Botanical Studies (2009) 50: 127-135.

*

Correspondence author: Dr. Kur-Ta Cheng, E-mail:

ktbot@tmu.edu.tw; Tel: +886-2-27361661 ext. 3169;

Fax: +886-2-27356689; Zheng-Tao Wang, E-mail:

wangzht@hotmail.com; Tel: +86-21-51322507; Fax:

+86-21-51322519.

INTRODUCTION

Radix Salviae Miltiorrhizae, commonly known as

Danshen in China, is one of the most important traditional

Chinese medicines and belongs to the Salvia genus. It

can stimulate blood circulation to remove blood stasis,

relieve restlessness and tranquilize the mind, and is

widely used in traditional Chinese medicinal preparation

to treat cardio-cerebral vascular diseases (Zhou et al.,

2005). The genus Salvia L. is composed of 78 species,

24 varieties and eight forma in China. Among them only

S. miltiorrhiza is listed as a botanical source of Danshen

in the Chinese Pharmacopoeia (2005). However, more

than 20 other species (including varieties and forma)

Comparison of rDNA ITS sequences and tanshinones

between Salvia miltiorrhiza

populations and Salvia

species

Hong XU

1, 3

, Zheng-Tao WANG

1,3,

*, Kur-Ta CHENG

2,

*, Tao WU

1,3

, Li-Hua GU

1,3

, and Zhi-Bi HU

1,3

1

Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, Institute of Chinese Materia Medica,

Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China

2

Department of Biochemistry, Taipei Medical University, Taipei, Taiwan

3

Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai, P. R. China

(Received April 28, 2008; Accepted October 16, 2008)

ABSTRACT.

Danshen is one of the well-known herbs classified as "blood-invigorating" in traditional

Chinese herbal medicine. Although Salvia miltiorrhiza is listed as the only botanical source of Danshen in the

Chinese Pharmacopoeia, more than 20 other Salvia species are also used as Danshen in China while about 10

Salvia species are employed as non-Danshen. In order to identify S. miltiorrhiza and related Salvia species,

and evaluate their quality, the rDNA ITS regions of ten Salvia species and twelve S. miltiorrhiza populations

were sequenced and compared, and tanshinones (including cryptotanshinone, tanshinone I and tanshinone

IIA) were quantitatively determined using the HPLC method. The nucleotide sequences of Salvia samples

showed obvious diversity, and each Salvia species and S. miltiorrhiza population were found to have a unique

sequence in the ITS region, so that they could be distinguished at the DNA level. Cluster analysis divided

the Salvia species into two main groups. Salvia miltiorrhiza and six other Danshen species had a closer

phylogenetic relationship and were differentiated from the non-Danshen species, results which corresponded

with their medicinal requirements. The HPLC data showed that tanshinones were present mainly in the roots of S.

miltiorrhiza and eight other Salvia species, including six Danshen species, and the contents were significantly

different in each of them. However tanshinones were not detected in the non-Danshen-S. deserta, and this

chemical variation can distinguish Danshen and non-Danshen species from each other. Although no direct

cluster analysis correlation between chemical and DNA data appeared, both types of analysis are important for

the quality evaluation of S. miltiorrhiza and related Salvia species.

Keywords: HPLC; rDNA; ITS; Salvia miltiorrhiza; Salvia; Tanshinones.

have been employed as Danshen in local areas in China

while about 10 species are described as non-Danshen and

are used to reinforce the kidney and lung, relieve toxic

heat, and subdue inflammation (Xiao et al., 1997; Guo

et al., 2002). This is confusing for consumers because

many species of Salvia resemble each other in shape,

and morphological differentiation is very difficult.

Tanshinones, the lipophilic diterpenoids quinines that

have been isolated from Danshen, are reported to be the

major bioactive components (Kim et al., 2002; Zhou et

al., 2006; Fu et al., 2007). Cryptotanshinone, tanshinone

I and tanshinone IIA are present in the greatest amounts.

In the Chinese Pharmacopoeia, the quantification of Radix

Salviae Miltiorrhizae is performed by determination

of tanshinone IIA with HPLC. However, this is not

sufficient to comprehensively evaluate the quality of the

herbal medicine because tanshinone IIA is not the sole

pharmaceutically active compound.

DNA-based polymorphism assay may offer an

alternative method of authenticating this herbal medicine.

mOleCUlAR BIOlOgy

128

Botanical Studies, Vol. 50, 2009

Here, the amplified ITS (internal transcribed spaces)

region of ten Salvia species, including S. miltiorrhiza and

six other Danshen species (S. digitaloides, S. przewalskii,

S. flava, S. trijuga, S. yunnanensis, S. bowleyana), one

non-Danshen species (S. deserta) and two non-medicinal

species (S. liguliloba, S. chienii) were sequenced and

compared to explore the possibility of using the ITS region

as a molecular marker to differentiate the Salvia species.

The contents of cryptotanshinone, tanshinone I and

tanshinone IIA were determined to evaluate the chemical

variation in Salvia species and S. miltiorrhiza populations.

mATeRIAlS AND meTHODS

materials

Fresh plants and dried crude drugs were collected from

different regions of China (Table 1) and identified by Dr.

Li-Hong Wu and Dr. Hong Xu of the Shanghai University

of Traditional Chinese Medicine. Voucher specimens

were deposited in the Shanghai University of Traditional

Chinese Medicine, Shanghai, China. Cryptotanshinone

(purity>97%), tanshinone I (purity>96%) and tanshinone

IIA (purity>96%) were purchased from National Institute

for the Control of Pharmaceutical and Biological Products

(P.R. China).

DNA Sequencing

Genomic DNA Extraction. Samples were subjected

to DNA isolation with a modified CTAB (cetyltrimethyl

ammonium bromide) protocol with additional purification

by DNA Purification Kit (Watson Biotechnologies, Inc.,

Shanghai, China).

PCR Amplification and Sequencing. The primers used

for the amplification of ITS were the P18S forward primer

(5¡¦-ATT GAA TGG TCC GGT GAA GTG TTC G-3¡¦)

and P26S reverse primer (5¡¦-AAT TCC CCG GTT CGC

TCG CCG TTA C-3¡¦). The PCR reactions followed those

of a previous report (Xu et al., 2006). The PCR products

were purified by a PCR Purification System (Watson, P.R.

China) and subcloned into a TA cloning vector pGEM-T

(Promega, USA) for sequencing by an ABI PRISM

TM

377

Genetic Analyzer (ABI, USA). Each species was tested on

two to three specimens from the same collection. Several

clones of each PCR product were sequenced to avoid

errors introduced by Ta q DNA polymerase.

Chemical analysis

The sample solution was prepared by ultrasonicating

the powders of the dried root using a mixture of

chloroform and methanol (8:1) and then analyzed by an

Agilent HP1100 liquid chromatograph (Hewlett Packard,

Table 1. Plant used in this study and their accession numbers in Genbank.

No. Taxon

Subgen.

Locality

Abbreviation Accession No. Medicinal parts and uses

1 S. digitaloides

Salvia Lijiang, Yunnan SD-YNLJ

DQ132869 Root used as Danshen

2 S. przewalskii

Salvia Lijiang, Yunnan SP-YNLJ

DQ132862 Root used as Danshen

3 S. flava

Salvia Lijiang, Yunnan SF-YNLJ

DQ132867 Root used as Danshen

4 S. trijuga

Sclarea Lijiang, Yunnan ST-YNLJ

DQ132870 Root used as Danshen

5 S. yunnanensis Sclarea Lijiang, Yunnan SY-YNLJ

DQ132866 Root used as Danshen

6-1 S. miltiorrhiza Sclarea Shangluo, Shanxi SM-SXSL

DQ132863 Root used as Danshen

6-2 S. miltiorrhiza

Zhongjiang, Sichuan SM-SCZJ

DQ132864 Root used as Danshen

6-3 S. miltiorrhiza

Shanghai

SM-SH

EU591975 Root used as Danshen

6-4 S. miltiorrhiza

Pinyi, Shandong SM-SDPY

EU591972 Root used as Danshen

6-5 S. miltiorrhiza

Yuncheng

,

Shanxi SM-SXYC

Root used as Danshen

6-6 S. miltiorrhiza

Yishui, Shandong SM-SDYS

Root used as Danshen

6-7 S. miltiorrhiza

Sheyang, Jiangsu SM-JSSY

EU591976 Root used as Danshen

6-8 S. miltiorrhiza

Bozhou, Anhui SM-AHBZ EU591964 Root used as Danshen

6-9 S. miltiorrhiza

Shanxi

SM-SX

EU591974 Root used as Danshen

6-10 S. miltiorrhiza

Binhai, Jiangsu SM-JSBH

Root used as Danshen

6-11 S. miltiorrhiza

Shandong

SM-SD

EU591973 Root used as Danshen

6-12 S. miltiorrhiza

Rugao, Jiangsu SM-JSRG

Root used as Danshen

7 S. bowleyana

Sclarea Linan, Zhejiang SB-ZJLA

EU592037 Root used as Danshen

8 S. deserta

Sclarea Urumuqi, Xinjiang SDE-XJWLMQ DQ132865 Non-Danshen, herb used to cause

diuresis, remove toxic heat and

resolve phlegm.

9 S. liguliloba Allagospadonopsia Linan, Zhejian SL-ZJLA

EU592036 Not used as herbal medicine

10 S. chienii Allagospadonopsia Huangshan, Anhui SC-AHHS DQ132868 Not used as herbal medicine

XU et al. ¡X rDNA ITS sequences and tanshinones of

Salvia

species

129

Palo Alto, CA, USA) with a Polaris C18 column (5 £gm,

250 ¡Ñ 4.6 mm). Elution was with methanol - water (75:25,

V:V) at 1 ml /min. The elution was monitored at 270 nm.

The three tanshinones were completely separated from

the other compounds. The detector response was linear

from 3.98¡Ñ10

- 3

to 5.373¡Ñ10

-1

£gg of cryptotanshinone

(y=4534.32824x+0.2926528, R=0.99999), 4.10¡Ñ10

-3

to

5.535¡Ñ10

-1

£gg of tanshinone I (y=3759.50369x+2.5238072

R=0.99999), and 4.50¡Ñ10

-3

to 6.075¡Ñ10

-1

£gg of tanshinone

IIA (y=5040.58974x+5.4577613, R=0.99999) was used

for the quantitative data. This method was sensitive and

accurate with good reproducibility.

Data Analysis

The sequences as well as the content of the tanshinones

were subjected to similarity matrix and cluster analyses

using the Clustal W programs (Multiple sequence

alignment programs, version 1.7), Molecular and

Evolutionary Genetic Analysis (MEGA, version 3.1), and

Numerical Taxonomy and Multivariate Analysis System

program package for PC (NTSYS-pc, version 2.1). The

sequences were aligned and compared using the Clustal

W programs, and the sequence divergence was analyzed

using the MEGA2 programs. The genetic dendrogram was

constructed by the Neighbor Joining (NJ) tree construction

method (Nei, 1987) using the MEGA2 programs.

The similarity index of the content of tanshinones

was constructed using similarity for interval data with

Euclidean distance [The Euclidean distance between

two points P= (p

1

, p

2

,¡K¡K, p

n

) and Q= (q

1

, q

2

¡K.., q

n

) is

defined as

n

2

2

2

2

1 1 2 2

n n

1

( ) ( ) ..... ( ) ( )

i i

i

p q p q

p q

p q



   

.

].

The chemical dendrogram was constructed by applying

the unweighted pair group method with arithmetic

averages (UPGMA) using NTSYS-pc programs. A Mantel

test with 1000 permutations was conducted by NTSYS-pc

to compare the chemical and genetic similarity matrices.

ReSUlTS

Sequence analysis

The ten Salvia species, including twelve S. miltiorrhiza

populations, were tested in this study. An approximately

700 bp fragment¡Xincluding the end of the l8S rDNA, the

beginning of the 26S rDNA, and the ITS1-5.8S rDNA-

ITS2 regions in their entirety¡Xwas specifically amplified

and sequenced for all samples. All specimens of the same

species from the identical collection place displayed iden-

tical sequences. Alignment of these sequences showed

that the 18S, 26S and 5.8S rDNA were highly conserved.

The ITS1 and ITS2 regions were more variable with the

inter-specific sequence divergence ranging from 1.33% to

19.4% in Salvia species, and from 0.22% to 3.17% in S.

miltiorrhiza populations (Figure 1, Table 2). It is clear that

genetic divergence in the rDNA ITS region exists among

Salvia samples, and each Salvia species and the population

of S. miltiorrhiza was found to have a unique sequence in

its ITS region, so its members can be easily distinguished

at the DNA level.

Based on the ITS1 and ITS2 sequence from Salvia

species, a NJ phylogenetic tree was constructed (Figure

2). The Salvia taxon clustered naturally into two separate

groups. Salvia miltiorrhiza, the six other Danshen

species, and the two non-medicinal species clustered

together, had a closer phylogenetic relationship, and were

differentiated from non-Danshen species. All members

of S. miltiorrhiza from different populations clustered

together. Based on the classification of Salvia in the Florae

Repubicae Sinicae (1977), the 10 studied Salvia species

should be grouped under three subgenus: Salvia, Sclarea

and Allagospadonopsia. The polygenetic tree of Salvia

species deduced from the ITS region, however, does not

totally match the classification based on morphological

characters. The five species of Sclarea, S. trijuga, S.

yunnanensis, S. miltiorrhiza, S. bowleyana, and S. deserta

were separated into different clades, as were S. liguliloba

and S. chienii of Allagospadonopsia. The three Subgen.

Salvia species considered, S. digitaloides, S. przewalskii,

and S. flava formed a strongly supported monophyletic

group.

Chemical analysis

The HPLC method was established for the simultaneous

determination of tanshinones in Salvia samples. Table 3

shows the summary results. Figure 3 shows the HPLC

chromatogram of S. miltiorrhiza from Shangluo, Shanxi

(SM-SXSL). It was noticed that tanshinones were

present mainly in the extracts from the dried roots of S.

miltiorrhiza and eight other Salvia species, including

six Danshen species and two non-medicinal species,

and the contents of the tanshinones were significantly

different with the highest in S. przewalskii and the lowest

in S. miltiorrhiza from Sheyang, Jiangsu province (SM-

JSSY). The contents of tanshinone IIA met the standard

of the Chinese Pharmacopoeia (>2 mg/g) in only four S.

miltiorrhiza populations (SM-SDPY, SM-SDYS, SM-

SD, SM-SX) and four other Danshen species (SD-YNLJ,

SP-YNLJ, ST-YNLJ, SY-YNLJ). The results suggest

that the quality of Danshen is not only relevant to the

different species, but also to the distribution of the species.

Tanshinones were not detected in non-Danshen species,

according to this chemical variation, Danshen species and

Non-Danshen can be distinguished from each other.

Based on the chemical similarity matrices, a

dendrogram was constructed by applying the UPGMA

method (Figure 4). Cluster analysis divided all the samples

into two main groups according to the content of one of

the tanshinones. The SP-YNLJ sample, characterized by a

relatively high content of tanshinone IIA (14.08 mg/g), was

the first main group. The second main group was divided

into two subgroups: (1) the SM-SD group, with a high

content of cryptotanshinone (7.021 mg/g) and (2) a second

subgroup that was divided into three further subgroups:

130

Botanical Studies, Vol. 50, 2009

XU et al. ¡X rDNA ITS sequences and tanshinones of

Salvia

species

131

Figure 1. Alignments of ITS 1 and ITS 2 sequences of Salvia species, ITS 1 corresponds to position 1~244 bp; ITS 2: 245~478

(Dots indicate identical nucleotide with SF-YNLJ; hyphens indicate alignment gap).

132

Botanical Studies, Vol. 50, 2009

Figure 2. NJ phylogenetic tree based on ITS1+ITS2 sequenc-

es from Salvia species (Bootstrap values are shown above the

b ran ches) .

(I) The four S. miltiorrhiza populations (SM-SXSL, SM-

SDPY, SM-SD, SM-SX) were clustered together in a

cryptotanshinone group (2.915~3.322 mg/g); (II) the

seven other S. miltiorrhiza populations and five Salvia

species (SF-YNLJ, SB-ZJLA, SC-AHHS, SL-ZJLA,

SDE-XJWLMU), characterized by low concentrations

of three tanshinones, were clustered together; and (III)

the tanshinone IIA group (5.368~6.427 mg/g), in which

three Salvia species (ST-YNLJ, SD-YNLJ, SY-YNLJ)

were clustered. Three S. miltiorrhiza populations from

Jiangsu Province were clustered in the same subgroup

with a relatively low content of three tanshinones. Two of

three Shandong populations, SM-SDPY and SM-SDYS

were clustered into the cryptotanshinone group, and the

third Shandong population, SM-SD was clustered into the

cryptotanshinone dominant group. The Shanxi populations

also clustered into the separate subgroup. In addition, the

tanshinones were obviously found at higher concentrations

in four Danshen species (ST-YNLJ, SD-YNLJ, SY-YNLJ,

SP-YNLJ) than those in S. miltiorrhiza.

Of the 2000 permutations, the Mantel test, conducted

Table 2. The percent of sequence divergence of ITS sequences of 21 samples in Salvia species with gaps deleted in each pairwise

comparison of sequences (%).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1

2 1.33

3 1.57 2.02

4 5.80 6.28 6.57

5 7.54 7.30 7.32 8.56

6 8.29 8.05 8.07 8.80 2.71

7 7.78 8.05 8.07 8.81 3.88 4.59

8 8.49 8.77 8.79 9.53 3.86 4.56 1.57

9 8.53 8.04 8.31 9.05 3.87 4.58 2.03 2.48

10 8.27 7.78 8.05 8.79 3.64 4.34 1.80 2.25 0.67

11 9.31 9.07 9.09 9.83 4.59 5.06 3.18 3.63 2.94 2.71

12 8.31 8.07 8.09 8.83 3.65 4.12 1.81 2.26 1.58 1.35 1.80

13 7.98 7.75 7.77 8.50 3.16 3.40 1.34 1.79 1.11 0.89 2.02 0.67

14 8.26 8.02 8.04 8.77 3.63 4.33 1.80 2.24 1.57 1.34 2.71 0.89 0.89

15 8.53 8.30 8.32 9.05 3.87 4.58 2.03 2.48 1.80 1.57 2.71 1.12 1.12 0.22

16 8.26 8.02 8.04 8.77 3.63 4.33 1.80 2.24 1.57 1.34 2.71 0.89 0.89 0.44 0.67

17 7.98 7.75 7.77 8.50 3.40 4.10 1.57 2.02 1.34 1.11 2.48 1.12 0.67 0.67 0.89 0.67

18 8.29 7.78 8.07 9.05 4.10 4.81 2.03 2.70 2.02 1.79 3.17 1.35 1.34 1.34 1.57 1.34 1.56

19 8.00 7.77 7.79 8.52 3.40 4.10 1.57 2.02 1.34 1.11 2.02 1.12 0.67 0.66 0.89 1.11 0.89 1.56

20 8.26 8.02 8.04 8.77 3.40 4.10 2.03 2.02 1.79 1.57 2.94 1.57 1.11 1.56 1.80 1.56 1.34 2.02 1.34

21 17.3 17.3 17.7 19.4 17.6 17.5 16.6 18.1 18.4 18.7 19.0 18.2 17.8 18.4 18.1 17.8 18.1 18.7 18.1 18.4

1, SF-YNLJ; 2, SD-YNLJ; 3, SP-YNLJ; 4, ST-YNLJ; 5, SY-YNLJ; 6, SL-ZJLA, 7, SC-AHHS; 8, SB-ZJLA; 9, SM-SD; 10, SM-

SDYS; 11, SM-SXYC; 12, SM-AHBZ; 13, SM-SH; 14, SM-SDPY; 15, SM-SX; 16, SM-JSRG; 17, SM-JSBH; 18, SM-JSSY; 19,

SM-SXSL; 20, SM-SCZJ; 21, SDE-XJWLMQ.

XU et al. ¡X rDNA ITS sequences and tanshinones of

Salvia

species

133

to analyze the relation between the genetic and chemical

content matrices, indicated a low correlation (R= 0.19760)

with no significant relation between the matrices (P=

0.8571).

DISCUSSION

Owing to the use of many Salvia species going under

the name Danshen, and of some species as non-Danshen in

China, it is necessary to authenticate the medicinal Salvia

species to insure its safe clinical application. However, the

traditional method of visual inspection is insufficient to

distinguish among the various Salvia species. Unlike other

traditional methods of authentication, the DNA sequencing

procedure is based on genotype rather than phenotype, so

the authentication is reliable, reproducible, and unaffected

by the physical form or physiological condition of the drug

sample. In angiosperm, ITS1 and ITS2 are rapidly evolv-

ing regions of nuclear ribosomal RNA genes (rDNA), and

they are variable in different species. Such sequences are

proposed to be useful in identification of both the medici -

nal plant and the botanical origins of the crude medicine

(Zhao et al., 2001; Lau et al., 2001; Chen et al., 2002; Shi-

ba et al., 2006; Xu et al., 2006). This is true even though

the commercial crude drugs, which are invariably sun-

dried and stored for long periods, and their genomic DNA

have the potential to degrade. It is probably to amplify

the ITS regions of the multi-copy rDNA repeat from their

DNA samples. In this study, the ITS region was success-

fully amplified and sequenced, whether the template DNA

used was isolated from fresh leaves or dried root. The

sequence divergence between the ITS regions in Salvia

species ranged from 1.33% to 19.4% and populations of S.

miltiorrhiza ranged from 0.22% to 3.17%. Therefore, ITS

regions could be adopted as a molecular marker for differ-

entiating Salvia species from one another and also popula-

tions of S. miltiorrhiza from each other. Cluster analysis in

the study divided the Salvia species into two main groups

according to ITS sequence. S. miltiorrhiza and six other

Danshen species were clustered together and separated

from non-Danshen species, which corresponded to differ-

ing medicinal requirements. However, the sequences did

not segregate the ten salvia species into the groups estab-

Table 3. Contents of tanshinones in the root of Salvia species.

Voucher Cryptotanshinone

(mg/g)

Tanshinone I

(mg/g)

Tanshinone IIA

(mg/g)

SM-SCZJ

1.098

0.133

0.246

SM-SDPY

3.322

1.276

2.968

SM-SXYC

1.348

0.407

0.810

SM-SDYS

3.140

1.067

2.502

SM-JSSY

0.404

0.104

0.111

SM-AHBZ

1.125

0.397

0.598

SM-SX

3.019

1.053

2.219

SM-JSBH

0.564

0.157

0.197

SM-SD

7.021

2.862

5.181

SM-JSRG

0.566

0.204

0.253

SM-SXSL 2.915¡Ó0.112 0.610¡Ó0.021 0.335¡Ó0.012

SM-SH 0.819¡Ó0.024 0.124¡Ó0.004 1.114¡Ó0.040

SP-YNLJ 6.428¡Ó0.186 1.590¡Ó0.049 14.08¡Ó0.309

SF-YNLJ 0.606¡Ó0.025 0.220¡Ó0.010 1.254¡Ó0.035

ST-YNLJ 1.581¡Ó0.059 1.575¡Ó0.055 6.427¡Ó0.221

SY-YNLJ 1.289¡Ó0.044 0.708¡Ó0.027 5.368¡Ó0.168

SD-YNLJ 1.214¡Ó0.033 1.587¡Ó0.043 6.218¡Ó0.210

SC-AHHS 0.115¡Ó0.003 0.121¡Ó0.004 0.916¡Ó0.034

SL-ZJLA 0.235¡Ó0.010 0.102¡Ó0.003 0.354¡Ó0.011

SB-ZJLA 0.724¡Ó0.016 0.522¡Ó0.017 0.837¡Ó0.035

SDE-XJWLMU ND

ND

ND

ND: not detected. Content: mean value (n=2) or mean value¡Ó

SD (n=3).

Figure 4. De ndrogram showi ng Eucl idea n distance ba sed

on tanshinones percentages of Salvia species using UPGMA

cluster analysis.

F i gure 3. HPL C ch roma tog ram of S. miltiorrhiza from

Sha ngluo, Shanxi (SM-SXSL). 1: cryptotanshinone; 2: tan-

shinone I; 3: tanshinone IIA.

134

Botanical Studies, Vol. 50, 2009

lished by morphological classification.

Chemical analysis showed high phenotypic variability

among the Salvia species and S. miltiorrhiza populations.

S. miltiorrhiza, six other Danshen species, and two non-

medicinal species closely resemble each other chemically

and are distinct from non-Danshen species. The tanshi-

none content divided the Salvia samples into two distinct

clusters, which are not in agreement with their geographic

origin or morphological classification. The Mantel test,

used to compare tanshinone content to the ITS sequence

matrices, indicated a low, non-significant relation be-

tween the matrices. The marked variations in the chemical

characteristics among the samples result not only from

genetic factors, but also from environmental factors. Ad-

ditionally, medicinal plants are processed for use as crude

drugs, causing some chemical constituents to change, so

it will be important to analyze the chemical composition

combined with some other non-genetic factor in the future.

However, the chemical variability found among the Salvia

miltiorrhiza populations on the one hand and the ITS se-

quence differentiation on the other may contribute to the

wide distribution of S. miltiorrhiza in China. Therefore,

it is necessary to preserve the good population resources

of S. miltiorrhiza and construct a good agriculture prod-

uct (GAP) system to produce high quality Danshen crude

drugs at the current stage. Chemical analysis also showed

that the content of tanshinones in the roots of some other

Danshen species are higher than in S. miltiorrhiza, which

might explain the reasoning behind their use as Danshen.

Further, their medicinal use could be validated through

future pharmacological research.

Acknowledgements. This work was supported by the

Shanghai Leading Academic Discipline Project (#Y0301)

and the Natural Science Foundation from the Shanghai

Municipal Education Commission (#04CB06).

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