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Journal of Chromatographic Science, 2017, 1–7
doi: 10.1093/chromsci/bmx076
Isolation of Flavonoids From Wild Aquilaria
sinensis Leaves by an Improved Preparative
High-Speed Counter-Current Chromatography
Mao-Xun Yang1,2,†, Yao-Guang Liang3,†, He-Ru Chen2,4,*,
Yong-Fang Huang5, Hai-Guang Gong5, Tian-You Zhang3, and Yoichiro Ito6
Department of Biomedicine, Zhongshan Torch Polytechnic, 60 ZhongShan Port Avenue, Zhongshan 528436, PR
China, 2Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601
Huangpu Avenue West, Guangzhou 510632, PR China, 3Guangdong Bless Biotechnical Development Co., Ltd, Area
A 4th Floor, Building 4, Science and Technology Garden, South China Modern Chinese Medicine Park, Zhongshan
528400, PR China, 4Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New
Drugs Research, 601 Huangpu Avenue West, Guangzhou 510632, PR China, 5College of Forestry, South China
Agricultural University, 483 Wushan Avenue, Guangzhou 510642, PR China, and 6Laboratory of Bioseparation
Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of
Health, Building 10, Room 8N230, 10 Center Drive, Bethesda, MD 20892-1762, USA
Author to whom correspondence should be addressed. Email:
These two authors contribute equally.
Received 11 March 2015; Revised 8 May 2017; Editorial Decision 19 July 2017
Four flavonoids including apigenin-7,4′-dimethylether, genkwanin, quercetin, and kaempferol
were isolated in a preparative or semi-preparative scale from the leaves of wild Aquilaria sinensis
using an improved preparative high-speed counter-current chromatography apparatus. The separations were performed with a two-phase solvent system composed of hexane–ethyl acetate,
methanol–water at suitable volume ratios. The obtained fractions were analyzed by HPLC, and the
identification of each target compound was carried out by ESI-MS and NMR. The yields of the
above four target flavonoids were 4.7, 10.0, 11.0 and 4.4%, respectively. All these four flavonoids
exhibited nitrite scavenging activities with the clearance rate of 12.40 ± 0.20%, 5.84 ± 0.03%, 28.10 ±
0.17% and 5.19 ± 0.11%, respectively. Quercetin was originally isolated from the Thymelaeaceae
family, while kaempferol was isolated from the Aquilaria genus for the first time. In cytotoxicity test
these two flavonoids exhibited moderate inhibitory activities against HepG2 cells with the IC50 values of 12.54 ± 1.37 and 38.63 ± 4.05 μM, respectively.
Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), a principal source
of the expensive agilawood, is distributed in the south China such as
Hainan, Guangxi, Guangdong, Fujian and Taiwan provinces. It is
one of the most valuable forest products currently known and
traded all over the world (1). Agilawood is of particular interest, but
becoming scarce year by year. However, the resource of leaves of
A. sinensis is abundant and available two quarters per year in southern China. Traditionally, these leaves are used in China for treatments for inflammation and anaphylaxis (2). They are also broadly
© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email:
used as a main component in several health foods including
A. sinensis tea, honey and flavor.
The ethanol extract from the leaves of A. sinensis was confirmed to
have analgesic, anti-inflammatory, and nitrite scavenging activities (3, 4).
Several previous studies have indicated that the main compounds
from the leaves of A. sinensis are flavonoids, benzophenone glycoside and triterpenoids. These compounds exhibited notable antinociceptive, anti-inflammatory, antioxidative, α-glucosidase inhibitory
and laxative activities (2, 5–10). Considering their various biological
activities, a large quantity of pure bioactive compounds (with a
focus on flavonoids) from the leaves of A. sinensis is needed for further pharmacological studies and industrial applications.
Traditional separation and purification methods of flavonoids
from the leaves of A. sinensis require multiple chromatographic
steps using silica gel, polyamide column, sephadex LH-20, preparative HPLC, etc. These methods are more or less non-green, tedious
and time consuming with a potential risk of loss of target compounds due to the highly irreversible adsorptive, contaminative and
denaturing effects of the solid matrix. High-speed counter-current
chromatography (HSCCC), a unique liquid–liquid partition chromatographic technique without solid matrix, can yield a highly efficient separation of a large amount of samples in several hours and
also permits introduction of crude samples directly into the separation column without extensive preparation (11). HSCCC has been
successfully applied to the isolation and purification of a number of
natural products including flavonoids (12–14). It is an effective and
economical separation technology especially for flavonoid-like compounds that can be adsorbed and lost in the solid-liquid chromatographic process. However, to our best knowledge, no report
has been published on the use of HSCCC for the isolation and purification of compounds from wild A. sinensis leaves.
Because of usually limited distribution space and relatively small
amount of stationary phase, usually only milligram to hundred milligram amounts of purified compounds can be obtained by HSCCC
apparatuses widely used at present. In order to increase the preparation quantity ranged from gram to ten gram by HSCCC, HSCCC
apparatus with a high β values was designed and assembled under
patents CN201310032823.8 and CN201320047321.8 by Prof Tian
You Zhang’s group in Guangdong, China (15, 16).
In the current study, four flavonoids (Figure 1) were successfully
purified from wild A. sinensis leaves by this improved preparative
HSCCC apparatus, and their anti-cancer activity was investigated.
Yang et al.
Chemicals and reagents
Silica gel (100–200 mesh) was purchased from Qingdao Ocean
Chemical Co. (Qing-dao, China); and pre-coated silica gel
HSGF254 thin layer chromatography (TLC) plates were obtained
from Jiangyou Silica Gel Development Co. (Yantai, China). High
performance liquid chromatography (HPLC) grade methanol
(MeOH) was from Merck Chemical Co. (Darmstadt, Germany).
Aanalytical grade n-hexane, ethyl acetate (EtOAc), MeOH, n-butanol,
petroleum ether (b.p. 60–90°C), acetone, sodium nitrite, sulfanilic
acid, N-ethylenediamine, citrate sodium, monosodium phosphate
and muriatic acid were purchased from Guangzhou Chemical
Reagent Co. (Guangzhou, China). All cell culture reagents were obtained from Invitrogen Co. (Carlsbad, CA, USA). Cell culture dishes
and plates were purchased from Corning Inc. (New York, USA).
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich Co. (St. Louis,
Human hepatocellular cancer cell lines (HepG2) were established
and maintained in our laboratory.
The present study utilized a GX-6L high-speed counter-current chromatograph equipped with a multilayer coiled separation column with
a total capacity of 1,000 mL (if six units are connected in series, the
total column capacity becomes 6.0 L); a manual sample injection
valve with a 20-mL or 50-mL sample injection loop (An engineering
HSCCC prototype, which designed and assembled under patents
CN201310032823.8 and CN201320047321.8, Guangdong, China);
and an HD-2000 ultraviolet detector (Jiapeng, Shanghai, China). An
LC-10Avp liquid chromatography (HPLC) system used was equipped
with a CTO-10ASvp column oven, a manual sample injection valve
(model 7725) with a 20-μL loop, and an SPD-10Avp ultraviolet
detector (Shimadzu, Kyoto, Japan); YMC-Pack ODS-A columns
(5 μm, 250 × 4.6 mm2 I.D.) for analytic purposes (YMC, Kyoto,
Japan). Identification of purified samples was carried out with an ESImass spectrometer a Finnigan LCQ Advantage MAX spectrometer
(Thermo Fisher Scientific, Waltham, MA, USA) and a nuclear
magnetic resonance (NMR) instrument from a Bruker AV-300 or
Bruker AV-400 (Bruker Biospin, Rheistetten, Baden-Württemberg,
Germany). A UV–spectrophotometer (Beijing’s General Instrument
Co., Ltd, China) and Microplate Reader (TECAN SpectraII Plate
Reader, Research Triangle Park, NC, USA) were also used.
Plant materials
The leaves of wild A. sinensis were collected from Sanxiang Town
of Zhongshan City, Guangdong Province, China in May, 2011. The
plant material was botanically authenticated by Prof Zhijian Feng in
College of Forestry, South China Agricultural University. A voucher
specimen (No. JNU-2267) was deposited in the herbarium of South
China Agricultural University.
Preparation of crude sample
Figure 1. Structures of the four flavonoids from the wild Aquilaria sinensis
Oven-dried leaves of wild A. sinensis (1.0 kg) were extracted thrice
each with 10.0 L of 70% acetone for 1 h under ultrasonication. The
combined extract was condensed under vacuum to give a syrupy
extract, which was then diluted with water to a total volume of
5 L and then partitioned successively with petroleum ether (5.0 L × 4),
Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative HSCCC Apparatus
EtOAc (5.0 L × 5), and n-butanol (5.0 L × 3). The combined layers of
each organic solvent were evaporated in vacuo to yield a petroleum
ether-soluble fraction (FP, 33.8 g), an EtOAc-soluble fraction (FE,
43.5 g), and a n-butanol-soluble fraction (FB, 21.7 g), respectively.
The FP was further subjected to silica gel column chromatography
using a gradient elution with petroleum ether/EtOAc (10:1–4:1, V/V)
to give two sub-fractions (FP1 and FP2) on the basis of TLC tracing.
Preparation of two-phase solvent system and sample
The two-phase solvent systems composed of n-hexane–EtOAc–
MeOH–water (HEMW) at various volume ratios were used for
HSCCC separation. Each set of solvent system was added to a separatory funnel and thoroughly equilibrated at room temperature
for 2 h. The upper phase and lower phase were separated and degassed by sonication for 30 min shortly before use. The sample solutions were prepared by dissolving FP1, FP2, FE or FB in the mobile
phase of the selected solvent system.
Selection of two-phase solvent systems
Successful separation by HSCCC largely depends upon the selection of
suitable two-phase solvent systems. In the previous research on separation of flavonoids, many different organic solvent systems were ever
selected, among which HEMW was used most frequently, 60% of the
reported solvent systems for the isolation of free flavonoids from plant
extracts where most flavonoids had suitable partition coefficient in the
above solvent system with different volume ratios (17, 18).
According to the above procedure, several different volume
ratios of HEMW solvent systems (Tables I and II) were made and
Table I. The K-Values of Apigenin-7,4′-Dimethylether and
Genkwanin Measured in Different Ratios of HEMW Solvent
Two-phase solvent
system HEMW
5:5: 5:5
5:2.5: 5:5
5:10: 5:5
5:5: 6:5
5:5: 7.5:5
5:5: 10:5
5:6: 6:5
5:7.5: 6:5
5:7.5: 7.5:5
tested in the present study. K-values of the target compounds were
measured to predict the retention volume of the tested systems.
HSCCC separation procedure
The stationary phase was pumped into the column from head to
tail. After the column was totally filled, the rotor was rotated at
400 rpm. Then, the mobile phase was pumped into the column at a
flow-rate of 10 mL/min until hydrodynamic equilibrium was
reached, when no further stationary phase was displaced from the
column. The volume of stationary phase displaced from the column
was noted to calculate the retention of the stationary phase in the
column. Then, 420 mg of FP1, 700 mg of FP2 and 780 mg of FE were
pumped into a 20-mL (2.0% of coil volume) sample loop and injected into the column through the injection valve. The effluent from
the tail end of the column was continuously monitored with a UV
absorbance detector at 340 nm. The data were recorded immediately
after sample injection. Fractions were collected manually when chromatographic peaks were detected. A 1-mL aliquot was taken from
each fraction and analyzed for quantity and purity by HPLC.
HPLC analysis and identification of HSCCC fractions
Each peak fraction of HSCCC was analyzed by RP-HPLC where
360 nm was chosen as the UV detection wavelength. Flow rate was
set at 1.0 mL/min. Identification of the HSCCC peak fractions was
based on the data of ESI-MS, 1H and 13C NMR.
Nitrite scavenging test
Anti-cancer activities of the four flavonoids were evaluated by the
nitrite scavenging activity assay using a UV spectrophotometer at a
wavelength of 544 nm performed as described previously (19). The
conditions were modified as follows: sodium nitrite (5 μg/mL),
naphthyl ethylene diamine dihydrochloride (0.2% w/v), sulfanilic acid
(0.4% w/v), reaction temperature 37°C, reaction time 30 min, citric
acid/sodium dihydrogenphosphate buffer solution at pH value of 3.0
or 7.0, and sample concentration of 3.0 mg/mL. The reaction mixture
(3 mL) containing sodium nitrite (2 mL), sample solution (0.5 mL) and
buffer solution (0.5 mL) (for adjusting the pH value of 3.0 or 7.0) was
incubated at 37°C for 30 min. After incubation, 0.5 mL of the reaction
mixture mixed with 2 mL of sulfanilic acid and allowed to stand for
5 min for completing diazotization. Then, 1 mL of naphthyl ethylene
diamine dihydrochloride was added, mixed and allowed to stand for
30 min at 37°C. A pink colored chromophore was formed in diffused
light. The absorbance of sample solutions was measured at 544 nm
against the corresponding blank solution (distilled water), and the %
scavenging value was computed according to the following formula:
Nitrite scavenging percentage =
Acontrol − Asample
× 100%
Table II. The K-Values of Quercetin and Kaempferol Measured in
Different Ratios HEMW Systems
Two-phase solvent system HEMW
5:4: 5:4
7:4: 5:4
5:6: 5:4
5:7: 5:4
5:5: 5:5
where, the Acontrol is the absorbance of solution without the addition
of sample solution.
Cytotoxicity test
Cytotoxic activities against cancer cells of the four flavonoids were evaluated using MTT assay, which was performed as described previously
(20) with Doxorubin (Dox) served as the positive control. Briefly, cells
were plated on 96-well plates at 3 × 103 cells per well for HepG2 cell
lines. After 48 h of exposure, the cells were stained with MTT.
Absorbance at 570 nm was used to measure with a multiplate reader.
Statistical analysis
All data were expressed as mean±SEM (standard error of mean). Results
were analyzed by one-way analysis of variance (ANOVA), and significant differences were determined by post-hoc Tukey test using SPSS 11.0
software, where differences were statistically significant at P < 0.05.
Preparative isolation of four flavonoids by HSCCC
The current study performed with an optimized HEMW two-phase solvent systems at the volume ratio of 5:7.5: 6:5 has achieved the preparative separation of apigenin-7,4′- dimethylether and genkwanin from
420 mg of FP1 and 700 mg of FP2 (Figure 2A–D), where 20 mg (4.7%)
of apigenin-7,4′-dimethylether with purity of 99.7%; and 70 mg (10%)
of genkwanin with purity of 93.1% were obtained each in a single HSCCC run. The detailed chemical structures of apigenin-7,
4′-dimethylether and genkwanin (Figure 1) were confirmed by the comparison of their NMR and MS with the data from literature (21, 22).
As shown in Figure 3A–C, quercetin (86 mg, 11.0%) with purity
of 99.4% and kaempferol (34 mg, 4.4%) with purity of 98.7% were
simultaneously obtained from 780 mg of FE at one HSCCC run by
modifying the HEMW two-phase solvent system at the volumn ratio
of 5:5: 5:5. The elucidation of their structures (Figure 1) was based
on the NMR and MS analysis combined with the data comparison
to the literature (23, 24).
Yang et al.
The K-value test of the target compounds indicated that the
HEMW two-phase system at a volume ratio of = 5:7.5: 6:5 was suitable for the separation of apigenin-7,4′-dimethylether and genkwanin (K values of 0.07 and 0.23, Table I); and that at 5:5: 5:5 was
suitable for the separation of quercetin and kaempferol (K values of
0.18 and 0.63, Table II).
In vitro cancer-preventing activity
As shown in Tables III, all the four flavonoids exhibited nitrite scavenging activities, in which quercetin is the most active compound at
the scavenging rate of 28.10 ± 0.17%. In contrast, kaempferol is the
poorest scavenger with scavenging rate of 5.19 ± 0.11%. The current determination was set in a condition with temperature at 37°C,
pH value of 3.0 and reaction time of 30 min.
In vitro cytotoxicity
Among the four flavonoids, Kaempferol and quercetin exhibited
moderate inhibitory activities against human hepatocarcinoma cells
(HepG2) with IC50 values of 12.54 ± 1.37, and 38.63 ± 4.05 μM
(Table I), respectively. However, 4′-dimethylether and genkwanin
showed only weak inhibitory activities against the growth of HepG2
cells, where their IC50 values were larger than 50 μM.
Preparative isolation of four flavonoids by HSCCC
Although HSCCC has been successfully applied to the isolation and
purification of many natural products including flavonoids (13, 14, 22),
Figure 2. HSCCC separations of FP1 and FP2. HSCCC chromatograms of FP1 (A) and FP2 (B): Experimental conditions: column volume: 1000 mL; phase system:
HEMW (5:7.5:6:5, V/V); stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention
of stationary phase: 77.6%. RP-HPLC profiles of apigenin-7,4′-dimethylether (C) and genkwanin (D). Column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); isocratic, eluant (V/V): (C) AcN:H2O:AcOH = 60:20: 2; (D) MeOH: 2% AcOH in water = 70:30. Note: The optional detection wavelengths of the prepartive HSCCC
apparatus matching UV absorbance detector are 220, 254, 280 and 340 nm, and 340 nm was chosed to HSCCC separations of the four flavonoids in this study.
Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative HSCCC Apparatus
Figure 3. HSCCC separation of FE. (A) HSCCC chromatogram. Experimental conditions: column volume: 1,000 mL; two-phase solvent system: HEMW (5:5:5:5, V/V);
stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention of stationary phase:
82.4%. (B and C) RP-HPLC profiles of quercetin (B) and kaempferol (C). Analytical conditions: column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); elution mode:
isocratic; eluant (V/V): (B) MeOH: 0.4% phosphoric acid in water = 55:45; (C) MeOH: 0.05% phosphoric acid in water = 65:35.
Table III. Nitrite Scavenging Activities and Growth Inhibitory
Activities to HepG2 Cells of the Four Flavonoilsa
Nitrite scavenging
rate (%)
HepG2 IC50 (μM)
12.40 ± 0.20
5.84 ± 0.03
28.10 ± 0.17
5.19 ± 0.11
12.54 ± 1.37
38.63 ± 4.05
0.17 ± 0.03
Data expressed as mean±SEM (standard error of mean) of three observation per sample.
– Means no data.
so far it has not been applied to the leaves of wild A. sinensiss. In
the present study separation conditions such as two-phase solvent
systems and operating procedures applied to the wild A. sinensis
leaves were successfully set up using an improved preparative
HSCCC apparatus. This prototype HSCCC apparatus can produce
excellent retention of stationary phase with a large sample loading
capacity and high chromatographic resolution. The compounds
with high purity can be obtained in hundred milligrams to grams
from one unit of HSCCC columns, and ten to hundred gram grade
samples (50 times injection volume of this study) can be separated if
six units are connected in series. Four flavonoids including apigenin7,4′-dimethylether, genkwanin, quercetin and kaempferol were
isolated in a preparative or semi-preparative scale with excellent stationary phase retention for all the tested solvent systems by this
improved preparative HSCCC apparatus used in this study, which
lay the foundation for the industrial production of these compounds. Here, it is necessary to point out that quercetin was originally isolated from the Thymelaeaceae family to which A. sinensis
belongs, and kaempferol was isolated for the first time from the
Aquilaria genus to which A. sinensis belongs (25).
In vitro anti-cancer activity
Sodium nitrite (SNT) is ubiquitous in the environment and can also
be formed from nitrogenous compounds by microorganisms present
in the soil, water, saliva and the gastrointestinal tract. SNT is widely
used in food and drug industries as a preservative. About 40% of
absorbed nitrite is excreted unchanged in the urine while the metabolism of the rest is not accurately known (26). When we ingest
nitrite, endogenous nitrosation may form N-nitrosocompounds
(NOCs) that have been observed to induce tumors of the kidney in
animals (27, 28). In the human body, primarily in the stomach,
nitrite can react with amines, amides or amino acids to produce
NOCs, most of which are potent animal carcinogens. Therefore,
scavenging ingested nitrite is probably one way to prevent carcinogenesis. As an example, vitamin C is traditionally used as a drug for
cancer prevention partially because it is an effective inhibitor of
NOCs formation (29, 30).
In our previous work, nitrite scavenging activities of the ethanol
extract from the leaves of A. sinensis were demonstrated (4),
although the bioactive constituents are unknown. Based on the full
understanding of flavonoids, we believe that some of those flavonoids should play a role as a nitrite scavenger. According to the
result of our study, all four flavonoids exhibited nitrite scavenging
activities. However, it should be noted that the current determination was set in a mimetic gastric environment. The mechanism of
nitrite scavenge by flavonoids is now under investigation in our
In vitro cytotoxicity
Kaempferol and quercetin have been reported to inhibit cancer
development through an anti-angiogenic mechanism (31, 32), in
which human ovarian cancer cells and hamster buccal pouch cells
were used. The present investigation indicated that both compounds
also exhibited moderate inhibitory activities against human hepatocarcinoma cells (HepG2). However, apigenin-7,4′-dimethylether and
genkwanin showed only weak inhibitory activities against the
growth of HepG2 cells. From the structural differences among the
four flavonoids (Figure 1), it is suggested that the methylation of
phenolic hydroxyl group(s) in the molecules might decrease their
anti-cancer activities. The aforementioned cancer inhibition of
kaempferol and quercetin may be explained on the basis of the antiangiogenic mechanism.
In summary, four bioactive flavonoids, namely apigenin-7,4′dimethylether genkwanin, quercetin, and kaempferol were successfully purified in a preparative or semi-preparative scale from the
leaves of wild A. sinesis under environment-friendly conditions by
an improved preparative HSCCC apparatus, with an HEMW solvent system at different volume ratios. Among those, quercetin was
originally isolated from the Thymelaeaceae family, while kaempferol
was isolated from the Aquilaria genus for the first time. All these
four flavonoids exhibited nitrite scavenging activities in a mimetic
gastric environment, in which quercetin was the best scavenger.
Among those flavonoids, quercetin and kaempferol exhibited moderate cytotoxic activities against HepG2. The current results partially disclose the bioactive constituents of the wild A. sinesis leaves.
Supplementary Data
Supplementary data are available at Journal of Chromatographic Science
This research was financially supported by the National Natural Science
Foundation of China (No. 81172982), the Foundation of Science and
2010A030100006 and 2013B020411003), and the Bureau of Science and
Technology of Zhongshan City (No. 2016B2166).
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