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Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb by High-performance Liquid Chromatography

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  • 南京理工大学
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Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb by High-performance Liquid Chromatography

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A simple reversed-phase liquid chromatographic method has been established for the simultaneous quantification of five bioactive flavonoids, i.e., taxifolin-7-O-rhamnoside, isoquercitrin, quercitrin, quercetin and kaempferol in Hypericum japonicum Thunb. Chromatographic separations were achieved on a Luna C 18 column (250 mm×4.6 mm, 5 µm) with a gradient of methanol and 0.5 % aqueous acetic acid (v/v) as mobile phase and UV detection at 350 nm. The assay was reproducible with overall intra- and inter-day variation of less than 4.8 %. The mean recovery of the method was 99.8 % ± 2.5 %. Using the optimized method, 19 samples were analyzed. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for Hypericum japonicum Thunb.
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Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb
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Asian Journal of Traditional
Medicines, 2007, 2 ( 2 )
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Simultaneous Determination of Five Bioactive Flavonoids in
Hypericum japonicum Thunb by High-performance Liquid
Chromatography
Juan Li, Bo Jiang, Xiao Liu, Juan Zhang, Xiaohui Chen, Kaishun Bi *
School of pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
A simple reversed-phase liquid chromatographic method has been established for the simultaneous quantication of ve bioactive
flavonoids, i.e., taxifolin-7-O-rhamnoside, isoquercitrin, quercitrin, quercetin and kaempferol in Hypericum japonicum Thunb.
Chromatographic separations were achieved on a Luna C18 column (250 mm×4.6 mm, 5 µm) with a gradient of methanol and 0.5 %
aqueous acetic acid (v/v) as mobile phase and UV detection at 350 nm. The assay was reproducible with overall intra- and inter-day
variation of less than 4.8 %. The mean recovery of the method was 99.8 % ± 2.5 %. Using the optimized method, 19 samples were
analyzed. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for Hypericum
japonicum Thunb.
Key words: Hypericum japonicum thunb (Tianjihuang); taxifolin-7-O-rhamnoside; isoquercitrin; quercitrin; quercetin; kaempferol; RP-HPLC
Introduction
Hypericum japonicum Thunb (Tianjihuang, TJH)
has been used as a Chinese medicine herb for the
treatment of bacterial diseases, infectious hepatitis,
gastrointestinal disorder, internal hemorrhage and
tumors [1-4 ]. For many years, Tianjihuang (TJH)
extracts has been formulated into an injection, which
exhibit a good therapeutic effect in treating acute and
chronic hepatitis [5] clinically.
Previous phytochemical studies of TJH have
demonstrated that it mainly contains flavonoids,
phloroglucinol derivatives, lactones, xanthonoids,
chromone glycosides and peptides [6-12]. Taxifolin-7-
O-rhamnoside, isoquercitrin, quercitrin, quercetin and
kaempferol are major components of TJH, and exhibit
brilliant pharmacological and biological activities.
For example, quercitrin and isoquercitrin showed
anticoagulation of activated partial thromboplastin time
(APTT) reagent, taxifolin-7-O-rhamnoside showed
promoting coagulation of APTT, and quercetin was
found to exert promoting coagulation of prothrombin
time regent [4]. Kaempferol has been reported to
affect cytochrome 450 activities in primarily cultured
rat hepatocytes [13]. Consequently it is essential to
explore a new method for the determination of these
avonoids.
LC method using UV detection has been reported
to determine the amount of quercetin in TJH, which
requiring of acid hydrolysis prior to separation [14].
HPLC method has been investigated for the ngerprint
qualitative analysis of TJH also, which can identify
and distinguish raw herbs of TJH from different
sources [15]. However, simultaneous quantitative
analysis of taxifolin-7-O-rhamnoside, isoquercitrin,
quercitrin, quercetin and kaempferol has not been
reported yet. In this study, a HPLC was used to
develop a determination method with high accuracy,
specicity and reproducibility for these avonoids in
TJH. Their structures and UVmax absorption values
were listed in Table 1. The developed method was
successfully applied to the quantification of five
bioactive constituents in 19 samples of TJH.
Experimental Section
Chemicals and materials
Methanol and acetonitrile were HPLC grade (Tedia
Company Inc.,USA). Acetic acid and phosphoric
acid were obtained from Kermel (Tianjing, China).
Commercial herb samples of TJH were purchased
* Author to whom correspondence should be addressed. Tel.:
+86-24-23986016; E-mail: Bikaishun@yahoo.com
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Compound UVmax (nm) Structure
Kaempferol 210,260,363
Quercetin 210,254,365
Quercitrin 210,259,353
Isoquercitrin 210,252,353
Taxifolin-7-O-rhamnoside 209,290,330
Table 1 Structures and λmax of ve bioactive avonoids in TJH
from drug stores or markets in different provinces of
China and authenticated by Qishi Sun, a Professor of
Pharmacognosy. A voucher specimen is deposited at
the herbarium of Shenyang Pharmaceutical University
(No. 5208), Liaoning, P.R. China. Quercetin and
kaempferol standards were purchased from National
Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). The standards of
taxifolin-7-O-rhamnoside, isoquercitrin and quercitrin
were isolated from the TJH by the author. Compared
with literatures, taxifolin-7-O-rhamnoside [8],
isoquercitrin [16], quercitrin [8] were fully characterized
using chemical and spectroscopic methods (UV, IR, 1H
NMR, 13C NMR, and MS). Purity analysis suggested
that their purities were all above 98 %.
Apparatus and chromatographic conditions
All separations were performed on a LC-2010
(Shimadzu, Japan) liquid chromatograph equipped
with a vacuum degasser, a quaternary pump, an auto
sampler, and a UV detector.
Chromatographic separation was carried out
on a Luna C18 column (250 mm×4.6 mm, 5 µm;
Phenomenex, USA) protected by a Luna C18 guard
column (7.5 mm×4.6 mm I.D.) at room temperature.
The mobile phase consisted of (A) methanol and (B)
0.5 % aqueous acetic acid (v/v) using a gradient elution
of 30 %–70 % A in 0-60 min. Detection was carried out
at 350 nm, the ow rate was kept at 1.0 mL·min-1 and
the injection volume was 20 µL.
Preparation of sample solutions
A 1 g powder of dried materials was extracted
with 30 mL 50 % methanol in an ultrasonic water bath
for 30 min. The extracted solution was ltrated through
analytical lter papers. The ltrate was evaporated at
Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb
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Asian Journal of Traditional
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40 ºC to dryness by vacuum. The extract was dissolved
in 25.0 mL methanol, and the suspended particles
were then ltrated through a 0.45 µm membrane lter.
Each sample was prepared with the above protocol for
HPLC analysis next.
Preparation of standard solutions
Stock solutions of five flavonoids were prepared
by dissolving five standard samples in methanol,
respectively. Calibration curves were established
based on six concentrations by diluting the stocking
solution with methanol in appropriate quantities.
Results and Discussion
Optimization of HPLC conditions
Diamonsil C18 column and Luna C18 column were
compared. The retention time did not vary signicantly
between two columns while the theoretical column
plates of Luna C18 column was higher than that of
Diamonsil C18 column for each analyte. Thus, Luna
C18 column was chosen for the analysis. The column
temperature was 25 ºC.
These five flavonoids had different polarity,
and they couldn’t be separated by isocratic elution.
In order to obtain good resolution, different mobile
phase compositions were also optimized including
methanol-water, methanol-aqueous acetic acid
and methanol-aqueous phosphoric acid were also
compared. The best resolution and sensitivity was
obtained by using the gradient elution of methanol-
aqueous acetic acid (0.5 %, v/v).
According to the absorption maxima of five
avonoids on the UV spectra with three-dimensional
chromatograms of HPLC-DAD detection, the
monitoring wavelength was performed at 350 nm. The
HPLC-DAD chromatogram was shown in Fig. 1. The
mobile phase was delivered with a flow rate of 1.0
mLmin-1. The chromatographic peaks and purity of
TJH were identified by comparing with the retention
time of each reference flavonoid and each UV
spectrum recorded. With the eluting solvent system
the theoretical column plates was more than 8000, the
tailing factor was less than 1.1 and the resolution was
greater than 1.5 for each compound. Chromatograms
of standard mixture (A) and TJH extract (B) were
shown in Fig. 2. The retention times of taxifolin-7-O-
rhamnoside, isoquercitrin, quercitrin, quercetin and
kaempferol were 11.5, 22.3, 25.7, 34.9 and 42.5 min,
respectively.
Optimization of extraction conditions
The optimal extraction solvent was determined
by adding 1.0 g powdered samples to 30 mL aliquots
of the following solvents: methanol, ethanol, ethyl
acetate and water. The results indicated that methanol
yielded the highest extraction concentrations. By
comparing extractions at 15, 30, and 60 min, it was
clear that five flavonoids were almost completely
extracted within 30 min.
As can be seen from Fig. 3, five curves showed
the fluctuation of extraction efficiency of different
concentrations of methanol. Finally, 50 % methanol
was chosen as the extraction solvent because the
flavonoids could be well resolved from background
Fig. 1 HPLC-DAD chromatogram of TJH extract
Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb
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Fig. 2 Chromatograms of standard mixture (A) and TJH extract (B).
(1) Taxifolin-7-O-rhamnoside; (2) Isoquercitrin; (3) Quercitrin; (4) Quercetin; (5) Kaempferol
Fig. 3 Extraction efciency of different concentration of methanol.
(A) Quercitrin; (B) Quercetin; (C) Isoquercitrin; (D) Taxifolin-7-O-rhamnoside; (E) Kaempferol.
Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb
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peaks with sufcient extraction efciency.
Method validation
-Linearity and LOD
The reference standards of five flavonoids were
accurately weighed and dissolved in methanol, and
then diluted to appropriate concentration ranges for
the construction of calibration curves. The calibration
curve for each compound was performed with
six different concentrations by plotting the peak
areas versus corresponded concentrations. All the
compounds showed good linearity in the concentration
range.
The limit of detection (LOD) under the chromato-
graphic conditions was determined by measuring the
magnitude of analytical background by injecting blank
samples and calculating the signal-to-noise (S/N) ratio
for each compound by injection a series of solutions
until the S/N ratio is more than 3 for LOD. The results
were shown in Table 2.
-Repeatability, precision and stability
Repeatability was determined by analyzing
six individual sample solutions that were prepared
from crude drug according to preparation of sample
Compound Regression equation arLinear range (µgmL-1) LOD (ngmL-1)
Taxifolin-7-O-rhamnoside y =1.123×10-4 x - 0.7667 0.9995 8.59-178 35
Isoquercitrin y =2.123×10-5 x - 0.2975 0.9992 4.60-94.7 25
Quercitrin y =1.906×10-5 x - 0.7787 0.9992 10.1-210 30
Quercetin y =1.917×10-5 x + 0.2485 0.9992 1.59-31.6 40
Kaempferol y =1.570×10-5 x + 0.2061 0.9990 0.883-12.8 27
Table 2 Linear relation between peak area and concentration (n=6)
solutions, and the relative standard deviations
were 2.4 % for taxifolin-7-O-rhamnoside, 1.9 %
for isoquercitrin, 2.2 % for quercitrin, 1.7 % for
quercetin, and 1.6 % for kaempferol.
The intra-day precision was determined by making
three repetitive injections of a standard mixture solution
(n =5), and inter-day precision was determined on
three consecutive days. The results were shown in
Table 3.
Stability was tested with standard solution and
sample solution which were stored at room temperature
and analyzed every 12 h within 3 days. The analytes
were found to be rather stable within 72 h (RSD < 3 %).
-Recovery
In the recovery test, it involved the addition of
known quantities of the mixed standard solution to the
known amounts of TJH samples. The fortied samples
were then extracted and analyzed with the proposed
HPLC method. The added standard solutions were
a x denotes the peak area and y denotes the concentration.
Compound Intra-day (n=5) Inter-day (n=3)
Mean ± SD ( µgmL-1) RSD (%) Mean ± SD ( µgmL-1) RSD (%)
Taxifolin-7-O-rhamnoside 87.5 ± 2.4 2.8 85.7 ± 4.1 4.8
Isoquercitrin 44.2 ± 1.0 2.3 44.0 ± 1.4 3.1
Quercitrin 88.6 ± 2.2 2.6 86.0 ± 2.7 3.1
Quercetin 12.8 ± 0.4 3.5 12.7 ± 0.3 2.6
Kaempferol 8.5 ± 0.2 2.9 8.5 ± 0.3 3.1
Table 3 Intra- and inter-day precision for the ve avonoids in Tianjihuang
Simultaneous Determination of Five Bioactive Flavonoids in Hypericum japonicum Thunb
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Asian Journal of Traditional
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Compound Added (µg·mL-1) Detected (µg·mL-1) aRecovery (%) bRSD (%)
Taxifolin-7-O-rhamnoside
6.85 6.51 95.0 1.4
13.70 13.39 97.7 3.0
20.76 21.39 103.0 2.0
Isoquercitrin
2.40 2.46 102.5 0.2
4.80 4.71 98.1 2.2
7.20 7.01 97.3 1.1
Quercitrin
8.60 8.62 100.3 1.7
16.40 16.07 98.0 0.8
26.20 26.68 101.9 0.5
Quercetin
4.00 3.97 99.2 2.3
8.00 8.19 102.4 2.0
12.00 12.39 103.2 2.6
Kaempferol
0.42 0.41 97.8 1.9
0.84 0.83 98.7 3.0
1.26 1.28 101.5 2.7
Table 4 Recoveries of the ve avonoids in TJH
a Calculated by subtracting the total amount after spiking from the amount in the herb before spiking. Data were means of there experiments.
b Calculated as detected amount/added amount×100 %. Data were means of there experiments.
prepared in the concentration range of calibration
curve with three different concentration levels (high,
middle and low) and triplicate experiments at each
level. The average recoveries were 98.6 % for taxifolin-
7-O-rhamnoside, 99.3 % for isoquercitrin, 100.1 %
for quercitrin, 101.6 % for quercetin and 99.3 % for
kaempferol. High recoveries suggested that there was
negligible loss of those five flavonoids during the
extraction process. (Table 4)
Application
The es t a b l i s h e d m ethod wa s su c c e s s fully
applied for the determination of five flavonoids in
different TJH samples. As shown in Table 5, there
were significant differences on the amounts of five
flavonoids among 19 samples. The most obvious
trend was that the amount of isoquercitrin varied
from 0.09 to 2.22 mg·g-1 in 19 samples with almost
24.7-fold variation. Obvious variation could be found
in other components as well, such as taxifolin-7-O-
rhamnoside and quercitrin which showed 22.5 and
11.7-fold variation, respectively. Many reasons may
contribute to the differences flavonoids level among
various samples, such as genetic variation, plant
origin, harvesting time and storage conditions. The
differences of the amounts of avonoids will inuence
quality of this herb and the therapeutic effect of the
injection. Thus, it is not overall to evaluate the quality
of TJH by determining single flavonoid, and it is
valuable to simultaneously determine these bioactive
avonoids.
Conclusion
An efficient method for the simultaneous
quantification of taxifolin-7-O-rhamnoside,
isoquercitrin, quercitrin, quercetin and kaempferol
in Hypericum japonicum thunb has been firstly
developed. Method validation studies conrmed that
this method was simple, sensitive and accurate. It was
superior to the previous methods and suitable for the
quality control of Hypericum japonicum Thunb.
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... The flow rate was at 0.8 mL/min with a 20 lL injection volume. The column oven temperature was set to 25°C, and the absorbance was monitored at 350 nm (Li et al., 2007). The extracts were analysed at least three times by HPLC. ...
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