410 J. Sep. Sci.

2010, 33, 410–421

Yizeng Liang1 Review Article

Peishan Xie2
Footim Chau 3
Chromatographic fingerprinting and related
1
Research Center of
Modernization of Chinese
chemometric techniques for quality control
Medicines, Central South
University, Changsha,
of traditional Chinese medicines
P. R. China
2
Zhuhai Chromap Institute of Development of chromatographic fingerprint (CF) and related chemometric methods and
Herbal Medicine Research, their applications to quality control of traditional Chinese medicines (TCMs) were discus-
Zhuhai, P. R. China
3
Department of Applied Biology sed. CF is essentially a kind of quality control method for TCMs (or Chinese herbal
and Chemical Technology, The medicines). Also, it is a quality-relevant-data high-throughput and integral tool to explore
Hong Kong Polytechnic chemically the complexity of TCMs. With the help of chemometrics, some difficulties in
University, Hung Hom,
Kowloon, Hong Kong
evaluation and analysis of CFs, such as calculation of information content, peak alignment,
pattern analysis, deconvolution of overlapping peaks, etc. could be well solved. To further
explore TCMs synergic quality, intensive study of CF coupled with chemometrics will create
Received October 13, 2009 the possibility to achieve the aim to reveal the working mechanisms of TCMs and to further
Revised November 29, 2009 control and strengthen TCMs’ intrinsic quality in a comprehensive manner.
Accepted November 29, 2009

Keywords: Chemometric application / Quality control / Traditional Chinese

medicines / Trends of chromatographic fingerprint development
DOI 10.1002/jssc.200900653

1 Introduction compounds can then be evaluated to determine not only the

It is well known that the holistic system of traditional Chinese
medicine (TCM) is featured by the integrity of the ingredients
contained in the Chinese herbal medicines (HM). This creates
a challenge in establishing quality control standards for raw
materials and the standardization of finished herbal drugs
because no single component can contribute to the total
efficacy [1, 2]. This difficulty has been acknowledged in the
draft of a Strategic Plan for Regional Traditional Medicine of the
World Health Organization (WHO) [3].
It is necessary to develop a type of quality assessment
system that adequately meets the complex characteristics of
TCM. Chromatographic fingerprint (CF) analysis represents
a rational approach for the quality assessment of TCM. It
utilizes chromatographic techniques, CE, GC, HPLC,
HPTLC, etc. [4] to construct specific patterns for recognition
of multiple compounds in TCMs. The entire pattern of
Correspondence: Professor Yizeng Liang, Research Center of
Modernization of Chinese Medicines, Central South University,
Changsha 410083, P. R. China
E-mail: yizeng_liang@263.net
Fax: 186-731-8825637
absence or presence of desired markers or activities but the
complete set of ratios of all detectable analytes [5].
Furthermore, with the help of hyphenated techniques, such
as GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-NMR, CE-
DAD, CE-MS, HPLC-NMR, etc. combined with resolution
methods recently developed in chemometrics, database
searching, and structural elucidation techniques, the quali-
tative and quantitative analysis of the main compounds
detected in CFs for TCM became possible [6–10]. CF
analysis of herbal drugs not only represents a comprehen-
sive qualitative approach for the purpose of species
authentication, evaluation of quality, and ensuring the
consistency and stability of herbal drugs and their related
products but also forms a comprehensive chemical base for
further research on working mechanism of TCMs.
In this paper, some aspects concerning recently devel-
oped chemometric techniques for CF and quality control of
TCM will be reviewed. Several examples presented in this
paper elucidate that CF combined with chemometrics is a
rational method for quality control of TCMs. The relation-
ship between chemical information acquired from finger-
print and the efficacy of TCMs can be explored with the
assistance of chemometrics.

Abbreviations: AMWFA, alternative moving window factor

analysis; CA, Chinese Angelica; CF, chromatographic
fingerprint; CR, Cnidium rhizome; DD, Dangguibuxue
decoction; HM, herbal medicine; PCA, principal component
analysis; pCR, Pericarpium Citri Reticulatae; pCRV,
Pericarpium Citri Reticulatae Viride; OP, orthogonal
projection; PLS, partial least squares; SL, Szechwan
Lovage; sPCR, secured principal component regression;
TCM, traditional Chinese medicine
2 Integral expression of TCM and pattern
analysis of CFs
In general, CF constructs a relative complete picture of
chemical components in TCMs to detect the product’s
‘‘phytoequivalence’’ [11]. The CF should feature the funda-
mental attributions of ‘‘integrity’’ and ‘‘fuzziness’’ so as to

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J. Sep. Sci. 2010, 33, 410–421
chemically represent the TCM investigated [12–15]. Thus,
the CF technique has been extensively accepted for quality
control of TCMs and the HMs with synergic features [16].
2.1 Information features of CFs of TCM
If one wants to obtain an informative fingerprint of TCMs, a
good extracting method and a chromatogram with good
resolution are needed. Thus, optimization of the extraction
method and operation procedure is an important task.
Suppose that we have obtained some fingerprints, the next
step will be to evaluate the information contents of the CFs
of HMs reasonably and efficiently. From the point of view of
multivariate, the information content of a chromatogram
with lots of peaks might be calculated by means of various
approaches [17–21]. In fact, a CF of a TCM could be
regarded as a continuous signal determined by its chroma-
tographic shape. Based on information theory, the informa-
tion content (F) for CFs of TCMs could be defined [22],
that is
X ! xi " ! xi "
U¼" P log P ð1Þ
xi xi
where xi is the real chromatographic response of the
chemical components involved in the CF under study.
Here, the normalization of xi divided by their sum is to
make the chromatogram investigated be of probability
property. In this way, Eq. (1) is exactly the expression of
Shannon entropy, that is, the information content
of the chromatogram investigated. Noting that in
information theory, if and only if xi with unchangeable
variance is characterized by normal distribution can its
information content F reach maximum [23]. Under an ideal
situation, all the chromatographic peaks in a chromatogram
can be separated completely and each peak confined to a
narrow zone might correspond to a normal distribution
profile [24]. A CF with all of peaks just completely separated
should be featured by maximal information content.
However, in reality, some peaks in a complex fingerprint
are almost inevitably overlapped with its adjacent one; such
overlapped peaks will surely show non-Gaussian normal
distribution and therefore undoubtedly cause a loss of the
information content. The method uses the whole chromato-
gram with a simple normalization, thus it is not necessary
to identify the retention time, peak intensity, peak width,
peak area, and/or peak height for all the peaks identified.
The calculation burden is reduced significantly. Moreover,
the theoretic background of the method is simple and
reasonable. Figure 1 shows such a situation discussed
above.
2.2 Peak alignment of CFs by chemometrics
The analysis of complex CFs can be quite challenging. When
one deals with several CFs jointly with all data points
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 411
obtained, some types of variation sources are likely confused
from one chromatogram to another. One of the variations is
imposed by the retention time shifts. For instance, the
retention time shifts happened in HPLC are caused by
the variation sources which might be due to (i) the
degradation of the stationary phase, especially, the low
stability of silica and silica-based supports at high pH
values and the collapse of C18 bonded phase because
of a high content of water in polar mobile phase eluted;
(ii) minor changes in mobile phase composition caused
by temperature and pressure fluctuations, variations
in flow-rate, and gradient dispersion; (iii) some problems
involved in the detectors, for example, a wavelength
shift in the UV spectrometer, a spectroscopic intensity
variation and the misalignment of the monochromator;
(iv) the column overloading on account of the great
injected amount or some components with high concentra-
tion; (v) the possible interaction between analytes; (vi) other
unknown shifts in the instrument. These variations
can cause imperfect alignment of the data when comparing
runs. Being able to determine which peaks appear reprodu-
cibly across chromatograms is difficult if the profiles are
misaligned. Furthermore, misalignment in chromatographic
data is rarely a simple shift. Most often, there is extension
and compression throughout the chromatogram at varying
points. A simple example of peak alignment is shown in
Fig. 2. Thus, it is important to choose an alignment protocol
that will compare points throughout the chromatogram.
Pattern recognition depends on the ability to directly compare
uniform data files. If the files are not aligned, a pattern
recognition algorithm may fail to recognize consistent signals
simply because they are not found at the same location each
time.
During the past decades, several kinds of useful
chemometric approaches have been developed for peak
synchronization in chromatographic profiles [25–38]. Some
of them corrected the retention time shifts by making
internal standards added or marker peaks coinciding in all
chromatograms under study [25, 27–29]. In Refs. [26,
30–32], some objective functions on the correlation between
the target and sample chromatograms were optimized
and then the sample chromatographic profiles were
aligned automatically with the target. In general, the
methods must be very efficient and elegant if the samples
investigated are quite similar in the concentration profiles of
the chemical components. However, if the concentration
profiles change greatly for the complex samples such as
HMs from the different habitats and/or from the various
harvest seasons, wrong results might be obtained by simply
seeking the optimal correlation coefficient between the
chromatograms.
More recently, some new techniques were developed in
chemometrics in peak alignment [33–38]. Among them,
from the point of view of information usage, the methods
can be roughly classified into two kinds. Some were trying
to use the information from spectrum to correct the chro-
matographic shifts, since the spectral information will be
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412 Y. Liang et al. J. Sep. Sci. 2010, 33, 410–421

Figure 1. Elucidation of infor-

mation contents (F) of the
chromatographic fingerprints
with different separation situa-
tions (A) and with different
extraction conditions (B). Plot
(A) a: 20 completely separated
peaks of information content 5
8.5334; b: 20 baseline sepa-
rated peaks of information
content 5 8.5334 (oversepara-
tion happened in plot a does
not increase the information
content of the fingerprint); c:
20 peaks with eight peaks over-
lapped of information content
equal to 8.3740 (information
content decreasing because of
peak-overlapping); d: 20 peaks
with 11 peaks overlapped of
information content equal to
8.2948 (information content
decreasing because of severe
peak-overlapping). Plot (B) a:
20 completely separated peaks
with rather uniform distribu-
tion of peak magnitudes of
information content 5 8.5333;
b: 20 completely separated
peaks with only few peaks
rather big of information
content 5 8.3989 (information
content decreasing because of
asymmetry distribution of
peak magnitudes); c: 25 almost
separated peaks of information
content 5 8.8085 (information
content increasing because of
more extracted compounds);
d: 16 almost separated peaks
of information content 5
8.1524 (information content
decreasing because of few
extracted compounds).

very useful for featuring the chromatographic peaks [37–38].
The other kind of methods were trying to fully use the
feature embedded in chromatograms with the help of
statistic techniques, such as analysis of variance feature
selection, Kalman tracking, and principal component
analysis (PCA) [33–35].
After such a correction of the chromatographic shifts of
the CFs resulted from HPLC, GC, CE, etc., the identity,
stability, and consistency of TCMs as well as the differ-
entiation of adulterants could then be done by similarity
analysis and chemical pattern recognition developed in
chemometrics.
2.3 Quality control of TCM based on pattern analysis
of CFs
By definition, a CF of a TCM, in practice, is a chromato-
graphic pattern of the extract of some common chemical
components of pharmacological activity or chemical char-
acteristics [39–41]. Several works using the CFs obtained by
HPLC or GC for quality control of HMs have been reported
with the help of similarity analysis and chemical pattern
recognition [42–50].
To identify Chinese Angelica (CA) from related herbs,
such as Japanese Angelicae Root (Angelica radix, JA), Szech-

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 410–421
Figure 2. Fingerprint 1 and fingerprint 2 before (A) and after (B)
correcting the retention time shifts by chemometric alignment.
wan Lovage Rhizome (Rhizoma chuanxiong, SL), and
Cnidium Rhizome (Cnidii rhizoma, CR), a HPLC fingerprint
of CA was developed based on the consistent chromato-
grams of 40 CA samples (Angelica sinensis (Oliv.) Diels). The
amount of senkyunolide A in CA was less than 30-fold of
that in SL and CR samples, which was used as a chemical
marker to distinguish them. Japanese Angelicae was easily
distinguished from CA, SL, and CR based on either chro-
matographic patterns or the amount of coniferyl ferulate
(see Fig. 3 for more details). No obvious differences exist
between SL and CR chromatograms except the relative
amount of some compounds, suggesting that SL and CR
might have very close relationship in terms of chemotax-
onomy [42].
Houttuynia cordata Thunb. is a commonly used Chinese
materia medica in either fresh herb or sun-dried one. A
comparative study by a modified GC-MS method coupled
with pattern recognition could effectively distinguish the
fresh herbs from the dry ones, even differentiate stems and
leaves [43]. A HPLC chromatographic pattern matching
successfully described the difference of whole chromato-
grams of raw and steamed roots of Panax notoginseng
quantitatively [44]. HPLC fingerprints of Pericarpium Citri
Reticulatae (pCR) and Pericarpium Citri Reticulatae Viride
(pCRV) were measured for deliberately collected 39
authentic samples and 21 commercial samples. The PCA
successfully distinguished the ‘‘mixed peels of Citrus’’
samples from authentic samples, partial least squares (PLS)-
linear discriminant analysis was then effectively applied to
class separation between authentic pCR and pCRV with
correct discriminating rate of 93.02% and prediction ability
of 86.67%, respectively [45].
The CF could also be used to address the quality stability
and consistency of the compound formula of TCM products.
A selective assessment was developed for monitoring the
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 413
manufacturing processes of a Chinese herbal preparation,
Qingfu Guanjieshu (QFGJS) capsule for assessing its
stability over time [46]. In fingerprinting analysis, the
chemical characteristics of four herbs in QFGJS (excluding
Radix Aconiti Lateralis Preparata) were present in the HPLC
chromatographic profile. The chemometric analysis results
showed that three batches of QFGJS products collected at 3
months intervals after production in the stability testing
were consistent [46]. Another example is Qingkailing (QKL)
injection. 2-D fingerprint combined with PCA classified
various samples from different manufacturers to solve the
quality survey of commercial QKL injections in the market
according to the fluctuation of their fingerprint profiles [47].
Also, the lot-to-lot consistency and inferior products of
Danshen Dropping Pill, which consisted of three Chinese
materia medica (Danshen, Sanqi, and borneol) can be
clearly determined by 2-D fingerprint with PCA and simi-
larity measurement [48].
The fundamental roles of fingerprinting analysis
aided by a pattern recognition software interface named
computer aided similarity evaluation have been brought
under four aspects in the work conducted by Xie’s team:
(i) species authentication (ginseng and its congeners);
(ii) batch-to-batch quality consistency TGP extract products);
(iii) in-process quality monitoring (tablets made from
immature fruit of Terminalia chebula); and (iv) differentiat-
ing adulterants from the true product (Ginkgo leaves extract
products) [49]. These facts suggested that CF is a rational
approach for assessing the problem of quality control of
TCMs.
To enhance the discriminating ability of pattern recog-
nition, some novel methods for better pattern analysis
were also developed. To deal with variable selection in
discriminating HMs, a chemometric approach concerning
forward selection and key set factor analysis using PCA was
developed. A case study for 79 herbal samples showed that,
the methods of forward selection were preferable to
determine the representative variables [51]. Secured princi-
pal component regression (sPCR) [52, 53] was originally
developed to detect and characterize the uncalibrated
spectral features. However, when the investigated objects
are diverse CFs in compositional distribution, it is not
friendly to use and the results are also not satisfactory. The
method called modified sPCR allows one to avoid the
above effects as much as possible [54]. The successful
application of modified sPCR to two real HMs of
Erigeron breviscapus from different geographical origins
and Ginkgo biloba from various sources or vendors
demonstrates that the method can detect reasonably unex-
pected features differing from the regulars or not being
modeled. Also, orthogonal projection (OP) technique was
applied to discriminant analysis of the HM Houttuynia
cordata Thunb. (HCT) and its final injection products. The
advantages of the OP technique were shown after compar-
ing with the conventional methods such as Mahalanobis
distance, and similarity comparison method. Three different
sources of medicinal material Houttuynia cordata Thunb.
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414 Y. Liang et al. J. Sep. Sci. 2010, 33, 410–421

Figure 3. HPLC chromatograms measured at UV 280 nm of (A) 40 Chinese Angelica samples; (B) four Japanese Angelicae Root samples;
(C) six Szechwan Lovage Rhizome samples; and (D) three Cnidium Rhizome sample. The peaks marked by numbers represent ferulic acid
(1), coniferyl ferulate (4), senkyunolide A (5), and Z-ligustilide (9), respectively.

Table 1. Total classification errors and incorrect rates of the test sets for ten times of calculations of the final injection products (letters A,
B, C, D, E and F denote different manufactures, respectively)

Different cross-validation procedure Total classification errors and incorrect rates

Mahalanobis distance Incorrect rates Similarity method Incorrect rates OP technique Incorrect rates

Total errors Total errors Total errors

A B C D E F A B C D E F A B C D E F

6-fold 3 5 3 3 4 5 19.2% 0 0 3 2 4 2 9.2% 0 0 0 0 0 0 0.0%

3-fold 7 9 5 6 9 10 21.9% 1 3 4 3 6 4 10.0% 0 0 0 1 1 0 0.95%
3-fold-bisa 16 20 19 18 23 21 27.2% 5 7 6 10 12 9 11.4% 0 0 1 2 4 3 2.3%

and its final injection products from six different manu- determination and identification of unknown samples
facturers were studied. The results are summarized shows it could be a powerful tool for quality control in HM
in Table 1. The good performance of OP technique in production [55].

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J. Sep. Sci. 2010, 33, 410–421
3 Further qualitative and quantitative
analysis of TCM fingerprints with the
help of hyphenated chromatographic
techniques and chemometrics
To understand bioactivities and possible side effects of active
compounds and to enhance quality control of TCMs, it
seems necessary for one to determine most of the
phytochemical constituents of herbal products. With the
development of analytical instruments, especially with the
development of the hyphenated chromatographic instru-
ments, such as GC-MS, HPLC-DAD, HPLC-DAD-MSn,
HPLC-NMR, CE-DAD, and CE-MS, the qualitative and
quantitative ability has been enhanced significantly, since
such instruments provide not only their separation ability
but also their qualitative ability with spectroscopic profiles.
It builds the chemical foundation for explanation of the
mechanism of pharmacological activity. Just like the
situation in metabolomics, as pointed out by Van de Greef,
‘‘the comprehensive identification of metabolites remains a
key challenge’’ [56]. Based on the CFs obtained, compre-
hensive identification of secondary metabolites of the TCMs
should be very momentous, and the assistance of chemo-
metric measurement is also indispensable.
3.1 Chromatogram/spectrum deconvolution of TCM
fingerprints by multivariate resolution
In general, the data generated by hyphenated instruments
are matrices with every row being a spectrum and every
column a chromatogram at some wavelength, wave number
or m/e unit. In common, the size of the data matrix such-
obtained is rather big. However, the matrix data obtained by
hyphenated chromatography has so-called dimension
advantages proposed by Booksh and Kowalski [57], which
will make resolution of overlapping chromatographic peaks
possible. Furthermore, the hyphenated technique might
enhance the chromatographic separation ability by the
additional spectral information, since one could easily find
some useful component selectivity with the help of
chemometric local rank analysis methods [58].
Chromatography is a powerful tool for analyzing
complicated system as TCM. However, as pointed out by
Giddings, relative to the maximum peak content or peak
capacity for closely spaced peaks, a random chromatogram
will never contain more than about 37% of its potential
peaks. Thus, the number of observed peaks is not the same
as the number of distinct chemical components [59]. On the
other hand, a Chinese herbal formula for treating the
common cold contains hundreds of chemical components,
and it seems impossible to get a baseline separation with
hundreds of analytes. As a result, identification of the purity
of a target peak cluster followed by resolution into pure
chromatograms and/or spectra is of great significance for
complex systems like TCMs. Fortunately, the recently
developed chemometrics resolution methods provide
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 415
powerful tools to accomplish this job [60–69]. With the help
of chemometric resolution methods, the qualitative and
relatively quantitative analysis for some CFs of volatile
components in TCMs obtained from GC-MS were also
reported [6, 7, 70–72]. When the boiling points of
compounds are close to each other, co-elution of two or
more different compounds is very possible although the
chromatographic conditions are optimized. In such a case,
by direct similarity searches in MS database alone is hard to
identify the compounds, which will even result in wrong
conclusions. Therefore, it is extremely necessary to resolve
the overlapping peaks by means of chemometric techni-
ques. With the help of chemometric resolution methods,
such as heuristic evolving latent projections and subwindow
factor analysis, determination of volatile components in
Cortex Cinnamomi [6], Notoptergium incium [71], Artemisia
capillaris [72], and peptic powder [7] were conducted by GC-
MS. Tentative identifications were performed by comparing
the retention time and mass spectra of samples with stan-
dards or/and earlier publications.
3.2 Comparing analysis between CFs with the help
of chemometrics
Over the last decades, many chemometric techniques
[73–84] have been successfully applied to solve the problems
arising from metabolic analysis, qualitative and quantitative
analysis of HMs, and others [77–82]. Also, SIM technique
was developed for the analysis of complex chromatographic
systems [83–84]. But the goals of identification and
quantification of all the chemical components in a complex
system are far from satisfactory [85].
Fortunately, in some cases, it is not necessary for the
researchers to resolve all the ingredients in complex systems,
but only to identify parts of them or find the difference and
similarities of components between two data of target
samples for purpose of quality control of TCMs. It is obvious
that the integrated investigation must be time-saving and
effective for obtaining the valuable information. In the
relationship between the two CFs, five possible relationships
of the chemical components between two different and
complex systems are firstly illustrated in Fig. 4. To analyze
the five possible relationships, a novel chemometric method
was developed. The method, named alternative moving
window factor analysis (AMWFA), could utilize the cross-
information hidden in the two systems to determine the
number of common components between them and even to
identify their corresponding spectra half-automatically. The
selective information in matrices X and/or Y can alter-
natively be employed for the identification of their common
components. Using component information in one system
to deduce the spectrum in the other related system is the
main advantage of AMWFA, since the same component
should have the same spectrum even if they exist in different
fingerprints. When no or only weak selective information
exists in them, strategy of sub-matrices extraction from data
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416 Y. Liang et al.
Figure 4. Schematic illustration of the five possible relationships
of chemical components contained in two different complex
chromatographic systems X and Y. The overstriking maculae in
X and Y denote different components.
X and/or Y can be used for identification with moving
window technique. From the results of comparative analysis
of volatile chemical components in ‘‘TCM drugs pair’’
rhizoma ligustici chuanxiong-radix paeoniae rubra and the
involved two single HMs and Angelica oral solution and its
plasma sample after oral intake to rabbit, powerful ability of
the method is shown [86]. Also, with the help of AMWFA,
the volatile components between stems and roots and also
among five Clematis species from China were studied and
analyzed by GC-MS. Tentative identification of the
compounds was assisted by comparison of temperature-
programmed retention indices with authentic samples [87].
Also, the similarities and differences of essential oil
components in pCRV and pCR were investigated by GC-MS
combined with the help of AMWFA. The essential oils from
pCRV and pCR significantly differed both qualitatively and
quantitatively. The main compound in the essential oils
from pCRV and pCR was D-limonene accounting for
65–83%. Essential oils from four samples of Rhododendron
were analyzed by GC-MS. A total of 128 volatile components
were identified temporarily with the help of retention indices
and chemometrics resolution method [88]. A compound
medicine of Dangguibuxuetang and its two involved single
medicines of Danggui (Radix Angelica sinensis) and Huangqi
(Radix Astragali) were compared for their relevant constitu-
ents. It was found that of twenty-one high peaks in Dang-
guibuxuetang, eight originate from Radix Angelica sinensis six
from Radix Astragali, and seven from both Radix Angelica
sinensis and Radix Astragali. Some new chemical ingredients
appeared in fingerprints of Dangguibuxuetang, and some
existing in those of single medicines contrarily disappeared
after the compatibility. It suggested that there might be
certain interactions of the chemical ingredients in
compound medicine besides their total sum effect of single
medicines [89].
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J. Sep. Sci. 2010, 33, 410–421
It is worth noting that the comparing analysis of CFs
could be used for tracing metabolic situation of TCMs. From
the point of view of exploring the mechanism about why
TCM works, this kind of research might be of great
importance. If one could trace the metabolic change of the
TCMs when they are taken into the body of animal/human
being, it will be very helpful for screening of active
component and/or the pharmacokinetics research of TCM.
Based on the metabolic fingerprinting technique and LC/
DAD-MS, a rapid screening and analysis of the multiple
absorbed bioactive components and metabolites of an oral
solution of Danggui and the Dangguibuxue decoction (DD)
in rabbit plasma after oral administration were developed
[90–92]. The results obtained from a comprehensive
comparative analysis of the fingerprints of the DD and its
metabolic fingerprints in rabbit plasma indicated that 46
components in the DD were absorbed into the rabbit’s body.
Of them, ten components were tentatively identified from
their MS and UV spectra and retention behaviors by
comparing the results with the reported literature. In addi-
tion, 21 components were only found in the metabolic
fingerprints, which suggested that they might be metabo-
lites of some components in the DD. This might be, in our
opinion, important not only for the pharmaceutical discov-
ery process and the quality control of crude drugs, but also
to be a helpful aid in getting to know the curative
mechanism of TCMs.
4 Exploration of relationship between
bioactivity and composition of TCM and
validation of their bioactive modes with
the help of chemometrics
With the chemical composition of TCMs at hand, the
exploration of relationship between the CFs and efficacy of
the HMs seems thus to be the important aspect for the
quality control of HMs. However, how to evaluate reason-
ably their relationship is obviously not a trivial task.
Recently, some work has been done along this research
direction with help of chemometrics [93–98].
The quantitative relationship between chemical
composition and decreasing cholesterol effect of Qi-Xue-
Bing-Zhi-Fang, a widely used HM in China, was investi-
gated. Quantitative composition–activity relationship
models generated by multiple linear regression, artificial
neural networks, and support vector regression exhibited
different capabilities of predictive accuracy. Moreover, the
proportion of two active components of Qi-Xue-Bing-
Zhi-Fang was optimized based on the quantitative compo-
sition–activity relationship model to obtain new formula-
tion. Validation experiments showed that the optimized HM
has greater activity [97]. Many herbal products are claimed to
have healing or protective effects, though often these activ-
ities are neither evaluated nor quantified. For instance, it
was shown that green tea exhibits protective effects against
several cancers introduced by chemical carcinogens as well
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J. Sep. Sci. 2010, 33, 410–421
as against atherosclerosis and coronary heart diseases [98].
These positive actions of green tea are related to its anti-
oxidant capacity and therefore it was tried to predict the total
antioxidant capacity of green tea from CFs by linear multi-
variate calibration techniques [99]. The predicted antioxidant
capacities can then be a measure for the protective effects
and for the quality of the tea. However, for chromatographic
data, e.g. fingerprints, only few multivariate calibration
applications are described. Recently, Dumarey et al. explored
linear multivariate calibration techniques to predict the total
antioxidant capacity of green tea from CFs. Also, van
Nederkassel et al. [95] predicted the total antioxidant capacity
of green tea from their CFs by the use of PLS [99, 100] and
uninformative variable elimination PLS [101]. More recent-
ly, using whole chromatographic profiles and measure-
ments of total bioactivity as input, a quantitative
pattern–activity relationship approach [102] was proposed as
a general method for providing two pieces of crucial infor-
mation about complex bioactive mixtures available: (i) a
model for predicting total bioactivity from the CF and (ii) the
features in the chromatographic profile responsible for the
bioactivity. The targeted approach makes information about
bioactivity available at the molecular level and provides
possibilities for assessment of HM possible beyond just
authentication and total bioactivity. As an example, the
antioxidant property of the HM Radix Puerariae lobatae was
measured through its reducing power toward a ferric ion
complex. A PLS model was created to predict the antioxidant
activity from the CF. Using the antioxidant activity as a
target, the most discriminatory projection in the multi-
variate space spanned by the chromatographic profiles was
revealed.
On the other hand, the mechanism of TCMs is usually
unknown, which also poses challenges to the pharmaceutical
and agrochemical industries [103–105]. Metabolic profile is a
very sensitive indicator of environmental influences and might
be used to detect and analyze changes of the total metabolic
state of microbe due to pathophysiological stimuli [106] [http://
www.touchbriefings.com/download.cfm?FileId 5 2386], which
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 417
makes it an attractive candidate for mode of action studies
[107]. Metabolic profile is being used to evaluate the pharma-
cological mechanism of the drugs or drug candidates. The
method is particularly useful when a moderate number of
different outcomes (e.g. modes-of-action, disease states) can be
pre-defined [108].
Using metabolomic profile and nine antibacterial
substances with known modes of action, the antibacterial
mode of berberine on Staphylococcus aureus was recently
investigated [109]. The working procedure is elucidated
briefly in Fig. 5. With the help of HPLC/ESI-MS, metabolic
profiles of S. aureus treated by berberine and nine anti-
bacterial substances with known modes of action (see
Table 2) were firstly acquired. After data pretreatment, those
profiles acquired were reduced into several MS vectors
containing 900 m/z values. Then, PCA was carried out upon
those metabolic profiles in order to classify those drugs
according to their mechanisms. From the result obtained by
PCA, the possible antibacterial mode of berberine was
explored. Also the antimicrobial roles of dihy-
drocucurbitacin F-25-O-acetate, one of the major compo-
nents in Hemsleya pengxianensis, on S. aureus were also
explored with the similar approach [110]. The result
obtained showed the mechanism may be to inhibit cell wall
synthesis.
Another example of score plot from PCA (the variance
of the first two PC being 89.5% of total variance of the data)
for exploring the antibacterial mode of TCM Aquilegia
oxysepala is shown in Fig. 6. From Fig. 6, one could see
clearly that 90 samples treated with different drugs and
controls are well-separated. Cefataxime, whose target is on
transpeptidases and carboxpeptidases, formed a distinct
cluster separated from the other antibiotics based on its
different mode of action. Acheomycin, lincolmensin,
erythromycin, chloromycetin, and streptomycin cluster
together. As known from Table 2, lincolmensin, erythro-
mycin, and chloromycetin have effects on 50S ribosomal
subunit; streptomycin and acheomycin act on 30S riboso-
mal subunit. In a word, the mode of action of those five
Figure 5. Working procedure
of validation of antibacterial
mode of traditional Chinese
medicine by metabolomics
and PCA.
www.jss-journal.com
418 Y. Liang et al. J. Sep. Sci. 2010, 33, 410–421

This may imply that the target of maguoflorine and A.

oxysepala on S. aureus is possibly similar to that of lincol-
mensin, erythromycin, chloromycetin, streptomycin, and
acheomycin, whose targets are protein. With almost the
same idea, several other HMs and their corresponding
active components were also investigated [118–120].

5 Concluding remarks

The parameters relevant to the quality of Chinese HMs

Figure 6. PCA projection of metabolic profile of controls and
cultures treated with Aquilegia oxysepala, genkwanin, apigenin,
maguoflorine, berberine, and nine antibiotics. Controls (&),
acheomycin (&), lincolmensin ( % ), erythromycin (m), chloro-
mycetin (&), streptomycin (&), cefataxime (v), vancomycin (&),
rifampicin ( & ), norfloxacin (&), A. oxysepala (&), genkwanin
(J), maguoflorine (&), apigenin (x), and berberine (v).
Table 2. Modes of actions of selected known antibiotics
acquired from CF are certainly a large dataset, which can
only be processed sufficiently and systematically through
chemometric calculation. It can explore deeply the inte-
grated quality information in whole to make the empirical
evidence logical, particularly when the compound data
derived from macro amount of samples collected are
subjected to a series analysis by means of chromatography
hyphenated with various detective measurements. Further-
more, the quality information serves as a ‘‘quality databank’’
in order to incorporate the phytochemical information with
biological activity of the herbals together so as to seek the
relationship between the CF and the synergic efficacy of the
TCMs [121–123]. Basically, chemical fingerprint analysis
aided by chemometrics can ensure the maintenance of the
consistent quality of samples for biological and pharmaco-
logical research.

Drug/class Function inhibited Molecular target This work is financially supported by the National Nature
Foundation Committee of P. R. China (Grant No. 20875104)
Chloromycetin Protein synthesis 50S ribosomal subunit and the international cooperation project on traditional Chinese
Streptomycin Protein synthesis 30S ribosomal subunit medicines of Ministry of Science and Technology of China
Acheomycin Protein synthesis 30S ribosomal subunit (2007DFA40680).
Erythromycin Protein synthesis 50S ribosomal subunit
Lincolmensin Protein synthesis 50S ribosomal subunit
Norfloxacin DNA replication/ Gyrase and topoisomerase IV The authors have declared no conflict of interest.
transcription
Rifampicin Transcription RNA polymerase
Cefataxime Peptidoglycan Transpeptidases and
synthesis carboxypeptidases
Vancomycin Peptidoglycan Cell wall peptidoglycan
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