全 文 ： 88 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn
Simultaneous determination of three main analytes of Murraya exotica
by HPLC
Bingyu Liu1, Chen Zhang1, Haining Lv1, Pengfei Tu1, Jianyong Xing2, Zhengzhou Han2, Yong Jiang1*
1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
2. China Resources Sanjiu Medical & Pharmaceutical Co. Ltd., Shenzhen 518110, China 
Abstract: Murraya exotica L. is one of the two official source species of traditional Chinese medicine Murrayae Folium et
Cacumen. At present, a rapid HPLC analysis method to simultaneously determine two major coumarins, hainanmurpanin (1) and
meranzin (2), and one main flavonoid, 3,5,6,7,3′,4′,5′-heptamethoxyflavone (3), from the leaves and twigs of M. exotica was
established. The analysis was performed on a DIKMA Spursil C18 column (4.6 mm×250 mm, 5 µm) at a flow rate of 1 mL/min,
using acetonitrile–H2O (5:5, v/v) as mobile phase. The column temperature was 25 ºC, and the detected wavelength was at 320 nm.
The three analytes (1–3) were separated well with good linearity, precision, stability and repeatability. The average recoveries
were in the range of 100.52%–101.97%, with RSD less than 1.73%. Twenty batches of M. exotica from different habitats were
detected, and the sum of the contents of 1–3 was in the range of 1.55–7.45 mg/g, respectively. Besides, the contents of these three
analytes were also determined for the samples from different medicinal parts (stems, lateral branches, mixture of twigs and
leaves) and different harvest times. The results showed that the contents of these three analytes in leaves and twigs are much
higher than those in the stems or lateral branches; the plants harvested from June or October contain more active compounds than
those from the other months. The above results proved that this high performance and simple HPLC assay can be readily utilized as
a practical method for the quality control of M. exotica.
Keywords: Murraya exotica L., Coumarin, Flavonoid, Quantification, HPLC
CLC number: R284 Document code: A Article ID: 1003–1057(2015)2–88–07
Received: 2014-12-11, Revised: 2014-12-30, Accepted: 2015-01-01.
Foundation items: National Natural Science Foundation of China
(Grant No. 81222051 and 81473106) and National Key Technology
R&D Program “New Drug Innovation” of China (Grant No.
2012ZX09301002-002-002 and 2012ZX09304-005).
*Corresponding author. Tel.: 86-10-82802719,
E-mail: yongjiang@bjmu.edu.cn 
http://dx.doi.org/10.5246/jcps.2015.02.010
1. Introduction
Murraya exotica L., known as “Jiulixiang” in Chinese,
is widely distributed in the southern China. As one of the
official source species of Murrayae Folium et Cacumen
listed in the Chinese Pharmacopoeia (2010 Edition),
this plant has been used as a febrifuge, astringent,
anti-dysenteric, and tonic agent, and also for treating
toothache and so on[1]. The previous chemical investi-
gation and pharmacological activity screening results
show that the main active components in M. exotica are
coumarins and flavonoids[2–13]. There have been a few
researches about the content determination of M. exotica
for its flavonoids or coumarins, but the quantitative
markers selection is not suitable, and the established
method is complex and with long separation time[14–16].






In our previous work, hainanmurpanin (1), meranzin (2),
two coumarins, and a flavonoid, 3,5,6,7,3′,4′,5′-hepta-
methoxyflavone (3) were found to be the three main
compounds in M. exotica (data not shown), and it was
reported that these coumarins and flavonoids have the
activities of anti-tumor, anti-inflammation, anti-fibrosis
and inhibiting arginase enzyme[17–20]. Therefore, in this
study, a rapid and validated HPLC method to simulta-
neously quantitative analysis of 1–3 was established in
order to efficiently control the quality of M. exotica from
different sources. The samples from different medicinal
parts, different harvest times, and different habitats were
determined, and the results supply the references for
the choice of medicinal parts, the harvest time of the
crude drugs, and of the planting areas.
2. Experimental
2.1. Materials, chemicals, and reagents
Acetonitrile used for HPLC was of chromatographic grade
(Tianjin Biaoshiqi Science&Technology Development
O OH3CO
OH3C
O
O
CH3H3C
O OH3CO
O
H3C CH3
O
OCH3
OCH3
OCH3
OCH3H3CO
H3CO
O
1 2 3
H3CO
Co. Ltd., Tianjin, China). Methanol was purchased from
Tianjin Damao Chemical Reagent Factory (Tianjin,
China). Deionized water was prepared in-house from
a Milli-Q plus water purification system (Millipore,
Bedford, MA, USA). The 0.22 µm filter membranes
used in the experiment were purchased from Xinjinghua
Co. (Shanghai, China).
Chemical reference substances of hainanmurpanin (1),
meranzin (2), and 3,5,6,7,3′,4′,5′-heptamethoxyflavone (3)
were isolated from M. exotica in our laboratory (Fig. 1).
Their structures were elucidated by comparison of their
spectroscopic data (ESI-MS, 1H, and 13C NMR) with
the published literature values[21–23]. The purities of 1–3
were all over 98.0% detected by HPLC-DAD.
Twenty batches of M. exotica, along with the samples
from different medicinal parts and different harvested
times were harvested from Guangdong, Guangxi, Fujian
and Hainan provinces. All the samples were identified
by Prof. Pengfei Tu to be M. exotica. The voucher
specimens are deposited at the Herbarium of the Peking
University Modern Research Center for Traditional
Chinese Medicine.
2.2. Reference solutions and sample preparation
References were accurately weighed, and dissolved in
methanol to obtain the stock solution of the mixture of
the three references (1–3) containing 230 µg/mL of 1,
450 µg/mL of 2, and 520 µg/mL of 3, which was stored
in the refrigerator at 4 °C prior to analysis.
The dried leaves of M. exotica were powdered by
a 40 mesh mill. Each sample (0.25 g) was accurately
weighted into a 100 mL flask, and 50 mL of 80%
methanol was accurately added. The mixture was extracted
by ultrasonification (power: 500 W; frequency: 40 kHz)
for 30 min. After the samples were cooled to room
temperature, the loss was made up by adding solvent
as needed. Then the extraction solution was filtered
through a 0.22 µm membrane, and an aliquot of 10 µL
of the filtrate was injected into the HPLC system for
analysis.
2.3. HPLC-DAD analysis
The analysis was carried out on an Agilent Series
1100 liquid chromatographic system (Agilent Technologies,
USA), equipped with an Agilent G1311A pump and
an Agilent G1316A photodiode array detector. The
analytical column was a DIKMA Spursil C18 column
(4.7 mm×250 mm, 5 µm). The chromatographic
data were recorded and processed using an Agilent
1100 workstation. The mobile phase was a mixture
of acetonitrile–water (5:5, v/v) and the flow rate was
set at 1.0 mL/min. Peaks were detected at 320 nm. The
injection volume was 10 µL and the column temperature
was maintained at 30 °C.
2.4. Method validation
2.4.1. Calibration curves, limits of detection and
quantification
Calibration curves were plotted using the concentrations
of each reference solution (X, mg/mL) versus the peak
area (Y). The limit of detection (LOD) and the limit of
quantification (LOQ) were determined at the signal-to-
noise (S/N) ratios of 3 and 10, respectively. LOD and
LOQ for each compound were gained by serial dilutions
of the stock solution.
2.4.2. Precision, repeatability, stability and recovery
In order to evaluate the precision, the intra- and
inter-day precisions were invested by analyzing the
mixed standard solution six times in the same day and
by repeating the experiments in three consecutive days.
To assure the repeatability, six replicates (S14) were
analyzed with the above established method. Stability
of sample solution was analyzed at 0, 2, 4, 6, 8, 12 h at
room temperature, respectively. The relative standard
89 Liu, B.Y. et al. / J. Chin. Pharm. Sci. 2015, 24 (2), 88–94
Figure 1. Chemical structures of compounds 1, 2, and 3.
90 Liu, B.Y. et al. / J. Chin. Pharm. Sci. 2015, 24 (2), 88–94
deviation (RSD) was used to evaluate the precision,
repeatability and stability.
The accuracy of the method was evaluated by recovery
tests. Around the same amount of the references were
added into a certain amount of the sample (0.125 g),
which had been determined previously. The recovery
was calculated according to the following formula:
recovery (%) = (observed amount – original amount)/
spiked amount × 100%. 
3. Results and discussion
3.1. Optimization of sample preparation
In order to achieve a satisfactory assay, extraction
conditions, including extraction methods (ultrasonification
and heat-reflux), extraction solvents (50%, 80%, and 100%
aqueous methanol), solvent volumes (25, 50, and 75 mL)
and extraction times (20, 30, and 40 min) were assessed
based on single factor experiments. After comparison, the
optimal sample preparation was to extract 0.25 g sample
powder with 50 mL of 80% methanol by ultrasonification
for 30 min at room temperature.
3.2. Optimization of chromatographic conditions
HPLC conditions including detection wavelength and
mobile phase were investigated for the optimization of
chromatographic separations. UV spectra of the three
analytes (1–3) were investigated and 320 nm was selected
as the optimal detection wavelength. All the analytes can
be separated well by using isocratic acetonitrile–water
(50:50, v/v) as mobile phase in a relatively short analytical
time (Fig. 2). Optimized chromatographic conditions were
achieved after several trials. Different HPLC parameters
including the mobile phases (methanol–water and
acetonitrile–water), column types (Agilent Eclipse XDB
C18 column, 250 mm×4.6 mm, 5 µm; DIKMA Diamonsil
C18 column, 250 mm×4.6 mm, 5 µm; DIKMA Spursil
C18 column, 250 mm×4.6 mm, 5 µm, and DIKMA
Inspire C18 column, 250 mm×4.6 mm, 5 µm), column
temperature (20, 25, and 30 °C) and flow rate (0.8, 1.0,
and 1.2 mL/min) were examined. Finally, an optimized
HPLC separation was developed on a DIKMA Inspire
C18 column at the flow rate of 1.0 mL/min, with column
temperature of 30 °C by considering the resolution,
baseline separation under different chromatographic
conditions.
3.3. Validation of the method
3.3.1. Calibration curves, limits of detection and
quantification
As shown in Table 1, good linearity and high
sensitivity under the optimal chromatographic conditions
were obtained, with r values greater than 0.999. The
LOD and LOQ of the three main analytes (1–3) were
0.68–1.23 µg/mL and 2.28–5.14 µg/mL, respectively.
Table 1. Calibration curves, linearity, LOD and LOQ for hainanmurpanin (1), meranzin (2), and 3,5,6,7,3′,4′,5′-heptamethoxyflavone (3)
Figure 2. Typical HPLC chromatograms of M. exotica (A) and the mixed references (B). 1: hainanmupanin; 2: meranzin; 3: 5,6,7,3′,4′,5′-
heptamethoxyflavone.
Compound tR (min) Regression equation r Linear range (mg/mL) LOD (mg/mL) LOQ (mg/mL)
1 6.35 Y = 20106X + 61.9790 0.9997 7.19–230 1.23 5.14
2 6.96 Y = 23047X + 190.2000 0.9994 14.06–450 0.88 3.52
3 9.05 Y = 30081X + 317.7000 0.9992 16.25–520 0.68 2.28
1200

1000

800

600

400

200

0
(A)
1 m
A
U

2
0 5 10 15
t (min)
1200

1000

800

600

400

200

0
(B)
1 m
A
U

2
0 5 10 15
t (min)
3 3
3.3.2. Precision, repeatability, stability and recovery
As demonstrated in Table 2, the RSD values of intra-
and inter-day precisions, repeatability, and stability were
all less than 1.73% for all analytes. The recoveries
of the three investigated analytes (1–3) ranged from
100.52% to 101.97% with RSD from 0.24% to 1.73%.
All data were collected and depicted in Table 3.
The validation results indicated that the developed
method is precise, accurate and sensitive enough for the
quantitative determination of the three main analytes
(1–3) in the leaves of M. exotica.
3.4. Determination of three main analytes in M. exotica
from different sources by HPLC
The leaves and twigs of M. exotica were the medicinal
parts in the Pharmacopoeia of the People’s Republic
of China (2010 Edition). In order to investigate the
amount of the main compounds from the different parts
of M. exotica, stems, lateral branches, twigs and leaves
were determined by the developed method. Higher
quantity of the analytes was found to be existed in the
mixtures of leaves and twigs, and little quantity was
present in the stems and lateral branches (Table 4).
Therefore, it is rational to choose leaves and twigs as
the medicinal parts of M. exotica. Besides, it is well
known that the quality of TCMs is also affected by
their harvest time, so the leaves and twigs of M. exotica
from different months were collected and tested. The
results are listed in Table 5. The amount of the main
substances in the leaves and twigs of M. exotica from
different months has imparity. The contents of the
analytes in the samples from June and October are
higher than those from the other months, and the contents
of active components in leaves are always higher
than those in twigs. From the aspect of protecting the
plants and making rational use of resources, it is a wiser
way to take June or October as the harvest time. The
amount and the trend of three analytes from different
months are clearly shown in Figure 3. After then, the
developed method was also applied to the simultaneous
determination of three analytes (1–3) in 20 batches of
M. exotica collected from different habitats. Each sample
91 Liu, B.Y. et al. / J. Chin. Pharm. Sci. 2015, 24 (2), 88–94
Compound
Precision (RSD, %)
Repeatability (RSD, %) Stability (RSD, %)
Intra-day Inter-day
1 0.18 0.23 1.01 0.23
2 0.14 0.33 1.17 0.21
3 0.12 0.27 1.12 0.17
Compound Original amount (mg) Spiked amount (mg) Observed amount (mg) Recovery (%) Average recovery (%) RSD (%)
1
0.2108 0.2000 0.4155 102.36
100.52 1.73
0.2108 0.2000 0.4151 102.16
0.2112 0.2000 0.4117 100.26
0.2116 0.2000 0.4079 98.12
0.2104 0.2000 0.4130 101.32
0.2120 0.2000 0.4098 98.88
2
0.3383 0.3327 0.6685 99.25
100.75 1.08
0.3383 0.3327 0.6700 99.70
0.3390 0.3327 0.6744 100.81
0.3397 0.3327 0.6761 101.11
0.3377 0.3327 0.6756 101.58
0.3404 0.3327 0.6799 102.07
0.4032 0.4028 0.8142 102.04
101.97 0.24
0.4032 0.4028 0.8132 101.80
0.4040 0.4028 0.8149 102.02
0.4048 0.4028 0.8156 102.00
0.4024 0.4028 0.8116 101.60
0.4056 0.4028 0.8178 102.33
3
Table 2. Precision, repeatability and stability of the three main analytes (n = 6)
Table 3. Recovery studies for the three analytes (1–3) (n = 6)
92 Liu, B.Y. et al. / J. Chin. Pharm. Sci. 2015, 24 (2), 88–94
was analyzed three times to determine the mean content
(%, w/w) and the results are listed in Table 6. Significant
variations of hainanmurpanin (1) (0.34–3.46 mg/g),
meranzin (2) (0–5.11 mg/g), and 3,5,6,7,3′,4′,5′-hepta-
methoxyflavone (3) (0–2.82 mg/g) were observed and
the content of 3,5,6,7,3,4,5-heptamethoxyflavone (3)
is much lower than that of two coumarins (1, 2). In
addition, contents of compounds 1–3 vary significantly
among the samples from different habitats. The content
of 1 of S18 is 9-fold higher than that of S10. The
content of 2 of S8 is 14-fold higher than that of
S20. Then the content of 3 of S16 is even 15-fold
higher than that of S9. And that also happened in
the samples from the same province. For example,
the content of 2 of S8 is 8-fold higher than that of
S10. Therefore it is of great significance to develop
an analytical method to control the quality of the
raw material of M. exotica. Among the samples
from different habitats, the total amount of compounds
1–3 from Maoming County, Shenzhen, Nanning, Tian’e
and Zhangzhou have higher quality than those from
other habitats.
Table 4. Contents of the three main analytes from stems, lateral branches, twigs and leaves of M. exotica (mg/g) (n = 3)
Table 5. Contents of the three main analytes in the leaves and twigs of M. exotica (S11) from different harvest times (mg/g) (n = 3)
“–”: Undetectable.
“–”: Undetectable.
Sample
1 2 3
Stems Lateral branches (×10-2) Twigs and leaves Stems Lateral branches (×10-2) Twigs and leaves Stems Lateral branches Twigs and leaves
S11 – 4.15±0.15 3.21±0.06 – – 2.26±0.02 – – 1.14±0.05
S14 – 9.04±0.11 1.17±0.03 – 7.35±0.10 1.97±0.07 – – 0.17±0.08
Sample
1 2 3
Leaves Twigs Leaves Twigs Leaves Twigs
January 8.45±0.03 2.29±0.11 5.22±0.02 0.50±0.01 8.54±0.06 0.06±0.11
February 8.61±0.07 2.05±0.08 2.28±0.03 – 10.42±0.06 –
March 3.85±0.10 3.79±0.10 2.77±0.12 0.96±0.04 10.45±0.08 –
April 3.94±0.02 4.61±0.09 2.93±0.11 0.35±0.05 8.88±0.02 –
May 4.60±0.02 4.22±0.05 3.34±0.04 2.77±0.04 11.23±0.03 0.31±0.07
June 13.53±0.09 6.08±0.07 10.78±0.02 4.31±0.06 13.11±0.11 0.86±0.10
July 9.60±0.08 5.15±0.05 3.93±0.03 3.06±0.06 10.24±0.02 0.37±0.06
August 10.68±0.10 4.19±0.13 4.57±0.09 3.75±0.09 6.25±0.12 –
September 11.47±0.12 3.92±0.02 5.16±0.02 1.35±0.11 9.27±0.06 –
October 16.42±0.04 5.25±0.06 21.50±0.10 1.34±0.03 11.48±0.07 0.03±0.06
November 10.54±0.03 4.55±0.04 9.27±0.07 1.79±0.03 13.35±0.04 –
December 7.76±0.05 3.96±0.11 8.46±0.02 0.19±0.04 11.77±0.03 –
Figure 3. Variation of the contents of three analytes (1–3) in leaves (A) and twigs (B) of M. exotica harvested in different months.
55
50
45
40
35
30
25
20
15
10
5
0
C
on
te
nt
(m
g/
g)

1 2 3 4 5 6 7 8 9 10 11 12
t (month)
Hainanmurpanin (1)
Meranzin (2)
3,5,6,7,3,4,5-Heptamethoxyflavone (3)
Total amount of the three analytes
(A)
20
18
16
14
12
10
8
6
4
2
0
C
on
te
nt
(m
g/
g)

1 2 3 4 5 6 7 8 9 10 11 12
t (month)
Hainanmurpanin (1)
Meranzin (2)
3,5,6,7,3,4,5-Heptamethoxyflavone (3)
Total amount of the three analytes
(B)
4. Conclusions
In this study, a rapid and validated HPLC method to
simultaneously determine two coumarins (1, 2) and one
flavonoid (3) from the leaves and twigs of M. exotica
has been established. The three main analytes (1–3)
were quantified with dependable linearity, LOD, LOQ,
precision, repeatability, stability and recovery within
20 min. The results strongly elicited that it is of great
importance to control the quality of M. exotica for the
contents variation in the samples from the different
harvest times, medicinal parts, and the habitats. Taking
June or October as the harvest time is more advantageous
than the other months. Leaves and twigs have been
selected as the medicinal parts illuminated in the
Chinese Pharmacopoeia for their higher quantities of
the active components. Moreover, Maoming County,
Shenzhen, Nanning, Tian’e and Zhangzhou cities have
higher quality of the three analytes than the others as
the possibly suitable planting areas. The established
quantitation method provides a significant means for
the quality control of M. exotica and for pharmaceutical
factories to choose high quality crude drugs.
Acknowledgements
This work was financially supported by the grants
from the National Natural Science Foundation of China
(Grant No. 81222051 and 81473106) and National
Key Technology R&D Program “New Drug Innovation”
of China (Grant No. 2012ZX09301002-002-002 and
2012ZX09304-005).
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HPLC方法同时测定九里香中的三个主要成分
刘冰语1, 章宸1, 吕海宁1, 屠鹏飞1, 邢建永2, 韩正洲2, 姜勇1*
1. 北京大学医学部 药学院 天然药物及仿生药物国家重点实验室, 北京 100191
2. 华润三九医药股份有限公司, 深圳 518110 
摘要: 九里香是传统中药九里香属植物的两个基原植物之一。本研究建立了一种快速、有效的HPLC分析方法, 能够
实现在20分钟内同时测定九里香中两个香豆素类化合物, 海南九里香内酯(1)和橙皮内酯(2), 以及一个多甲氧基黄酮类
化合物, 3,5,6,7,3′,4′,5′-七甲氧基黄酮(3)。色谱条件为采用DIKMA Spursil C18 色谱柱, 乙腈–水(50:50, v/v)等度洗脱, 柱温
25 ºC, 流速1 mL/min, 320 nm检测。三个主要成分分离度良好, 在测试范围内线性关系良好, r>0.999; 精密度、重复性、
稳定性良好; 平均加样回收率在100.52%–101.97%之间, RSD值小于2%。采用该方法对20批不同产地的九里香药材进行
含量测定, 三个主要成分的含量和在1.55–7.45 mg/g。此外, 还对九里香不同部位如主茎、侧枝、嫩枝和叶, 及不同采收期
的药材进行了测定。结果表明, 三个主要成分在嫩枝及叶中的含量明显高于主茎或侧枝中, 所以药典规定以九里香的
叶及嫩枝作为药用部位是合理的; 六月或十月为最佳采收期; 产自广东茂名市、深圳市、广西南宁市、天峨市和福建
漳州市的九里香中主要活性成分的含量高于其他产地。该方法具有高效、简便的特点, 可用于九里香药材及其相关产品
的质量控制。
关键词: 九里香; 香豆素; 黄酮; 含量测定; 高效液相色谱