Food and Chemical Toxicology 48 (2010) 2656–2662

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Antioxidant activities of leaf extract of Salvia miltiorrhiza Bunge and related

phenolic constituents
Yuan Zhang, Xing Li, Zhezhi Wang *
Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource
Development of Endangered Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710062, China

a r t i c l e i n f o a b s t r a c t

Article history: Leaf of Salvia miltiorrhiza Bunge, the waste part during the root harvest, is rich in health-promoting phen-
Received 27 September 2009 olics and is a novel resource of natural antioxidants. The acetone and methanol extracts of leaves (AL and
Accepted 24 June 2010 ML, respectively) of S. miltiorrhiza were evaluated by various in vitro antioxidant assays. The total
phenolic contents of AL and ML were 39.0 ± 1.13 and 54.3 ± 1.1 mg gallic acid equivalents/g extract
tested, respectively. EC50 of ML was 7.0 ± 0.28 lg/mL in DPPH radical scavenging assay and 246.5 ±
Keywords: 10.35 lg/mL in superoxide radical quenching assay. It was also found that ML has prominent effects
Salvia miltiorrhiza Bunge
on the inhibition of linoleic acid oxidation (93.2%), which was equivalent to the positive control, butyl-
Leaf
Antioxidant activity
ated hydroxytoluene (BHT, p > 0.05), and was significantly higher than a-tocopherol (VE, p < 0.05). The
Phenolic compound reducing power of leaf extracts was as strong as roots (p > 0.05). HPLC and correlation analysis show that
HPLC salvianolic acid B and rosmarinic acid constitute the most abundant phenolic compounds. They are the
major contributors to antioxidant activities. The results suggested that S. miltiorrhiza leaves could be
considered as a new potential source of natural phenolic antioxidants for food, pharmaceutical, cosmetics
or nutraceutical industries.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Recently, intense attention has been devoted to natural sources
of phenolic antioxidants, not only for the prevention and treatment
of various diseases caused by oxidative damage, but also for
improving the shelf life of food products (Matkowski et al., 2008;
Tepe, 2008; Zhao et al., 2007). It has been demonstrated that
plants, especially the time-honored traditional Chinese medicines
(TCMs) contain high amounts of natural phenolic antioxidants
which have been identified as a free radical or active oxygen scav-
engers (Socha et al., 2009; Zhang and Wang, 2009).
Salvia miltiorrhiza Bunge is a perennial herb in the Labiatae
family (Li and Hedge, 1994). Its dry root or rhizome, designated
as Danshen in Chinese, is officially listed in the Pharmacopoeia of
the People’s Republic of China (Pharmacopoeia Committee of the
People’s Republic of China, 2005). Danshen is one of the most pop-
ular traditional herbal medicines in China. There are many tradi-
tional Chinese medicine preparations containing Danshen, such
as Fufang Danshen tablets, Compound Danshen dripping pills,
Abbreviations: AL, acetone extract of leaves; AR, acetone extract of roots; GAE,
gallic acid equivalents; ML, methanol extract of leaves; MR, methanol extract of
roots; TP, total phenolic content.
* Corresponding author. Tel.: +86 29 85310260; fax: +86 29 85310546.
E-mail address: zzwang@snnu.edu.cn (Z. Wang).
Danshen injection and Xiangdan injection. Danshen and its prepa-
rations have been widely used for the treatment of cardiovascular,
cerebrovascular, hyperlipidemia, and acute ischemic stroke dis-
eases (Li et al., 2009). The active constituents of Danshen have been
divided into two groups: one is lipid-soluble tanshinones, and the
other is water-soluble phenolic acids. Up to date, more than 20
phenolic acids isolated from Danshen have been well studied (Li
et al., 2009). The structures of major phenolic acid compounds
are shown in Fig. 1. Pharmacological research indicated that phe-
nolic acids from Danshen exhibited remarkable antioxidant activ-
ity (Huang and Zhang, 1992; Matkowski et al., 2008; Zhao et al.,
2008).
Due to its diverse pharmacologic properties, Danshen, pro-
cessed from the roots of S. miltiorrhiza, is ranked as one of the most
important commercial herbs in China. Its annual output is around
5000–7000 tons. As we know, the leaf biomass of S. miltiorrhiza
constitutes a considerable proportion of the whole plant. However,
large amounts of leaves are discarded as waste during root harvest
time. Although the antioxidant activity of S. miltiorrhiza roots is
well known, scarce information of the antioxidant activity of other
organs, especially the leaves could be obtained (Li and Wang, 2009;
Matkowski et al., 2008). In addition, the qualitative and quantita-
tive analyses of phenolic acids as well as their contribution to
the overall antioxidant properties of leaf extracts have not been
studied in detail to date.

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.06.036
 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662
Fig. 1. Structures of six phenolic acids in S. miltiorrhiza.
As part of our studies on novel source exploitation from tradi-
tional Chinese medicines, this study presents a first comprehensive
analysis of the contents of phenolic compounds and the in vitro
antioxidant activities of acetone and methanol extracts from leaves
of S. miltiorrhiza. The results were also compared with the corre-
sponding root extracts, and two major water-soluble phenolic
acids, rosmarinic acid and salvianolic acid B.
2. Materials and methods
2.1. Chemicals and reagents
Folin–Ciocalteu’s phenol reagents, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
ascorbic acid (VC), a-tocopherol (VE), butylated hydroxytoluene (BHT), dihydronic-
otinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitroblue
tetrazolium chloride (NBT), b-carotene, linoleic acid, Tween 40, gallic acid, (-)-epi-
catechin, and rutin were purchased from Sigma–Aldrich (St. Louis, USA). Sodium
carbonate (Na2CO3), sodium nitrite (NaNO2), aluminium chloride (AlCl3), sodium
hydroxide (NaOH), ferrichloride (FeCl3), potassiumferricyanide (K3Fe(CN)6), trichlo-
roacetic acid (TCA), 30% hydrogen peroxide, and HPLC grade methanol were pur-
chased from the Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). Milli-Q water
(Millipore, Bedford, MA) was used in all work. Standards of Danshensu, proto-
catechuic acid, protocatechualdehyde, caffeic acid, rosmarinic acid, and salvianolic
acid B were purchased from the National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, China). All standards were prepared as stock solu-
tions in methanol. Stock working solutions of the standards were stored in darkness
at 18 °C. All other chemicals and solvents are of analytical grade and purchased
from Xi’an Chemical Reagent Company (Xi’an, China).
2.2. Plant materials
S. miltiorrhiza Bunge were collected from the Qinling Mountain, Shaanxi Prov-
ince (northwest of China) before the harvest stage (Sep., 2006) at 600 m altitude.
A voucher specimen (No. DS060805) was identified by Professor Yi Ren and depos-
ited in the Key Laboratory of the Ministry of Education for Medicinal Resources and
Natural Pharmaceutical Chemistry, China.
2.3. Preparation of the extracts
The air-dried leaves and roots of S. miltiorrhiza (50.00 g) were ground to a fine
powder in a mechanical grinder with a 2 mm diameter mesh and then successively
extracted with petroleum ether, acetone, and methanol in a Soxhlet apparatus for
6 h. After filtration of each solvent, the organic phases were independently concen-
trated under a vacuum by evaporating to dryness. The acetone and methanol ex-
tracts were stored in a refrigerator for further analysis. The colour and extraction
efficiency (%) of acetone and methanol extracts was shown in Table 1. Each sample
was extracted in triplicate.
2.4. Determination of total phenolics (TP)
TP were measured using a modified colorimetric Folin–Ciocalteu method (Luo
et al., 2004). Aliquots of test samples (1.0 mL, 1.0 mg/mL) were mixed with
1.5 mL of 20% Na2CO3 and incubated at room temperature for 2 min. After the addi-
2657
tion of 500 lL 50% Folin–Ciocalteu’s phenol reagents and 7 mL deionized water, the
reaction tube was incubated for another 2 h at room temperature after which the
absorbance was read at 760 nm. Measurements were carried out in triplicate and
calculations were based on a calibration curve obtained with gallic acid (24–
64 lg/mL). The total phenolics were expressed as milligrams of gallic acid equiva-
lents (GAE) per gram of extract. The calibration equation for gallic acid was
y = 0.0122x + 0.0016 (r2 = 0.9996).
2.5. Analysis of individual phenolic compounds by analytical RP-HPLC
Qualitative and quantitative analysis of the phenolic compounds in the extracts
were performed according to the procedure described as follows. Different extracts
were separated using a reversed-phase HPLC column on a SHIMADZU LC-2010A sys-
tem equipped with an autoinjector, a UV detector, and LC-Solution software (SHIMA-
DZU, Kyoto, Japan). Spectral data were detected at 280 nm during the whole run. A
SHIMADZU (5 lm, 150  4.6 mm) column (SHIMADZU, Kyoto, Japan) was used for
phenolic compound separation at 30 °C. The mobile phase was composed of solvent
(A), 0.5% acetic acid water solution, and solvent (B), methanol. The solvent gradient
was as follows: 0–30 min from 10% B to 25% B; 30–45 min from 25% B to 35% B; 45–
60 min from 35% B to 50% B. A flow rate of 1.0 mL/min was used, and 10 lL of 20 mg/
mL extracts were injected. Samples and mobile phases were filtered through a
0.22 lm filters prior to HPLC injection. Each extract was analyzed in triplicate.
2.6. Determination of antioxidant activities
The antioxidant activities of rosmarinic acid, salvianolic acid B, acetone and
methanol extracts were evaluated using five antioxidant test systems. DPPH radical
scavenging activity was carried out according to the method of (Brand-Williams
et al., 1995) with minor modifications. The superoxide radical scavenging activities
were measured by the method of (Robak and Gryglewski, 1988). The b-carotene–
linoleic acid bleaching assay was carried out according to the method of described
by (Socha et al.,2009) with some modifications. The ferric reducing/antioxidant
power of the extracts was determined according to the method of (Oyaizu, 1986).
Detailed methodology is presented in our previous research paper (Zhang and
Wang, 2009).
2.7. Stability of the extracts
The colour of acetone and methanol extracts was stable during storage for
12 months at 25 °C. The antioxidant activities of these stored extracts were com-
pared with that of freshly prepared extracts. No significant difference (p < 0.05)
was observed using the five antioxidant tests mentioned above.
2.8. Statistical analysis
EC50 values from the in vitro data were calculated by regression analysis. Each
experiment was repeated three times. The data were expressed as means ± stan-
dard error (SE) and analyzed using SPSS (version 13.0). One-way analysis of vari-
ance (ANOVA) and Tukey multiple comparisons were carried out to test any
significant differences between the means. Differences between the means at the
5% confidence level were considered significant. Correlation coefficients (r) to deter-
mine the relationship between variables were calculated using the Bivariate corre-
lation statistical function.
2658 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662
Table 1
Colour, extraction efficiency (%), total phenolic (TP) and phenolic acid contents of extracts from S. miltiorrhiza.a
AL
Colour Light green
Extraction efficiency (%)b 11.27 ± 0.12ac
TP (mg of gallic acid/g extract) 39.0 ± 1.13a
Phenolic compounds
Danshensu (lg/mg) 0.28 ± 0.02a
Protocatechuic Acid (lg/mg) 0.07 ± 0.01a
Protocatechualdehyde (lg/mg) 0.03 ± 0.01a
Caffeic Acid (lg/mg) 0.66 ± 0.06a
Rosmarinic Acid (lg/mg) 4.56 ± 0.31a
Salvianolic Acid B (lg/mg) n.d.
Abbreviations: AL, acetone extract of leaves; ML, methanol extract of leaves; AR, acetone extract of roots; MR, methanol extract of roots.
a
Each value is presented as the mean ± standard error (n = 3).
b
The extraction efficiency is expressed as the % of acetone or methanol-extractable dry weight of plant material.
c
Values followed by the same letter are not significantly different (p > 0.05).
d
n.d., Not detected.
3. Results and discussion
3.1. Total phenolic contents of various extracts
Since Salvia species are known to be rich in phenolics, the con-
tents of total phenolics (TP) in the acetone and methanol extracts
from leaves (AL and ML, respectively) of S. miltiorrhiza were inves-
tigated (Table 1). They were also compared with the corresponding
root extracts (AR and MR, respectively). The amounts of TP in dif-
ferent S. miltiorrhiza extracts varied widely, ranging from 39.0 to
54.3 GAE/g. ML had the highest TP content (54.3 GAE/g). It was
similar to MR (p > 0.05), but significantly more than other samples
(p < 0.05). No difference of TP content between AL and AR (p > 0.05)
were observed. The leaf extracts (AL + ML) contain as much pheno-
lic content (p > 0.05) as that of root extracts (AR + MR), and were
93.3 and 96.5 GAE/g, respectively. Our data demonstrate the high
total phenolic contents from leaf extracts of S. miltiorrhiza for the
first time. The positive correlation between phenolic content and
antioxidant potential of various plant extracts have been well dem-
onstrated in prior reports (Socha et al., 2009; Zhang and Wang,
2009). Therefore, the high content of total phenols in leaf extracts
of S. miltiorrhiza indicated the strong antioxidant properties of this
part. The results confirmed the possibility of recovering high
amounts of phenolics with antioxidant properties from leaves of
S. miltiorrhiza.
3.2. Qualitative and quantitative analyses of individual phenolic
compounds in leaf extracts
In the past 50 years, the chemical constituents from roots of S.
miltiorrhiza have been well studied (Li et al., 2009). However, the
phenolic components from leaves of S. miltiorrhiza have been
poorly investigated. In the present study, qualitative–quantitative
analysis of the leaf and root extracts from S. miltiorrhiza was per-
formed by HPLC (Table 1). The chromatographic profiles of pheno-
lic composition of leaf extracts were shown in Fig. 2. It can be
noticed that satisfactory separation with good resolution can be
achieved by the optimized HPLC condition. A total of six phenolic
compounds (danshensu, protocatechuic acid, protocatechualde-
hyde, caffeic acid, rosmarinic acid, and salvianolic acid B, Fig. 1)
were identified by comparison to the retention times and UV spec-
tra of authentic standards analyzed under identical analytical con-
ditions. The quantitative data were calculated from their respective
calibration curves (Table 2). Under the optimized HPLC condition,
rosmarinic acid was present in high levels in both AL and ML
(4.56 ± 0.31 lg/mg and 4.72 ± 0.40 lg/mg, respectively), while sal-
vianolic acid B was rich only in ML (14.34 ± 0.47 lg/mg). Traces of
ML AR MR
Green Light red Red
18.66 ± 0.33b 15.18 ± 0.22b 16.09 ± 0.13b
54.3 ± 1.18b 42.9 ± 1.89a 53.6 ± 0.70b
1.20 ± 0.01b 0.35 ± 0.01a 0.28 ± 0.01a
0.05 ± 0.01a 0.06 ± 0.01a n.d.d
0.02 ± 0.00a 0.03 ± 0.01a n.d.
0.59 ± 0.04a 0.38 ± 0.03b n.d.
4.72 ± 0.40a 15.19 ± 0.93b 0.7 ± 0.07c
14.34 ± 0.47a 3.77 ± 0.17b 18.07 ± 1.16a
danshensu, protocatechuic acid, protocatechualdehyde, and caffeic
acid were also detected, but they were about one-tenth or even
just one-hundredth to that of salvianolic acid B and rosmarinic
acid. Therefore, salvianolic acid B and rosmarinic acid were the
main components of the leaf extracts (Table 1), which were evalu-
ated as phenolic controls in successive antioxidant tests. Salvianol-
ic acid B is the predominant ingredient when Danshen is processed
traditionally by extraction with water. It is responsible for many of
therapeutic actions of Danshen, especially the significant antioxi-
dative and free radical scavenging effects and to protect against
ischemia–reperfusion injuries in heart and brain (Huang and
Zhang, 1992; Zhao et al., 2008). Rosmarinic acid has been reported
to have a number of biological activities in vitro, such as antiviral,
antibacterial, anti-inflammatory, anticarcinogenic, antithrombotic
and antioxidant (Petersen and Simmonds, 2003). The presence of
rosmarinic acid in medicinal plants has beneficial and health-pro-
moting effects (Petersen and Simmonds, 2003). Several patents for
rosmarinic acid have been granted, particularly within the phar-
maceutical, cosmetic and food industries (Achamlale et al., 2009).
With the constantly increased market demand, the price for phe-
nolic crude extract of S. miltiorrhiza is rising. However, a large
amount of leaves is discarded as waste during the root harvest.
Our present study revealed that, compared with the corresponding
root extracts, leaf extracts of S. miltiorrhiza exhibited considerable
properties not only in the quality but also in the quantity of sal-
vianolic acid B and rosmarinic acid. It appears of interest to evalu-
ate the potential of this valuable resource for the production of
natural phenolic antioxidants, from a waste utilization perspective.
As far as our literature survey could ascertain, no information ex-
ists about the utilization of leaves of S. miltiorrhiza. From this point
of view, our study can fulfill an important deficiency in the litera-
ture and be a starting point for the exploration of the potential re-
sources of phenolic antioxidants.
3.3. Antioxidant activities of the extracts
Due to the complex nature of plant extracts and the diverse
mechanism of protective effects, antioxidant activity evaluation
using any single method seems to be rather unrealistic (Verhagen
et al., 2003). Accordingly, both the hydrophilic and lipophilic anti-
oxidant assays were employed to assess the antioxidant effects of
the various extracts of S. miltiorrhiza in this study (Fig. 3, Table 3).
Of these, DPPH radical scavenging assay, superoxide radical
quenching assay, b-carotene–linoleic acid system and reductive
potential are the most commonly used for the determination of
antioxidant activities of plant extracts.
 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662 2659

Fig. 2. HPLC chromatograms of (A) acetone extract of S. miltiorrhiza, (B) methanol extract of S. miltiorrhiza, and (C) mixture of standard phenolic compounds at 280 nm. Peak:
(1) danshensu, (2) protocatechuic acid, (3) protocatechualdehyde, (4) caffeic acid, (5) rosmarinic acid, and (6) salvianolic acid B.

3.3.1. DPPH radical scavenging activities and ML exhibited high scavenging activity toward DPPH (91.2%,
As shown in Fig. 3A, the DPPH radical scavenging activities of 86.8%, 90.9% and 91.2%, respectively) at 0.08 mg/mL. EC50 values
four extracts of S. miltiorrhiza were dose-dependent. AR, MR, AL were found to be the least in salvianolic acid B, followed by ros-
2660 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662

Table 2
Linear calibration curves for the HPLC-UV analysis of the phenolic compounds of extracts from S. miltiorrhiza.

No. Phenolic compounds Retention time (min)a Equation of linear regression R2 Linearity range
(peak area-concentration) (mg/ml)
1 Danshensu 7.30 ± 0.10 y = 5E 08x + 6E 05 0.9978 0.01–1
2 Protocatechic acid 8.96 ± 0.17 y = 2E 08x + 1E 05 0.9993 0.01–1
3 Protocatechualdehyde 11.82 ± 0.13 y = 1E 08x + 7E 06 0.9997 0.01–1
4 Caffeic acid 20.80 ± 0.08 y = 1E 08x + 4E 05 0.9702 0.01–1
5 Rosmarinic acid 53.34 ± 0.19 y = 8E 09x + 3E 05 0.9997 0.01–1
6 Salvianolic acid 57.21 ± 0.17 y = 3E 07x 0.0007 0.9999 0.01–1
a
Each value is presented as mean ± standard error (n = 3).

A BHT VC VE AL ML AR MR
B 100.0 VC AL ML AR MR
100.00

80.0

Scavenging effect (%)

80.00
Scavenging effect (%)

60.00 60.0

40.00 40.0

20.00 20.0

0.00 0.0
0.6 10.0 20.0 80.0 0.10 0.20 0.50 1.00
Concent ration (µg/mL) Concentration (mg/mL)

BHT VE AL ML
AR MR Control
D 5.0 BHT AL ML AR MR
C 0.7 4.5
4.0
0.6
Absorbance (470nm)

3.5
Absorbance (nm)

3.0
0.5
2.5

0.4
2.0
1.5
0.3 1.0
0.5
0.2 0.0
0 20 40 60 80 100 0.01 0.10 0.30 0.50
Time of incubation (min) Concentration (mg/mL)

Fig. 3. (A) The DPPH radical scavenging activities of VC, VE, BHT and extracts of S. miltiorrhiza (0.0009–0.1200 mg/mL). (B) The superoxide radical scavenging activities of VC
(0.005–0.100 mg/mL) and extracts of S. miltiorrhiza (0.050–1.000 mg/mL). (C) Antioxidant activities of VE, BHT, control and extracts of S. miltiorrhiza (2 mg/mL) obtained using
the b-carotene–linoleic acid assay. (D) Reducing power of BHT and extracts of S. miltiorrhiza (0.01–0.50 mg/mL). Each value is presented as the mean ± standard error (n = 3).
The vertical bars indicate standard errors, where they exceeded the symbol size.

marinic acid, VC, BHT, ML, AR, MR, VE and AL (Table 3). It seemed The scavenging abilities of leaf extracts (AL and ML) on superoxide
that ML was superior to all extracts tested and was similar to VE radicals were a little weaker than root (Table 3). In addition, the
(p > 0.05) with regard to scavenging abilities, while AL was sub- scavenging activities of all the extracts were found to be lower
stantially weaker than the rest. However, Matkowski et al. re- (p < 0.05) than that of VC, which is considered to be a potent super-
ported that the EC50 value of methanol extract of leaves (17.14 ± oxide radical scavenger. However, rosmarinic acid and salvianolic
2.20 lg/mL) was significantly lower than roots (12.14 ± 0.50 lg/ acid B, the two major phenolic compounds of leaf extracts, possess
mL) in DPPH free radicals (Matkowski et al., 2008). The results sug- comparable radical scavenging abilities with VC (p > 0.05). In the
gested that our extraction procedures could concentrate more ac- DPPH and superoxide radical scavenging assay, the activity differ-
tive compounds in methanol extract by successively eluting with ences between pure phenolic acids and crude leaf extracts were 2–
petroleum ether, acetone, and methanol. As active compounds 7 folds, suggested that purification of active phenolic ingredients in
present in medicinal plants are in low concentrations, applying leaves of S. miltiorrhiza should be needed.
selective solvents for the extraction procedure is recommended.
In addition, the considerable differences may also depend on the
3.3.3. b-Carotene–linoleic acid assay
distinct habitat in which the plant has been collected.
As shown in Fig. 3C, all of the extracts were capable of inhibiting
the bleaching of b-carotene by scavenging linoleate-derived free
3.3.2. Superoxide radical scavenging activities radicals. The absorbance of the control at 470 nm decreased to a
In fact, as shown in Fig. 3B, all the extracts showed good super- minimal value of 0.246 after 80 min, while that of the extracts
oxide radical scavenging activities (87.66%–97.20%) at 1.0 mg/mL. were still between 0.31 and 0.615. These results indicated that
 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662 2661

Table 3 about 70% of the whole plant, around 3500–4900 tons of leaves
Antioxidant activities of extracts from S. miltiorrhiza and references measured by are discarded as waste during root harvest time. As far as our liter-
different assays.a
ature survey could ascertain, data on the antioxidant activities of S.
Plant EC50 in EC50 in Inhibition of Reducing miltiorrhiza leaves are scarce. In our previous study, the chemical
Extracts DPPH superoxide linoleic acid powerb composition, antimicrobial and antioxidant activities of the essen-
radical radical oxidation (%)
(lg/ml) (lg/ml)
tial oil in leaves of S. miltiorrhiza were evaluated (Li and Wang,
2009). The biological activity tests indicated that the oil may be
AL 13.9 ± 0.16ac 259.7 ± 5.02a 81.6 ± 1.15a 1.7 ± 0.07a
ML 7.0 ± 0.28b 246.5 ± 10.35a 93.2 ± 0.77b 2.6 ± 0.04a
considered as a potent natural agent in food preservation. Matkow-
AR 7.6 ± 0.35b 104.3 ± 1.50b 97.5 ± 0.17b 2.5 ± 0.06a ski et al. have also confirmed that leaves of S. miltiorrhiza grown in
MR 7.9 ± 0.18b 122.3 ± 1.29b 71.7 ± 0.06c 2.7 ± 0.08a Poland show strong antioxidant activity (Matkowski et al., 2008).
BHT 4.5 ± 0.23c n.a.d 90.3 ± 0.19b 3.3 ± 0.44b These two reports are the unique representatives of the
VC 3.7 ± 0.05c 32.7 ± 0.83c n.a. n.a.
phytochemical composition and biological activities of leaves of
VE 8.0 ± 0.46b n.a. 82.9 ± 0.29a n.a.
Rosmarinic 3.2 ± 0.11c 35.6 ± 0.58c 84.2 ± 0.82a 3.1 ± 0.01b S. miltiorrhiza in the literature. However, both the chemical compo-
acid nents and biological activities of medicinal plants may be consider-
Salvianolic 2.9 ± 0.13c 33.6 ± 0.63c 90.5 ± 0.96b 3.5 ± 0.02b ably different. They are dependent on the habitat, development
acid B stage and collected season. Our study can fulfill the literature defi-
Abbreviations: AL, acetone extract of leaves; ML, methanol extract of leaves; AR, ciency. Compared with the corresponding root extracts, leaf ex-
acetone extract of roots; MR, methanol extract of roots. tracts of S. miltiorrhiza possess considerable amounts of total
a
Each value is presented as mean ± standard error (n = 3). phenolics, similar phenolic composition and significant antioxi-
b
Absorbance at 700 nm (0.5 mg/ml).
c dant activities. Salvianolic acid B and rosmarinic acid were the pre-
Column wise values with same letter indicate no significant difference
(p > 0.05). dominant phenolic acids in the leaf extracts of S. miltiorrhiza. In
d
n.a., Data is not available. many in vitro and in vivo studies, salvianolic acid B and rosmarinic
acid have been reported to possess higher antioxidant activities
than vitamins and synthetic antioxidants and to protect against
all of the extracts could significantly inhibit oxidation of linoleic ischemia–reperfusion injuries in heart and brain (Achamlale
acid. Thus, our results implied that leaves of S. miltiorrhiza contain et al., 2009; Huang and Zhang, 1992; Petersen and Simmonds,
the same valuable antioxidants as roots. The antioxidant activities 2003; Zhao et al., 2008). Our data is consistent with the previous
of the extracts were decreased in the following order: AR > ML > studies. Among the four antioxidant assays, both salvianolic acid
salvianolic acid B > BHT > rosmarinic acid > VE > AL > MR. The re- B and rosmarinic acid exhibited stronger antioxidant activities
sults showed that ML had prominent effects on the inhibition of than that of positive control (VC, VE or BHT). They are excellent
linoleic acid oxidation, which were significantly (p < 0.05) higher scavengers for free radicals, both cation radicals such as DPPH
than the rosmarinic acid and VE. There were no statistical differ- and anion radicals like superoxide. They are also involved in the
ences among ML, salvianolic acid B and BHT (p > 0.05). According inhibition of linoleic acid oxidation and function as reducing
to the b-carotene bleaching data, AR and ML as mixture were pre- agents.
ponderant in a complex heterogeneous medium than the pure phe- Taking the cardioprotective and antioxidant application of phe-
nolic compounds and positive controls. This suggested that nolic acids from S. miltiorrhiza into consideration, our study on
synergies among the antioxidants in the crude extracts may occur phytochemical contents and antioxidant activities of S. miltiorrhiza
and the leaf extracts have potential use as an antioxidative preser- leaf, provide new scientific information for the further develop-
vative in emulsion-type systems. ment of modern herbal medicines. Also, the efficient, inexpensive
and environmentally rational utilization of Danshen industry
3.3.4. Ferric reducing/antioxidant power assay wastes (leaves of S. miltiorrhiza) is of undisputed importance for
The reducing power of all samples was proportional to their higher profitability and minimal environmental impact.
concentrations (Fig. 3 D). The absorbance was similar at 0.01 mg/
mL (0.206–0.311) and diverged with the increase of the reaction 3.4. Comparison between different antioxidant assays
concentration to 0.5 mg/mL (1.743–3.512). According to Table 3,
the reducing power of leaf extracts was as strong as roots in this The results of TP contents, HPLC analysis and the different anti-
system (p > 0.05), suggesting their remarkable potency to donate oxidant assays used in the present investigation were compared to
electron to reactive free radicals, then convert them into more sta- and correlated with each other. There are significant linear rela-
ble non-reactive species and finally terminate the free radical chain tionships between DPPH radical scavenging activity and reducing
reaction. However, they were still slightly less effective than that of power in all extracts (r = 0.949, p < 0.01). The correlation of TP
rosmarinic acid, salvianolic acid B and BHT (Table 3). Our data is and salvianolic acid B contents was very high (r = 0.907, p < 0.01).
consistent with the previous studies (Matkowski et al., 2008). Salvianolic acid B also showed a positive correlation with DPPH
Antioxidants are widely needed to prevent deterioration of scavenging activity (r = 0.693, p < 0.05) and reducing power
many oxidisable goods, such as food, cosmetics, pharmaceuticals (r = 0.748, p < 0.01). Notably, a stronger correlation was observed
and plastics. The growing interest in the replacement of synthetic between ability to inhibit linoleic acid oxidation and rosmarinic
antioxidants by natural ones has fostered research on identifying acid contents (r = 0.804, p < 0.01). Grzegorczyk, Matkowski and
new antioxidants from plant sources (Moure et al., 2001). Special Wysokinska found out the polyphenols (represented by rosmarinic
attention is focused on the extraction of antioxidant compounds acid) in S. officinalis were more efficient in an aqueous environment
(mainly phenolics) from inexpensive or costless sources from agri- (Grzegorczyk et al., 2007). However, according to the present re-
cultural and industrial wastes. As reviewed previously, numerous sults, salvianolic acid B may be the most powerful antioxidant
antioxidants could be extracted from those residual sources component for DPPH scavenging activity and reducing power in
(Moure et al., 2001). accordance with previous reports (Zhao et al., 2007, 2008), and ros-
Danshen, processed from the roots of S. miltiorrhiza, belongs to marinic acid primarily exerts its effect through the inhibition of
one of the most popular traditional herbal medicines in Asian linoleic acid oxidation, which is in line with the study of Tepe
countries. The annual production of Danshen is around 5000– (2008). The variation in correlation coefficients among different
7000 tons. Since the leaf biomass of S. miltiorrhiza constitutes antioxidant assays indicated that a single assay is not sufficient
2662 Y. Zhang et al. / Food and Chemical Toxicology 48 (2010) 2656–2662
to evaluate the antioxidant activity. Therefore, two or more meth-
ods should always be employed in order to evaluate the compre-
hensive antioxidant activity.
4. Conclusion
This study is a first report on the antioxidant activities and
phenolic composition of the leaves of S. miltiorrhiza, from a waste
utilization perspective. Compared with the corresponding root ex-
tracts, leaf extracts of S. miltiorrhiza possess considerable amounts
of total phenolics, similar phenolic composition and significant
antioxidant activities. HPLC and correlation analysis show that sal-
vianolic acid B and rosmarinic acid constitute the most abundant
phenolic compounds. They are the major contributors to antioxi-
dant activities. In light of these valuable bioactivities, leaves of S.
miltiorrhiza considered as waste materials have good commercial
potential to be utilized as promising natural antioxidants in the
food, pharmaceutical or cosmetic industries, not only for the low
cost but also for the large amounts available.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are grateful to Dr. Jeremy T. Lundholm (Biology
Department/Environmental Studies Program, Saint Mary’s Univer-
sity) for improving the manuscript. This work benefited from
financial support from the ‘‘Foundation for Excellent Doctor Degree
Dissertation” (S2008YB04) of Shaanxi Normal University and the
10–11th ‘‘five-year-technique-project” by the Ministry of Tech-
nique and Science (2006BAI06A12-04), PR China.
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