Study on genetic polymorphism of genus Acorus and its
application for standardization of Acori Graminei Rhizoma
Name of dispatched researcher（派遣研究者名）

Lanlan FAN

Instructor（指導教官，研究指導者）

Shao-Qing CAI. Ph. D.

Affiliation of Instructor（所属）

Peking University

Host Collaborator（受入研究者）

Katsuko Komatsu

Introduction
Acori Graminei Rhizoma is originated from the rhizome of Acorus gramineus Solander
and has been used as analgesic, sedative, stomachic and anthelmintic drugs in Japan. While
the rhizome of A. calamus Linn. also has been used as a stomachic in Asian countries, but
its demand is few in Japan. Although Acori Graminei Rhizoma is imported from China, thebotanical source of this drug mainly used in China is not A. gramineus but A. tatarinowii Shott[1]. In China, there are four Acorus species including above 3 species [2]. The rhizome of Acorus
species has been reported to have sedative, anticonvulsant, enhancing memory, lowering blood
lipids, regulating heart rate, stomachic, antispasmodic, antimicrobial, anticancer, antiepileptic
activities besides toxicity [1]. For the safe and efficient uses of Acori Graminei Rhizoma or Acori
Tatarinowii Rhizoma, the standardization is necessary in China and Japan. Until now a lot ofchemical studies on the rhizomes of A. calamus were performed to demonstrate several variations
on composition [3-5]. However, comparative study between the rhizomes of A. gramineus and A.
tatarinowii is few because the correct identification of two drugs as well as plants is very difficult.
In order to find out molecular marker for identification and further to clarify their chemicalcomposition, the study on genetic polymorphism of genus Acorus is carried out. Until now, wehave investigate the ITS sequences of 41 plant specimens belonging to the 3 Acorus species
which were collected from Anhui, Zhejiang and Chongqing in China and 17 crude drug samples
from markets of Japan and China. The results indicated significant genetic polymorphism in ITS
sequences of A. tatarinowii and A. gramineus [6]. Eight types of ITS sequences (types T1~T8)
were found in A. tatarinowii and three types (G1~G3) in A. gramineus.
In the present study, we collected plant specimens from Guangxi, China and crude drug
samples from Japan, China and Korea, subsequently analyzed their ITS sequences andincorporated the results to the previous achievement, in order to obtain more evidences forclarification of genetic variations for standardize Acorus Rhizoma.

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the results to the previous achievement, in order to obtain more evidences for clarification ofgenetic variations for standardize Acorus Rhizoma.

Materials and Methods
Materials
Methods
Plant andand
crude
drug samples

Plant and crude drug samples

Fifteen plant specimens which were identified as A. gramineus by ourselves were collected

Fifteen plant specimens which were identified as A. gramineus by ourselves were collectedfrom Guangxi Province, China (Table 1, Fig. 1), and six crude drug samples collected from markets

from Guangxi Province, China (Table 1, Fig. 1), and six crude drug samples collected fromof China
Japan and
Korea
Vouchers
are depositedat the MuseumofMuseum
Materia Medica,marketsof ChinaJapan
and(Table
Korea2).(Table2). Vouchers
are depositedat theof Materia
Medica,Instituteof Natural
Toyama, Japan.
Instituteof Natural
Medicine,Medicine,
UniversityUniversityof Toyama,ofJapan.

Table 1 Plant materials of A. gramineus and their types of ITS sequences.
Type of ITS

Code No.

Location of Collection

Date of collection

SL1

Shanglin, Guangxi, China

2012.10.

N3

SL2Shanglin, Guangxi, China

2012.10.

N1

L1

Longan, Guangxi, China

2012.10.

N4

L2Longan, Guangxi, China

2012.10.

N4

L3Longan, Guangxi, China

2012.10.

N5

L4Longan, Guangxi, China

2012.10.

N1

L5Longan, Guangxi, China

2012.10.

N3

L6Longan, Guangxi, China

2012.10.

N5

L7Longan, Guangxi, China

2012.10.

N1

L8Longan, Guangxi, China

2012.10.

N5

L9Longan, Guangxi, China

2012.10.

N4

L10Longan, Guangxi, China

2012.10.

N1

L11Longan, Guangxi, China

2012.10.

N1

L12Longan, Guangxi, China

2012.10.

N1

Form 2-2（様式２－２）
L13
Longan, Guangxi, China

2012.10.

N2

sequence

3

Fig. 1 Plant Samples from Shanglin (left) and Longan (right), Guangxi, China
Table 2 Crude drug materials and their types of ITS sequences, as well as identification－ 76 －
results
TMPW

Type ofFig. 1 Plant Samples from Shanglin (left) and Longan (right), Guangxi, China
Table 2 Crude drug materials and their types of ITS sequences, as well as identification
results
No.

Name

TMPW
No.

Produced area

1

セキショウブ

26407

Anhui, China

2

石菖蒲

26537

Sichuan, China

3

石菖蒲

26600

Anhui, China

4

石菖蒲

26617

Jiangxi, China

5

石菖蒲

27715

Guangxi, China

6

石菖蒲

26926

Jeju, Korea(Wild)

Type of
ITS
sequence

Identity

T3

A. tatarinowii

T8

A. tatarinowii

C1

A. calamus

Qizhou, Zhejiang, China

T1

A. tatarinowii

Nanning, Guangxi, China

G3

A. gramineus

Seoul, Korea

T1

A. tatarinowii

MarketTochimoto Tengaido,
Osaka, JapanTCM market,
Chongqing, China
Huangshan, Anhui,
China

Genomic
DNA
Extraction
Genomic
DNA
Extraction

Total DNA of dry leaves (40-80 mg) was extracted using DNeasy Plant Mini Kit (QIAGEN,

Total DNA of dry leaves (40-80 mg) was extracted using DNeasy Plant Mini Kit (QIAGEN,

Germany), and crude drugs (120-200 mg) using Blood & Cell Culture DNA Mini Kit (25) (QIAGEN,

Germany), and crude drugs (120-200 mg) using Blood & Cell Culture DNA Mini Kit (25) (QIAGEN,

Germany). DNA quality and quantity were determined by electrophoresis on 1% agarose gel

Germany).
DNA quality
and quantity
were determinedby electrophoresis
on 1% agarose gel stained
stainedby UltraPower
DNA/RNA
Safedye(Gellex Internationalltd., Japan).
by UltraPower DNA/RNA Safedye (Gellex International ltd., Japan).
Polymerase Chain Reaction (PCR) Amplification
Primers for amplification of ITS regions are listed in Table 3 and their locations are

Polymerase
Chain Reaction (PCR) AmplificationForm
2-2（様式２－２）

indicated in Fig. 2. The PCR was performed in a 25 μL reaction mixture, containing 0.4 mM ofPrimers
amplificationof ITS1×PCR
regionsbuffer
are listed
Table
3 and their locations
are Life
indicatedeach dNTP,
0.4formMof each primer,
andin0.5
U KOD-Plus-Neo(Toyobo
Science,
Japan)
1followed
μLPCR
of templateDNA.ofThethermalforandamplificationasfollows:
in Fig.
2. Thewas
performed
in95
a 25
mixture,containing
ofwere
each
dNTP,
for
5 and
min,
by 35
cycles°C μL
forreaction
30cycles, 52conditions°C for 50
s,
680.4
°CmM
for 60s, andfinal
95 0.4°C formin,
followed
35 cyclesof 95
forU30KOD-Plus-Neo
s, 52 °C for 50(Toyobos, and 68°CScience,for 60 s,Japan)
and final
mM5 ofeachprimer,by
1×PCR
bufferand°C0.5Lifeextension at 68 °C for 20 min. 2 μL of resulting PCR-amplicon was detected by 1.0% agarose gel

extension at 68 °C for 20 min. 2 μL of resulting PCR-amplicon was detected by 1.0% agarose geland 1 μL of templateDNA.
The thermal
cycle
conditions
amplification
were
follows:
95 °C
electrophoresis
and theremainedpart waspurifiedusingforWizard®
SV GelandasPCR
Clean-Upelectrophoresis and the remained part was purified using Wizard® SV Gel and PCR Clean-Up

System(Promega,U.S.A.).
U.S.A.).The
Theamplification
amplification strategy
showedin Fig.
2. 2.
System(Promega,
strategywas
wasshowedin Fig.
4

Fig. 2 Location of primers used in PCR amplification and sequencing of ITS region

Table 3 Sequences of primers used in this study
Primer namePrimer sequences (5′→3′)－ 77 －

Length (bp)

Forward primer
ITS-1F

TCCACTGAACCTTATCATTTAG

22

Fig. 2 Location of primers used in PCR amplification and sequencing of ITS region

Table 3 Sequences of primers used in this study
Primer namePrimer sequences (5′→3′)

Length (bp)

Forward primer
ITS-1F

TCCACTGAACCTTATCATTTAG22

In 18S-25S-5′F

TCTCGCATC GATGAAGAACG

20

ITS5

GGAAGGAGAAGTCGTAACAAGG

22

In-18S-25S-3′R

GACTCGATGGTTCACGGGATTCT

23

18S-25S-3′R

CCATGCTTAAACTCAGCGGGT

21

ITS4

TCCTCCGCTTATTGATATGC

20

Reverse primer

Sequence Determination

Sequence Determination

The purified PCR products served as template, sequencing reaction were performed using

The purified PCR products served as template, sequencing reaction were performed using thethe Big Dye Terminator V3.1 (Applied Biosystems, U.S.A.) with the same primers as in PCR

Big Dye Terminator
V3.1(Applied
Biosystems,
U.S.A.)
with condition
the same wasprimers
as forin PCR
amplificationand several
insideprimers.
The thermal
cycling
96 °C
1 min,
severalcycling
condition
was 96 °C
for 1 min, using
25
25 amplification
cycles of 96 and°C for10 s,inside
50 °Cprimers.
for 5 s The
andthermal
60 °C for4 min.
After further
purification
TM
Bigcycles
Dye of
XTerminator
U.S.A.),
the supernatants
were
96 °C for 10 s,Purification
50 °C for 5 sKit
and (Applied
60 °C for Biosystems,
4 min. After further
purification
usingBigDye

injected to andTManalyzed on an automated DNA sequencer (ABI Prism 3100-Avant Genetic

XTerminator

Purification Kit (Applied Biosystems, U.S.A.), the supernatants were injected to andAnalyzer, Applied Biosystems, U.S.A.). The obtained sequences were edited and aligned by the

analyzed on an automated DNA sequencer (ABI Prism 3100-Avant Genetic Analyzer, Applied

AutoAssemble program (Version 1.3.0, Applied Biosystems, U.S.A.), and adjusted manually

Biosystems,
U.S.A.). The obtained sequences were edited and aligned by the AutoAssemble
wherenecessary.
The boundaries
of ITS1,
5.8SBiosystems,
and ITS2 regions
assignedmanuallyby comparison
with a reference
program(Version 1.3.0,
AppliedU.S.A.),were
and adjusted
where necessary.
sequence in Genbank FJ874939.
Results and Discussion

5

The nucleotide sequences of rDNA ITS region of fifteen plant samples from Guangxi, China,
and six crude drug samples collected from markets of China, Japan, and Korea were determined
and compared. Amplification products (~800 bp fragment) of the whole ITS region (ITS1-5.8SITS2) were successfully obtained by using the total DNA from plant specimens as template.
However, it is difficult to amplify the whole ITS region in the case of crude drug samples, due
to severe degradation of the extracted total DNA. Therefore, we separately amplified the ITS15.8S(5’-end) and 5.8S(3’-end)-ITS2 regions for the crude drug samples, subsequently decided theirsequences and finally assembled them as one piece for comparison.

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difficult to amplify the whole ITS region in the case of crude drug samples, due to severe
degradation of the extracted total DNA. Therefore, we separately amplified the ITS1-5.8S(5’-end)
and 5.8S(3’-end)-ITS2 regions for the crude drug samples, subsequently decided their sequences andfinally assembled them as one piece for comparison.

Fig. 3 Comparison of ITS sequences
FJ874939: A reference ITS sequence of A. gramineus registered in GenBank. Dot means the same
nucleotide as that in the 1st line of the FJ874939gra sequence. R=A & G, W=A & T, Y=C & T.

As for the fifteen plant specimens collected6 from Guangxi, China, the whole lengths of the
ITS region were sequenced except for four samples (SL1, L5, L6 and L7) in which about 100 bp
sequence at the 5’-end of ITS1 region were unreadable. Five types of sequences (type N1 ~ N5)
were detected and none of them was identical to our previously decided sequences (types G1~G3
and types T1~T8). Though BLAST search in GenBank, these sequences showed high homology
to a reference sequence of A. gramineus (Accession No. FJ874939) in Genbank. The sequencescomparison is shown in Fig. 3. Four sites of nucleotide substitutions at alignment positions 44th,
309th, 513rd and 514th, and one site of indel at positions 36th were observed among them. In
addition, additive nucleotides (double peaks in sequencing electrophorogram) were detected at
the nucleotide positions 309th, 513rd and 514th in the types N2~N5 sequences, presented as
R[Guanine (G) & Adenine (A)], W[A & Thymine (T)] and Y[T & Cytosine (C)], which might infera hybrid origin of such specimens. Type N1 was a putative pure line sequence.
A phylogenetic tree based on ITS sequences of the 3 Acorus species, including type N1 and－ 79 －

origin of such specimens. Type N1 was a putative pure line sequence.
A phylogenetic tree based on ITS sequences of the 3 Acorus species, including type N1 and ourpreviously determined sequences, was reconstructed by UPGMA method (Fig. 4). Three main clades
g3
FJ874938gra
FJ874943tat
FJ874939gra
FJ874944tat

A. gramineus

N1
AF209785tat
g2
g1
t5
t9
t8
t6
FJ874940gra
t7
AF468820grat4

A. tatarinowii

AF468818tat
t1
t2
FJ874941gra
t3
FJ874945tat
FJ874935cal
c1

A. calamus

0.005

Fig. 4 Phylogenetic tree based on ITS sequences from the three Acorus species(T1~T8: ITS sequences of Acorus tatarinowii, G1~G3: ITS sequences of A. gramineus, C1: ITS
sequences of A. calamus. FJxxxxxx and AFxxxxxx: Accession No. of ITS sequences of Acorus
species registered in GenBank.

our previously determined sequences, was reconstructed by UPGMA method (Fig. 4). Three

7 a basic position, showed far relationship frommain clades were formed. A. calamus, located at
the other two species; A. tatarinowii and A. gramineus were clustered into two clades. Therefore,
we tentatively named them as A. calamus-, A. tatarinowii- and A. gramineus-clades. Type N1
belonged to the A. gramineus-clade, which was consistent with our identification.
Six crude drugs were analyzed and their ITS sequence were compared with our previously
obtained data. Sample No. 1 (TMPW No. 26407), which was produced in Anhui Prov., China
and available in Japanese market, possessed type T3 sequence. Therefore, it was identified as
deriving from A. tatarinowii. Four samples (No. 2~5) collected from various locations in China
showed types T8, C1, T1 and G3 sequences; thus their botanical origins were identified as A.
tatarinowii, A.calamus, A. tatarinowii and A. gramineus, respectively. Although these 4 crude
drugs were sell as “ShiChangpu (Sekisyoubu)” in markets, their botanical sources are completely
different, which must result in unstable and unexpected therapeutic effects. Especially, thesample No. 3, which was collected for local market in Anhui Prov., China, should be Changpu(Syoubu), but not “ShiChangpu (Sekisyoubu).” Sample No. 6 which was collected from Seoul,
Korea and was claimed to be from wild source growing in Jeju Island, revealed type T1 sequence.
Therefore, it was identified as A. tatarinowii.

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This study found out several new types of ITS sequences of A. gramineus, proving moreevidences for clarification of genetic variations of Acorus Rhizoma, and demonstrated the ITS
sequence was useful and powerful for authentication of Acorus Rhizoma.
Acknowledgement
The author is very grateful to Professor Katsuko Komatsu and assistant professor Shu Zhu(Department of Pharmacognosy, Institute of Natural Medicine, University of Toyama, Japan) for
offering good platform for this research. The author is grateful to all of the laboratory members in
Department of Pharmacognosy, Institute of Natural Medicine, University of Toyama. This work
was supported by 2012 GRANT-IN-AID for general research II program, Institute of Natural
Medicine, University of Toyama, Japan.
References
1. Committee, C.P., Pharmacopoeia of the People's Republic of China. 2010 Edition ed. Vol. Part I.
2010, Beijing: Chemical Industry Press. p. 85.
2. Committee, E., Flora Reipublicae Popularis Sinicae. 1979, Science press: Beijing. p. 4-8.
3. Duvall, M.R., et al., Phylogenetic analysis of rbcL sequences identifies Acorus calamus as theprimal extant monocotyledon. Proc Natl Acad Sci USA, 1993. 90(10): 4641-4644.
4. Sugimoto, N., et al., DNA profiling of Acorus calamus chemotypes differing in essential oil
composition. Biol Pharm Bull, 1999. 22(5): 481-485.
5. Goremykin, V.V., et al., Analysis of Acorus calamus chloroplast genome and its phylogenetic
implications. Mol Biol Evol, 2005. 22(9): 1813-1822.
6. 小川 瞬：Acorus 属植物の遺伝子多型と石菖蒲の基原に関する研究，修士論文，2010.

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