全 文 ： 药学学报 Acta Pharmaceutica Sinica 2010, 45 (8): 1067−1070 · 1067 ·




Molecular authentication of Valeriana jatamansi by
ARMS based on 5S rDNA sequence
DIAO Ying1, 2, DUAN Wei-tao3, LIN Xian-ming4, LIAO Chao-lin4, HU Zhong-li3*, WANG You-wei1, 2*
(1. College of Life Science and Technology, Chongqing University of Arts and Sciences, Chongqing 402168, China;
2. College of Pharmacy, Wuhan University, Wuhan 430072, China; 3. Key Lab of the Ministry of Education for Plant
Developmental Biology, College of Life Science, Wuhan University, Wuhan 430072, China;
4. Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi 445000, China)
Key words: Valeriana jatamansi; 5S rDNA; phylogenetic relationship; ARMS; identification
CLC number: R931.5 Document code: A Article ID: 0513-4870 (2010) 08-1067-04
基于 5S rDNA 序列利用 ARMS 技术鉴定蜘蛛香
刁 英 1, 2, 段卫涛 3, 林先民 4, 廖朝林 4, 胡中立 3*, 王有为 1, 2*
(1. 重庆文理学院生命科学与技术学院, 重庆 402168; 2. 武汉大学药学院, 湖北 武汉 430072; 3. 教育部植物发育生
物学重点实验室, 武汉大学生命科学学院, 湖北 武汉 430072; 4. 湖北省农业科学院中药材研究所, 湖北 恩施 445000)

摘要: 蜘蛛香 (Valeriana jatamansi) 在我国西南和东北地区作为一种传统的草药用来治疗消化不良和风湿
病。蜘蛛香在化学活性成分和药效方面都与缬草属其他药用植物存在较大差别。为了研究缬草属植物之间的亲
缘关系以及寻找一种能够有效方便地将蜘蛛香与其他缬草属植物区别开的方法, 本文对 5 种缬草属药用植物的
5S rDNA 序列进行了扩增和测序。测序的结果表明扩增的 5S rDNA 片断, 长度在 307 bp 到 355 bp 之间。系统发
育分析表明, 在所测试的 5 种缬草属植物中, 中国缬草 (V. pseudofficinalis) 与宽叶缬草 (V. officinalis var. latifolia)
亲缘关系最近, 而长序缬草 (V. hardwickii) 与其他 4 种材料的遗传距离最远。根据蜘蛛香 5S rDNA 序列的 SNP
位点设计了 6 条诊断型引物, 通过等位基因特异 PCR (amplification refractory mutation system, ARMS) 分析, 其
中一条引物可以将蜘蛛香与其他 4 种缬草属植物区别开。此外, 利用这条特异引物, ARMS 技术还能够成功地鉴
别出复方中药中是否含有蜘蛛香。由此可见, 这种方法不仅可以用以鉴别蜘蛛香的基原植物, 同时还能用于蜘蛛
香商业产品的鉴别。
关键词: 蜘蛛香; 5S rDNA; 系统发育关系; ARMS; 鉴定

Genus Valeriana (Valerianaceae) comprises over
250 species[1]. Dried rhizome of Valeriana jatamansi
Jones, commonly known as ‘Zhizhuxiang’ in Chinese is
a folk herbal medicine, which was firstly recorded in
‘Diannan Bencao’ and described in detail in ‘Bencao
Gangmu’[2]. In the trade market, the Valeriana herbs
from different Valeriana species such as V. officinalis

Received 2010-04-03.
*Corresponding author Tel: 86-27-68753606, Fax: 86-27-68753611,
E-mail: huzhongli@whu.edu.cn
Tel: 86-27-68759879, Fax: 86-27-68759010,
E-mail: wyw931@yahoo.com.cn
var. latifolia, V. pseudofficinalis and V. jatamansi are
considered to have actions of sedation and hypnosis.
The species V. jatamansi growing in Southwest and
Northeast China has been used mainly to treat dyspepsia
and rheumatism for a long time and is now growing
rare throughout the distributing area. Recently,
pharmacological and clinical experiments have proved
that the chemical components are very complex among
the Valeriana species and their medicinal effects are
quite different[3]. So they must be carefully identified
and used. However, it is difficult to distinguish
·研究简报·
· 1068 · 药学学报 Acta Pharmaceutica Sinica 2010, 45 (8): 1067−1070

V. jatamansi from other Valeriana species, because of
their similarities in morphology and histology.
Compared with the morphological and chemical
methods, molecular genetic methods, especially those
PCR-based methods, have been proved stable, reliable
and efficient to identify the herbal medicines. The
ARMS (amplification refractory mutation system) is
also described as allele-specific PCR, which could detect
single base mutations. Now, ARMS have successfully
been used to authenticate herb medicines such as Panax
ginseng[4], Alisma orientale[5] et al. In higher eukaryotes,
the 5S rDNA unit consists of a 120 bp conserved 5S
rRNA gene coding region and non-transcribed spacer
(NTS) of various size. Such diversity of the NTS in
different species can be used as an identification basis[6].
In this study we established the technique of ARMS
based on 5S rDNA sequence, to identify V. jatamansi
from other Valeriana species and herbal medicines
efficiently and conveniently and ascertain the phylogenetic
relationship among five Valeriana species.

Materials and methods
1 Plant materials
The five tested species distributing in Southwest
China included V. jatamansi, V. pseudofficinalis C. Y.
Cheng et H. B. Chen, V. officinalis Linn. var. latifolia
Miq., V. hardwickii Wall. and V. flaccidissima Maxim.
All the specimens were collected and cultivated in
Institute of Chinese Herbal Medicines, Hubei Academy
of Agricultural Sciences. Plant materials were authen-
ticated by Prof. Lin (Hubei Academy of Agricultural
Sciences) and the vouchers were kept in Wuhan University.
Young leaves were dried in silica gel and used as
research materials. Detailed messages of specimens
were depicted in Table 1. In addition, for the herbal
medicine preparations, three items available in the
marketplace were bought from Aierkang pharmacy
(Wuhan) and used in ARMS analysis, including
XiangGuoJianXiaoPian (sugar coated tablet, produced
by Yunnan Yunhe Medicine Co., Ltd., batch number:
07020602), a resource containing the powder of roasted
Rhizoma Valeriana jatamansi, and RenShenGuiPiWan
(honeyed pill, produced by Jilin Shuangshi Medicine
Co., Ltd., batch number:080101) and XiaoYaoWan
(watered pill, produced by China Resources Sanjiu
Medical & Pharmaceutical Co., Ltd., batch number:
090301), the resources without Rhizoma Valeriana
jatamansi.
2 Extraction of genomic DNA, PCR amplification,
cloning and data analyzing
The dry leaves and herbal medicine preparations
were ground to fine powders in liquid nitrogen.
Genomic DNA was extracted using the DNA extraction
kit (Beijing Tiangen Co., Ltd). According to Fukui
et al.[7], the complete 5S rDNA NTS region were
amplified by using primers of NF (5’-GATCCCATCAG
AACTCCGAAG-3’) and NR (5’-CGGTGATTTAGTG
CTGGTAT-3’). Each 25 μL PCR mixture contained:
50 ng of total genomic DNA, 400 mmol·L−1 of each
dNTP, 1U Taq polymerase (Dingguo Biotechnology Co.,
Ltd.), 1×PCR buffer, 2.0 mmol·L−1 MgCl2, and 0.4
mmol·L−1 of each primer. Samples were amplified
through 30 thermal cycles in MJ-PTC100TM (Eastwin
Life Sciences, Inc.), and each cycle consisting of 94 ℃
for 30 sec, 55 ℃ for 30 sec, and 72 ℃ for 1 min. The
PCR products were extracted from gels, cloned into
pGEM-T vector (Promega), and at least three clones
of each specimen were sequenced on an ABI3730XL
sequencer (Sunbiotech Co., Ltd, Beijing, China). All
sequences were submitted to GenBank. Sequences
were aligned and clustered using Clustal X[8] and
DNAMAN Version 4.0 software, respectively.
3 ARMS analysis
Based on the nucleotide substitutions in the NTS
of V. jatamansi, six reverse diagnostic primers with
complementary 3’-residues were designed for ARMS
analysis, separately (Table 2). All the corresponding
forward primers were NF. The PCR procedure of
ARMS analysis was the same as the PCR amplification
of 5S rDNA region and the annealing temperature was
optimized within the range of 55−60 ℃. The genomic

Table 1 Detail messages of tested Valeriana species
Species Voucher Genbank accession number Size of 5S rDNA sequence
V. jatamansi VVJ080501 EU926613−EU926615 307−342 bp
V. pseudofficinalis VVP080502 EU926616−EU926617 330−331 bp
V. officinalis var. latifolia VVO080503 EU926605−EU926607 330−331 bp
V. hardwickii VVH080504 EU926603−EU926604 361−355 bp
V. flaccidissima VVF080505 EU926608−EU926612 308−351 bp

DIAO Ying, et al: Molecular authentication of Valeriana jatamansi by ARMS based on 5S rDNA sequence · 1069 ·


DNA isolated from the herbal drugs of XiangGuoJian-
XiaoPian, RenShenGuiPiWan and XiaoYaoWan were
tested with ARMS analysis in the same PCR condition
using the selected diagnostic primer. PCR products were
checked on a 1.2% agarose gel and were visualized by
ethidium bromide staining under UV. Negative controls
were performed along with each PCR amplification.
Three independent experiments for ARMS were tested
to verify the reproducibility of the methods.

Table 2 Diagnostic primers for ARMS analysis of V. jatamansi.
Bold nucleotides are complementary with V. jatamansi specially.
Forward primer was the same as NF (5’-GATCCCATCAGAACT
CCGAAG-3’)
Reverse primer name Nucleotide sequence (5’–3’)
zhiR1 CTCGCATCACGGCAACCAAACGCTC
zhiR2 TCGCATCACGGCAACCATACGCT
zhiR3 TCGCATCCTGGCATCCATTCGTT
zhiR4 GCATCACGGCAACCAAACGCTG
zhiR5 GCATCACGGCAACCAAACGCTT
zhiR6 CTCGGCAACCAAACGCTCC

Results and discussion
The PCR product contained partial 5S rRNA gene
and complete NTS sequences. Size differences of
PCR products were not detected under agarose gel
electrophoresis. According to the sequencing results,
different 5S rDNA sequences of each tested species
were selected for the further studies. The sequence
analysis of different clones found that the coding
sequences of 5S rRNA gene were conserved, but the
NTS sequence varied in the tested species. The NTS
region varied from 189 to 225 nt in V. jatamansi, 212−
213 nt in V. pseudofficinalis, 212−213 nt in V. officinalis
var. latifolia, 237−244 nt in V. hardwickii and 190−233
nt in V. flaccidissima. It seems that NTS divergence
within one species was comparably stable for V. flac-
cidissima, V. hardwickii and V. jatamansi among the
five tested species. In the phylogenetic tree, NTS
sequences from V. flaccidissima, V. hardwickii and
V. jatamansi clustered into a subclade, separately, while
those of V. pseudofficinalis and V. officinalis var. latifolia
nested together (Figure 1). This result suggested
V. pseudofficinalis and V. officinalis var. latifolia
had the close relationship among the tested species.
V. hardwickii showed the longest branch in the cluster
tree which indicated it had the farthest genetic distance
to the other four species.

Figure 1 Phylogenetic tree constructed from the 5S rDNA NTS
sequences. A scale bar showing the distance of 0.05 is at the
top left

For ARMS analysis, the same nucleotide substitu-
tions in different clones of V. jatamansi were found at
position 171 (EU926613), 167 (EU926614) and 174
(EU926615), respectively. Six diagnostic primers
were designed for ARMS analysis on the basis of the
SNP site. In order to ensure amplification specificity,
one strong destabilizing mismatch (G/C pair replaced
by G/T pair) was deliberately introduced into primer
zhiR5 at the 1st nucleotide from 3’ terminus[9]. The
five species were amplified with the six specific
primers, respectively. Theoretically, only V. jatamansi
should be successfully amplified. Expectedly, primer
zhiR5 could amplify the fragment approximate 200 bp
in V. jatamansi with the annealing temperature 58 ℃
and no PCR product was observed in the other four
tested species at the same time (Figure 2a). The rest of
diagnostic primers could not amplify the expected DNA
fragment in all the PCR amplifications. Therefore,
these five primers could not be used for authentication
of V. jatamansi except primer zhiR5. Three herbal
medicine preparations from market were used to test
the reliability of ARMS method using primer pair
zhiR5 and NF. The sample containing V. jatamansi
showed the positive fragment as expected after the PCR
amplification, while no PCR product was found in
the other two herbal medicine preparation which not
containing the resource of V. jatamansi (Figure 2b).
These results suggested this ARMS analysis could
be used not only to authenticate the genuine plant of
· 1070 · 药学学报 Acta Pharmaceutica Sinica 2010, 45 (8): 1067−1070

V. jatamansi from Valeriana species but also identify
V. jatamansi from the herbal medicine preparations.


Figure 2 Authentication of V. jatamansi from Valeriana species
(a) and compound herbal medicines (b) by ARMS analysis with
diagnostic primer zhiR5 and NF. M: DL2000 DNA marker,
1: V. jatamansi, 2: V. hardwickii, 3: V. officinalis var. latifolia,
4: V. flaccidissima, 5: V. pseudofficinali, 6: XiangGuoJianXiaoPian,
7: RenShenGuiPiWan, 8: XiaoYaoWan, C: Negative control

In this study, 5S rDNA sequence is proved to be a
useful species-specific marker for phylogenetic analysis
of Valeriana species. Thus we could get deep insight
of the relationship among the species on DNA level
besides the morphological analysis. V. pseudofficinalis
was the most widely used for sedation or hypnosis in
oriental traditional medicine. Because of the genetic
properties and phylogenetic relationships, V. officinalis var.
latifolia could be the substitution of V. pseudofficinalis,
but V. jatamansi must be doubted for this medicinal
usage. On the other hand, molecular authentication of
the herb medicines has many advantages than other
methods relied upon morphological and histological
inspections, such as reliability, convenience and
precision. 5S rDNA variation owing to insertions/
deletions, minirepeats and pseudogenes afford the
potential possibility to characterize the herbal medicines.
Moreover the tested materials have lost some of their
original botanical features, so it is more difficult to be
identified by the traditional methods. So SNP analysis
with ARMS technique can be used to specifically
identify V. jatamansi out from other Valeriana species
and herbal medicines.
Acknowledgments: The authors wish to thank Prof.
Yunchun Song (Wuhan University) for grammatical suggestion.
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