全 文 ：热带亚热带植物学报　2013， 21(3)： 259~265
Journal of Tropical and Subtropical Botany
Salvia miltiorrhiza has been widely used for
prevention and treatment of coronary heart disease[1].
Studies on chemical constituents of S. miltiorrhiza were
mainly focused on lipophilic compounds. However,
more attention has been paid to phenolics in recent
years due to their pharmacological activities[2–3], of
which some phenolics have been identified[2], such
as salvianolic acid A – K, danshensu and rosmarinic
acid. Chemical fingerprinting developed in these
years has been widely used in quality control of
不同来源丹参药材酚酸类成分的分析研究
盛东峰*, 李俐俐
(周口师范学院生命科学系， 河南 周口 466001)
摘要： 为比较不同来源的丹参(Salvia miltiorrhiza)药材的酚酸类成分，采用化学指纹图谱和定量分析的方法，对不同来源丹参药
材中的酚酸类成分进行了系统分析。结果表明：产地、采收期、病害、根色、根的粗细以及药材部位等因素尽管对丹参酚酸类成
分绝对含量的影响比较大，但对各成分相对含量的影响较小；不同来源丹参药材酚酸类成分指纹图谱相似性较高；8 月份采收
的药材，丹酚酸 B 含量较高；病害能够显著降低丹酚酸 B 的积累；与白根和褐色根相比，砖红色根中的丹酚酸 B 含量较高；根越
粗，丹酚酸 B 含量也越高。这为丹参药材的品质评价和资源利用提供了依据。
关键词： 丹参； 酚酸类成分； 高效液相色谱； 指纹图谱； 质量评价
doi: 10.3969/j.issn.1005–3395.2013.03.012
Comprehensive Investigation of Phenolics in Salvia miltiorrhiza by
HPLC Fingerprinting and Marker Compounds
SHENG Dong-feng*, LI Li-li
(Department of Life Science, Zhoukou Normal University, Zhoukou 466001, China)
Abstract: In order to compare the phenolics in Salvia miltiorrhiza with different sources, the phenolics extracted
from different sources were studied by chemical fingerprinting method, and salvianolic acid B as marker molecule
was quantitatively analyzed. The results showed that the absolute amounts of phenolics in S. miltiorrhiza roots
were seriously affected by different factors, such as origin, harvest time, root disease, root color, root diameter,
and the part of plant. However, these factors had little influence on relative amounts of phenolics. The chemical
fingerprinting of phenolics derived from different sources had high similarity. The content of salvianolic acid
B harvested in August was the highest, and which was significantly reduced by diseases, such as root rot and
nematode. Contents of phenolics in the brick red roots were the highest, compared to puce and white roots. The
bigger the diameter, the higher contents of phenolics were. So, these could provide the basis for quality evaluation
and resource utilization of S. miltiorrhiza.
Key words: Salvia miltiorrhiza; Phenolics; High performance liquid chromatography; Chemical fingerprinting;
Quality assessment
Received： 2012–11–22　　　　Accepted： 2013–01–30
This study was supported by Henan Natural Science Foundation (2010B230013).
* Corresponding author. E-mail: shengdongfeng@126.com
260 第21卷热带亚热带植物学报
traditional Chinese medicine (TCM)[4]. Quality
assessments of S. miltiorrhiza have been reported
with the method of fingerprinting[5–10] and quantitative
analysis of marker compounds[11]. Whereas, most
of them focused on the roots of S. miltiorrhiza
and analyzed by HPLC[5], high-speed counter-
current chromatography (HSCCC)[6], thin layer
chromatography (TLC)[7], capillary electrophoresis
(CE)[8], coulometric electrode array detector (CEAD)[9]
and mass spectrography (MS)[10–11]. These methods
were considered to be feasible for quality assessment.
However, the quality of S. miltiorrhiza is difficult to
control, because it is usually influenced by climate,
disease, geography, morphological character and
harvest time. Fingerprinting is insufficient for quality
assessment of TCM since it is not quantitative, while
quantitative analysis is necessary to evaluate the
amounts of the main active compounds. The method of
chromatographic fingerprint together with quantitative
analysis of marker compounds was confirmed as a
useful approach for quality assessment of TCM[12].
By this method, quality of lipophilic components in
S. miltiorrhiza has been assessed and the influences
on the quality has been comprehensively investigated
by impact factors including origin, harvest time, root
disease, root diameter, root color and different part
of S. miltiorrhiza. In this paper, based on phenolic
components in S. miltiorrhiza, the quality of S.
miltiorrhiza will be assessed and the factors will be
investigated.
1 Materials and methods
1.1 Chemicals and Materials
HPLC grade methanol was purchased from
Fisher Scientific (Fairlawn, NJ, USA). Analytical
grade ethanol was from Ante Biochemistry (Suzhou,
China). HPLC-quality water was generated from
a UPW ultrapure water system purchased from
Shanghai Ultrapure Technology (Shanghai, China).
Salvia miltiorrhiza was purchased from markets
(Table 1), in which DB-1 to DB-10 were collected
from GAP base in five counties of Shangluo City.
Salvianolic acid B were purchased from the National
Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China).
1.2 Sample preparation
Samples of S. miltiorrhiza were dried at 60℃,
comminuted into powder, and sieved through a 0.45 mm
screen. The powder (0.5 g) was extracted in water bath
at 90℃ for 4 h with 25 mL deionized water. 1 mL of
water-extract was added into 4 mL ethanol, and then
the extract was centrifuged at 12000 ×g for 15 min
and finally filtered through a 0.45 µm millipore filter.
Stock solutions of salvianolic acid B were
prepared in methanol and were diluted to the desired
concentration.
1.3 Chromatography
HPLC was performed with a Waters (Milford,
MA, USA) binary pumpand photodiode array
detector (PAD). The column was a Waters SunFire C18
(250 mm × 4.6 mm, 5 μm particle). Data were acquired
and processed by use of Empower 2 Software. Separation
was achieved by eluting with a linear gradient
prepared from methanol and water (containing 0.01%
H3PO4). The gradient was: t = 0 min 15% methanol;
t = 30 min, 40% methanol; t = 40 min, 55% methanol;
t = 60 min, 75% methanol. The flow rate was 1 mL
min–1, the column temperature 30℃, and the sample
size 20 μL. The effluent was monitored between
190 nm and 500 nm by use of the PDA.
2 Results and discussion
2.1 Method validation
The extract of sample 1# was injected five times.
The repeatability of the method was assessed by
analysis of five independently prepared extracts
of sample 1#. The precision of the analysis was
determined by replicate analysis of the same extract of
sample 1# over a period of 48 h. The relative standard
deviation (RSD) values of retention time and peak
area of salvianolic acid B were calculated. The results
revealed the method performed well. The detector
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Table 1 Samples tested
No. Sample code Source Sample description Collection time (M / Y)
1 DB-1 Shangluo, Shaanxi Whole root 11 / 2006
2 DB-2 Shangluo, Shaanxi Whole root 11 / 2006
3 DB-3 Shangxian, Shaanxi Whole root 11 / 2006
4 DB-4 Shangxian, Shaanxi Whole root 11 / 2006
5 DB-5 Luonan, Shaanxi Whole root 11 / 2006
6 DB-6 Luonan, Shaanxi Whole root 11 / 2006
7 DB-7 Zhashui, Shaanxi Whole root 11 / 2006
8 DB-8 Zhashui, Shaanxi Whole root 11 / 2006
9 DB-9 Danfeng, Shaanxi Whole root 11 / 2006
10 DB-10 Danfeng, Shaanxi Whole root 11 / 2006
11 DA-1 Shaanxi Whole root 11 / 2006
12 DA-2 Shanxi Whole root 11 / 2006
13 DA-3 Hebei Whole root 11 / 2006
14 DA-4 Jiangsu Whole root 11 / 2006
15 DA-5 Shandong Whole root 11 / 2006
16 DA-6 Beijing Whole root 11 / 2006
17 DA-7 Anhui Whole root 11 / 2006
18 DP-1 Shangluo, Shaanxi Leaf 05 / 2006
19 DP-2 Shangluo, Shaanxi Petiole 05 / 2006
20 DP-3 Shangluo, Shaanxi Stem 05 / 2006
21 DP-4 Shangluo, Shaanxi Petal 05 / 2006
22 DP-5 Shangluo, Shaanxi Receptacle 05 / 2006
23 DP-6 Shangluo, Shaanxi Rachis 05 / 2006
24 DP-7 Shangluo, Shaanxi Seed 05 / 2006
25 DP-8 Shangluo, Shaanxi Root periderm 05 / 2006
26 DP-9 Shangluo, Shaanxi Root xylem 05 / 2006
27 DP-10 Shangluo, Shaanxi Root phloem 05 / 2006
28 DT-1 Shangluo, Shaanxi Root velamen* 05 / 2006
29 DT-2 Shangluo, Shaanxi Root velamen 06 / 2006
30 DT-3 Shangluo, Shaanxi Root velamen 07 / 2006
31 DT-4 Shangluo, Shaanxi Root velamen 08 / 2006
32 DT-5 Shangluo, Shaanxi Root velamen 09 / 2006
33 DT-6 Shangluo, Shaanxi Root velamen 10 / 2006
34 DT-7 Shangluo, Shaanxi Root velamen 11 / 2006
35 DT-8 Shangluo, Shaanxi Root velamen 12 / 2006
36 DD-1 Shangluo, Shaanxi Whole root suffered from nematodiasis 05 / 2006
37 DD-2 Shangluo, Shaanxi Whole root suffered from root rot 05 / 2006
38 DC-1 Shangluo, Shaanxi Sable root 05 / 2006
39 DC-2 Shangluo, Shaanxi White root 05 / 2006
40 DJ-1 Shangluo, Shaanxi Root head 11 / 2006
41 DJ-2 Shangluo, Shaanxi Root diameter > 0.8 cm 11 / 2006
42 DJ-3 Shangluo, Shaanxi Root diameter at 0.5 – 0.8 cm 11 / 2006
43 DJ-4 Shangluo, Shaanxi Root diameter at 0.3 – 0.5 cm 11 / 2006
44 DJ-5 Shangluo, Shaanxi Root diameter at 0.2 – 0.3 cm 11 / 2006
45 DJ-6 Shangluo, Shaanxi Root diameter at 0.05 – 0.2 cm 11 / 2006
46 DJ-7 Shangluo, Shaanxi Fibrous roots diameter < 0.05 cm 11 / 2006
*: Root velamen including periderm and xylem.
盛东峰等：不同来源丹参药材酚酸类成分的分析研究
262 第21卷热带亚热带植物学报
response was linearly correlated with concentration of
salvianolic acid B with a range of 55.0 – 220.0 μg mL–1.
The regression equation was y = 1020000x + 74200
(r = 0.9997).
2.2 Assessment of S. miltiorrhiza roots from the
same origin
Shangluo was an important GAP origin of S.
miltiorrhiza. In this study, ten batches of S. miltiorrhiza
from Shangluo in Shaanxi Province were collected
and analyzed. Fifteen common peaks of phenolic
components at 288 nm were obtained according to the
consistency of retention time and spectra of the peaks
(Fig. 1). A standard fingerprinting representing the
mean values of the RRT and RPA of all the samples
was constructed according to SFDA (State Drug
Administration of China 2000). Correlation coefficient
was employed to evaluate the values of similarity
between the chromatograms of samples and the
standard fingerprinting. The results showed the values
of correlation coefficient were all more than 0.99. It
indicated that the relative contents of phenolic
components in the samples from GAP base of S.
miltiorrhiza were consistent.
Salvianolic acid B is the main phenolic component
in S. miltiorrhiza. Peak area of salvianolic acid B takes
up more than 60% of total peak areas. Its contents were
inconsistent in 10 batches of S. miltiorrhiza from GAP
base. The highest content reached to 4.851%, and the
lowest content was just 2.106%. Our previous report
showed that all the ten batches of samples had good
quality on lipophic components[13]. However, Table
2 showed that samples DB-6, 7, 8 had bad quality on
phenolic components. It demonstrated that quality of S.
miltiorrhiza should be assessed according to their usage
and components.
2.3 Assessment of S. miltiorrhiza roots from different
origins
The roots of S. miltiorrhiza were collected from
seven provinces in China and analyzed. The quantitative
analysis showed that the contents of salvianolic acid
B in seven samples were inconsistent. The highest
content reached to 6.594%, and the lowest was only
3.891%. However, All of them were more than 3%,
which was the lowest statute content. The similarity
values among seven samples were more than 0.99,
which indicated the origin had more influence on
the absolute amounts than the relative contents of
phenolics components.
Fig. 1 Fingerprinting of the phenolics components at 288 nm in ten batches of Salvia miltiorrhiza from Shangluo GAP base. Peak 9 was salvianolic
acid B.
第3期 263
2.4 Assessment of different parts of S. miltiorrhiza
In the experiment, water extracts from different
parts of S. miltiorrhiza, including petal, receptacle,
rachis, seed, leave, petiole, stem, root periderm, root
xylem, and root phloem, were analyzed. The results
showed that the phenolic components were mainly
distributed in root xylem and root phloem. However，
The other parts also contained some phenolic
components. These were different from lipophilic
components[13]. For example, the content of danshensu
(3,4-dihydroxyphenyllactic acid) was the highest in
receptacle and leave, and then in root phloem, petiole,
rachis, and petal. Protocatechualdehyde and salvianolic
acid B contents were the highest in root phloem,
and then in root periderm, root xylem, and petal.
Noticeably, areas of peaks 3 – 6 were the biggest in
petal and receptacle. For example, area of peak 6 in
petal and receptacle was 15 and 7 times more than
that in root phloem, respectively. They might be good
resources for the other medicines that mainly contain
components 3 – 6. The similarity analysis showed that
the green parts were similar, petal, receptacle, and
seed were similar, and the underground parts were
similar. In origin of S. miltiorrhiza, only the roots
were used and the other parts were discarded or burned
out. Our results gave a good guidance for exploring and
utilizing the other parts of S. miltiorrhiza.
2.5 Assessment of S. miltiorrhiza roots obtained at
different harvest times
In the study, roots of S. miltiorrhiza were collected
every month from May to December. The dynamic
changes in three main peaks were compared (Fig.
2). Fig. 2 showed that changes in peak 6 and 8 were
unapparent and the curve of peak 9 fluctuated from
May to December. However, they all reached to
maximum in August and the similarities among the
samples were all more than 0.9. It demonstrated that the
relative contents of the components in S. miltiorrhiza
harvested at different times were consistent. These
results were consistent with lipophilic component[13].
2.6 Assessment of S. miltiorrhiza suffered from
different diseases and different colors
Roots of S. miltiorrhiza suffered from root rot and
nematode disease were collected. The results showed
that all of the 15 components could be detected in the
two kinds of ill roots, but their contents were lower
than those in normal roots. However, area of peak
8 in root rot was 6 times more than that in normal
roots. It indicated that root rot might accelerate the
accumulation of peak 8.
Table 2 Contents of salvianolic acid B in different Salvia miltiorrhiza samples
Sample No. Salvianolic acid B (%) Sample No. Salvianolic acid B (%)
DB-1 3.714 DA-5 6.594
DB-2 3.824 DA-6 3.891
DB-3 4.851 DA-7 4.577
DB-4 4.063 DD-1 1.734
DB-5 3.052 DD-2 2.513
DB-6 2.706 DC-1 2.772
DB-7 2.106 DC-2 4.411
DB-8 2.505 DJ-1 2.310
DB-9 3.631 DJ-2 4.253
DB-10 3.865 DJ-3 2.792
DA-1 5.178 DJ-4 2.725
DA-2 4.647 DJ-5 2.772
DA-3 6.235 DJ-6 1.920
DA-4 4.231 DJ-7 0.917
盛东峰等：不同来源丹参药材酚酸类成分的分析研究
264 第21卷热带亚热带植物学报
In this study, quality of buff, sable and brick red
roots was also studied. The number of phenolic
components in three kinds of roots was the same.
However, their contents were different. The total peak
area and areas of peak 2, 4, 8 and 9 were the highest
in brick red roots. Areas of peak 5 – 7 were the highest
in buff roots. Sable roots had the worst quality.
2.7 Assessment of S. miltiorrhiza roots with different
diameters
The dried roots from Shangluo GAP base were
divided into seven groups according to root diameters
(Table 1). The results showed that all of the 15 peaks
were detected in all seven groups of roots. Similarity
analysis showed that the correlation coefficient for the
seven samples were all more than 0.9. It indicated that
the relative contents of fifteen components in seven
groups of roots were consistent. However, peak areas
in seven groups were quite different (Fig. 3), only
peak 9 was seriously influenced by root diameter.
The quantitative analysis showed that the thicker the
diameter was, the better the quality was (Table 2),
Fig. 3 Peak areas of phenolic components in different groups of Salvia miltiorrhiza. DJ-1 to DJ-7 were seven groups of S. miltiorrhiza roots according
to diameter. P3, P6, P8 and P9 was the peak 3, 6, 8 and 9, respectively.
Fig. 2 Dynamic accumulation of main phenolic components in roots of Salvia miltiorrhiza. P6, P8 and P9 was the peak 6, 8 and 9, respectively.
第3期 265
which was different from lipophilic component[13].
3 Conclusions
In our previous report, quality of lipophilic
components in S. miltiorrhiza has been assessed by
HPLC fingerprinting and quantitative analysis[13].
By the method, quality of phenolic components was
also assessed in this paper. The standard fingerprinting
including 15 common peaks at 288 nm was established,
in which salvianolic acid B was determined. The
influences of climate, disease, geography, morphological
character, and harvest time on the quality of S.
miltiorrhiza were also comprehensively investigated.
The relative contents of phenolic components were
consistent under different conditions as well as
lipophilic components. However，the influences on
the absolute amounts of phenolics and tanshinones
were different. For example, contents of lipophilic
components were the highest in DJ-3 and DJ-6, but
those of phenolics were the highest in DJ-2.
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盛东峰等：不同来源丹参药材酚酸类成分的分析研究