homoisoflavone typicalvone A (1), together with two known compounds, n-heneicosane and p-hydroxy
benzoic acid, were isolated from Aspidistra typica Baill. The structure of the new compound was elucidated by sepctral
techniques, viz. 1D, 2D NMR spectra and HR-ESI-MS. Compared with berberine hydrochloride, typicalvone A showed moderate
anti-bacterial activities, especially against the Gram-negative Paeudomonas aeruginosa ATCC-9027 and Escherichia coli
ATCC-25922, with MBC values of 1000 and 500 µg/mL, respectively. In addition, it exhibited weak inhibitory activities against
the tumour cells.
Keywords: Aspidistra typica Baill., Homoisoflavone compound, Antibacterial activity, Antitumor activity
CLC number: R284 Document code: A Article ID: 1003–1057(2016)9–700–04
Received: 2016-04-15, Revised: 2016-05-10, Accepted: 2016-05-26.
Foundation item: The Project of Department of Science in Sichuan
Province (Grant No. 14ZA0003).
*Corresponding author. Tel.: +86-028-86291470,
E-mail: liangxiaoxia@sicau.edu.cn
http://dx.doi.org/10.5246/jcps.2016.09.079
1. Introduction
Aspidistra typica Baill. is widely distributed in Western
China, and it has been used as a traditional food at
the Dragon Boat Festival for thousands of years. In
addition, it also has a long history of application in
medical practice, which is mainly used for the analgesic
treatment and detoxification[1–5]. Previous phytochemical
investigations of Aspidistra typica Baill have led to the
isolation of some kinds of saponin constituents[6,7].
There are no other reports on constituents of this plant.
As the principal active components, flavonoids have
potent biological activities, such as antioxidant [8],
antibacterial[9] and antitumor[10]. Our investigation on the
flavonoids led to the isolation of a new homoisoflavone,
typicalvone A (1) together with two known compounds,
n-heneicosane and p-hydroxy benzoic acid. Here we
described the isolation and structure elucidation of
the new compound, as well as the evaluation of its
antibacterial and antitumor activities.















2. Materials and equipments
2.1. General experimental procedures
Optical rotations were recorded on a Perkin-Elmer
341 polarimeter. UV measurements were obtained
from a Shimadzu UV-1201 spectrophotometer. IR
spectra were obtained on a Nicolet FT-IR 200SXY
spectrophotomer. 1H and 13C NMR were determined in
CDCl3, with TMS as the internal standard, on a Varian
Unity INOVA 600/54 NMR spectrometer. MS spectra
were measured on Finnigan LCQ and Micromass Auto
Ultima-Tof spectrometer. Silica gels G and H (Qindao
Marine Chemical Factory, China) were used for TLC
and column chromatography, respectively.
Figure 1. Structures of compounds 1–3.
OH
OHO
1
4
O
OH
OHOH
O
CH3
O
1
2
3
4
5
7
8
9
1
3
46
121
1
2
3
Short communication
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2.2. Plant materials
The root barks (5.55 kg) of Aspidistra typica Baill.
were collected in March, 2014 in Ya‟an City of Sichuan
Province, China, and authenticated by Qiaojia Fan of
Sichuan Agricultural University. The voucher specimen
(No. 20140316-1) has been deposited at the Pharmacy
Department, Sichuan Agricultural University.
2.3. Extraction and isolation
The dried root barks of Aspidistra typica Baill (5.55 kg)
were sliced into small pieces and extracted using 70%
EtOH (3×20 L, ultrasound-assisted extraction for 1 h) at
room temperature to get a residue (201.97 g), which was
then suspended in water and successively partitioned
with petroleum ether (3×2 L), CHCl3 (3×2 L), EtOAc
(3×2 L) and n-butanol (3×2 L) to yield petroleum extract,
CHCl3 extract, EtOAc extract and n-butanol–soluble
extract (24.3 g, 21.1 g, 13.43 g, 111.0 g, respectively).
The CHCl3 extract (21.1 g) was subjected to separation
over a silica gel column (12 cm×100 cm, 100–200 mesh,
250 g) and eluted with a gradient solvent system of
CHCl3–methanol to yield five subfractions (F1–F5).
Compound 1 (25 mg) was purified by repeated
chromatography on silica gel from F2 (5.4 g).
Typicalcone A (1): Yellow oil; –17.96 (c 1.00,
MeOH); IR (KBr) cm–1: 3436, 2926, 1712, 1637,
1473, 1372, 1294; UV (CHCl3): λmax (log ɛ) 290 nm;
1H (600 MHz, CDCl3) and
13C-NMR (150 MHz, CDCl3)
(spectroscopic data in Table 1); ESI-MS m/z (%): 353
[M+Na+] (100); HR-ESI-MS m/z: 331.1172[M+H]+;
calcd C18H19O6, 331.1189.
2.4. Antibacterial activity in vitro
The antibacterial activities of typicalvone A were
screened by the micro-dilution method[11] using berberine
hydrochloride as a comparator. The tested compounds
were dissolved in DMSO to prepare the stock solutions
at a concentration of 4 mg/mL. The tested compounds and
reference drug were prepared in Mueller-Hinton broth
(Guangdong Huaikai Microbial Sci. & Tech. Co. Ltd.,
Guangzhou, Guangdong, China) by two-fold serial dilution
to obtain the desired concentrations (1000, 500, 250,
125, 62.5, 31.3, 15.6 μg/mL). The bacterial suspension
was adjusted with sterile saline to a concentration of
5×105 CFU. These dilutions were inoculated and incu-
bated at 37 ºC for 16–18 h.
2.5. Cell growth inhibition assay
Cell growth inhibition was assessed using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay[12]. All compounds were dissolved in
DMSO, and the final concentration of DMSO in the
culture medium was controlled at less than 0.1% (v/v).
Tumor cells were seeded in 96-well plates with at a
density of 1×105 cells/well. After incubation with the
compounds for 48 h, MTT solution (2.5 mg/mL in
PBS) was added (10 μL/well), and the plates were
incubated for an additional 4 h at 37 ºC. The produced
formazan crystals were dissolved in 100 μL of DMSO,
and the optical density of the solution was determined
at 492 nm using a microplate reader (TECAN, Austria).
Growth inhibition was estimated based on the optical
density of the solution.
3. Results and discussion
3.1. Structural elucidation of compound 1
Compound 1 was obtained as yellow oil, –17.96
(c 1.00, MeOH). The molecular formula was determined
as C18H18O6 by the pseudo molecular ion peak at m/z
331.1172 [M+H]+ in positive HR-ESI-MS, indicating
the presence of 10 degrees of unsaturation. The proton
signals of 1H NMR spectra and the carbon signals of
the 13C NMR spectra (Table 1) revealed the basic
skeleton of a homoisoflavone. In the 1H NMR spectra,
1,4-para-substituted phenyl group appeared as doublets
at δH 7.06 (2H, d, J 7.8 Hz, H-2′,6′) and 6.77 (2H, d,
J 7.8 Hz, H-3′,5′), a penta-substituted phenyl group
appeared as one singlet at δH 6.14 (1H, s), a methoxy
group appeared as one singlet at δH 3.83 (3H, s), and
a methyl group appeared as one singlet at δH 2.01 (3H, s).
The only oxygenated methylene showed two doublets
20
D[α]
20
D[α]
702 Liang, X.X. et al. / J. Chin. Pharm. Sci. 2016, 25 (9), 700–703
with the 2J coupling constant resolved at δH 4.26, 4.04
(2H, ABq, J 13.2 Hz, H-2). A typical feature of methylene
protons attached to a phenyl group was observed at
δH 2.93 (2H, dd, J1 12 Hz, J2 16 Hz, H-9).
The 13C NMR spectra of compound 1 were used to
further define some of the above functionalities. One
methoxy carbon (δC 56.0, 7-OCH3), one methyl carbon
(δC 7.22, 8-CH3), one oxygenated phenyl carbon (δC
162.2, C-5), two quaternary carbon signals (δC 105.2,
C-8; δC 100.1, C-4a) and one tertiary carbon signal (δC
92.5, C-6) were distinguished, which were confirmed
by the HMBC correlations between 6-H (δH 7.06) with
C-5 (δC 162.2), C-7 (δC 166.6), C-8 (δC 105.2), C-4a (δC
100.1); 8-CH3 with C-7 (δC 166.6), C-8 (δC 105.2), C-8a
(δC 105.2); 7-OCH3 with C-7 (δC 166.6). A quaternary
carbon signal at δC 198.4 revealed the keto structure in
a homoisoflavone skeleton. Two oxygenated carbon
signals δC 71.6 (s) and δC 72.1 (t) as well as one methylene
carbon signal at δC 40.6 (t) were located at C-3, C-2 and
C-9, respectively, on the basis of HMBC correlations
between 2-H (δH 4.26, 4.04) with C-4 (δC 198.5), C-9
(δC 40.6), C-8a (δC 158.7). The 1,4-para-substituted phenyl
carbons (δC 131.8, C-2′; δC 131.8, C-6′; δC 115.3, C-3′;
δC 115.3, C-5′) were determined based on the HMBC
correlations between 2′-H (δH 7.06) with C-3′ (δC 115.1),
C-4′ (δC 154.8); 3′-H (δH 6.77) with C-1′ (δC 126.2),
C-4′ (δC 154.8); 5′-H (δH 6.77) with C-1′ (δC 126.2),
C-4′ (δC 154.8); 6′-H (δH 7.06) with C-4′ (δC 154.8),
C-5′ (δC 115.1). The absolute configuration of C-3
was assessed by the negative sign of its specific
rotation ( –17.96), indicating the (S) configuration
at C-3[13,14]. Finally, the structure of typicalvone A
was elucidated as 1. We, for the first time, isolated a
homoisoflavone from Aspidistra typica Baill.
Based on the above-mentioned analyses, compound 1
was elucidated as (3S)-5-methyl-7-methoxy-8-hydroxyl-
3-(4-hydroxybenzyl) chroman-4-one, a novel structure
named typicalvone A. Table 1 shows the 1H NMR and
13C NMR assignments interpreted by HMBC spectra.
3.2. Antibacterial effects
The antibacterial activity and antitumor activity of
typicalvone A were evaluated in the present study. In order
to verify the antibacterial activity of typicalvone A,
berberine hydrochloride was selected as a control for
comparison. Typicalvone A exhibited moderate anti-
bacterial activity against Gram-positive Staphylococcus
aureus ATCC-25923, Micrococcus luteus CMCC-28001,
and Gram-negative Paeudomonas aeruginosa ATCC-9027,
Escherichia coli ATCC-25922 bacterial strains, with
MBC (minimum bactericidal concentration) values of
500 μg/mL, 500 μg/mL, 1000 μg/mL and 500 μg/mL,
respectively, exhibiting a slightly better antibacterial
activity compared with berberine hydrochloride (Table 2).
Figure 2. Key 1H-1H COSY and HMBC correlations of typicalvone A.
O
OH
OHOH
O
CH3
O
HMBC ( H→C)
1H-1H COSY
Position δC δH mult (J = Hz) Position δC δH mult (J = Hz)
1 / / 9 40.6 2.93 dd (16, 12)
2 72.1
α 4.26, ABq (13.2)
β 4.04, ABq (13.2)
1′ 126.2 /
3 71.6 / 2′ 131.7 7.06 d (7.8)
4 198.5 / 3′ 115.1 6.77 d (7.8)
4a 100.1 / 4′ 154.8 /
5 162.2 / 5′ 115.1 6.77 d (7.8)
6 92.5 6.14 s 6′ 131.7 7.06 d (7.8)
7 166.6 / 7-OCH3 56.0 3.83 s
8 105.2 / 8-CH3 7.27 2.01 s
8a 158.7 / 5-OH / 11.35, s
Bacterial strains
MBC (μg/mL)
Berberine hydrochloride Typicalvone A
Staphylococcus aureus
ATCC-25923
250 250
Micrococcus luteus
CMCC-28001
250 250
Paeudomonas aeruginosa
ATCC-9027
>1000 500
Escherichia coli
ATCC-25922
500 500
20
D[α]
Table 1. The NMR data (1H: 600 MHz; 13C: 150 MHz) of typicalvone A
(in CDCl3).
Table 2. The MBC results of typicalvone A.
703 Liang, X.X. et al. / J. Chin. Pharm. Sci. 2016, 25 (9), 700–703
3.3. Antitumor effects
Cisplatin was selected as a control for comparison.
The preliminary screening by MTT assay against five
tumor cell lines (human promyelocytic leukemia HL-60,
lung cancer cell A-549, hepatocellular carcinoma cell
SMCC-7721, breast cancer cell MCF-7 and colon cancer
cell SW480) indicated that typicalvone A had weak inhibi-
tory activities against the tumor cells (all IC50>40 μM).
4. Conclusions
In summary, a new homoisoflavone compound was
first isolated from the root of Aspidistra typica Baill.
As a potent antibacterial agent, it showed moderate
antibacterial activities compared with berberine hydro-
chloride, especially against the Gram-negative bacteria.
Meanwhile, it also had weak inhibitory activities against
the tumor cells.
Acknowledgements
This work was financially supported by the project
of Department of Science in Sichuan Province (Grant
No. 14ZA0003).
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从卵叶蜘蛛抱蛋中分离得到的一个具有潜在抗菌活性的高异黄酮类化合物
梁晓霞*, 孔令茜, 何敏
四川农业大学 动物医学院 天然药物研究中心, 四川 成都 611130
摘要: 本文从卵叶蜘蛛抱蛋(Aspidistra typica Baill) 中分离得到一个新的高异黄酮类化合物卵叶酮A (1) 及两个已知
化合物正二十一烷、对羟基苯甲酸, 并通过波谱技术确证其结构。卵叶酮A表现出良好的抗菌活性, 其中对革兰氏阴性菌
绿脓杆菌ATCC-9027和大肠杆菌 ATCC-25922的抑菌效果优于盐酸小檗碱, 其最小杀菌浓度分别为1000 μg/mL和500 μg/mL,
但该化合物对肿瘤细胞增殖抑制活性不甚明显。
关键词: 卵叶蜘蛛抱蛋; 高异黄酮类化合物; 抗菌活性; 抗肿瘤活性