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Institute of Chinese Materia Medica, China Academy of Chinese Medical Science, Beijing 100700, China
Department of Chinese Medicine, Nanyang Medical College, Nanyang, Henan 473000, China
a r t i c l e
i n f o
Article history:
Received 6 December 2014
Received in revised form 5 March 2015
Accepted 8 March 2015
Available online 17 March 2015
Keywords:
Descurainia sophia (L.)
High-performance liquid chromatography
with diode array detector ngerprints
Multivariate statistical analysis
Processing mechanism
Plantago depressa Willd
a b s t r a c t
The seeds of Descurainia sophia (L.) (short for DSS below), with a long history of medicinal utilization in
China, have attracted the attention of many Chinese medicine practitioners for the potent efcacy. In the
present study, the raw and processed DSS were differentiated by several chemometrics methods based
on HPLC ngerprints. Moreover, peaks which were mainly responsible for the differentiation between
raw and roasted DSS were found. Therefore, the method of the chromatographic ngerprints combined
with multivariate statistical analysis was effective and reasonable in orientating chemical constituents
which were mainly responsible for the differentiation between raw and roasted materials, thus shedding
light on illustrating the processing mechanism. Whats more, this method can also be applied in the
identication of authenticity.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Descurainia sophia (L.) Webb ex Prantl, an annual dicot belonging
to family Brassicaceae (Cruciferae), has been used in folk medicines
for the treatment of throat diseases, measles and smallpox in the
middle Asia [1,2]. In China, its seeds (DSS) have been extensively
exploited to relieve cough, prevent asthma, reduce edema and promote urination for thousands of years [3,4]. However, parallel to
most Chinese medicinal herbs, DSS have been encountering the
challenges of varietal complexity and counterfeit incorporation. In
view of the morphological specicity and high similarity of adulterants, it seems inaccessible that either morphological characteristics
or microscopic characteristics could be able to distinguish them
accurately and handily.
Currently, the chromatographic ngerprint technique plays an
important role in the quality control of Chinese medicinal herbs,
Abbreviations: DAD, diode array detector; DSS, the seeds of Descurainia sophia
(L.); HCA, hierarchical cluster analysis; PCA, principle components analysis; PLSDA, partial least squares discriminant analysis; PDS, the seeds of Plantago depressa
Willd.; RDSS, the roasted seeds of Descurainia sophia (L.); TCM, traditional Chinese
medicine.
Corresponding author at: Institute of Chinese Materia Medica, China Academy
of Chinese Medical Science, No. 16 Nanxiaojie, Dongzhimennei Ave., Beijing 100700,
China. Tel.: +86 10 64033301; fax: +86 10 64033301.
E-mail address: wangzhuju@sina.com (Z. Wang).
http://dx.doi.org/10.1016/j.jpba.2015.03.010
0731-7085/ 2015 Elsevier B.V. All rights reserved.
as principle components analysis (PCA), hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLS-DA).
According to the statistical results combined with the chromatographic ngerprints, peaks responsible for discrimination between
raw and processed DSS were found and how they change in the
course of processing was also analyzed.
As mentioned above, there are plenty of adulterants of DSS in
medicinal markets. Meanwhile, DSS are mixed with the seeds of
other medicinal herbs as a result of the huge price difference [19].
In this study, HPLC ngerprints from the seeds of Plantago depressa
Willd. (PDS) were also obtained to compare with that of DSS, and
HCA was also conducted to differentiate them. Moreover, in order
to explore the inuence on the quality of PDS caused by varying
adulterating degree, chromatographic ngerprints of mixtures of
different proportions of DSS and PDS were performed.
2. Materials and methods
2.1. Materials and reagents
21 batches of raw DSS were purchased from different Chinese
medicinal herbs suppliers in the Chinese herbal medicine market
located in Bozhou, Anhui Province (Samples 19) and Anguo, Hebei
Province (Samples 1021), and authenticated by professor Suiqing
Chen, Department of Pharmacognosy of Henan University of TCM.
Then, RDSS (Samples 2235) were produced by roasting raw DSS
(chosen randomly from 21 batches of raw samples) according to the
processing method described in Chinese Pharmacopeia. In addition, 10 batches of PDS (Samples 3645) were obtained from the
Chinese herbal medicine market in Anguo where these seeds have
been found occasionally mixed with DSS. After the careful identication and authentication of these 10 batches of samples based
on their difference in morphological and microscopic characteristics, it turned out that only samples 38, 44, 45 were unadulterated
while the remaining were more or less mingled with DSS. All the
samples were stored in a dry ambience at constant temperature to
minimize any changes through degradation, and all the raw and
roasted voucher specimens were deposited in institute of Chinese
Materia Medica, China Academy of Chinese Medical Science.
Phosphoric acid (guaranteed reagent) was purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetonitrile and methanol for HPLC analysis were supplied by
ThermoFisher Scientic Inc. (Shanghai, China). The water used
was ltered with the solvent lter (Tianjin Jinteng Experiment
Equipment Co., Ltd., Tianjing, China).
2.2. Sample preparation
The tested samples were crushed into powder with a pulverizer
for 1 min, and passed through a 65 m-mesh sieve. Each sample powder (1.0 g) was weighed accurately and reuxed in 20 mL
80% methanol solution for 30 min. The residue was sifted from the
extract, then this solution was ltered with a 0.45 m microporous
membrane, and a 5 L aliquot of the ltrate was injected for HPLC
analysis. The extract solutions of all the samples were summited
for HPLC analysis within 24 h after preparation to maintain the
samples stability.
2.3. Chromatographic conditions
All HPLC analyses were performed with a Dionex U-3000
series (Shanghai, China) equipped with a SR-3000 Solvent Rack,
a LPG-3400SDN Quaternary Pump, a WPS-3000SL Auto sampler,
an injector with a 100 L loop, a TCC-3000RS Column compartment, a DAD-3000RS detector and Chromeleon 7 chromatography
workstation. A Thermo Scientic Acclaim TM 120-C18 column
Fig. 1. The chromatographic ngerprints of DSS and PDS samples: (A) DSS; (B) RDSS; (C) PDS sample 43 and (D) PDS sample 45.
and 1%) were compared, and the data indicated that the optimal
condition was acetonitrile1% phosphoric acid aqueous solution.
3.3. Methodology validation
RRT and RPA of ten characteristic peaks were calculated for
the estimation of precision, repeatability and stability, and the
results were as follows: precisionthe relative standard deviations (RSD) of RRT and RPA were found not to exceed 0.41% and
2.69% respectively; repeatabilitybelow 0.19% and 2.77% respectively; and stabilityless than 0.27% and 4.04% respectively. Thus,
all results indicated that the quality of the studied samples and the
HPLC-DAD measurements were stable and under control.
Fig. 2. (A) HCA of DSS and RDSS samples and (B) HCA of DSS and PDS samples.
Fig. 3. PCA biplots PC1-PC2 (57.38% data variance explained) of DSS and RDSS samples (the red circles represent 52 variables; the blue circles stand for 35 samples).
(For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)
Fig. 4. (A) PLS-DA score scatter plot of DSS and RDSS samples; (B) PLS-DA loading scatter plot of DSS and RDSS samples; (C) VIP (variable importance for the project) plot of
PLS-DA of DSS and RDSS samples.
Fig. 5. Chromatographic ngerprints of PDS mixed with different percentages of DSS (S1: 100% PDS; S2: 10% DSS + 90% PDS; S3: 20% DSS + 80% PDS; S4: 30% DSS + 70% PDS;
S5: 40% DSS + 60% PDS; S6: 50% DSS + 50% PDS; S7: 60% DSS + 40% PDS; S8: 70% DSS + 30% PDS; S9: 80% DSS + 20% PDS; S10: 10% DSS + 90% PDS; S11: 100% DSS).
incorporated into PDS, the front peaks belonging to DSS have gradually become the main and strong peaks. Consequently, these
samples were likely to produce completely different medicinal
effects from those pure PDS when applied in clinic. As a result, it
is necessary to establish the chromatographic ngerprints of mixtures of different proportions of DSS and PDS. That will provide
varying adulterating degree for reference more intuitively and
accurately.
4. Conclusion
In summary, whether the unsupervised HCA and PCA or supervised PLS-DA, both proved to be satisfactory for matching and
discriminating the chromatographic ngerprints of DSS and RDSS.
The results of PCA and PLS-DA indicated that peaks 26, 24, 4, 25,
22, 32 played dominating roles. Obviously, many new components
emerged after roasting. The accurate orientation of these peaks
makes it possible to explain the dissimilarity of efcacy fundamentally, shining a spotlight for revealing processing mechanism.
Similarly, this method can also be applied in the identication of
authenticity, which can detect the divergence among every sample more effectively and precisely, making up for the subjectivity
of morphological identication and inconvenience of microscopic
identication. The developed method was demonstrated to be simple, sensitive and reproducible, and will be widely adapted to other
Chinese medicinal herbs.
Acknowledgments
The authors are grateful to professor Suiqing Chen for authentication of all the samples.
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