Pharmaceutical and Biomedical Analysis of Terpene Constituents in Salvia miltiorrhiza
ABSTRACT The present paper reviews the most relevant experimental data on miltirone, a diterpene pigment and one of the major active constituents with phenanthrene-quinone structure of the roots of Salvia miltiorrhiza. Radix Salvia miltiorrhizae Bunge (Labiatea, Laminaceae), a native plant in China, is listed in the Chinese Pharmacopoeia with the name of Danshen and used in Chinese folk medicine for the treatment of different pathologies. In fact, two types of major bioactive components in Dan-shen, including water soluble phenolic acids and lipophylic diterpenoid quinines, have the effectiveness in treating coronary heart disease, heart-stroke, cerebrovascular diseases, menstrual disorders, miscarriage, hepatitis and insomnia. Moreover, recent studies demonstrated the ability of different extracts of S. miltiorrhiza to suppress alcohol drinking in selectively bred Sardinian alcohol-preferring (sP) rats. Administration of miltirone to sP rats produced similar results, suggesting that it is the likely active constituent of S. miltiorrhiza responsible for the reducing effect of its extracts on alcohol intake. However, the study of S. miltiorrhiza components is very complex, therefore selective and efficient analytical methods are required to simultaneously determine their structures for further study of the pharmacological effects and to control the quality of the preparations in which are contained. Currently, there are many methods for the determination of phenolic compounds and diterpenes among which, highperformance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) has been shown to be a powerful method for separation and identification of individual molecules in complex samples. In the proposal review, we will report an overview about the main results concerning the analysis of the main diterpenes constituents of S. miltiorrhiza in medicinal preparations and biological fluids, and our recent data on quantitative determination of miltirone in rat plasma.
- [show abstract] [hide abstract]
ABSTRACT: Still little is known about the cellular mechanisms that contribute to the attenuated proliferation of aortic smooth muscle cells under the influence of the oxidative stress factors such as homocysteine (Hcy). Thus, we aimed to evaluate whether Salvia miltiorrhiza Bunge (Labiatae), a Chinese medicinal herb widely used in folk medicine for therapy of variety of human cardiovascular disorders would modulate this Hcy promoted growth effect in model animal aortic cells system. The Salvia miltiorrhiza roots aqueous extract (SMAE) containing 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenyl lactic acid and salvianolic acid B, as confirmed by narrow-bore HPLC analyses with binary gradient elution was used in variable concentrations for the treatment of the rat aortic smooth muscle A10 cells under Hcy stimulation. Two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry was applied for the elucidation of protein changes characterizing the response of the rat A10 cells into the Hcy-induced oxidative stress. This study showed that a low dose (0.015 mg/mL) of the SMAE significantly inhibited growth (>60%, p<0.05) of the Hcy stimulated rat A10 cells. In addition, concentration of intracellular reactive oxygen species (ROS) obviously decreased in the rat A10 cells after its incubation with SMAE in terms of catalase increasing activity. Next, marked down-regulation of protein kinase C beta-1 (PKC beta-1) and phosphorylated mitogen-activated protein kinase (p-MAPK) expression suggest that observed inhibitory effect of the polyphenol-rich SMAE on the Hcy-induced growth of rat A10 cells was realized via the PKC/p44/42 MAPK-dependent pathway. The intensity changes of 10 protein spots in response of the rat A10 cells to the Hcy-induced oxidative damage as alpha-4-tropomyosin, vimentin, F1F0-ATP synthase (beta subunit), glucose regulated protein 75 (GRP75), actin (fragment), prohibitin, capping protein, plakoglobin, endoplasmic reticulum protein (ERp29), and peptidylprolyl isomerase A (PPIase A), were detected with statistical significance (p<0.05). Meanwhile, it was showed that used here SMAE resist carbonylation of specific cytoskeleton and chaperone proteins as vimentin, alpha-4-tropomyosin and GRP75, respectively, leading to phenotype transformations in the rat A10 cells. These data suggest that applied here SMAE exerts its protective effect through circulating ROS suppression and subsequent modulation of protein carbonylation in rat aortic smooth muscle A10 cells. Redox-proteomics protocol highlighted in this study may be applicable in facilitating the assessing potential novel molecular therapeutic targets to reduce cardiovascular risk related with elevated Hcy levels in various human populations and elucidating new mechanisms through which protein functions can be regulated by the redox status with the use of naturally occurring antioxidants.Journal of ethnopharmacology 05/2009; 124(3):463-74. · 2.32 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A novel strategy to trace the origins of commercial pharmaceutical products has been developed based on the direct chemical profiling of the pharmaceutical products by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS). Besides the unambiguous identification of active drug components, various compounds present in the matrixes are simultaneously detected without sample pretreatment, providing valuable information for drug quality control and origin differentiation. Four sources of commercial amoxicillin products made by different manufacturers have been successfully differentiated. This strategy has been extended to secerning six sources of Liuwei Dihuang Teapills, which are herbal medicine preparations with extremely complex matrixes. The photolysis status of chemical drug products and the inferior natural herd medicine products prepared with different processes (e.g., extra heating) were also screened using the method reported here. The limit of detection achieved in the MS/MS experiments was estimated to be 1 ng/g for amoxicillin inside the capsule product. Our experimental data demonstrate that DAPCI-MS is a useful tool for rapid pharmaceutical analysis, showing promising perspectives for tracking the entire pharmaceutical supply chain to prevent counterfeit intrusions.Analytical Chemistry 10/2010; 82(19):8060-70. · 5.70 Impact Factor
Current Pharmaceutical Analysis, 2008, 4, 249-257
1573-4129/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.
Pharmaceutical and Biomedical Analysis of Terpene Constituents in Salvia
Antonella De Palma1, Rossana Rossi1, Mauro Carai2, Claudia Cabras2, Giancarlo Colombo2, Lolita
Arnoldi3, Nicola Fuzzati3, Antonella Riva3, Paolo Morazzoni3 and Pier L. Mauri1,*
1Institute for Biomedical Technologies, Proteomics and Metabolomics Unit - CNR, via Fratelli Cervi , 93, 20090 Segra-
te (Milan), Italy
2Institute of Neuroscience - CNR, viale Diaz, 182, I-09126 Cagliari, Italy
3Indena S.p.A., Viale Ortles, 12, 20139 Milan, Italy
Abstract: The present paper reviews the most relevant experimental data on miltirone, a diterpene pigment and one of the
major active constituents with phenanthrene-quinone structure of the roots of Salvia miltiorrhiza. Radix Salvia miltiorrhi-
zae Bunge (Labiatea, Laminaceae), a native plant in China, is listed in the Chinese Pharmacopoeia with the name of Dan-
shen and used in Chinese folk medicine for the treatment of different pathologies. In fact, two types of major bioactive
components in Dan-shen, including water soluble phenolic acids and lipophylic diterpenoid quinines, have the effective-
ness in treating coronary heart disease, heart-stroke, cerebrovascular diseases, menstrual disorders, miscarriage, hepatitis
Moreover, recent studies demonstrated the ability of different extracts of S. miltiorrhiza to suppress alcohol drinking in se-
lectively bred Sardinian alcohol-preferring (sP) rats. Administration of miltirone to sP rats produced similar results, sug-
gesting that it is the likely active constituent of S. miltiorrhiza responsible for the reducing effect of its extracts on alcohol
However, the study of S. miltiorrhiza components is very complex, therefore selective and efficient analytical methods are
required to simultaneously determine their structures for further study of the pharmacological effects and to control the
quality of the preparations in which are contained.
Currently, there are many methods for the determination of phenolic compounds and diterpenes among which, high-
performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) has been
shown to be a powerful method for separation and identification of individual molecules in complex samples.
In the proposal review, we will report an overview about the main results concerning the analysis of the main diterpenes
constituents of S. miltiorrhiza in medicinal preparations and biological fluids, and our recent data on quantitative determi-
nation of miltirone in rat plasma.
Keywords: Miltirone, S. miltiorrhiza, Diterpenes, Tanshinones, Liquid chromatography mass spectrometry.
Salvia miltiorrhiza Bunge (Labiatea, Laminaceae) is an
annual sage plant, that mainly grows in hilly areas of China
and neighboring countries. Radix Salvia miltiorrhiza Bunge,
also known as Dan-shen, is one of the most commonly used
medicinal herb in China and is officially listed in the Chinese
Pharmacopeia . Its dried roots are widely adopted in
traditional Chinese medicinal preparations for the treatment
of several pathologies due to their better performance and
fewer side effects as confirmed in the long-time clinical use
[2,3]. A great number of finished herbal products containing
Dan-shen as its major component have been developed and
marketed widely in China for the treatment above all of car-
diovascular diseases [4-6]. As reported in this review in re-
*Address correspondence to this author at the Institute for Biomedical
Technologies - CNR, Proteomics and Metabolomics Unit, Via Fratelli
Cervi, 93, 20090 Segrate (MI), Italy; Tel: +39 02 26422728; Fax: +39 02
26422770; E-mail: email@example.com
cent years many efforts have been made by different groups
and with the support of different kind of instruments to cre-
ate selective and efficient analytical methods for the identifi-
cation and the structural characterization of the constituents
of S. miltiorrhiza [7,8]. In particular, it was found that the
bioactive constituents of S. miltiorrhiza cover two chemical
types: water soluble phenolic acids and lipophilic diterpenoid
quinones [9,10]. The water soluble phenolic acids in S.
miltiorrhiza include single phenolic and polyphenolic acids.
The single phenolic acids mainly include protocatechuic
acid, ptotocatechuic aldehyde, caffeic acid, isoferulic acid
and Danshensu (3,4- diydroxyphenyllactic acid). The poly-
phenolic acids are mainly the conjugate of danshensu, caffeic
acid or its dimer. Other polyphenolic acids are rosmarinic
acid, lithospermic acid and salvianolic acid A-G. In recent
years, the water-soluble components (including phenolic
acids and their esters) have attracted increasing attention
because of their antioxidant , ant-platelet aggregation
, antitumor , antithrombosis, and antiviral activities
250 Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 De Palma et al.
The lipophilic diterpenoid quinones, know as tanshinones
in S. miltiorrhiza, include tanshinone I, tanshinone IIA, cryp-
totanshinone and miltirone. It has been reported that tanshi-
nones possess pharmacological actions of anti-inflammation,
dilating coronary arteries, increasing coronary flow and pro-
tecting the myocardium against ischaemia [16,17]. In addi-
tion, many works also reported that tanshinones have exhib-
ited significant cytotoxic, antibacterial, anti-dermatophytic,
anti-neoplastic and anti-platelet aggregation activities
All these above mentioned pharmacological activities
have been widely described in literature, so the present paper
briefly reviews the most recent findings on the effects of S.
miltiorrhiza extract on voluntary alcohol intake in animal
models of alcoholism. In particular we focused our attention
on miltirone, the active ingredient of S. miltiorrhiza that is
probable responsible for this effect but for which few struc-
tural and pharmacokinetics studies are documented.
In addition, we report an overview about analytical
methods concerning the assay of S. miltiorrhiza components.
2. ANTI – ALCOHOL PROPERTIES OF S. miltiorrhiza
EXTRACTS AND MILTIRONE
Different lines of experimental evidence have demon-
strated that extracts from the dried roots of S. miltiorrhiza
reduced alcohol drinking in Sardinian alcohol-preferring (sP)
rats, one of the few rat lines selectively bred worldwide for
high alcohol preference and consumption. Specifically, acute
or repeated administration of S. miltiorrhiza extracts have
been found to: (a) delay the acquisition of alcohol drinking
behavior in alcohol-naive rats given alcohol under the home-
cage 2-bottle “alcohol vs water” choice regimen (specifi-
cally, exposure to the 2-bottle choice regimen started imme-
diately after the first administration of the S. miltiorrhiza
extract) ; (b) reduce alcohol intake under the 2-bottle
choice regimen in rats which were alcohol-experienced at the
time of extract administration (a model of the “active drink-
ing” phase of human alcoholism) [21,22]; (c) suppress the
temporary increase in alcohol intake occurring after a period
of deprivation from alcohol (a phenomenon named “alcohol
deprivation effect”, which has been proposed to model the
relapse episodes occurring in human alcoholics) .
A more recent study investigated whether miltirone is the
active ingredient responsible for the reducing effect of S.
miltiorrhiza extracts on alcohol intake . To this aim, a
first experiment compared the effect of four different ex-
tracts of Salvia miltiorrhiza, varying as to the content of
miltirone, on alcohol intake in sP rats. Specifically, 100
mg/kg of four extracts containing approximately 0%, 2%,
3%, and 7% miltirone, respectively, were acutely adminis-
tered to alcohol-experienced sP rats exposed to the 2-bottle
choice regimen. Notably, the reducing effect of the different
S. miltiorrhiza extracts on alcohol intake positively and sig-
nificantly correlated with their miltirone content (r=0.9833).
Subsequent experiments tested the effect of pure milti-
rone on alcohol intake in sP rats . Specifically, miltirone
was administered at doses (0, 2.5, 5, and 10 mg/kg, i.g.)
comparable to its content in the 100 mg/kg dose of the four
extracts tested in the correlation experiment (see above). In
the first experiment, the repeated daily administration of
miltirone resulted in a significant and dose-dependent delay
in acquisition of alcohol drinking behavior in alcohol-naive
sP rats. Miltirone effect on alcohol intake was completely
selective, as daily water intake was compensatorily higher in
miltirone than vehicle-treated rats, while daily food intake
was virtually unaltered by treatment with miltirone. In the
second experiment, the acute administration of miltirone to
alcohol-experienced sP rats (i.e., rats exposed to the 2-bottle
choice regimen for several weeks before the experiment with
miltirone whose alcohol drinking behavior was well consoli-
dated at the time of the miltirone experiment) resulted in a
significant and dose-dependent reduction in alcohol intake.
Miltirone-induced reduction in alcohol intake was fully
compensated by an increase in water intake; conversely, food
intake was not altered by miltirone treatment. The results of
these two experiments closely replicate those previously
collected with S. miltiorrhiza extracts, strengthening the hy-
pothesis that miltirone is likely the active ingredient of S.
miltiorrhiza responsible for the reducing effect of its extracts
on alcohol intake.
In terms of the mechanism of miltirone action, the anx-
iolytic effect of miltirone  might play a role in the drug
capacity to reduce alcohol intake in sP rats, as: (a) anxiety is
an inherent trait likely predisposing sP rats to high alcohol
drinking; (b) alcohol is likely consumed by sP rats to self-
medicate anxiety . Accordingly, it can be proposed that
the anxiolytic effect of miltirone may have substituted for the
anxiolytic effect of alcohol usually sought by sP rats, result-
ing in a less urgent need of alcohol and, in turn, in the ob-
served reduction of alcohol intake.
Previous work in rats demonstrated that S. miltiorrhiza ex-
tracts produced significant decrements in blood alcohol lev-
els when alcohol was given intragastrically but not intraperi-
toneally, suggesting that S. miltiorrhiza extracts exerted an
inhibitory action on alcohol absorption from the gastrointes-
tinal tract [21,22]. This study was subsequently extended to
miltirone, in an attempt to evaluate whether also this effect
of S. miltiorrhiza extracts was due to miltirone. To this aim,
sP rats were initially treated with vehicle or 10 mg/kg milti-
rone (i.g.) and then with 1 g/kg alcohol, given intraperito-
neally or intragastrically in two independent groups of rats.
Blood samples were collected from the tip of the rat’s tail at
different time intervals after alcohol administration. Simi-
larly to the results of the experiments testing the S. miltior-
rhiza extracts, miltirone administration resulted in a pro-
nounced reduction in blood alcohol levels when alcohol was
given intragastrically, and lack of any effect when alcohol
was administered intraperitoneally. At present, it is unknown
whether, and eventually to which extent, the decreasing ef-
fect of S. miltiorrhiza extracts and miltirone on alcohol ab-
sorption may impact alcohol intake.
3. STRUCTURAL CHARACTERIZATION OF MAIN
COMPOUNDS S. miltiorrhiza
In chemical studies over several decades, it has been
found that there are a variety of diterpenoids, phenolics, fla-
vonoids, triterpenoids and sterols in Radix S. miltiorrhiza
Pharmaceutical and Biomedical Analysis of Terpene Constituents Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 251
Up to now, bioactivity researches on this herb have
showed that the two major interesting compounds are the
phenolic acids and the diterpenes. Moreover, to evaluate the
fingerprint of these constituents it was necessary to develop
analytical methods that can simultaneously analyze both
water-soluble and non-polar bioactive compounds.
In recent years, liquid chromatography hyphenated with
tandem mass spectrometry (LC-MS/MS) has been success-
fully applied to evaluate the structures of the constituents of
different herbal extracts and in literature it is possible to find
many works on the multi-component fingerprint of S.
As an example Yang et al.  studied the fragmentation
behavior of tanshinones and applied HPLC/ESI–MSn to their
structural characterization. The authors of this work isolated
11 reference compounds with diterpenoid structure from
Dan-shen extract and introduced them into the electrospray
ionization source (ESI) by continuous infusion.
For each of these reference compounds the [M+H]+ ions
was selected as precursor ion to produce MS/MS spectra, in
which the most abundant product ion was then selected for
further MSn analysis. On the basis of these mass spectra ob-
tained it was possible dividing diterpenes constituents of S.
miltiorrhiza into 6 groups according to their chemical struc-
tures and fragmentation patterns. In fact, tanshinones have
either a furano-orthonaphthaquinone or a furano-para-
naphtaquinone skeleton and the different position of ?-
conjugation extensions result in different fragmentation
pathways. The rules deduced by this study were successfully
implemented in the identification and structural characteriza-
tion of 27 tanshinones, including miltirone. The fragmenta-
tion pathway of miltirone described by Yang et al. , was
confirmed in our laboratory (Fig. 1). In particular, the
MS/MS spectrum (collision energy 40% and isolation width
1), obtained from the miltirone molecular ion ([M+H]+ 283.1
m/z), gives a prominent ion product at 265.1 m/z, due to the
loss of H2O, and a less intense product ion at 223.1 m/z, due
to the loss of propylene from 265.1 m/z. Then 223.1 m/z lost
another CO group to m/z 195.1, that subsequently lost CH3
to give m/z 181. A loss of propylene (283.1 to 241.1 m/z)
was also observed in the MS/MS spectrum (Fig. 2).
The MS3 spectrum of the 265.1 m/z ion produces frag-
ment 223.1 m/z, due to a loss of propylene group. In the MS3
spectrum of 265.1 m/z it is possible to observe the presence
of a less intense ion at 237.1 m/z due to the loss of CO group
On the basis of our previous experience , about the
possibility to obtain quantitative data using flow injection
coupled to tandem mass spectrometry (FI/ESI-MS/MS), we
have also developed an analytical method to assay miltirone
(IDN 5477, supplied by Indena S.p.a, Milan, Italy). In particu-
lar, a volume of 1μL for each standard concentration of
miltirone (10-100 ng/mL) was injected into the ion trap mass
spectrometer (LCQDECA, Thermo Electron Corporation, San-
Josè, CA, USA) and its molecular ion was isolated (width 1)
and fragmented (collision energy 40%). In this way, it was
possible to monitor the product ion 265.1 m/z, increasing the
LOD sensitivity and to observe a linear relationship between
the peak area (average of quadruplicate injection) and the
injected amounts of miltirone, with a regression coefficient
(r2) around 0.95 (25, 50, 75, 100, 200 ng/mL). This prelimi-
nary study evidences that it is possible to analyze each sam-
ple (replication n=5) in short time (3-4 min) with a limit of
detection (LOD) around 5 ng/mL, corresponding to 20 fmol
injected (signal-to-noise S/N=5). In this way a rapid evalua-
tion of miltirone is possible, of course, using LC-MS/MS
method, both reproducibility and regression coefficient are
better (see section 5).
4. ANALYTICAL METHODS FOR QUALITY CON-
TROL OF S. miltiorrhiza EXTRACTS
Most herbal medicines are composed of complex costitu-
ents and proper methods are required for quality control of
them to ensure their stability, efficiency and safety. As de-
scribed above, phenolic acids and diterpenes are the main
bioactive compounds present in a S. miltiorrhiza extract.
The most published methods for analyzing these com-
pounds are mainly on liquid chromatography as separation
method and on UV or MS detectors as identification ones
(see Table 1). High-performance liquid chromatography
coupled to diode array detection (HPLC-DAD) is used to
achieve the fingerprinting analysis of the components from
plant extracts . For example in the study proposed by Ma
et al.  is developed a method that, using HPLC combined
to UV detection, permits the quality evaluation of Dan-shen
through simultaneous determination of four phenolic acids
and three diterpenes in only 60 min of analysis time. Li et al.
 developed a rapid, sensitive and reproducible ultraper-
Fig. (1). Chemical structure of Miltirone with the proposed fragmentation pathway.
252 Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 De Palma et al.
Fig. (2). a) ESI-MS and b) ESI-MS/MS spectra of [M+H]+ at 283.1 m/z.
Fig. (3). MS3 spectrum of m/z 265.1 (283.1 ? 265.1).
Pharmaceutical and Biomedical Analysis of Terpene Constituents Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 253
Table 1. Summary of Main Methods Used for the Analysis of Phenolic Acids and Diterpenes in Salvia miltiorrhiza Extracts
Reference Extraction Method Separation System Detector
Methanol : dichloromethane
(4:1, v/v) for 60 min by
HPLC- Alltech, Allitima
C18column (2.1 mm x 150
ESI positive 4 diterpenes N.A.
Methanol : chloroform (7:3,
v/v) for 30 min by sonica-
HPLC- an Agilent Zorbax
C18column (250 x 4.6
UV 4 diterpenes
0.02 - 0.05
Methanol : chloroform (7:3,
v/v) for 60 min a 100°C
HPLC- an Agilent Zorbax
C18column (250 x 4.6
27 diterpenes N.A.
 65% Ethanol
HPLC- Inrtsil ODS3 col-
umn (250 x 4.6 mm, 5?m)
 Methanol for 60 min a 75°C
HPLC- Agilent Zorbax
SB-C18column (250 x 4.6
4 phenolic acids
< 0.9 ng/mL
0.2 - 0.6
70% Methanol for 30 min by
HPLC- Agilent Zorbax
XDB-C18column (3 x 50
mm, 1.8 ?m)
70% Methanol for 1h in an
HPLC- Agilent Zorbax
C18column (250 x 4.6
9 phenolic acids
0.02 - 0.09
0.02 - 0.06
 Ethanol for 10 min a 100°C
UPLC- UPLC BEH C18
column (50 mm x 2.1 mm
i.d., 1.7 ?m )
PDA 10 diterpenes
0.02 - 0.21
N.A.= Not Available
formance liquid chromatography (UPLC) method for the
identification of 10 diterpenoid compounds in S. miltiorrhiza
for the first time, among which miltirone. In addition to
chromatography (including thin-layer chromatography, TLC,
gas chromatography, GC, high-performance chromatogra-
phy, HPLC), high-performance capillary electrophoresis
(HPCE) has recently been developed as an effective method
for the quality control of traditional Chinese medicine
(TCM). In the last years an application of capillary electro-
phoresis, non-aqueous capillary electrophoresis (NACE)
, has been developed rapidly and has proven to be a
promising approach for the separation of a large range of
compounds. As an example, Gu et al.  compared NACE
with high-speed counter chromatography (HSCCC) [36-38],
in the development of fingerprint of S. miltiorrhiza Bunge.
The authors of this work observed that both HSCCC and
NACE, with their own advantages and disadvantages were
necessary to investigate the complicated costituents tradi-
tional Chinese plants. Unfortunately, the assignment of
peaks, corresponding to the active constituents and toxic
ingredients is usually difficult due to the unavailability of
reference standard. In fact, isolation and purification of some
constituents from S. miltiorrhiza are not easy due to their
instability under normal conditions or to their presence in
trace amounts [4,28]. Therefore, the development of simple
and rapid methods for the analysis of diterpenes and phenolic
acids is of great significance for the quality control of Dan-
shen. High-performance liquid chromatography/mass spec-
trometry (HPLC/MS) equipped with electrospray ionisation
(ESI) ion source is one of the most powerful technique for
the rapid identification of constituents of plant extracts
[39,40]. Furthermore, the mass spectrometer is a sensitive
and selective detector and allows detection of minor or even
trace amounts of constituents from a microscale sample.
As reported by Hu et al. and Li et al. [28,41], the MS re-
sponse of phenolic acids is more sensitive using negative ion
mode while the detection of diterpenoid quinones is more
sensitive in positive ion mode. Zeng et al.  report the
identifications of phenolic compounds in S. miltiorrhiza ex-
tracts using an HPLC separation, based on an Ivertsil ODS3
analytical column (250 x 4.6 mm i.d., 5 ?m; GL Science,
Japan) coupled to UV photodiode-array detector and triple
quadrupole MS. The use of the negative ion mode combined
to the MS/MS analysis lead to the complete characterization
254 Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 De Palma et al.
of 28 phenolic acids. In this case, the most abundant con-
stituents resulted rosmarinic acid and lithospermic acid de-
As another example Zhu et al.  used a 30 min HPLC
separation, based on a reversed-phase C18 column, coupled
to a mass spectrometer equipped with a TOF analyzer or to
an ion trap analyzer for characterizing simultaneously 22
phenolic acids and 18 diterpenes constituents from Radix S.
miltiorrhiza. Specifically, the authors used three different
detectors to obtain three specific goals: UV detector to opti-
mize chromatographic conditions, TOF-MS to obtain accu-
rate molecular weights and ion trap MS to perform fragmen-
tation of molecular ions and to confirm structural identifica-
tions. In this way, coupling electrospray ionization mass
spectrometry (ESI-MS) with HPLC, it was possible to detect
many target components in a complex mixture with an high
sensitivity and a fast screening compatibility. The HPLC
method coupled to UV and ESI-TOF/MS developed by Cao
et al.  confirms the possibility to simultaneusly evaluate
phenolic acids and diterpenes. In particular, this work de-
scribes the qualitative and quantitative determination of 9
major phenolic acids and 6 main diterpenoids in 21 samples
of S. miltiorrhiza.
In our laboratory, we have used a modified HPLC
method to perform stability studies of three tanshinones.
Three purified tanshinones (Tanshinone IIA, Miltirone, Tan-
shinone I) were separately treated for 24 hours at reflux in
95% ethanol solution. Each sample was analyzed by HPLC-
PDA/ESI-MS employing a Spectra System P4000 pump
system controller equipped with a Spectra System UV 6000
LP DAD detector and a LCQ mass spectrometer (Thermo
Electron Corporation, SanJosè, CA, USA). The chroma-
tographic column was an Agilent Zorbax XDB C8 (250 x 4.6
mm, 5 ?m). The detection wavelength range was set from
215 to 600 nm, the flow rate was 1.0 ml/min and the column
temperature maintained at 35°C. The mobile phases con-
sisted of water (eluent A), methanol (eluent B) and tetrahy-
drofuran (eluent C). Gradient elution was as follow: initially
25% B and 10 % C at 0 min, linearly changing to 82% B and
3% C in 50 min, then to 90% of B and 10% of C in two min,
maintaining 90% of B and 10% of C in the last four minutes
of the run. After each analysis, the initial solvent conditions
were maintained for 12 min to re-equilibrate the system for
Tanshinone IIA heated at reflux in 95% ethanol yields
two main degradation peaks at Rrt 0.64 (Rrt: relative reten-
tion time normalized to standard, tanshinone IIA) and 0.91
(respect to tanshinone IIA). The peak (at Rrt 0.64) possess-
ing the ions [M+Na+] at m/z 333 and [2M+Na+] at m/z 643
was identified as hidroxytanshinone which was previously
described as an oxidation product of tanshinone IIA [43, 44].
The other degradation product (Rrt 0.91) having [M+Na+]
and [2M+Na+] ions at m/z 361 and 699 respectively, and a
fragment ion at m/z 293 [M+H-C2H5OH]+ was tentatively
identified as ethoxytanshinone.
About stability studies, miltirone (95% ethanol) resulted
stable at 50°C for 24h; while at 60°C for 24h recovery was
around 80%. Stability decreased rapidly when miltirone was
kept in solid state at 80°C (recovery around 35%).
Miltirone heated at reflux in 95% ethanol showed the
presence of three new peaks at Rrt 0.67, 0.91 and 1.01 (re-
spect to miltirone). The MS spectrum of peak at Rrt 0.67
exhibited the [M+Na+] and [2M+Na+] ions at m/z 321 and
619 respectively, and a fragment ion at m/z 281 [M+H-H2O].
This peak was tentatively identified as hydroxymiltirone (5-
nthrene-3,4-dione). The peak at Rrt 0.91 had the [M+Na+]
and [2M+Na+] ions at m/z 349 and 675 respectively and a
fragment ion at m/z 281 [M+H-C2H5OH]+. This degradation
product was tentatively identified as ethoxymiltirone. To
further prove this hypothesis, Miltirone was heated at reflux
for 24 h in 95% methanol. Two degradation products were
observed at Rrt 0.67 and 0.81 (respect to miltirone). The
peak at Rrt 0.67 was already identified as hydroxymiltirone.
The HPLC-MS spectrum of the second peak exhibited the
[M+Na+] and [2M+Na+] ions at m/z 335 and 647 respectively
and a fragment ion at m/z 281 [M+H-CH3OH]+ due to the
loss of methanol, confirming the influence of the solvent on
the structure of the main degradation product. The compound
eluting at Rrt 1.01 exhibited a different UV spectrum of both
miltirone and the other degradation products with maxima at
277, 362 and 410 nm. Its MS spectrum had the [M+H+] ion
at m/z 299. The UV and the MS spectra allowed to identify
this degradation product as Arucadiol  already described
in Salvia argentea.
When tanshinone I was heated at reflux in 95% ethanol
no degradation products were observed.
From these data it is possible to conclude that in hydroal-
coholic solutions, tanshinones possessing a 8,8-dimethyl-
5,6,7,8-tetrahydro-phenanthrene-3,4-dione moiety, such as
tanshinone IIA and miltirone, give rise to degradation prod-
ucts stemming from hydroxylation and alkoxylation in posi-
tion 5. This is in agreement with the literature  which
reports that the photoxidation of tanshinone IIA yields hy-
droxytanshinone and 6,7,8,9-tetrahydro-1,6,6-trimethylfuro
[3,2-c]naphth[2,1-e]oxepine-10,12-dione. In addition, when
the oxidation occurs in ethanol or methanol the correspond-
ing ethers are the major degradation products.
5. PHARMACOKINETIC ANALYSIS
Concerning pharmacokinetic studies some works are re-
ported in literature [46-50]. Specifically, Park et al.  de-
veloped a sensitive and reliable LC-MS/MS method for as-
saying the plasma levels of four tanshinones in rats using
oral administration of an enriched extract of S. miltiorrhiza
and a liquid-liquid extraction as sample clean-up method.
LC-MS/MS methodology was also applied by Li et al. 
to analyze plasma level of tanshinone IIA and cryptotanshi-
none after oral administration of total tanshinones with a
dose of 150 mg/Kg. These methods don’t determine the
quantitative presence of miltirone in biological samples or
tissue, even if this diterpene is described to be one of the
main active compound of S. miltiorrhiza . For this reason,
we have developed an analytical method, based on the use of
liquid chromatography coupled to tandem mass spectrometry,
to evaluate plasma levels of miltirone after acute oral admini-
stration of an enriched miltirone extract with a dose of 20
mg/Kg (IDN 5477, supplied by Indena S.p.A, Milan, Italy). In
particular, the plasma samples of treated rats (Colombo
Pharmaceutical and Biomedical Analysis of Terpene Constituents Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 255
Fig. (4). Typical calibration curve obtained by injecting miltirone standard solutions.
Fig. (5). a) Extracted ion chromatogram of 265.1 m/z in rat plasma sample collected at 60 min. after acute oral administration of miltirone
enriched extract and b) its related MS/MS spectrum at 16.7 min.
et al. 2006)  were collected for a period of 12 hours (0, 30,
60, 120, 180, 360, 720 min) in heparinized syringes and cen-
trifuged at 2000 x g for 10 min at 4°C. Each collected fraction
(300μL) was extracted with the same volume of ethylacetate
and, after centrifugation at 2000 x g for 2 min, the supernatant
was evaporated to dryness under vacuum and the residue was
dissolved in 150μL of 0.1% formic acid (Mauri et al. 2006)
. In this case, it was not possible to apply the FI/ESI-
MS/MS approach (see section 3) because plasma samples are
more complex than S. miltiorrhiza extracts and a chroma-
tographic step is necessary to separate miltirone from plasma
impurities. In particular, 10?L of plasma extracted with ethy-
lacetate were injected, using a 1090 Hewlett Packard pump, in
a Vydac MS C18 column (1 i.d. x 150mm, 5?m, 300 A°, All-
tech Ass. Inc., Deerfield, USA) and eluted by an acetonitrile
gradient (eluent A, 0.1% formic acid in water; eluent B, 0.1%
formic acid in acetonitrile; 0-2 min 15% B, 2-7 min from 15 to
55% B, 7-25 min from 55 to 95% B). The flow rate was
70 ?L/min. Detection of miltirone was performed by means of
a LCQDeca ion trap mass spectrometer (Thermo Electron
Corporation, San Josè, CA, USA), equipped with an ESI ion
source. ESI parameters were optimized by flow injection of
miltirone standard solution. LC-MS/MS analyses were carried
out in the positive ion mode, with isolation (width 1) of milti-
rone molecular ion (m/z 283.1), its fragmentation (energy
40%) and monitoring of fragment ions 265.1, 223 and 241
m/z. The content of miltirone in plasma samples was obtained
by external standardization from calibration curves prepared
by injecting miltirone standard solutions (15 - 500 ng/mL)
(Fig. 4). In this range all plots were linear (r2 = 0.996). In
plasma samples, miltirone resulted stable at 4°C for 24h. Fig.
(5) reports a typical chromatogram of rat plasma sample (col-
256 Current Pharmaceutical Analysis, 2008, Vol. 4, No. 4 De Palma et al.
lected at 60 min) after acute oral administration of miltirone
enriched extract. Miltirone was identified both from the reten-
tion time and positive on line mass spectra (full and fragmen-
tation spectra). The maximum plasma concentration of milti-
rone was observed to be around 90 ng/mL around 60 min (Fig.
6) and AUC resulted around 16 mg*min/mL.
S. miltiorrhiza extract has used for the treatment of dif-
ferent diseases, but in the recent years its interest is increased
in relation to the effects on Sardinia alcohol-preferring rats.
In fact, different experiments evidenced the reduction of
alcohol drinking behaviour due to the administration of S.
miltiorrhiza extract. Specifically, miltirone seems to be re-
sponsible to decrease alcohol intake in sP rats due to its anx-
In order to develop future investigations, concerning bio-
active actions of S. miltiorrhiza compounds, it is of primary
importance to get analytical methods for characterizing them
in complex matrices, such as plasma. In the recent years dif-
ferent analytical methods have been proposed for character-
izing both water soluble (such as phenolic acids) and lipo-
philic (such as terpenes) compounds present in Salvia
miltiorrhiza extracts. Specifically, the methods reported in
literature are mainly based on liquid chromatography cou-
pled to tandem mass spectrometry by means of an electros-
pray ion source. However, these methods do not include
miltirone assay. For this reason we have developed two ana-
lytical approaches to be able to identify and quantify milti-
rone in different matrices. In particular, flow injection elec-
trospray mass spectrometry (FI-ESI-MS) method can be used
for a rapid and simple analysis of miltirone in S. miltiorrhiza
extract. It is suitable for automation and can be useful for
routine quality control of pharmaceutical preparations. On
the contrary, for pharmacokinetic studies concerning milti-
rone it is necessary to use LC-MS/MS approach to allow
quantification of this bioactive compound in plasma samples,
due to the presence of many impurities.
In conclusion, there are useful analytical methods for
characterizing and assaying the different components of S.
miltiorrhiza in pharmaceutical preparations and plasma sam-
ples. These methods can be used to develop future investiga-
tions concerning the major biological activities of the com-
ponents of S. miltiorrhiza.
EIC = Extracted ion chromatogram
HPLC = High performance liquid chromatography
Limit of detection
MS/MS = Tandem mass spectrometry
Time of flight
UPLC = Ultra performance liquid chromatography
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Received: 26 March, 2008 Revised: 24 June, 2008 Accepted: 27 June, 2008