J.Xiang's HOMEPAGE

A Rapid High-Performance Liquid
Chromatographic Method for Quantitative
Analysis of Antidiabetic-Active Components
in Anemarrhena asphodeloides Rhizomes

Abstract
Extracts of herbs with promising antidiabetic capacity, used in traditional medicine, have been
analyzed by high-performance liquid chromatography (HPLC) coupled with diode-array
detection (DAD). Optimization of the chromatographic conditions is discussed. Two kinds of
xanthone glycoside were successfully separated and quantified in herbs from seven regions.
The neomangiferin and mangiferin content of herbs from different regions varied greatly. The
sensitivity, selectivity, accuracy, precision, and ruggedness of the proposed method were
excellent, and the method can be used for quality control of Anemarrhena asphodeloides
rhizome raw material.
Keywords
Column liquid chromatography
Solvent extraction
Mangiferin and neomangiferin
Anemarrhena asphodeloides rhizome
Introduction
Traditional Chinese medicines (TCM)
have been used to treat human disease in
China for centuries. Many have complicated
constituent profiles and the quality
of the constituent herbs is easily affected
by several factors, including highly variable
species, environmental conditions,
harvest time, storage, and processing.
Work on quality control and quantitative
analysis of the active components in
traditional and herbal medicines has
therefore become necessary. In this work
we selected one of the most important
medicinal and economic plant materials,
Anemarrhena asphodeloides rhizome, to
establish a better method of analysis of
xanthone type compounds.
A. asphodeloides rhizome (Chinese
name: zhi-mu), a member of the Liliaceae
family, grows in China, Korea, and Japan
and has been widely used in TCM for its
antidiabetic, antipyretic, anti-inflammatory,
and diuretic properties. Mangiferin
and neomangiferin (Fig. 1), natural xanthone
glycosides which have been shown
to have important and broad pharmacological
activity, including antidiabetes [1],
antioxidant [2], anti-inflammatory [3], and
antiobesity [4] action, have been reported
to be present in A. asphodeloides rhizome
[5, 6]. Identification and determination
of these compounds will therefore
play an important role in evaluation of the
efficacy, safety, and therapeutic consistency
of A. asphodeloides rhizome and its
medical preparations.
Mangiferin has also been found in
some ferns and in more than a hundred
species of higher plants. Methods for
determination of mangiferin in some
medical plants have been reported,
including thin-layer chromatography with
densitometry [7] and high performance liquid
chromatography [8, 9]. To the best of
our knowledge, however, a method for
simultaneous determination of neomangiferin
and mangiferin in A. asphodeloides
rhizome has not been reported. Considering
the importance of mangiferin and
neomangiferin in this material, in this research
we have developed an HPLC
method for rapid (10 min) separation
and determination of mangiferin and
neomangiferin in extracts of A. asphodeloides
rhizome. Optimization of the chromatographic
conditions is discussed.
Experimental
Materials and Reagents
Mangiferin and neomangiferin were
obtained from Department of Pharmacognosy,
Second Military Medical University;
the purity of the two compounds
was 99%. Anemarrhena asphodeloides
rhizome was collected from seven regions
of China. The samples were authenticated
and voucher specimens were deposited in
the Department of Pharmacognosy, Sec-
2005, 61, 633–636
DOI: 10.1365/s10337-005-0563-2
0009-5893/05/06
2005 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH
Short Communication Chromatographia 2005, 61, June (No. 11/12) 633
ond Military Medical University. The
samples were powdered to 40 mesh and
stored at 25C in air-tight containers until
further use. Acetonitrile, HPLC grade,
was purchased from Fisher Scientific
(Springfield, USA). Redistilled water was
used to prepare all standard solutions. All
other chemicals and reagents used were of
analytical grade unless indicated otherwise.
Identity and Purification
of Neomangiferin
and Mangiferin
To ensure the purity of neomangiferin
and mangiferin, the two compounds were
chromatographed by RP HPLC using
the conditions described below. The 99%
purity was confirmed by normalization
of peak area. The melting points of neomangiferin
andmangiferinwere 230 ± 2C
and 260 ± 2C, respectively.
Apparatus
Chromatography was performed with
Waters (Massachusetts, USA) model 515
binary gradient equipment, a model 996
diode-array detector, a Millennium32 chromatography
workstation, and a temperature-
control module, equipped with a
Rheodyne LLC (USA) model 7725 injection
valve with 20-lL sample loop. Compounds
were separated on a 200 mm · 4.6 mm i.d.,
5 lm particle, Hypersil C18 column (Dalian
Elite Analytical Instrument Company,
China).
HPLC Conditions
Optimum HPLC separation was achieved
by use of 10% acetonitrile in aqueous
phosphate buffer at a flow rate of
1.0 mL min)1. The buffer solution was
0.05 M monosodium phosphate adjusted
to pH 3.2 with phosphoric acid. The detection
wavelength was 317 nm and the
temperature was 40C.
Preparation of Sample
Solutions
Anemarrhena asphodeloides rhizome powder
(approx. 0.5 g) was weighed accurately
into a 25-mL tube and extracted with
25 mL methanol, with sonication, for
20 min. The resulting mixture was centrifuged
at 4500 rpm for 5 min and the
supernatant was transferred to a 50-mL
volumetric flask. The solid residues were
further extracted with 20 mL methanol,
with sonication, for 10 min, and centrifuged
as described above. The extracts
were combined, diluted to 50 mL with
methanol, and filtered through a 0.45 lm
Nylon syringe filter (Millex-HN, Ireland)
before injection for HPLC analysis.
Preparation of Stock
and Standard Solutions
A combined stock solution was prepared
by dissolving 20.3 mg neomangiferin and
29.6 mg mangiferin in 100 mL methanol
in a volumetric flask. A series of standard
solutions was prepared by quantitatively
transferring 0.1, 0.4, 0.8, 1.6, and
2.0 mL of the combined stock solution
to 10-mL volumetric flasks and diluting
to volume with mobile phase. The ranges
of concentration for the two compounds
were 2.03–40.50 lg mL)1 for
neomangiferin and 2.96–59.21 lg mL)1
for mangiferin.
Results and Discussion
Method Validation
System Suitability Test
The system suitability test was performed
by making six replicate injections of each
of the standard solutions and measuring
the resolution and asymmetry factors of
the peaks of the analytes of interest. For
pharmaceutical applications the RSD of
peak area should be <2.0% and resolution
should be >1.5 between adjacent
peaks for all the analytes of interest. The
values obtained met these requirements.
Peak asymmetry was 1.02 for neomangiferin
and 1.04 mangiferin.
Linearity
Linearity was determined by constructing
two calibration plots. For each plot
standard solutions were prepared at five
concentration levels and chromatographed.
Peak area (A) and concentration
(C) for each compound were subjected to
regression analysis to calculate the
calibration equations and correlation
coefficients. The regression equations
obtained for the two compounds were
A ¼ 48004C ) 23845 (r ¼ 0.9997, n ¼ 5)
for mangiferin and A ¼ 34537C ) 16860
(r ¼ 0.9995, n ¼ 5) for neomangiferin.
The linear ranges were 2.0–40.5 lg mL)1
for neomangiferin and 2.96–59.2 lg mL)1
for mangiferin. The results show there was
excellent correlation between peak area
and concentration for each compound.
Quantitation Limit
The quantitation limit is the lowest
concentration of a compound that can be
accurately and precisely quantified.
Typically this is ten times the noise level.
The LOQ of each compound was determined
experimentally by performing six
injections of each at concentrations near
Fig. 1. The molecular structures of mangiferin and neomangiferin
634 Chromatographia 2005, 61, June (No. 11/12) Short Communication
the LOQ. The LOQ were 2.0 lg mL)1 for
neomangiferin and 2.96 lg mL)1 for
mangiferin; the chromatogram obtained
is shown in Fig. 2.
Accuracy
The accuracy of the method was tested by
determining the recovery at three levels.
Known amounts of neomangiferin and
mangiferin were added to 0.5 g powdered
Anemarrhena asphodeloides rhizomes
(source Bozhou, Anhui) and the samples
were extracted as described above.
Recovery of mangiferin and neomangiferin
is shown in Table 1.
Precision
System precision is a measure of the
method variability that can be expected if
a given analyst performs the analysis at
three different concentrations. It was
determined by performing three replicate
analyses of each standard solution at
three different concentrations. The RSD
values for neomangiferin and mangiferin
are shown in Table 2.
The method precision was determined
by preparation and analysis of five replicate
sample solutions in one batch. The
RSD was used to evaluate the precision
of the method. The RSD values obtained
were 1.87% for neomangiferin and
2.28% for mangiferin, indicative of the
good precision of the method.
Optimization of the
Chromatographic Conditions
In neutral and slightly basic media,
flavonoids are partially deprotonated.
Establishing mobile phase pH was,
therefore, the first stage of optimization
of the mobile phase. To maintain mangiferin
and neomangiferin in the neutral
form, the pH of the aqueous component
of the mobile phase was set at 3.2 by use
of phosphate buffer for the separation.
On the basis of the structures, solubility,
and acid–base properties of the
two compounds, the method was established
with a C18 column and a mobile
phase containing acetonitrile and aqueous
phosphate buffer. The first mobile
phase was a 10:90 binary mixture of
acetonitrile and buffer. When the same
binary mixture in different proportions
(15:85, 10:90, 5:95) was tested it was
found that as the proportion of acetonitrile
in the mobile phase was reduced the
retention times of two compounds gradually
increased. As a result, a mobile
phase comprising a 10:90 (v/v) acetonitrile–
0.05 M phosphate buffer (pH 3.2)
binary mixture was finally adopted to
achieve reasonable retention. Resolution,
RS, of 3.29 for neomangiferin and 4.27
for mangiferin was achieved with retention
factors, k, of 0.91 ± 0.05 for neomangiferin
and 2.20 ± 0.04 for
mangiferin, results which met the analytical
criteria discussed above.
The detection wavelength was chosen
as a compromise between the
absorption maxima of the two compounds
and those of other interfering
components of the mobile phase. Taking
all things into consideration, a
wavelength of 317 nm was finally selected
for detection; this proved reasonable
for simultaneous determination
of neomangiferin and mangiferin in
A. asphodeloides rhizome.
With these chromatographic conditions
baseline resolution was achieved
with reasonable retention times and
symmetrical peaks for the two compounds.
Typical chromatograms obtained
from a standard mixture of the
two compounds and from a sample extract
are shown in Fig. 3.
Application of the Method
The validated LC method was used to
determine the amounts of the two
compounds in A. asphodeloides rhizome
collected from seven regions of China,
mainly the Hebei and Shanxi provinces in
inner Mongolia and China, respectively.
Fig. 2. Chromatogram obtained at the quantitation limit, acquired at 317 nm by HPLC–DAD,
using a 200 mm · 4.6 mm i.d., 5 lm particle, Hypersil C18 column. The mobile phase was 10:90 (v/
v) acetonitrile–0.05 M monosodium phosphate buffer (pH 3.2). Compounds: 1 ¼ neomangiferin,
2 ¼ mangiferin
Table 1. Recovery of mangiferin and neomangiferin from Anemarrhena asphodeloides rhizome
(n = 3)
Neomangiferin Mangiferin
Amount added (lg) 259.84 324.8 389.76 378.88 473.6 568.32
Amount found (lg) 249.76 312.98 379.16 363.08 457.54 562.41
Recovery (%) 96.12 96.36 97.28 95.83 96.61 98.96
RSD (%) 1.82 1.46 1.75 1.95 1.62 1.71
Table 2. System precision for neomangiferin and mangiferin at different concentrations (n = 5)
Neomangiferin Mangiferin
Concentration (lg mL)1) 8.12 16.24 32.48 11.84 23.68 47.36
RSD (%) 0.62 0.48 0.37 0.43 0.45 0.32
Short Communication Chromatographia 2005, 61, June (No. 11/12) 635
Some of the results obtained are listed in
Table 3. The results revealed the high
variability of the neomangiferin and
mangiferin content of samples from the
different regions of China.
Conclusions
The results described above showed the
proposed LC method was very suitable
for rapid determination of xanthone glycosides
in extracts of A. asphodeloides
rhizomes from different regions. This
work also showed that HPLC was a
powerful technique for study of xanthone
compounds in the complex extracts obtained
from medicinal plants.
Acknowledgement
The authors thank the Science Foundation
of Shanghai Municipal Commission
of Science and Technology for financial
support of this work (Grant 01Dj9012).
References
1. Miura T, Ichiki H, Iwamoto N, Kato M,
Kubo M, Sasaki H, Okada M, Ishida T,
Seino Y, Tanigawa K (2001) Biol Pharm
Bull 24(9):1009–1011
2. Yoshikawa M, Ninomiya K, Shimoda H,
Nishida N, Matsuda H (2002) Biol Pharm
Bull 25(1):72–76
3. Leiro J, Garcia D, Arranz JA, Delgado R,
Sanmartin ML, Orallo F (2004) Int Immunopharmacol
4(8):991–1003
4. Yoshikawa M, Shimoda H, Nishida N,
Takada M, Matsuda H (2002) J Nutr
132(7):1819–1824
5. Aritomi M, Kawasaki T (1969) Tetrahedron
Lett 12:941–944
6. Hong YF, Han GY, Guo XM (1997) Acta
Pharm Sinica 32(6):473–475
7. Nedialkov P, Kitanov G, Tencheva J (1998)
Acta Pharm 48:211–214
8. Jurgenliemk G, Nahrstedt A (2002) Planta
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(2003) Eur Food Res Technol 216:270–273
Fig. 3. Chromatogram, acquired at 317 nm by HPLC–DAD, from a standard solution (a) and
from a hydroalcoholic extract of Anemarrhena asphodeloides rhizome (b), obtained by use of a
200 mm · 4.6 mm i.d., 5 lm particle, Hypersil C18 column. The mobile phase was 10:90 (v/v)
acetonitrile–0.05 M monosodium phosphate buffer (pH 3.2). Compounds: 1 ¼ neomangiferin,
2 ¼ mangiferin
Table 3. Mangiferin and neomangiferin content (mg kg)1) of Anemarrhena asphodeloides rhizome
from different regions (n = 3)
Region Neomangiferin Mangiferin
Bozhou, Anhui 780 ± 8.3 810 ± 9.4
Pingdu, Shandong -* 420 ± 5.2
Jishan, Shanxi 30 ± 0.6 1120 ± 10.1
Jiyuan, Henan 90 ± 1.2 1120 ± 11.4
Xifeng, Ganshu 10 ± 0.3 1010 ± 9.7
Hohhot, Inner Mongolia 750 ± 8.4 720 ± 8.2
Harbing, Heilongjiang 30 ± 0.7 810 ± 7.6
*Not detected
636 Chromatographia 2005, 61, June (No. 11/12) Short Communication

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