Chinese Medicine, 2013, 4, 125-136
Published Online December 2013 (http://www.scirp.org/journal/cm)
Open Access CM
The Prospect of Application of Extractive Reference
Substance of Chinese Herbal Medicines
Peishan Xie1,2*, Shuangcheng Ma3*, Pengfei Tu4, Zhengtao Wang5, Erich Stoeger6, Daniel Bensky7
1Macau University of Science and Technology, Macau, China
2Guangdong UNION Biochemical Development Co. Ltd., Guangzhou, China
3National Institute for Food and Drug Control, Beijing, China
4Peking University Modern Research Center for Traditional Chinese Medicine, Beijing, China
5Shanghai University of Traditional Chinese Medicine, Shanghai, China
6Plantasia GmbH, Oberndorf, Austria
7Independent Scholar, Seattle, USA
Email: *firstname.lastname@example.org, email@example.com, *firstname.lastname@example.org
Received September 21, 2013; revised October 26, 2013; accepted November 9, 2013
Copyright © 2013 Peishan Xie et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The emerging development of Extractive Reference Sub stance (ERS) is a methodology that meets the needs for quality
control for Chinese Herbal Medicines (CHM) and respects the holistic viewpoint of Traditional Chinese Medicine
(TCM) and its clinical use of multiple ingredients with synergistic effects. The convention of using just a selected few
Chemical Reference Substances (CRS) cannot adequ ately assess the quality of intact CHM. A validated chemical spec-
trum of an ERS provides the global characteristics in order to more specifically identify and assess targeted CHM. This
paper describes the fundamental concepts, potential significance, and basic criteria of ERS, along with methods of
preparation and calibration. Given the diversity of CHM, the various problems that will occur in establishing the proper
process of ERS will need to be so lved in a step by step manner. The ERSs of Ziziphi spinosae semen and ERS of Frit-
illaria thunbergii bulbus are given as examples of the development of ERS and demonstrate why we are optimistic
about the utility o f this approach.
Keywords: Extractive Reference Substance (ERS); Chinese Herbal Medicine (CHM); ERS R & D Strategy;
Holistic Quality Control
The “reference substances” are indispensable substances
for assessing the quality of Chinese herbal medicines
(CHM) and their products. Currently, th ere are two kinds
of available reference substances applied to Pharmaco-
poeia of People’s Repub lic of China (ChP)—Herbal Ref-
erence Substance (HRS) and Chemical Reference Sub-
stance (CRS). HRS is used for microscopic and thin-
layer chromatographic identification, while CRS is used
for chromatographic identification and quantitative de-
termination. Decades of practical experience have dem-
onstrated the positive roles of HRS and CRS in routine
CHM quality control; however some drawbacks have
also become clear. While HRS is unequivocally neces-
sary for microscopic identification, it is unsatisfactory for
chemical identification (e.g., TLC identification) because
of the fluctuation of the chemical composition between
different individual substances. As for CRS, in addition
to its merits for the attribution of the corresponding che-
mical compounds distributed in the herbal drugs, it acts
as an external standard for assay of the target component
. The primary issues are limited varieties, less speci-
ficity for holistic identification of the complex composi-
tion of CHM, and expense and waste of natural resources.
Furthermore, as widely argued, any arbitrarily selected
CRS (chemical marker) is almost irrelevant to the syner-
gic efficacy of an individual herbal drug, much less com-
plex formulated herbal products [2-5]. The high cost and
enormous waste of resources for obtaining a single pure
CRS cannot be ignored. To make one gram of the pure
chemical extract often requires dozens of kilograms of
the herbal drug and an enormous amount of organic sol-
vents. The final products of CRS are so costly that it is
prohibitively expensive for testing some low-cost herbal
P. S. XIE ET AL.
drugs. The higher purity is needed, the lower yield will
be obtained, and the more expensive it will be for ob-
taining a single pure CRS. Moreover, the real signify-
cance of a few arbitrarily selected CRS for quality con-
trol of a multi-ingredient CHM is questionable at best.
There is a consensus at present that the conventional QC
approach must be changed. Recently, simultaneous de-
termination of multiple components by HPLC has made
rapid progress due to the increasingly sophisticated chro-
matography and MSn detection technologies, and much
more information relevant to the quality has been found
[6-9]. However, the aforementioned principle problems
on routine quality control still remain. On the other hand,
the growing QC requirement of herbal drugs is challeng-
ing the drawbacks of CRS. So the adoption of the limited
extractive reference substances (ERS) (“powdered ex-
tract” in USP) by the United States Pharmacopoeia (USP)
aims to coordinate expediently with the QC requirement
of the corresponding Dietary supplements in the USP.
The ChP has now initiated the ERS program as a candi-
date of reference substances in the upcoming editio n. So-
me researchers call the ERS as “Substitute referen ce sub-
stance” . From the perspective of researchers, the use
of ERS is not just an alternative reference substance, but
an advanced approach for meaningful and comprehen-
sive quality control to match the synergistic traits of
CHM. As the secondary metabolites in the herbals, the
bioactive ingredients compose the chemical pattern play-
ing the role of fingerprint for iden tifying each taxonomic
plant species; hence regardless of what isolated chemical
markers are chosen as the targets for analysis, they will
likely not be relevant to the synergistic mechanisms in-
volved in the holistic approaches of TCM [11,12]. A full-
view of a chemical profile of an ERS of the given CHM
through chromatographic separation may sufficiently
provide the evidence to assess the samples qualitatively
and semi-quantitatively (see below). Therefore, ERS can
be developed as a methodology for comprehensively
oriented QC reference substances. To have the ERS of
CHM mature into a fully formed method of quality con-
trol and identification, the criteria for developing the
ERS of CHM need to be developed in a methodical
manner. This paper presents the proposal and method-
ologies for how this can come to fruition.
2. Prerequisites for Establishment of ERS of
Since the 1960s, the concept and the practice of quality
control of Chinese herbal medicines (CHM) in the
Chinese Pharmacopoeia (ChP) has followed the model of
European herbal drugs in western pharmacopoeias like
the British Pharmacopoeia (BP). In the 1960’s, there
were no other examples on how to formulate a rea-
sonable quality standard for herbal medicines that could
serve as a precedent. While at that time the only feasible
way was to emulate the quality standard monograph of
chemical medicines, this concept an d strategy for quality
control was doomed as it was rooted in the reductionist
mindset of a single-compound-oriented analysis. Com-
mon sense tells us no one chemical ingredient can be
responsible for the synergic efficacy of a herbal drug.
One of the traits of CHM is of diverse curative effects in
the context of the composition of the various formulas.
This makes it impossible to pinpoint a single specific
bioactive molecule as being responsible for the efficacy
of a given CHM. In recent years, along with the rapid
development of multivariable analytical technologies,
facilities and algorithms, the publication on simultaneou s
determination of multiple ingredients in CHM has ex-
ploded. The new techniques remain very expensive and
will tend to exhaust the expensive and scarce chemical
references substances without any more ability to truly
appreciate and control the inherent quality of CHM, par-
ticularly if theses approach come into effect in routine
QC. Such a prospect leads to anxiety in the herbal medi-
cine industries although such kind of research would be
welcomed in academic circles. Other open-minded herbal
analysts have considered how to develop appropriate
strategies that utilize multivariable analysis pragmaticall y
in a holistic manner. It is well known that specific chemi-
cal patterns in a plant can be revealed by chromato-
graphic analysis. The acquired chromatographic profile
represents the unique character for the given species
which is called “chromatographic fingerprint”. The chro-
matographic fingerprint generated from authentic species
sample can be recognized as the criterion for identifi-
cation of the target species. Once the criterion is establi-
shed, the chromatographic fingerprint of a standardized
Extractive of the given species is being able to act as the
Extractive Reference Substance (ERS). To keep the full-
view of the fingerprint (peak numbers, peak-peak ratio,
integrated peaks area of the column chromatography
(HPLC, GC etc.) or the total color image of the Planar
chromatography (HPTLC) as a whole is very effective
for chemical identification and assessment of the inherent
quality, particularly at the stage we are at present with a
lack of sufficient knowledge about the chemical bioacti-
vity of CHM. We cannot assume at present which peaks
in the fingerprint are indispensable, which are comple-
mentary and which are inessential and can be ignored in
most cases when they only exist in trace amounts. More-
over, as the understanding of how to use CHM is the
result of experience accumulated over thousands of years,
the holistic approach of traditional Chinese Medicine
(TCM) must be respected. Given this, it is impossible to
pinpoint any single molecule for being responsible for
the diverse effects of a complex CHM formula, the prin-
ciple way in which CHM is used TCM practitioners. Un-
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P. S. XIE ET AL. 127
derstanding the fundamental characteristics of CHM is
the cornerstone of development of ERS of CHM.
3. ERS—The Brand—New Reference
Substance in Holism Manner for
Assessing the Quality of CHM
Unlike HRS and CRS, the ERS of CHM has not yet
achieved the status of a standard in spite of such limited
adoption by the USP and ChP. In part, this is due to the
suspicion that it is not yet truly feasible to be widely im-
plemented. This is a normal concern at the early stages of
development of any technique. Th ere will be a process of
trial and error until the method has been validated and
shown to be reproducible. It will not become the panacea
protocol for QA/QC of CHM. But we should take an
optimistic attitude toward its research and development
potential, as it will bring a new approach to the field of
CHM’s QC/QA: a chromatographic fingerprint as a ref-
erence that makes comprehensive quality control possi-
ble. The majority of the TCM manufacturers have always
manufactured Chinese proprietary products using crude
drugs as the starting materials. Given this, inconsisten-
cies of the quality of the final products can be predicted.
Over the last decade, some new emerging Chinese medi-
cine industries have adopted new technologies and are
instead using the herbal drug extracts as the starting ma-
terials. That is certainly a good beginning to improve
QC/QA. Meeting the need for effective qu ality control of
new products, ERS is also a powerful reference sub-
stance for the in-p rocess QC. ChP get ready to launch the
program of ERS of herbal medicines for upcoming edi-
tion of the Pharmacopoeia, which will undoubtedly rap-
idly drive forward the development of ERS.
The adva ntages of the ERS of CHM include:
• It can reflect the total detectable chemical characteris-
tics of the herbal drug from a holistic view.
• It can accordingly ensure the consistent distribution
of chemical ingredients batch-to-batch of the CHM
• The chemically attributed components in the ERS can
easily be used as an available alternative for some
known CRS for identification, while the intact che-
mical profile can provide a much more detailed qual-
• The integration value of the ERS can be used as a
simple semi-quantitative analysis for quick reference.
• It is a cost/effective reference substances in quality
control of CHM
4. Basic Requirements of ERS
Any kind of reference substance must meet the four basic
universal requirements: Authenticity, Specificity, Con-
sistency and Stability (ASCS). Focusing on the ERS, the
primary requirements would be defined as follows: 1)
The chromatographic fingerprint could reflect that of the
original herbal drug, for example, the TLC image (fin-
gerprint) of ERS basically conforms to that of the origin-
nal drug. This fullfils the requirements for authenticity
and specificity; 2) The physical appearance of the ERS
must be robust, because most herbal drugs contain vari-
ous hygroscopic components that are prone to soften and
become sticky, so that the dry-powdered ERS deform.
Therefore, keeping the ERS’s appearance consistent is
very important. The proper extraction technology should
be carried out carefully to balance the diverse ingredients
and principle components and be comparable with the
chromatographic profiles of the original crude drugs, as
well as maintaining a consistent appearance for the final
ERS products. In order to fulfill the real needs, the ERS
for identification and for full functionality (qualitative
and quantitative) needs to take into account all four as-
pects of “ASCS”, dealing with each aspect in turn to en-
sure that this project dev elops steadily.
Authenticity (A): Logically, the most basic source of
the authenticated species of an herbal drug is from its
original natural habitat. In reality, some of the so-called
exemplary habitats (daodi in Chinese) have been migrat-
ing over time. For example, the exemplary habitat of the
famous species of Di-Huang (Rehmannia radix) was ori-
ginally in the Shanxi province of the north-west zone of
China in ancient times, but it ha d migrated to Henan pro-
vince in the Central plains zone of China by the early
Ming Dynasty (1368-1644 AC). So nowadays, people
know Henan province (Huaiqing county) as the exem-
plary habitat of Di-Huang. In the wake of increasing de-
mand in recent decades, the area where Di-Huang is cul-
tivated has been extended to a rather wide region. Fur-
thermore, some Good Agriculture Practice (GAP) bases
have been established beyond the original region. On the
other hand, some of the original exemplary habitats of
some Chinese herbal drugs have suffered environmental
contamination resulting is a significant loss of quality.
Additionally, as farmers have migrated to the cities, so-
me areas that used to grow herbal drugs have been left
uncultivated. So me examples of co mmonly-used Chinese
herbal drugs that are still cultivated in the traditional
exemplary habitats like Dang-Gui (Angelicae sinensis ra-
dix), Chuan-Xiong (Chuanxiong rhizoma), Bai-Zhi (An-
gelicae dahuricae radix), Gan-Cao (Glycyrrhizae radix et
rhizoma) and Fu-Zi (Aconiti lateralis radix praeparata).
Wild-crafted Chinese herbal drugs have gradually de-
clined due to overharvesting along with a decrease in
their habitats, to the point where some have become en-
dangered species. Therefore, we need to let go of any
dogma regarding exemplary habitats given the reality of
the situation today. While it is still preferable to collect
samples of a given species from its traditional exemplary
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P. S. XIE ET AL.
habitat if possible, often this is no longer practical and
the next best approach is to obtain sufficient samples
from the wholesale herbal drug markets in the main cities
to ensure that they represent the main stream of the her-
Ensuring authenticity is the first priority when dealing
with the samples collected from outside the exemplary
habitats. The basic testing should be carried out accord-
ing to the standard in Chinese Pharmacopoeia including
subjective observation of the appearance, the taste and
smell, as well as chemical identification, testing and as-
say if necessary. Only the qualified samples can be in-
volved in the list of candidates. It is worth noting that
different samples from different habitat may have quite
different chemical compositions. Sometimes, we may
look at an unexpected astonishing picture in individual
cases  (Figure 1).
Specificity (S): The ERS produced from the candidates
of the given species must present a chemical ingredient
pattern similar to the original crude drug in order to be
considered of adequate quality. It has been well known
that the most practical approach to this is to conduct
chromatographic fingerprint analysis. Thin-layer chroma-
tography method is preferred, as instant comparison is
possible via the picture-like TLC image. A rough esti-
mate can be done rapidly of the similarity among the
sample images on the same plate via comparative ob-
servation of the bands numbers, band positions (Rf va-
lues), color and intens ity (Figure 2); scanning profiles of
the HPTLC images via corresponding digital software
can be further comparative observation in detail and
make more convincing assessment by similarity analysis
 (Figures 3 and 4). HPLC fingerprint can be also
applied for identification if necessary. Generally the
validated chemical fingerprint of the ERS is sufficient to
meet this basic requirement of an authentic herbal drug.
Epim e dium sagittatum
(Habi tat Guizhou,)
Epimediu m sagittatu m
(Habitat Anh ui).
Spe cies-ide n tifie r region
Figure 1. HP LC fin gerp rin t o f E pim edii h e rba (Yin-Yang-Huo)
grown in different habitats. (2) epimedin A, (3) epimedin B,
(4) epimedin C, (6) icariin. The three samples from the An-
hui province showed none of the main bioactive flavonoids
detected in “species Identifier region” of its HPLC finger-
S 1-4 01 02 03 04 05 06 07 08 09 10 11
Figure 2. HPTLC fluorescence image of Bupleuri Radix
(Chai-Hu). (S1): saikosaponin f; (S2) saikosaponin b2; (S3)
saikosaponin a; (S4) saikosaponin d; (01) Bupleurum
chinense DC.; (02) B. scorzonerifolium Willd; (03) B. longi-
radiatum Turcz; (04) B. bicaule Helm; (05) B. polyclonum Y.
Li et S. L. Pan; (06) B. wenchuanense Shan et Y. Li (07) B.
marginatum Wall. ex DC. var. stenophyllum (Wolff) Shan et
Y. Li; (08) B. falca t um L; (09) B. yi nchowe nse Shan et Y. Li;
(10) B. simithii Wolff. var. parvifolia Shan et Y. Li; (11) B.
tenue Huch. -Ham. ex D. Don. *Samples (01)-(09) roots; (10)
(11) aerial parts.
Region A Region B Region C
saikosaponin c + f
Figure 3. The digital scanning profile of the HPTLC fluo-
rescence image of Bupleuri radix (Chai-Hu). (A): main sai-
kosaponins region; (B): inter-species identifier region; (C)
Consistency (C): A worrisome problem for ERS is in-
consistencies between the chemical profiles of different
batches. Therefore it is often a necessity to blend differ-
ent batches of the ERS for adjustment of th e ratio to rea-
ch a relatively consistent compo sition of the chemical in-
gredients based on the established common pattern of the
chromatographic fingerprint and the semi-quantifiable
data of the ERS.
Stability (S): The ERS must be sufficiently stable dur-
ing the storage period. The tests for stability should
therefore be conducted rigorously. Particularly, the phy-
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P. S. XIE ET AL. 129
010 20 30 40 50 60 70
1 Std Fingerprint
B. chinense 18
B. scorzonerifolium 2
Sam pl e
B. longiradiatum 5
m erchandise 33
B. polyclonum 2
B.marginatum var. stenophyllum 1
B. yinchowense 1
B. sim ithii Wolff. Var 1
B. falcatum 1
B. wenchuanense 1
Figure 4. Similarity analysis of HPTLC fingerprint of Chai-
sical appearance of the ERS must be well preserved. Ge-
nerally, stickiness or soften ing of the appearance of ERS
that occurs during storage are common problems. The
solution to such problems will rely on proper extractive
technologies, packaging, and storage environments. Ge-
nerally, some clean-up procedures have to be done in
most cases during the preparation process, so we should
as far as possible balance what needs to be removed and
what needs to be retained. For example, proteins, sugars,
some tannins might generally be removed during the
In brief, the acceptable ERS should conform to the fol-
lowing requi rements:
• A qualified ERS must be produced from authentic-
cated samples of the reliable habitat and include suf-
ficient samples from various sites. Consistent raw
material is necessary.
• The detected chemical composition in the ERS must
be as similar as possible to that in the original herbal
drug. It means that the appearance of the HPTLC im-
age or the major composition of the HPLC profile
should be fairly similar to that of original herbal
• The chemical ingredients inter-batches must be gen-
erally consistent (it works when comparing between
the HPLC fingerprint or the HPTLC image). If large
amount of samples need to be compared concomi-
tantly, the similarity analysis might be conducted,
then the similarity between the batch es of ERS shou ld
be assumed of >0.9 (calculated by cosine or corre-
lated coefficient) among batches.
• The physical state of the ERS must be stable during
storage. The extractives of most CHM are quite hy-
groscopic. This is the major problem during storage
to which particular attention must be paid during the
production stage. The volatile o ils in the herbal drugs
will be very unstable once it is extracted by e.g.,
steam-distillation. Special approach for its stability
need to be de veloped .
• A fully functional ERS requires calibration of the
contents of the specified markers (cf. Section 8).
5. Preparation of ERS of CHM
In principle, aqueous extraction is the closest simulation
of aqueous decoction practiced in TCM. However, the
traditional aqueous TCM decoction contains not only wa-
ter-soluble components but also some insoluble substan-
ces in suspension as the decoction is ingested without
prior fine filtration. A clear aqueous solution of the her-
bal drugs made in the laboratory, therefore, is not equiva-
lent to the TCM decoction. Using 60% - 70% methanol
or ethanol extraction is the reasonable protocol to appro-
ximate the real-world decoctions. The alternative option,
if necessary, is to prepare two kinds of extracts; lipo-
philic and hydrophilic fractions depending on the need.
Some special cases with special extraction procedure
should be done according to those methods that have
been developed previously.
The preparation procedure of ERS includes extraction,
concentration and the final formation of the ERS. In
addition to the convention al extraction methods, the eco-
friendly extraction methods including ultrasonic, pre-
ssurized-solvent speed extraction, smashing tissue ex-
traction, and High-performance, High-pressure, diffe-
rentially Low-temperature Successive Extraction (HHLS).
This last can could be selected if the method can bear the
scale of the bulk products. Some clean-up steps need to
be conducted with careful and reasonable measures. No
matter which method is adopted, in-process quality con-
trol must be conducted. Which method of extraction is to
be selected for use will depend on the properties of the
particular herb to tailor-make the ERS of targeted herbal
drug which is also a matter of trial and error. One final
step is the stability test. Needless to say, the storage
location should have a low temperature, low humidity,
and be dark. In brief, no matter which preparation me-
thod was selected, the ultimate concern would focus on
whether can the final ERS products guarantee the four
essential principles—Authenticity, Specificity Consis-
tency, and Stability (ASCS).
6. Establishing the Specification of the ERS
The ERS of CHM must be certified. The typical charac-
teristics should be validated with TLC, HPLC or GC fin-
gerprint. The standard procedure and methodology should
also be validated. The characteristics of the ERS and the
chromatographic fingerprint are presented together with
the certification of its equivalen ce with the crude drug of
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P. S. XIE ET AL.
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the original species (raw material). The specification of
ERS includes the name of the entity, the equivalent ratio
of ERS/raw material, description, identification, test,
assay (for full-functional ERS) and storage.
converted to the equivalent amount of the raw material as
per the extraction ratio. The ratio of the ERS to the raw
material is approximately 1:14 (g, ERS/g, crude drug).
It contains spinosin 14 mg/g; jujuboside A 10.5 mg/g;
jujuboside B 5.5 mg/g (Figure 5).
Referring to the volatile ingredients (essential oils) of
the herbal drug, particularly the monoterpenes or ses-
quiterpenes, the chemical pattern of which are unstable,
the chemical pattern would seriously fluctuate and the
end product will be hard to finalize, so it is almost im-
possible to provide usable ERS of volatile ingredients of
herbal drugs. A tentative fresh-prepared the extractive as
reference by using a small sealed pouch pack of the
powdered authentic raw material in reserve might be an
option (Solven t ext rac t i on should be feasibl e ) .
1) Thin-layer chromatography identification (ChP
2010 Ed) (Figure 6).
The test is not valid unless the visible color TLC
image of the ERS sample shows high similarity (>0.9,
correlation coefficient). light grayish brown to bluish
green bands of juju boside A (Rf ca. 0.16) and jujuboside
B (Rf ca. 3.3) to that of the crude drug sample solution;
in addition, several other weak saponin bands as well as
the residual seed oil band on the front of the image also
appear. Under UV 366 nm light, a light yellowish blue
fluorescence band corresponds to spinosin CRS (Rf ca.
0.44), accompanied by one just above the spinosin band
an d three weak fluorescence bands lower than the spinosin
band as those appeared in the crude drug sample solution.
7. Examples of the Process and Application
The ERS of Suan-Zao-Ren (Ziziphi spinosae semen) and
Zhe-B ei-Mu (Fritillariae thunbergi bulbus) are used here
as examples to demonstrate a basis for the establishment
of the ERS of CHM.
7.1. ERS of Ziziphi spinosae Semen)
(ERS Suan-Zao-Ren) 7.1.3. C onsistency
More than ten batches of ERS of Ziziphi spinosae semen
have been ch ecked qualitatively and quantitatively.
Monograph of Chinese herb s “Extractive Reference Sub-
stances” (ERS). Carry out the method for HPLC fingerprint (optional)
ERS of Ziziphi spinosae sem en ( defatted).
ERS of Suan-Zao-Ren (defatted)—(SZR-ERS). Assay Chromatographic Conditions: According to the
method in ChP 2010 ed .
Code number: SZR-RSE 2012-02 (ERS 3).
7.1.1. Definition 7.2. ERS of Fritillariae thunbergi Bulbus)
(ZBM-ERS) (ERS Zhe-Bei-Mu)
Defatted extract of Ziziphi spinosae semen for use as the
Extractive Reference Substance (ERS) of Ziziphi spino-
sae semen. The ERS of Ziziphi spinosae semen (Chinese
name: Suan-Zao-Ren) is a light grayish brown dry pow-
der, which is 70% ethanol extraction of defatted seeds of
Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F.
Chou (Rhamnaceae). The amount of the final product is
Monograph of Chinese herb s “Extractive Reference Sub-
ERS of Fritillariae thunb e rgii bulbus.
ERS of Zhe-Bei-Mu—(ZBM-ERS).
Code number: ZBM-ERS 2012-04.
Jujuboside-a jujuboside-b spinosin
Figure 5. The chemical structures of main saponins and flavonoid in Ziziphi spinosae semen (Suan-Zao-Ren).
P. S. XIE ET AL. 131
S1 1 2 3 4 5 6 7 8 9 10 11
S2+3 1 2 3 4 5 6 7 8 9 10 11
Figure 6. HPTLC images of Ziziphi spinosae semen (Suan-Zao-Ren). Above: fluorescence image of the flavonoids; Lower:
visible color image of the saponin: (S1) spinosin; (S2) jujuboside A; (S3) jujuboside B; (1) ERS of defatted Ziziphi spinosae
semen; (2)-(8) (10) commercial samples of Ziziphi spinosae semen; (9) (11): Adulterant (Ziziphi mauritianae semen). The
HPTLC fluorescence (flavonoids) and visible color (saponin) images of the ERS of Ziziphi spinosae semen show that the au-
thenticity and specificity of the ERS of Ziziphi spinosae semen are acceptable for a reference substance.
Figure 7. HPLC-DAD fingerprint of Ziziphi spinosae semen (above) and the ERS sample (lower). Peak (1): spinosin; (2): ju-
juboside (a); (3) jujuboside (b). The fingerprint can be divided into four sections for easy reorganization of the fingerprint’s
intact features. (a) alkaloids section; (b) flavonoids section; (c) saponin section; (d) seed oil section.
Extract of Fritillariae thunbergii bulbus (EFTB) for use
as Extractive Reference Substance (ERS of Fritillariae
thunbergii Bulbus). The EFTB is light yellowish gray dry
powder, which is 70% ethanol extraction of the bulb of
Fritillaria thunbergii Miq. The amount of the final
product is converted to the equivalent amount of the bulb
raw material as per the extraction ratio. The ratio of the
ERS to the raw material is approximately 1: 10 (g/g).
It contains peimine 5.7 mg/g; peiminine 5.0 mg/g
Carry out the method for thin-layer chromatography
(ChP 2010 Ed.) (Figures 9 and 10).
Criteria: should demonstrate the same TLC pattern to the
HRS’s HPTLC image in which peimine (S1) and
peiminine (S2) dominate with another light brownish
orange band just above the band of peimine when spray-
ed with Dragendorff reagent and viewed under white li-
ght. The fluorescence image of EFTB generated by
spraying 10% sulfuric acid ethanol solution observed
under UV 366 nm is also very similar to that of the crude
drug which consisted of about 10 fluorescence bands.
That means the EFTB can be used as a reference sub-
stance for identification.
Confirmation: For testing the feasibility of this process,
11 batches of Fritillariae thunbergii bulbus (FTB) were
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P. S. XIE ET AL.
Figure 8. Chemical structure of alkaloids—peimine and pei-
(a) (b) (c) (d)
S 1 2 S 1 2 S 1 2 S 1 2
Figure 9 HPTLC images of ERS of Fritillarae thunbergii
bulbus (Zhe-Bei-Mu) (1) and crude drug of Fritillarae
thunbergii bulbus (2). (a): visualized by spraying Dragen-
dorff reagent, (b): fluorescence under UV 365 nm after
spraying sulfuric acid reagent, (c): the invert color image of
(b); (d): visible image generated by spraying sulfuric acid
reagent under white light. The main alkaloids—peimine (S1
band 6*, Rf ca. 0.46; and peiminine (S2 = band 8, Rf
ca.0.65) stained brown bands with Dragendorff reagent.
*The location of Peimine is very near to band 6 or they even
overlap. The intact Fluorescence image consisted of mainly
10 blue or grayish blue fluorescence bands combined with
the visible alkaloids image (a) constructed the characteristic
fingerprint of FTB, the ERS of FTB (EFTB) provided very
similar image with the crude drug. The legible invert color
image (c) transformed from the fluorescence image (b)
aided for more distinct observation.
S ERS 1 2 3 4 5 6 7 8 9 10 11
S ERS 1 2 3 4 5 6 7 8 9 10 11
Figure 10. Application of ERS of Fritillariae thunbergii
bulbus to 10 batches of Chinese herbal medicine—crude
drug of Fritillariae thunbergii bulbus (FTB) T: 21 RH: ℃
50%. (a)Fluorescence image after spraying 10% sulfuric
acid/ethanol solution; (b) Visible color image of plate after
spraying Dragendorff’s reagent. (S1) = peimine; (S2) = pei-
minine; Track (1) ERS of FTB; (2)-(11) commercial crude
drug—FTB The images show that the authenticity and spe-
cificity of the ERS of FTB are acceptable for a reference
analyzed with the EFTB on the same plate, sample
preparation and application was carried out quantita-
tively; the result showed the pattern of EFTB’s image
was basically as same as that of the crude drug FTB.
HPLC fingerprint of ERS of FTB provided a rather
simple profile. As the main aklaloids, peimine and pei-
minine were clearly separated. It can be quantitafied by
external standard method for determining their contents
8. Quantification Issue of ERS
The use of herbal reference extractive (=ERS) instead of
pure reference substance for quantitative analysis is con-
troversial. The issue in question is the nonequivalence
between the herbal extractive reference and the corre-
sponding pure substance due to the uncertain assigned
value of the analyte, chromatographic resolution and
stability . Most analysts have also taken for granted
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P. S. XIE ET AL. 133
Figure 11. HPLC fingerprint of 11 batches of FTB express
high similarity (cosine > 0.9) compared with ERS of FTB
(S1: peimine, S2: peiminine).
that one should determine a single chemical marker pre-
cisely and accurately in a herbal drug as one does the
chemical pharmaceuticals. This would be true if ERS for
herbal quality control was synonymous with the CRS for
QC of pure chemical medicine or natural single com-
ponent product. However, the situation of CHM is com-
pletely different. Determining a chemical marker for the
purpose of use in TCM has to take into consideration the
fact that any chemical marker is just a few parts per
thousand or even a few parts per ten thousand, when the
intake dose of a prescribed herbal medicine is generally
10 - 30 g, even bigger, per potion as part of a compound
formula that commonly uses 6 - 12 ingredients. This
situation is obviously co mpletely differen t than those that
inolve pure chemical pharmaceuticals. In other words, it
makes no sense to treat the herbal drug exactly the same
as a chemical medicine . This brings us to a funda-
mental question: what is the real important of deter-
mining accurately and precisely such a minute amount of
one or few chemical marker(s) in a herbal medicine
(except for toxic ingredien ts)?
We suggest that it is necessary to work out the appro-
priate and meaningful quantitative measurements in re-
gards to Chinese herbal medicines. In fact, all accepted
external standard quantitative determinations rely on the
chromatographic integrated raw data (peak area). Why
not use the integr ated peak area und er quantitative ope ra-
tion conditions to rapidly estimate the semi-quantity of
the contents of the all peaks or appointed peaks at the
same time as performing the chromatographic finger p rin t?
This would be an easy, rapid, and economical approach.
The raw data is rough but practically reliable and useful.
One example of its utility is demonstrated by the possi-
bility of semi-quantitativ e estimation of the bioactiv e fla-
vonoids in Epimedium leaves through chromatographic
integrated data. It is well known that the C-8-prenylated
flavonoids are special bioactive contents in Epimedium
spp. with the main ones being epimedin A, epimedin B,
epimedin C and Icariin, (ABCI). A set of integrative
peaks area of ABCI acquired from HPLC fingerprint de-
monstrated the ratio of ABCI peaks being concordant
with the precisely determined content by external stan-
dard assay  (Figure 12). Other examples—Coptidis
Rhizoma and Ginseng Radix are shown herewith the
same expected results (Figures 13 and 14). The only
issue with this is how to set the measurement unit to
make it generally acceptable. Furthermore, it would be
possible for the quantitative determination of some ap-
pointed peaks by means of calibrated ERS, in which
some known chemical components were determined by
external standards . Of course, a good resolution of
the target peak and good reproducibility is the prerequi-
site for this method’s utility. There is an example for
comparison of the quantitative determinations between
using CRS and ERS to determine the contents of spino s in,
jujuboside A and jujuboside B in Ziziphi spinosae semen
(Suan-Zao-Ren). The primary results seem acceptable in
terms of herbal medicines (Table 1).
On the quantitative analysis, Helliwell practically sug-
gested that the key con sideration on the qu antification by
using Herbal Drug Preparation Reference Standard
(RES), the content of specified constituent(s) is not an
absolute value, but an assigned value determined by a
specified method . Our testing results by using the
aforementioned method exemplified the feasibility of
such a suggestion.
Our argument is that application of ERS is a more
meaningful strategy for identification and semi-quanti-
fication for herbal drug quality assessment. It is true that
there is still work to be done to improve the preparation
and application of ERS to QA/QC for chromatographic
identification and rapid quantifiable estimation and see
how it can be performed in a cost-effective way. There
are of course other problems that will need to be solved
through trial and error, so that it is necessary to go for-
ward one step at a time. As the saying goes that “the per-
fect can be the enemy of the good enough ”.
The ERS of CHM is a new methodology as well as a
subject of much debate. The first issue might be what the
criterion is for pragmatic useful ERS. The essential fea-
ture of the qualified ERS for identification is that it
should be able to represent consistently the detected in-
tact characteristics of the original crude drug expressed
by the chromatographic fingerprint. The chemical attri-
bution of the elements in the profile can be pinpointed by
advanced technologies combined with available CRS.
Considering the variety and complexity of CHM, it
would be unwarranted hurdle at the early stage if over-
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P. S. XIE ET AL.
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Total content of ABCI (%)
E.b. E.k. E.s E.p. E.w.
Integrated ABCI peaks Area of Epimedium spp.
E.b. E.k. E.s.E.p. E.w.
Figure 12. The comparative results of the contents of the total four C-8-prenylated flavonoids (ABCI) in epimedi herba. Light:
External standard method; Right: the HPLC integrated peaks area.
Figure 13. Comparison between integrated raw data (peak area) and external standard determination for assessing the qual-
ity of main ginsenosids in Extractive Ginseng.
Figure 14. Comparison of the quantitative assessing between integrated raw data (peak area) and external standard method
of five alkaloids in Extract of Coptidis. A rapid estimation of the contents of the five alkaloids, reading HPLC peak area value
is comparable with the conventional external standard determination.
emphasizing the desire of chemical attribution in an ERS;
successive exploration requires sustainable research. To
be useful ERS will need to be done in a flexible manner
so that it can be tailored to different situations for various
types of samples. A “one-size fits all” would be imprac-
tical. The practical, economical, and relatively easily
accomplished nature of ERS is a factor pushing this ap-
proach forward, particularly for the commercial and in-
dustrial field of CHM. We need to keep in mind that the
conceptual integrity of the medicine and the fuzziness of
many problems will direct the research and application of
ERS works in concert with effective quality control of
P. S. XIE ET AL. 135
Table 1. Table type styles (Table ca ption is indispensa ble ) .
the content (% crude drug) a
By CRS By ERS RSD
spinosin 0.0632 0.0634 0.16
Jujuboside A 0.0523 0.0531 0.76
Jujuboside B 0.0268 0.0287 3.47
aUsing the routine external standa rd determination method.
CHM. Preparing a qualified practical and economical
ERS of CHM requires a certain amount of expertise and
diligence. Referring to the full-functional ERS, a rapid
quantifiable estimation of the bioactive fraction of the
whole using acquired integration data from the chroma-
tographic fingerprint is a practical issue. Calibrating the
appointed target peaks in the ERS to serve as CRS to
determine the sample tested is worth investigating.
There is a fundamental difference in outlook between the
antagonistic-oriented approach of single chemical phar-
maceuticals and the orientation of TCM towards balan-
cing the human body’s function. Strategies of meaningful
quality control need to take into account the complexity
of CHM. No work into the safety and efficacy can afford
to ignore the synerg ic action exerted by multi-ing redients
in the herbals according to TCM constructs. Research
and application of ERS, a stand ardized extractive with its
detectable chemical pattern for a given species, are be-
coming a new trend for reference substances used for
herbal medicine quality control. The criteria on the ERS
of CHM should pursue the Authenticity, Specificity,
Consistency and Stability (ASCS) in a holistic manner.
Overcoming the inertia generated by dogma needs to be
done as soon as possible to achieve real meaningful qua-
lity control of Chinese herbal medicines.
We appreciate our team members, Shuai Sun, Li Shao,
He Li, Ruiyin Wang, Longgang Guo, Tao Kang and
Xiaofeng Li, for their participation in the experiments on
preparation and quality analysis of ERS cited in this pa-
per. We also thank David Glyn Pinder for his editing
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ChP = Pharmacopoeia of the Peoples Republic of China
CHM = Chinese Herbal Medicine
TCM = Traditional Chinese Medicin e
CRS = Chemical Reference Substance
HRS = Herbal Reference Substance
ERS = Extractive Reference Substance
USP = The United States Pharmacopoeia
EuP = The European Pharmacopoeia