antioxidant activity of salvia tchihatcheffii leaf extracts

M. Uygun, D. A. Uygun, K. Aktaş, C. Özdemir, M. Özkan, A. A. Karagözler, / Ege Uni. J. of Faculty of
Sci., Vol. 36, (1/2), 2012, 18-33
(REFEREED RESEARCH)
ANTIOXIDANT ACTIVITY OF SALVIA
TCHIHATCHEFFII LEAF EXTRACTS
Murat Uygun1, Deniz Aktaş Uygun1*, Kamuran Aktaş2, Canan Özdemir2, Mustafa
Özkan3, A. Alev Karagözler1
1
Department of Chemistry, Faculty of Science and Arts, Adnan Menderes University, 09010 Aydın,
Turkey
2
Department of Biology, Faculty of Science and Arts, Celal Bayar University, 45047 Manisa, Turkey
3
Department of Biology, Faculty of Science and Arts, Ahi Evran University, 40100 Kırşehir, Turkey
Received: 10.05.2012
Accepted: 30.11.2012
Abstract: Salvia tchihatcheffii (S. tchihatcheffii) is an endemic plant to Ankara province of Turkey. The
leaves of Salvia (Lamiaceae) species have a reputed use in traditional medicine and they are known as
“adaçayı” in the regions where they grow and consumed as a hot drink. In the present study, the
antioxidant activities of the diethyl ether (E), ethanol (A), water (W) and hot water (S) extracts of S.
tchihatcheffii leaves were investigated by ferric thiocyanate (FTC) and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) free radical scavenging methods. S extract exhibited high antioxidant activity (96.9 %) with FTC
contain the highest amount (262.
suggest that, leaves of the S. tchihatcheffii may consider as a natural source of antioxidants and proline.
Keywords: Salvia tchihatcheffii, adaçayı, antioxidant activity, DPPH, proline
1. INTRODUCTION
An imbalance caused by overproduction of oxidants leads to oxidative stress,
resulting in damage to large biomolecules such as lipids, DNA and proteins. Oxidative
damage increases the risk of degenerative diseases such as cancer and cardiovascular
diseases. Antioxidants reduce oxidative damage to biomolecules by modulating the
effects of reactive oxidants [1]. In brief, antioxidants can prevent or inhibit oxidation
*
Corresponding Author:
Tel: +90 256 2128498
Fax: +90 256 2135379
E-mail: [email protected]
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M. Uygun, D. A. Uygun, K. Aktaş, C. Özdemir, M. Özkan, A. A. Karagözler, / Ege Uni. J. of Faculty of
Sci., Vol. 36, (1/2), 2012, 18-33
by: 1) decreasing localized O2 concentrations so that oxidation is less likely to occur; 2)
preventing initiation reactions by scavenging free radicals which are capable of directly
abstracting H from molecules; 3) binding transition metal ion catalysts such as Cu and
Fe to prevent generation of initiating free radicals; 4) decomposing peroxides to prevent
their conversion to further active and initiating radical species; 5) reacting with chainpropagating radicals such as the peroxyl and alkoxyl radicals to prevent continued H
abstraction from fatty acid side chains [2].
Natural and synthetic antioxidants are added in the foods for preventing detrimental
deterioration. The synthetic antioxidants such as butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT) are already in use as food preservatives, but their
safety and toxicity are being questioned [3, 4]. Therefore, there is still great need for
novel and safe antioxidant substances [5].
Plants (fruits, vegetables, medicinal herbs, etc.) may contain a wide variety of free
radical scavenging molecules, such as phenolic compounds (e.g. phenolic acids,
flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds
(alkaloids, amines, betalains), vitamins, terpenoids (including carotenoids), and some
other endogenous metabolites, which are rich in antioxidant activity [6].
Salvia L. belongs to the family Lamiaceae, representing a diverse cosmopolitan
assemblage of nearly 1000 species [7]. The genus comprises at least 500 species in
Central and South America, 250 species in Central Asia and the Mediterranean and 90
species in Eastern Asia [8]. Turkey is a major centre of diversity for Salvia in Asia [9].
Since the most recent reviews of the genus in Turkey, four new species have been
described and the total has now reached 90. Forty seven of these Salvia species in
Turkey are endemic [10].
The leaves of Salvia (Lamiaceae) species have a reputed use in traditional medicine.
They are known as “adaçayı” in the regions where they grow and consumed as a hot
drink. The most common sage in the world is S. officinalis L. [11]. Salvia species and
their essential oils are used in food flavoring, pharmaceuticals and in perfumery. Salvia
species have been reportedly used in folk medicine for wound healing and in alleviating
stomach, liver and rheumatism pains and for treating the common cold in the form of
infusion and decoction [12].
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Sci., Vol. 36, (1/2), 2012, 18-33
Antioxidant activities of some Salvia species were reported in recent studies [13 –
19]. The main objective of this study was to investigate the antioxidant activities of S.
tchihatcheffii extracts. To evaluate the antioxidant activity of the extracts, inhibition of
lipid peroxidation, radical scavenging activities, total phenolic contents, flavonoid
contents, reducing powers and proline contents were measured and correlation among
this parameters were also investigated.
2. EXPERIMENTAL
Chemicals
Ethyl alcohol, diethyl ether, HCl, FeCl2.4H2O, FeCl3.6H2O, glacial acetic acid,
methanol and toluene were obtained from Merck (Darmstadt, Germany). Phosphate
buffer solution (PBS), ammonium thiocyanate, Folin Ciocalteu’s Reagent (FCR),
butylated hydroxytoluene (BHT) and proline were obtained from Sigma (Steinheim,
Germany). Linoleic acid, Na2CO3, NaH2PO4.2H2O, Na2HPO4, K3Fe (CN)6, 2,2diphenyl-1-picrylhydrazyl (DPPH), rutin trihydrate, 5-sulfosalicylic acid dihydrate,
ninhydrin were obtained from Fluka (Buchs, Switzerland). Trichloroacetic acid (TCA),
Phosphoric acid was from Carlo Erba (Rodano, Italy); gallic acid, NaOH, AlCl3.6H2O
was from Riedel-de Haen (Czech Republic). Ascorbic acid was from Panreac
(Barcelona, Spain). Deionized double distilled water was used for all assays.
Plant material
Salvia tchihatcheffii (Fisch. & Mey.) Boiss. was collected from the vicinity of
Polatlı/Ankara Province of Centre Anatolia/Turkey in May and identified by botanist
Dr. Mustafa Özkan in the Department of Biology at Ahi Evran University. Some
samples were used for analysis; some were dried as herbarium sample and stored in
Celal Bayar University Herbarium (CBUH 15276).
Plant extracts
Dried leaves of S. tchihatcheffii were finely powdered in a grinder. The powder was
passed through a sieve in order to maintain particle size unity (300 microns). Three
solvents (diethyl ether, ethanol and water) with distinct polarity (dielectric constants
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4.3, 24.3, and 78.5, respectively) were employed sequentially for the preparation of
plant extracts as described our previous work [20]. For this reason, 10 grams of
powdered plant was mixed with 250 ml of diethyl ether and extracted in a Soxhlet
apparatus at +40 °C until the solvent in the sample compartment became colorless. The
extraction unit was covered in order to prevent light deterioration during extraction. The
resulting solution was decanted to the flask of a rotary evaporator (IKA RV 05 basic
1B) and evaporated to dryness. This diethyl ether extract was coded as (E) sample and
stored in a desiccated container at +4°C until used. The residue obtained from diethyl
ether extraction was washed with aliquots of ethanol until the extract became colorless.
Then, the extracts were filtered, pooled and evaporated down to dryness using rotary
evaporator at +40°C. This ethanol extract was coded with letter (A) and stored in a dry
place at +4 °C. The residue from ethanol extract was mixed with 20x distilled water and
heated for 2-3 minutes and filtered immediately through a filter paper. The filtrate was
freeze dried (Labconco Freezone 6; 0.04 mbar; -50°C) for 96 hours. This extract was
coded with letter (W) and stored in a dry place at +4 °C.
S. tchihatcheffii is traditionally consumed as a hot drink and it known as “adaçayı”.
Therefore, it was decided to include a direct hot water extract in this study. For this, 10
g of plant powder was mixed with 20x volume of water and boiled for 10 minutes and
filtered hot. Then the filtrate was freeze dried. The residue was coded with letter (S) and
stored in a dry place at +4 °C.
Total antioxidant activity determination
Total antioxidant activity was measured by ferric thiocyanate (FTC) method
described by Saha et al. [21]. For this, 4 mg of plant extract was dissolved in
appropriate solvent and mixed with 4.1 ml linoleic acid emulsion (2.5 %, v/v). Eight ml
of PBS (0.04 M; pH 7.4) and 3.9 ml of water was added to the mixture and incubated at
+40°C and assayed with 24 h intervals for 96 hours. For the assay, 100 µl of the
reaction mixture was mixed with 9.7 ml of ethyl alcohol, 100 µl of NH4SCN (30 %,
v/w) and FeCl2 (20 mM in 3.5 % hydrochloric acid) solutions. The mixture was
vortexed and absorbance at 500 nm was read after 3 min using a Shimadzu 1601 UVVis spectrophotometer. Absorption of reaction mixture with no plant extract was
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measured as control. For the positive control, sample was replaced with BHT. Total
antioxidant activity was calculated as % inhibition according to the following equation:
Percent inhibition = [(A0-A1) / A0] x 100, where A0 is the absorbance of the control
reaction and A1 is the absorbance of the sample.
DPPH radical scavenging assay
Radical scavenging activity of plant extracts against 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical was determined spectrophotometrically described by Brand-Williams et
al. [22]. Briefly, 1 ml of 0.1 mM DPPH in ethanol was mixed with 3 ml of extract
solution with different dozes (5-250 µg/ml) and the mixture was vortexed. The samples
were kept in the dark for 30 min at room temperature and then the decrease in
absorption at 517 nm was measured. Control was used for this test, as a negative control
containing all regents except the sample. Ascorbic acid, BHT and rutin were used as
positive controls. DPPH radical scavenging activity was expressed using the formula: %
DPPH radical scavenging activity = [(A0-A1) / A0] x 100 where A0 was the absorbance
of the control and A1 was the absorbance of the sample.
Determination of total phenolic content
Total phenolic contents (TPC) of the extracts were assayed according to FolinCiocalteu method [23]. Briefly, stock solutions were prepared by dissolving 10 mg of
plant extract in 10 ml ethyl alcohol or water. 300 µl of this stock solution was added to
45.7 ml of water. Then, one ml of FCR was added to the mixture. After 3 min 3 ml of
Na2CO3 (20 % w/v) solution was added to the mixture. The mixtures were incubated for
2 h in the dark at room temperature. Absorption at 760 nm was measured. The amount
of total phenolic compounds was calculated as mg of gallic acid equivalents (GAE)
from the calibration curve of gallic acid standard solution.
Determination of flavonoid content
The flavonoid content was measured by the method applied by Quettier-Deleu et al.
[24]. For this, 1 ml of extract solution was mixed with 1 ml of 2 % (w/v) methanolic
solution of AlCl3.6H2O. After 10 min the absorbance was measured at 430 nm. Rutin
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(Rt) was used as standard flavonoid and the results were expressed as mg rutin
equivalent (RtE) / gram extract.
Reducing power of the extracts
The reducing powers of the extracts from S. tchihatcheffii were determined according
to the method of Oyaizu [25]. Stock solutions were prepared by mixing 10 mg of extract
with 10 ml of extraction solvent. Stock solution (1 ml) was mixed with 2.5 ml of
phosphate buffer (0.2 M; pH 6.6) and 2.5 ml of K3Fe(CN)6 (1% w/v). The mixture was
incubated at +50°C for 20 minutes and 2.5 ml of TCA (10% w/v) was added. After
centrifugation 2.5 ml of the supernatant was added to the tubes containing 2.5 ml
distilled water and 0.5 ml FeCl3.6H2O (1% w/v). The absorbance of resulting solution
was measured at 700 nm using water as blank. A control was also prepared replacing
water with plant extract. Ascorbic acid (AA) was used as standard and reducing power
was expressed as % ascorbic acid.
Proline analysis
Proline analysis was performed according to Bates [26]. Diethyl ether extract was
exempted from this test since proline is practically insoluble in diethyl ether. Briefly, 50
mg of extract was homogenized in 10 ml sulfosalicylic acid (3 % w/v) and filtrated
through filter paper. Two ml of the filtrate was mixed with 2 ml acid ninhydrin solution
(1.25 g ninhydrin + 30 ml glacial acetic acid + 20 ml 6 M H3PO4) and 2 ml of glacial
acetic acid and kept at +100 °C for 1 h. Then the reaction was stopped by transferring
the mixture to an ice bath. Four ml of toluene was added to the mixture and vortexed for
15-20 seconds. The toluene phase was aspirated and absorbance at 520 nm was
measured using pure toluene as a blank. A calibration curve was prepared with pure
proline. Results were expressed as µg proline/gram extract.
Statistical analysis
All results were means ± SD of three parallel measurements. Analysis of variance
was tested by two-way ANOVA procedure. Significant differences between means were
determined by Mann-Whitney test. P values < 0.05 were regarded as significant.
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3. RESULTS AND DISCUSSION
A variety of tests expressing antioxidant potency of food components have been
suggested. These can be categorized into two groups: assays for radical scavenging
ability and assays that test the ability to inhibit lipid oxidation under accelerated
conditions. The features of an oxidation are a substrate, an oxidant and an initiator,
intermediates and final products. Measurement of any of one of these can be used to
assess antioxidant activity [27]. Figure 1 shows the total antioxidant activity of the
various extracts from S. tchihatcheffii as measured by the FTC method after 72 h
incubation. The ferric thiocyanate test determines the antioxidant activity with the
measurement of the amount of peroxides formed in a linoleic acid emulsion of
antioxidant during incubation [28]. The hot water extract (S) exhibited the highest (96.9
%) inhibition of linoleic acid peroxidation. Diethyl ether extract (E), ethanol extract (A)
and water extract (W) showed 23.5 %, 89.2 % and 91.5 % inhibition, respectively.
Standard synthetic antioxidant BHT (4 mg) caused 97.1 % inhibition of linoleic acid
peroxidation. As seen in Figure 1, total antioxidant activity of hot water, water and
ethanol extracts of S. tchihatcheffii were very similar to BHT. High antioxidant activity
of hot water extract supports its use against cold in folk medicine.
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100
90
80
Inhibition (%)
70
60
50
40
30
20
10
0
E
A
W
S
BHT
Extracts (4 mg)
Figure 1. Total antioxidant activities of S. tchihatcheffii extracts determined by FTC method. E: diethyl
ether extract; A: ethanol extract; W: water extract; S: hot water extract; BHT: butylated hydroxytoluene.
Data present means  SD (n=3). Results demonstrate the inhibition at 72 h.
The effect of antioxidant on DPPH radical scavenging was thought to be due to their
hydrogen donating ability or radical scavenging activity. When a solution of DPPH is
mixed with that of a substance that can donate a hydrogen atom, then this gives rise to
the reduced form diphenypicrylhydrazine (non radical) with the loss of this violet color
[29]. Figure 2 demonstrates DPPH scavenging activity, expressed in percents, caused by
different concentrations of S. tchihatcheffii extracts. In a scale of 5-250 µg of extracts in
a total volume of 4 ml, the highest percent scavenging activity (92.2 %) was observed
with 50 µg/ml ethanol extract (A) in the presence of 0.025 mM DPPH (final
concentration). However, DPPH scavenging activity is best presented by IC 50 value,
defined as the concentration of the antioxidant needed to scavenge 50 % of DPPH
present in the test solution. Lower IC50 value indicates higher antioxidant activity. The
water extract (W) of S. tchihatcheffii provided the highest radical scavenging activity
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with the lowest IC50 value of 4.95  1.24 µg/ml. DPPH scavenging activity of extracts
increased in the order of W > S > A > AA > Rt > BHT > E.
100
DPPH scavenging activity (%)
90
80
70
60
50
AA
40
BHT
Rt
30
E
20
A
W
10
S
0
0
50
100
150
200
250
Extract concentration (mg/mL)
Figure 2. Radical scavenging activities of S. tchihatcheffii extracts and standards measured by DPPH
method at different concentrations. E: diethyl ether extract; A: ethanol extract; W: water extract; S: hot
water extract; BHT: butylated hydroxytoluene; AA: ascorbic acid; Rt: Rutin; [DPPH] final = 0.025 mM.
Phenolic antioxidants are products of secondary metabolism in plants, and the
antioxidant activity is mainly due to their redox properties and chemical structure,
which can play an important role in chelating transitional metals, inhibiting
lipoxygenase and scavenging free radicals [30, 31]. It revealed there is a relationship
between the antioxidant ability and total phenol contents. For the determination of total
phenolics content generally employs Folin and Ciocalteu’s phenol reagent. Phenolic
compounds react with FCR only under basic conditions. Dissociation of a phenolic
proton in basic medium leads to a phenolate anion, which is capable of reducing FCR in
which the molybdate in testing system is reduced forming a blue colored molybdenum
oxide with maximum absorption near 750 nm. The intensity of blue coloration produced
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is proportional to the total quantity of phenolic compounds present in the testing
samples [32]. Gallic acid was used as the standard to represent all phenolics present in
S. tchihatcheffii extracts. Figure 3 exhibits gallic acid equivalents of total phenolic
contents of all extracts. As displayed, S extract carried highest total phenolics and the
rest of the extracts followed the order of W > A > E. Total phenolic contents of extracts
calculated from regression equation of calibration curve (A760nm = 0.0008[Gallic acid]mg
+ 0.0001) and expressed in mg GAE/g extract were 262.50  4.17, 251.39  4.81,
197.22  6.36, 15.28  2.41 for S, W, A and E as listed in Table 1. There was a
significant linear correlation (R2 = 0.96) between total phenolics content and antioxidant
capacity (FTC). This significant linear correlation between these two variables has been
obtained in many studies [33]. The correlation between DPPH radical scavenging
activity (IC50) and total phenolic content was no linear (R2 < 0.95).
90,00
Total phenolics ( mg GAE)
80,00
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
E
A
W
S
Exract (300 mg)
Figure 3. Total phenolics content of S. tchihatcheffii extracts expressed as gallic acid equivalent (GAE).
E: diethyl ether extract; A: ethanol extract; W: water extract; S: hot water extract. Data present means 
SD (n = 3).
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Flavonoids are a class of secondary plant phenolics found ubiquitously in fruits and
vegetables, as well as food products, which act as pharmacological active compounds in
many medicinal plants. Many of the biological actions of flavonoids have been
attributed to their powerful antioxidant properties [34]. Flavonoid content of the extract
was listed in Table 1. However, we didn’t found correlation between antioxidant
activity and flavonoid content.
Sample
Diethyl ether extract
(E)
Ethanol extract (A)
Water extract (W)
Hot water extract (S)
Ascorbic acid (AA)
Butylated
hydroxytoluene
(BHT)
Rutin (Rt)
IC50 (µg/ml)
Total phenolicsd
(mg GAE/g
extract
b,c
Total flavonoids
(mg RtE/g
extracts)
EC50 (µg/ml)c
Proline contentd
(µg/g extract)
193.92  9.68
15.28  2.41
42.98  0.29
119.06  5.82
-
a
6.48  0.46
4.95  1.24a
5.72  0.37a
8.92  1.86
197.22  6.36
251.39  4.81
262. 50  4.17
-
116.53  0,79
35.76  0.37
38.61  0.10
-
9.88  0.18
8.58  0.21
8.34  0.17
2.96  0.15
2192.83  121.03
6076.55  52.75
6274.22  150.98
-
176.42  7.13
-
-
-
-
34.87  3.57
-
-
-
-
a
Significantly lower (p < 0,05) than Ascorbic acid, BHT and Rutin.
b
Final DPPH concentration was 0.025 mM.
c
Correlation coefficient between IC50 and EC50 was R2 = 0.99.
d
Correlation coefficient between total phenolics and proline content was R2 = 0.99.
Table 1. IC50 values, total phenolics, total flavonoids, EC50 values and proline content of S. tchihatcheffii
extracts
The reductive potential of a compound reflects its ability to act as an electron donor.
The electron donor reacts with free radicals, converts them to more stable products, and
finally terminates radical chain reactions. The reductive capacity of a compound is
recognized as a significant indicator of its potential antioxidant activity [31]. Figure 4
shows the absorbance values as a measure of the reducing power of S. tchihatcheffii
extracts compared to ascorbic acid in a concentration range 2.5-25 µg/ml. The EC50
value is the effective concentration at which the absorbance is 0.5 and it is used for to
express the reducing power. EC50 value of S. tchihatcheffii extracts were presented in
Table 1. Lower EC50 value represents better reducing power. The hot water extract (S)
of S. tchihatcheffii provided the highest reducing power with the lowest EC50 value of
8.34  0.17 µg/ml. Reducing power of extracts increased in the order of AA > S > W >
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A > E. A high correlations were observed between reducing power and antioxidant
activity determined by DPPH (R2 = 0.99) and FTC (R2 = 0.99).
1,60
1,40
Absorbance (700 nm)
1,20
1,00
0,80
E
A
0,60
W
S
0,40
AA
0,20
0,00
0
5
10
15
20
25
Extract (mg/ml)
Figure 4. Reducing power of different concentration of S. tchihatcheffii extracts compared to ascorbic
acid. E: diethyl ether extract; A: ethanol extract; W: water extract; S: hot water extract, AA: ascorbic acid.
Data present means  SD (n = 3).
The role of proline in proline-linked pentose phosphate pathway in plants is studied
and it is concluded that plants with high proline content exhibit high concentration of
phenolic compounds [35]. Therefore, proline content of edible plants may be accepted
as a measure of its antioxidant capacity [20]. The proline content of S. tchihatcheffii
extracts is presented in Table 1. The hot water extract (S) of S. tchihatcheffii showed the
highest proline content (6274.22  150.98 µg/g extract) and there was a high correlation
between total phenolic and proline contents of A, S and W extracts (R2 = 0.99).
Phytochemical investigations have shown that Salvia species are mainly rich in
diterpenoids and triterpenoids as well as in flavonoids and other phenolic compounds
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[18]. The phenolic content of S. tchihatcheffii extracts was found to be well correlated
with their antioxidant activity. Especially, the hot water extract has high total phenolic
and proline content. Also it showed high antioxidant activity (FTC and DPPH) and high
reducing power. The results of this study suggest that S. tchihatcheffii is a powerful
source of natural antioxidants.
4. CONCLUSIONS
In this study, we reported, for the first time, potential antioxidant capacity of S.
tchihatcheffii extracts. The results indicated that S. tchihatcheffii extracts were the most
potent since it exhibited high radical scavenging activity against DPPH and high
inhibition of lipid peroxidation. Positive correlation was established between total
phenolics and inhibition of lipid peroxidation. Especially, the presence of high levels of
phenolic compounds in the hot water extract may have contributed to the observed high
antioxidant activity. Reducing power is one important mechanism for action of
antioxidants. A high correlation was observed between reducing power and antioxidant
activity (FTC and DPPH). A newly dawn area in antioxidant activity researches is
proline content of plants. There was a high correlation between total phenolic and
proline contents of A, S and W extracts in our study.
S. tchihatcheffii is consumed as a hot drink (tea) by local inhabitants and may
provide a health benefit beyond basic nutrition. The results presented here strongly
suggest that the antioxidant capacity of S. tchihatcheffii should be considered as an
important feature and examinated with additional in vivo research.
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