142

Notes

Biol. Pharm. Bull. 32(1) 142—146 (2009)Vol. 32, No. 1

Identification of Cistanche Species by Chemical and Inter-simple Sequence
Repeat Fingerprinting
Hai Ming SHI,a Jing WANG,a Meng Yue WANG,a Peng Fei TU,*,b and Xiao Bo LI*,aaSchool of Pharmacy, Shanghai Jiao Tong University; Shanghai 200240, P. R. China: and b Department of Natural
Medicines, School of Pharmaceutical Sciences, Peking University; Health Science Center, Beijing 100083, P. R. China.
Received April 28, 2008; accepted October 30, 2008; published online November 4, 2008
Herba Cistanches is a common traditional Chinese medicine that has been used to reinforce the vital function of kidney and induce laxation for more than two thousands years. Four Cistanche species were found as
Herba Cistanches in China herbal markets, including C. deserticola, C. tubulosa, C. salsa and C. sinensis.
Phenylethanoid glycosides, particularly echinacoside and acteoside, are considered as active ingredients in Cistanche species. The contents of these compounds showed variation in different species and geographical sources.
Standard chemical fingerprints were generated from each of four Cistanche species, which could be identification
markers. In genetic analysis of Cistanche species, ninety-four inter-simple sequence repeat (ISSR) primers wereused for polymerase chain reaction (PCR) amplification, and of which eight primers were found to be sufficient
to distinguish different Cistanche species. As a result, the chemical fingerprint combined with the genetic fingerprint for distinction of Cistanche species could serve as markers for quality control of Herba Cistanches.
Key words

authentic identification; Cistanche; fingerprint; HPLC; inter-simple sequence repeat

Herba Cistanches, the stems of Cistanche species (Orobanchaceae), is a common traditional Chinese medicine witha reputable name as “Desert Ginseng”. Herba Cistanches was
first recorded in Shen Nong Bencao Jing in ca. 100 B.C. as a
top grade and used to treat various diseases including impotence, female infertility, morbid leukorrhea, profuse metrorrhagia, cold sensation in the loins and knees, and chronic
constipation in the aged.1) Nowadays, it is reported to enhance sex and learning behavior,2) and to possess the sedativeand vasorelaxant effects.3,4) Besides its common uses in
China, Herba Cistanches is also used as a healthy food supplement for men’s care in Japan and Southeast Asia. Therefore, the demand for Herba Cistanches is increasing. Four
Cistanche species occur in Northwest China, including C.
deserticola Y. C. MA, C. tubulosa (SCHRENK) WIGHT, C. salsa(C. A. MEY.) G. BECK and C. sinensis G. BECK. The Chinese
Pharmacopoeia (2005 edition) recorded that Herba Cistanches should be the dried fleshy stems of C. deserticola
and C. tubulosa. However, C. salsa and C. sinensis were
found in the local markets in China, which could serve as
substitutes of Herba Cistanches due to the shortage of C. deserticola and C. tubulosa.5)
Although the aforementioned four Cistanche species
belong to the same genus, the chemical compositions ofthese plants were different.6,7) Phenylethanoid glycosides aremain chemical ingredients in Cistanche species, and theiramounts showed variation among different species.8) The ingredients associated with the pharmacological activities of
Herba Cistanches are echinacoside and acteoside, which havesignificant vasorelaxant, neuroprotective, hepatoprotective
effects.4,9—11) The levels of these two constituents are commonly used as quality control markers of Herba Cistanches.
Identification of the specific Cistanche species is difficult
if it was only based on the pharmacognostical characteristics.
To ensure the quality of Herba Cistanches, several approaches have been applied for the differentiation of Cistanche species. Tomari et al. has distinguished C. deserticola,
C. salsa and C. tubulosa by comparing the nucleotide sequences of the plastid rps2 gene and the intergenic spacer re∗ To whom correspondence should be addressed.

gion between rpl16 and rpl14.12) Jiang et al. has developed a
HPLC- Diode Array Detector (DAD)-MS fingerprint method
to comparatively analyze Cistanche species.13)
In this study, we determined the chemical and genetic distinctions of stems derived from C. deserticola, C. tubulosa,C. salsa and C. sinensis. Chemically, we developed a simple
and valid fingerprinting method by HPLC-UV to distinct
four Cistanche species, and the level of echinacoside andacteoside in different species was also presented. Genetically,
inter-simple sequence repeat (ISSR) fingerprint was used to
identify different species of Cistanche.
MATERIALS AND METHODS
Plant Materials A total of 32 individuals were sampledfrom 3 sites throughout Northwest of China, which are primary distribution areas of Cistanche plants, included Xinjiang, Neimenggu, and Ningxia Autonomous Region (Table1). They were collected in different date and area, and identified by one of us (Prof. Pengfei Tu). Voucher specimens were
deposited at School of Pharmacy, Shanghai Jiaotong University. Each plant sample was cut to two parts from length
wise. One part was stored at 20 °C until DNA extraction;
the other part was cut to ca. 4 mm slides, then put in 70 °C
water to kill enzymes, dried in cool for HPLC analysis.
DNA Extraction and Polymerase Chain Reaction(PCR) Amplification Genomic DNA of samples was isolated from their fresh tissues using an extraction protocol by
DNeasy Plant mini Kit (QIAGEN Inc.). Purified DNA wasadjusted to a final concentration of 20 ng/m l for use. PCR
amplification was performed in a total volume of 25 m l reaction solution. Each reaction was composed of 60 ng of genomic DNA, 1PCR buffer, 2 mM MgCl2, 200 m M dNTP,
0.2 m M primer and 1 unit Taq DNA polymerase (Sangon Inc.,
Shanghai, China). Amplification was carried in a PTC-100
thermocycler (MJ Research Inc.) under the following conditions: a hot start step of 5 min at 94 °C, followed by 10 cyclesof 50 s at 94 °C, 1 min at 59 °C, 1 min at 72 °C using a touchdown program with 0.5 °C annealing temperature decrements

e-mail: xbli@sjtu.edu.cn; pengfeitu@vip.163.com© 2009 Pharmaceutical Society of Japan

January 2009
Table 1.

143

Contents of Echinacoside and Acteoside from Different Species of Herba Cistanches
Content (mg/g)
SpeciesC. deserticola (Cde-1)
C. deserticola (Cde-2)
C. deserticola (Cde-3)
C. deserticola (Cde-4)
C. deserticola (Cde-5)
C. deserticola (Cde-6)
C. deserticola (Cde-7)
C. deserticola (Cde-8)
C. deserticola (Cde-9)
C. deserticola (Cde-10)
C. tubulosa (Ctu-1)
C. tubulosa (Ctu-2)
C. tubulosa (Ctu-3)
C. tubulosa (Ctu-4)
C. tubulosa (Ctu-5)
C. tubulosa (Ctu-6)
C. tubulosa (Ctu-7)
C. tubulosa (Ctu-8)
C. tubulosa (Ctu-9)
C. tubulosa (Ctu-10)
C. salsa (Csa-1)
C. salsa (Csa-2)
C. salsa (Csa-3)
C. salsa (Csa-4)
C. salsa (Csa-5)
C. salsa (Csa-6)
C. sinensis (Csi-1)
C. sinensis (Csi-2)
C. sinensis (Csi-3)
C. sinensis (Csi-4)
C. sinensis (Csi-5)
C. sinensis (Csi-6)

Geographical origins

Jimusaer, Xinjiang
Yongning, Ningxia
Alashan, Neimenggu
Alashan, Neimenggu
Guejina, Neimenggu
Alashan left, Neimenggu
Alashan left, Neimenggu
Alashan right, Neimenggu
Alashan right, Neimenggu
Alashan right, Neimenggu
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yutian, Xinjiang
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Yanchi, Ningxia
Alashan left, Neimenggu
Alashan left, Neimenggu
Alashan left, Neimenggu

per cycle until 54 °C was attained, followed by 30 cycles of
94 °C for 50 s, 54 °C for 1 min, 72 °C for 1 min and then aterminal extension at 72 °C for 8 min, followed by cooling at
4 °C to stop the reaction. The PCR products were stored at
20 °C until electrophoresis. The genomic DNA extracts
were run in 1% agarose gels, PCR products were electrophoresed in a mixed 2% agarose, 0.5 m g/ml EB, photographed using a BIODoc-ItTM System (UVP Inc.). Gene
Ruler 1 kb DNA ladder plus (Sangon Inc., Shanghai, China)
was used as a marker to determine the size of the ISSR fragments. All these conditions used throughout the study foreach primer were identical.
Data Analysis The amplification products were scoredin terms of a binary code as present (1) or absent (0), each ofwhich was treated as a unit character regardless of its intensity. A genetic identity matrix, using the Nei & Li’s coefficient, was constructed using the computer program PopGen
v.1.31. Phenetic dedrogram, based on the genetic identity
matrix comparing individuals, were constructed using theunweighted pair group method of arithmetic averages(UPGMA, PopGen v.1.31).
Sample Preparation and HPLC Analysis Before use,
the materials were dried to constant weight in an oven at
50 °C (Yihen, Shanghai, China) then pulverized and sieved(60 meshs). A 1.0-g powder of sample was extracted with
30.0 ml methanol in an ultrasonic water bath for 30 min. The
extracted solution was centrifuged at 12000 rpm for 3 min,the supernatant was filtrated through a 0.2 m m membrane fil-Harvesting time
Echinacoside

Acteoside

1.1
1.7
1.5
1.7
1.9
1.4
1.5
2.2
2.0
2.3
31.8
60.3
65.2
48.1
85.4
45.2
46.8
51.9
119.9
45.7
4.7
4.8
4.2
5.2
4.4
6.8
—
—
—
—
—
—

4.8
6.4
6.2
9.8
6.4
5.8
7.0
6.8
15.2
11.6
10.4
8.6
22.1
8.6
22.1
15.9
7.3
13.9
25.4
14.3
5.5
6.7
6.3
6.7
7.9
8.2
5.3
6.5
8.4
4.5
5.2
4.0

April 2005
June 2003
June 2003
June 2003
May 2004
May 2004
May 2004
May 2004
May 2004
May 2004
November 2002
November 2002
November 2002
November 2002
November 2002
May 2004
November 2002
November 2002
November 2002
May 2004
May 2004
May 2004
May 2004
May 2004
June 2003
June 2003
May 2003
May 2003
May 2004
June 2003
June 2003
June 2003

ter before injection. Each sample was prepared with theabove protocol for HPLC analysis. HPLC analysis was performed on a Shimadzu LC 2010A liquid chromatograph system (Shimadzu Co., Japan) consisting of a quaternary pump,a column oven, an autosampler. Samples were separated on a
Shim-pack VP-ODS C18 (4.6 mm i.d.250 mm, 5 m m, Shimadzu Co., Japan). The mobile phase consisted of 0.095%
phosphoric acid in deionized water (A) and 0.095% phosphoric acid in acetonitrile (B) using gradient program of 0—
12% (B) in 0—8 min, 12—16% (B) in 8—20 min, 16—20%(B) in 20—35 min, 20—25% (B) in 35—55 min, 25—30%(B) in 55—70 min, 30—0% (B) in 70—90 min. The flow rate
was 1.0 ml/min, the injection volume was 10 m l and column
temperature was maintained at 25 °C. UV detector was set at
334 nm for acquiring chromatograms.
RESULTS AND DISCUSSION
To develop a fingerprint for Herba Cistanches, an optimized strategy for HPLC conditions was performed. Analysis with methanol had longer duration than those with acetonitrile. The acetonitrile–water system had the same analytical time as the acid acetonitrile–acid water system, but theformer had a poor resolution. So the 0.095% phosphoric acidin acetonitrile–0.095% phosphoric acid in water system waschosen for its well baseline resolution and suitable duration
for analysis. In order to obtain a sufficiently large number ofdetectable peaks on the HPLC chromatogram, the UV spec-

144

Fig. 1.

Vol. 32, No. 1

Structures of Echinacoside (A) and Acteoside (B)tra of samples were investigated and 334 nm was selected as
detection wavelength.
The major constituents of Herba Cistanches are echinacoside and acteoside, which could be determined by HPLC
analysis (Fig. 2). The HPLC calibration curves of echinacoside and acteoside exhibited good linearity in a range from 1
to 22 m g. The correlation coefficients of these two compounds were 0.9999. The precision test within the same sample of chemical markers (n6) showed the relative standard
deviation (RSD) values of 0.92% (echinacoside), 0.93%(acteoside). The short-term (12 h) and long-term (24 h) repeatabilities of echinacoside and acteoside were calculated
for six determinations (n6). The RSD of short (or long)term repeatability for echinacoside and acteoside was 1.35%(1.24%) and 1.82% (1.19%), respectively. The recovery experiment was carried out to evaluate the accuracy of themethod. Known amounts of echinacoside and acteoside wereadded to the herbal sample and were extracted by using theultrasonication method accordingly; the extracted material
was subjected to analysis, and the contents of these compounds were calibrated. The average recoveries of the tested
chemicals were 96.9% (echinacoside) and 96.4% (acteoside)(n9). The contents of the two compounds in the samples
were quantified and the results were shown in Table 1 withthe mean values of three replicate injections. C. tubulosa
contained much more 12-fold amount of echinacoside and 2fold amount of acteoside when compared with other three
Cistanche species. Meanwhile, the content of two constituents is some different in various batches of Cistanche
species, which is due to the variation of habitat, climate, circumstances, collecting time and soil condition.
In the present study, 10 batches C. deserticola, 10 batches
C. tubulosa, 6 batches C. salsa, 6 batches C. sinensis wereanalyzed to evaluate similarities, respectively. The correlation coefficients obtained are listed in Table 2. The results indicated that the similarities of C. deserticola were relatively
low, which means the secondary metabolites of C. deserticola would vary in different locations. C. tubulosa were generally consistent except two samples showed some differences. The results of C. salsa samples were found resembling

Fig. 2. Standard Fingerprint of Cistanche Species: C. deserticola (A), C.
tubulosa (B), C. salsa (C), C. sinensis (D)Table 2.
Species

The Similarities of HPLC Fingerprint from Different Cistanche

No. Similarities

No. Similarities

No. Similarities No. Similarities

Cde-1
Cde-2
Cde-3
Cde-4
Cde-5
Cde-6
Cde-7
Cde-8
Cde-9
Cde-10

Ctu-1
Ctu-2
Ctu-3
Ctu-4
Ctu-5
Ctu-6
Ctu-7
Ctu-8
Ctu-9
Ctu-10

Csa-1
Csa-2
Csa-3
Csa-4
Csa-5
Csa-6

0.775
0.843
0.733
0.980
0.693
0.918
0.843
0.868
0.792
0.903

0.989
0.982
0.926
0.896
0.970
0.877
0.959
0.962
0.988
0.959

0.952
0.893
0.974
0.924
0.915
0.958

Csi-1
Csi-2
Csi-3
Csi-4
Csi-5
Csi-6

0.959
0.984
0.887
0.929
0.943
0.856

Data analysis was performed by the professional software named Traditional Chinese
Medicine Chromatographic Analyzing and Data Administration System (National Institute for the Control of Pharmaceutical and Biological Products) for evaluating the similarity of different chromatograms by calculating the correlation coefficient.

to each other and C. sinensis samples were somewhat different. It is well known that the standard HPLC fingerprint must
be representative of authentic herb. For this purpose, the one
with the biggest similarity was selected as the standard fingerprint. Accordingly, Cde-4 (C. deserticola), Ctu-1 (C.

January 2009
Table 3.

145

Sequences of 8 Polymorphic Primers Used for ISSR Fingerprint and the Amplified DNA Products

No.

Primersequence

No. ofamplifiedbands

No. ofpolymorphic
bands

No.

Primersequence

No. ofamplifiedbands

No. ofpolymorphic
bands

1
2
3
4

(AG)8CT(AG)8CA(GA)8CT(GA)8CC

11
15
14
9

11
15
14
9

5
6
7
8

(AG)8CC(GA)8C(AG)8TC(GA)8T

13
10
11
13

13
10
11
13

tubulosa), Csa-3 (C. salsa) and Csi-2 (C. sinensis) were selected as the standard fingerprints for different Cistanche
species with 10, 7, 12 and 6 characteristic peaks, respectively. By comparison with standards, peaks with the retention time at 46.5 and 60.2 min were identified as echinacoside and acteoside. From Fig. 2, it is found that the fingerprint of C. deserticola was similar to C. salsa. Meanwhile, C.
tubulosa can be differentiated from C. deserticola and C.
salsa by comparing the relative contents of echinacoside andacteoside, which were dominant in C. tubulosa. Furthermore,echinacoside was the common peak in C. tubulosa, C. deserticola and C. salsa, which were not present in C. sinensis.
Therefore, the presence of echinacoside can be distinguished
C. sinensis from other Cistanche species.
As in ISSR fingerprints, ninety-four primers (synthesized
by Sangon Inc., Shanghai, China) were tested for the identification of Cistanche plants, of which eight primers, namely
primer 1—8 (Table 3), produced clear and reproducible
bands. A total of 96 amplification bands were generated,which were all polymorphic ones. The loci numbers varied
from 9 to 15 per primer (Table 3), with fragment size ranging
from 200 to 2000 bp (Fig. 3). The eight ISSR fingerprints obtained using primers 1—8 were showed in Fig. 3. Primer 1
revealed a potential specie-specific band (Fig. 3A) for C.
sinensis (lane 3; about 1800 bp). Primer 2 could be used to
identify two Cistanche species (Fig. 3B), one specific bandfor C. deserticola (lane 1; about 2100 bp) and two for C.
tubulosa (lane 4; about 1600 bp and 1300 bp). It is obvious tofind a specific band for C. salsa (lane 2; about 2000 bp) byusing primer 3 (Fig. 3C). Primer 4 could identify C. deserticola with a specific band (lane 1; about 1500 bp) (Fig. 3D).
The specific bands included one for C. deserticola (lane 1;
about 1100 bp), one for C. tubulosa (lane 4; about 510 bp),
which were obtained using the primer 5 (Fig. 3E). Primer 6
could identify C. deserticola with a specific band (lane 1;
about 1200 bp) (Fig. 3F). The specific bands included one forC. salsa (lane 2; about 450 bp), one for C. tubulosa (lane 4;
about 220 bp), which were obtained using the primer 7 (Fig.
3G). Primer 8 could identify C. sinensis (lane 3; about
1600 bp) and C. tubulosa (lane 4; about 1400 bp) with specific bands (Fig. 3H). Complete distinctness of four species
examined was achieved by the amplification pattern fromprimers 1—8, different species showing the presence of aband from those where the band was absent. To authenticate
Cistanche species efficiently, an unweighted pair groupmethod using arithmetic average (UPGMA) dendrogram(Fig. 4) was obtained by comparing the similarity among thefour Cistanche species basing on primer 8. The dendrogram
clearly distinguished the examined Cistanche species into
four groups. In the first cluster, there are six C. sinensis samples with three sub-groups. The second cluster consisted of

Fig. 3.

ISSR Fingerprint Patterns Using the Primers 1—8 (A—H)

Lanes 1—4 represent C. deserticola, C. salsa, C. sinensis, C. tubulosa, respectively.
Lane N: Blank. Lane M: 1 kb Plus DNA Ladder.

ten C. tubulosa samples with three sub-groups. Six C. salsa
samples with two sub-groups formed the third cluster. The
remaining ten C. deserticola samples formed the fourth cluster, which consisted of three sub-groups. From the UPGMA
clustering results, C. deserticola and C. salsa showed theclose affinity with a similar index value of 0.58. Meanwhile,
C. tubulosa showed genetic relationship with C. deserticola
and C. salsa at a similar index value of 0.44. In contrast, C.
sinensis showed distant relationship with other three Cistanche species at a similar index value of 0.36. Interestingly,
the cluster results by ISSR fingerprint is in accordance withthe results generated by chemical fingerprint.
CONCLUSION
Precise identification of crude drugs is a prerequisite forchemical and pharmacological investigations of traditional

146

Fig. 4.

Vol. 32, No. 1

UPGMA Drendrogram on the Basis of ISSR Fingerprints of Primer 8

Chinese medicine and for their clinical applications. HPLC
fingerprint has played more and more important role for
identification and quality control of TCM, which has beenused as an effective tool for Cimicifuga species identification.14) Molecular genetic methods have several advantages
over classical authentication by using morphological andchemical analyses,15) and effectively applied to discriminate
Astragalus species.16) In case of Cistanche species here, bothchemical fingerprint and genetic analysis are used for identification, and the results show that chemical and genetic
methods are two rather distinct analyses, but their results arein line with each other suggesting the unique characteristic of
the species. Thus, both chemical and genetic methods could
be used as tools in identifying the correct species of Cistanche. Meanwhile, our present study provides a powerfulevidence for the rationality of C. tubulosa using as a new
natural source of Herba Cistanches in Chinese Pharmacopoeia (2005 edition). In addition, our results suggest that
C. sinensis should not be as substitute of C. deserticola andC. tubulosa due to the deficiency of echinacoside.
Acknowledgements We thank the National Natural Science Foundation of China (Grant No. 30371042) for financial
support of this work. We are grateful to Dr. Christopher X.
Lu for reading the manuscript.

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