全 文 ：444 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 2008 年 11 月 第 6 卷 第 6 期








Simultaneous Determination of Four Active Compo-
nents in Swertia by RP-HPLC

TIAN Luan-Yuan1, CHEN Jia-Chun1*, HUANG Feng-Jiao2, FANG Jin-Bo1
1Tongji School of Pharmaceutical Sciences, Huazhong University of Science and Technology, Wuhan 430030;
2Wuhan Institute of Pharmaceutical Industry, Wuhan 430061, China
【ABSTRACT】 AIM: To simultaneously determine four anti-hepatitis components in Swertia. METHOD: A new
HPLC method using ultraviolet absorbance detection with detected wavelength at 254 nm was presented for the simul-
taneous determination of four active components, namely swertiamarin, mangiferin, swertianolin and bellidifolin in 7
species of Swertia plants (Gentianaceae). The samples were separated and detected on the column of Alltima C18(250
mm× 4.6 mm, 5 μm) under the condition of gradient elution. RESULTS: The limit of detection (S/N = 3) was less
than 0.86 μg · mL−1 and the limit of quantification ( S/N = 10 )was less than 1.73 μg · mL−1. All calibration curves
showed good linear regression within test ranges. The RSD for intra- and inter-day of four investigated compounds were
lower than 2% and 4.6%, respectively. The mean recovery was between 97.94% and 99.84%. The method was estab-
lishded for the simultaneous determination of the four anti-hepatitis constituents relating to different kinds of chemical
structures in Swertia plants. CONCLUSION: The method was successfully applied to quantify the four active compo-
nents in different parts of the herb and to identify the specific species of genus Swertia.
【KEY WORDS】 Swertia; HPLC-UV; Swertiamarin; Mangiferin; Swertianolin; Bellidifolin
【CLC Number】 Q917 【Document code】 A 【Article ID】1672-3651(2008)06-0444-06
doi: 10.3724/SP. J. 1009.2008.00444
In China, the genus Swertia (Gentianaceae)
comprises about 80 species among which 30 are well
known as the folk traditional medicines. About 13
species have been registered in the provincial book as
folk medicine in China[1]. The seven species of Swer-
tia plants involved in this study have been widely
used to treat hepatitis in the folk of China and are
registered in the standards of medicinal materials of
Hubei and Yunnan provinces in China. Chemical in-
vestigations on genus Swertia has resulted in the iso-
lation of four main kinds of metabolites: triterpenoids,
flavonoids, iridoid glycosides and xanthone deriva-
tives[2-5].
Swertiamarin, mangiferin, swertianolin and bel-
lidifolin were isolated from S. punicea Hemsl. and
were useful for the treatment of liver diseases[6-9].
There were analytical methods for the quantitative

【Received on】 2008-02-26
【Foundation Item】 The project was supported by the National
Natural Science Foundation of China (No.30271590).
【*Corresponding author】 Chen Jia-Chun: Prof., Tel: 86-27-
83692482, Fax: 86-27-83692793, E-mail: homespringchen@126.
com
evaluation of medicinal Swertia plants and their
preparations by determining only one component or
several components with similar molecular struc-
tures[10-16], but there is no reported method for the
simultaneous determination of the four anti-hepatitis
compounds relating to different kinds of chemical
structure in different plants of genus Swertia.
The present study developed and validated an HPLC
method for the simultaneous determination of four
active anti-hepatitis components, including swertia-
marin, mangiferin, swertianolin and bellidifolin in
Swertia (Fig. 1). The method was successfully applied
to determine and compare with the contents of 7 spe-
cies of Swertia plants and different parts of S. punicea
for the first time. It is sensitive, convenient and reli-
able to evaluate and identify the quality of medicinal
Swertia plants.
1 Materials
Seven species of plant samples, Swertia punicea
Hemsl., S. kouitchensis Franch., S. bifolia Batal., S.
cincta Burk., S. macrosperma (C.B.Clarke) C.B.
Clarke, S. diluta (Turcz.) Benth. et Hook. f. and S.
TIAN Luan-Yuan, et al. /Chinese Journal of Natural Medicines 2008, 6(6): 444−449
2008 年 11 月 第 6 卷 第 6 期 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 445



Fig. 1 Chemical structures of 4 investigated compounds

erythrosticta Maxim. of medicinal plants of genus
Swertia (Gentianaceae) and four samples of different
parts, root, stem, leaf and flower, of the plant of S.
punicea Hemsl were collected at the flowering time in
Hubei and Sichuan provinces of China and identified
by Professor Chen Jia-Chun, one of the authors. The
voucher specimens were deposited in the Department
of Pharmacognosy, Tongji School of Pharmaceutical
Sciences, Huazhong University of Science and Tech-
nology, Wuhan, China.
Four reference compounds, swertiamarin(1).
Mangiferin (2) and swertianolin(3) were isolated and
purified from water extract of S. punicea Hemsl and
bellidifolin(4) from ethyl acetate extract of S. punicea
Hemsl by repeated silica gel column chromatography
in our lab. The purity of all compounds was more
than 98% (determined by HPLC). The structures were
confirmed by their spectral data (UV, IR, MS, 1H and
13C NMR) compared with the data from litera-
tures[17-21].
Methanol for HPLC was obtained from Hanbang
Science & Technology (Nanjing, China) and deioni-
zed water was prepared using YARON SZ-93
(Shanghai, China). All other solvents for extraction
were from Nanjing Chemical Factory (Nanjing,
China).
2 Methods
2.1 Apparatus and chromatographic conditions
All analyses were performed on an Agilent 1100
series (Agilent Technologies, USA). HPLC system
was equipped with a vacuum degasser, a quaternary
pump, an autosampler, a colum compartment, a ultra-
violet absorbance detector and a ChemStation. The
analytical column used in the experiment was Alltima
C18 (250 mm × 4.6 mm, 5 μm) protected by guard
column Alltima C18 (12.5 mm × 4.6 mm, 5 μm). The
column temperature was maintained at 30℃. The
ultraviolet absorbance detection was performed at 254
nm.
The gradient solvent system was employed as a
mobile phase consisting of water containing 0.05%
(V/V) phosphoric acid (A) and methanol (B) at the
flow rate of 1 mL·min−1. The course of the gradient
was 20%-80% B in 50 min, followed by 80%-100% B
in 5 min and then washing column with 100% B for a
further 1 min and returned to 20% B in 2 min; finally,
reconditioning the column with 20% B isocratic for 5
min. The injection volume was 20 μL.
2.2 Limits of detection and quantification
The stock solutions of 2.97 mg·mL−1 of 1, 0.32
mg·mL−1 of 2, 0.62 mg·mL−1 of 3 and 0.49 mg·mL−1
of 4 were prepared in methanol. The LOD and LOQ
were determined by injections of solutions obtained
by successive two-fold dilution of the stock solutions
in methanol. The limits of detection (LOD) and quan-
tification (LOQ) under the chromatographic condi-
tions were separately determined in six replicate de-
terminations at signal-to-noise ratio (S/N) of 3 and 10,
respectively. Table 1 shows the data of LOD and LOQ
for each investigated compounds.
2.3 Linearity range and calibration curves
Methanol mixed standard solution containing

Table 1 Calibration results, LOD and LOQ values of the investigated compounds
Analytes Calibration curve r2 Test range (μg·mL−1) LOD (μg·mL−1) LOQ (μg·mL−1)
Swertiamarin(1) y = 594.29 x+ 127.34 0.999 4 2.93~29.29 0.86 1.73
Mangiferin(2) y = 4 014.1 x+ 274.10 0.999 6 0.32~3.16 0.07 0.35
Swertianolin(3) y = 3 405.4 x+ 627.12 0.999 4 0.62~6.15 0.06 0.24
Bellidifolin(4) y = 6 533.1 x+ 2388.9 0.999 9 0.48~4.80 0.25 0.49

TIAN Luan-Yuan, et al. /Chinese Journal of Natural Medicines 2008, 6(6): 444−449
446 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 2008 年 11 月 第 6 卷 第 6 期

four reference compounds were prepared and diluted
to appropriate concentrations for the construction of
calibration curves. A calibration curve for each com-
pound was performed with six different concentra-
tions by plotting the peak area versus the concentra-
tion of each analyte.
2.4 Coefficient of variation, repeatability and recov-
ery
For intra-day coefficient of variation (CV) assay,
the mixed standard solutions was analyzed for six
replicates within 1 day. For the inter-day CV assay,
the solutions were examined in duplicates for three
consecutive days. The intra-day CV and inter-day CV
were expressed by the relative standard deviation
(RSD). The deviation from the nominal concentration
was defined as accuracy.
The repeatability of the method was evaluated
by analyzing the same sample (n =5) of the well-pro-
portioned pulverized plant of S. punicea Hemsl which
was extracted and analyzed using the method de-
scribed in Section 2.6. The content differences of the
investigated compounds in the five samples were ex-
pressed by RSD.
The extraction recovery was carried out by add-
ing a mixed standard solution of the four investigated
compounds to a certain amount (0.15 g) of pulverized
whole plant of S. punicea Hemsl. The mixture was
extracted and analyzed using the method described in
Section 2.6. Six replicates were performed for the test.
Table 3 shows the recoveries of the 4 investigated
compounds.
2.5 Preparation of sample solutions for analysis
The seven species of Swertia plants and different
parts (root, stem, leaf, flower) of S. punicea Hemsl.
were powdered by a mixer respectively. Appropriate
weight of each sample was accurately weighed and
then extracted twice with 10 mL methanol in an ul-
trasonic bath at 45 for 30 min℃ , respectively. The
extract filtered through NO.1 Xinhua filter paper
(Hangzhou Paper Factory, China) was evaporated to
dryness, and the residue was dissolved with methanol
into 10 mL volumetric flask. The sample solution was
filtered through 0.45 μm syringe membrane filter
(Type Millex-HA, Millipore, USA) prior to HPLC. A
sample of 20 μL was injected into the HPLC column.
Chromatographic peaks of the samples were identi-
fied by comparing the retention time with that of the
standard compounds and were subsequently quanti-
fied using the external standard method.

Table 2 Coefficient of variation of the investigated compounds (n=6)
Intra-day Inter-day
Analytes
Accuracy (%) RSD (%) Accuracy (%) RSD (%)
Swertiamarin (1) 100.9 0.5 98.5 3.0
Mangiferin (2) 99.6 0.3 102.0 2.4
Swertianolin (3) 101.8 1.5 100.7 4.5
Bellidifolin (4) 100.3 1.7 99.7 4.6

Table 3 Recoveries for the assay of the investigated compounds in S. punicea Hemsl.
Analytes Original (mg) Spiked (mg) Found (mg)a Recovery (%)b RSD (%)
Swertiamarin(1) 4.00 2.97 6.94 99.05 2.66
Mangiferin(2) 0.24 0.32 0.56 99.92 3.82
Swertianolin(3) 0.63 0.62 1.24 98.44 2.76
bellidifolin(4) 0.89 0.49 1.37 97.88 2.78
a The data were present as average of three determinations.
b Recovery (%) = 100×(amount found−original amount)/amount spiked

3 Results and discussion
3.1 Optimization of HPLC conditions
The UV maximum absorption of all compounds
was detected at 254 nm obtained from UV spectra.
Several trials with various proportions metha-
nol-water or acetonitrile-water system were chosen as
mobile phase, but the separation was not satisfactory.
Eventually, it was found that methanol-water gradient
solvent system provided a better separation. In the
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2008 年 11 月 第 6 卷 第 6 期 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 447

course, gradient methanol increased from 20% (V/V)
to 80% in 50 min, followed by 80% to 100% in 1 min
at flow rate 1 mL·min−1. The presence of acid in mo-
bile phase could improve the peak trailing of com-
pounds, so adding phosphoric acid was considered on
the basis of methanol-water gradient solvent system.
Subsequently, the gradient solvent system consisting
of water containing 0.05% (V/V) phosphoric acid and
methanol was employed as the optimal mobile phase.
3.2 Calibration curves and limits of detection and
quantification
Table 1 shows the calibration results, LOD and
LOQ values analyzed by HPLC-UV method. The
calibration curves for four investigated compounds
were given by the equations, where Y indicates the
peak area and X represents the concentration in
μg·mL−1. All calibration curves show good linear re-
gression (r2 >0.999 4) within the ranges of investi-
gated concentrations.
The LOD values from the HPLC-UV method
for 1-4 were 0.86, 0.07, 0.06 and 0.25 μg·mL−1, re-
spectively. The LOQ values for 1-4 were 1.73, 0.35,
0.24 and 0.49 μg·mL−1, respectively.
3.3 The coefficient of variation, repeatability and
recovery
The coefficients of variation for the measure-
ments of the four compounds are shown in Table 2.
The accuracy values were between 98.5% and 102.0%.
The intra-day coefficients of variation (CVs) ex-
pressed as RSD were below 1.7%. The RSDs of in-
ter-day coefficient of CVs were below 4.6%.
The repeatability for the developed method pre-
sented as RSD (n=5) was less than 0.48%, 0.46%,
1.48%, 1.66% for compounds 1-4, respectively.
The mean recoveries and the RSD of the inves-
tigated 1-4 were shown in Table 3. The recovery val-
ues of the compounds studied ranged from 97.88% to
99.92%. The results indicated that the method was
reliable and repeatable.
3.4 Analysis of samples
The typical chromatograms of the extracts of 7
Swertia plants (Gentianaceae) and different parts of S.
punicea Hemsl. are shown in Fig. 2, and the contents
of four compounds are given in Table 4.
Traditionally, the identification of the specific
species of genus Swertia (Gentianaceae) is carried out
by sophisticated botanist through careful inspection of
the morphological and histological characteristics[22].
However it is difficult to make a decision when the
crude herb is processed and its appearance or texture
is damaged. Therefore, it is important to establish a
simple and reliable technique to unambiguously dis-
tinguish one species from another. This work showed
that the chemical variation is to comprehensively
identify and quantify specific species of genus Swer-
tia (Gentianaceae) in terms of the occurrence and/or
relative concentration of the four active components
by comparing the overall HPLC profiles of species of
genus Swertia. In such case, the present HPLC
method could serve as a better analytical tool to dis-
tinguish between different species of genus Swertia
collected in different geographical locations.
The quantitative analysis results showed that the
contents varied largely depending on the different
botanical parts of S. punicea Hemsl. In whole plant of
S. punicea Hemsl., the first, second, third and fourth
constituents with highest content were 1(24.33
mg·g−1), 4(7.87 mg·g−1), 3(5.30 mg·g−1)and 2(1.67
mg·g−1), respectively. In different parts of S. punicea
Hemsl., the highest content of these four compounds
were observed in the flower, followed by the leaf,
stem and root. This suggests that the whole plants
with flowers should be regarded as the best selection
when Swertia plants are used for the treatment of
hepatitis.
This newly validated HPLC-UV method not
only facilitates quality control and identification of
medicinal Swertia plants, but also could advance the
systematic evaluation of the quantity of the other spe-
cies in genus Swertia (Gentianaceae). Furthermore,
we may use this method to find more useful species
of Swertia by comparing the contents of four active
components.
4 Conclusion
A simple and sensitive HPLC-UV detection
method for the simultaneous determination of the four
anti-hepatitis constituents relating to different kinds
of chemical structures in Swertia plants has been de-
veloped for the first time. The method was found to
be accurate and precise. The proposed separation and
detection procedures were successfully applied to the
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448 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 2008 年 11 月 第 6 卷 第 6 期






Fig. 2 HPLC chromatograms of (A)Mixed standards (1, 1.485 mg·mL−1; 2, 0.160 mg·mL−1; 3, 0.310 mg·mL−1; 4, 0.245 mg·mL−1) and
methanol extracts (B) root of S. punicea, (C) stem of S. punicea, (D) leaf of S. punicea, (E) flower of S. punicea, (F) whole plant of S.
punicea, (G)S. kouichensis Frach, (H)S. bifolia Batal, (I)S. cincta Burk, (J)S. macrosperma C.B.Clarke, (K)S.diluta,(L)S. erythrosticta
Maxim. 1, Swertiamarin; 2, mangiferin; 3, swertianolin; 4, bellidifolin

Table 4 Contents of the investigated compounds in Swertia (n=3)
c (mg·g-1)
Samples
Sample weight(mg) Swertiamarin(1) Mangiferin(2) Swertianolin(3) Bellidifolin(4)
Root of S. punicea 976.86 ± 6.28 6.72 0.42 0.74 0.67
Stem of S. punicea 479.21 ± 4.16 25.00 0.60 1.99 2.54
Leaf of S. punicea 127.13 ± 3.70 99.60 3.22 3.61 7.81
Flower of S. punicea 99.01 ± 1.67 128.94 8.86 7.75 21.22
S. punicea Hemsl 251.56 ± 0.98 24.33 1.67 5.30 7.87
S. kouichensis Frach 250.21 ± 2.13 11.23 1.32 1.33 10.32
S. bifolia Batal 254.06 ± 1.03 0.94 1.58 0.15 0.32
S. cincta Burk 251.89 ± 4.12 tr 0.69 2.69 1.70
S. macrosperma C.B.Clarke 252.35 ± 3.22 tr 1.66 tr 1.98
S.diluta 253.44 ± 1.92 16.43 tr tr tr
S. erythrosticta Maxim 250.78 ± 3.11 tr tr tr 3.15
tr,content was below the LOQ

quantification of swertiamarin, mangiferin, swertia-
nolin and bellidifolin in Swertia plants collected from
various locations. Moreover, this method is very
suitable for the identification of the medicinal Swertia
plants when the four components mentioned above
are chosen as chemical markers. This method may be
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2008 年 11 月 第 6 卷 第 6 期 Chin J Nat Med Nov. 2008 Vol. 6 No. 6 449

of value for routine quality assurance and standardi-
zation of the bioactive compounds from the raw ma-
terial, extracts or formulations containing Swertia
punicea Hemsl.
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高效液相法同时测定獐牙菜属植物中四种活性成分
田峦鸢 1, 陈家春 1*, 黄风娇 2, 方进波 1
1 华中科技大学同济医学院药学院, 武汉 430030;
2 武汉医药工业研究所, 武汉 430061
【摘 要】 目的：同时测定獐牙菜属植物中四种抗肝炎活性成分的含量。方法：用高效液相色谱法对獐牙菜苦苷、
芒果苷、当药醇苷和雏菊叶龙胆酮四种抗肝炎活性成分进行了分析。结果：在选定的色谱条件下四种成分分离良好, 在检
测浓度范围内线性良好。日内和日间精密度分别小于 2% 和 4.6%, 加样回收率为 97.94%~99.84%。建立了獐牙菜属植物
中四种不同化学结构类型抗肝炎活性成分的含量测定方法。结论：本方法简单、准确, 能成功的的运用于同时测定獐牙菜
属植物不同部位中獐牙菜苦苷、芒果苷、当药醇苷和雏菊叶龙胆酮四种抗肝炎活性成分的含量以及獐牙菜属植物的鉴定。
【关键词】 獐牙菜; HPLC-UV; 獐牙菜苦苷; 芒果苷; 当药醇苷; 雏菊叶龙胆酮

【基金项目】 国家自然科学基金资助项目(No. 30271590)