Topical application of Salvia officinalis hydroethanolic leaf extract improves wound healing process

Abstract

Salvia officinalis L. (common sage) is a popular herb in the mint family, Lamiaceae. To our knowledge, literature
regarding the wound healing properties hydroethanolic extract of Salvia officinalis is scarce. Here, we tried to evaluate the in vitro antioxidant properties and in vivo wound healing activity of the hydroethanolic extract of S. officinalis. About 105 healthy Wistar rats were inflicted with wound by excision and incisionand were randomly divided into five experimental groups: Group I, as control; Group II, received placebo; groups III-V treated with 1, 3 and 5% S. officinalis hydroethanolic leaf extract, respectively. Thehydroethanolic leaf extract of Salvia officinalis showed the highest total flavonoid and phenolic content and antioxidant capacity. Topical application of S. officinalis extract, especially higher dose, significantly (P <0.05) increased the percentage of wound contraction, a period of re-epithelialization, breaking strength ratio and upregulated hydroxyproline content versus control group. Additionally, S. officinalis significantly (P <0.05) increased the new vessel formation and Fibroblast distribution. Our results showed that S. officinalis, especially S. officinalis 5%, were significantly promoting wound healing effect and can be considered as an appropriate compound for clinical application in wound care.

Full text

Indian Journal of Experimental Biology
Vol. 55, February 2017, pp. 98-106
Topical application of Salvia officinalis hydroethanolic leaf extract improves
wound healing process
Sirvan Karimzadeh & Mohammad Reza Farahpour*
Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran
Received 13 January 2015; revised 22 November 2015
Salvia officinalis L. (common sage) is a popular herb in the mint family, Lamiaceae. To our knowledge, literature
regarding the wound healing properties hydroethanolic extract of Salvia officinalis is scarce. Here, we tried to evaluate the
in vitro antioxidant properties and in vivo wound healing activity of the hydroethanolic extract of S. officinalis. About 105
healthy Wistar rats were inflicted with wound by excision and incisionand were randomly divided into five experimental
groups: Group I, as control; Group II, received placebo; groups III-V treated with 1, 3 and 5% S. officinalis hydroethanolic leaf
extract, respectively. Thehydroethanolic leaf extract of Salvia officinalis showed the highest total flavonoid and phenolic
content and antioxidant capacity. Topical application of S. officinalis extract, especially higher dose, significantly (P <0.05)
increased the percentage of wound contraction, a period of re-epithelialization, breaking strength ratio and upregulated
hydroxyproline content versus control group. Additionally, S. officinalis significantly (P <0.05) increased the new vessel
formation and Fibroblast distribution. Our results showed that S. officinalis, especially S. officinalis 5%, were significantly
promoting wound healing effect and can be considered as an appropriate compound for clinical application in wound care.
Keywords: Antioxidant activity, Common Sage, Herbal
In regenerative medicine, the main challenge is
improving tissue regeneration in the body through
therapeutical manipulations and manage scar formation.
The main purpose of this phenomenon is to make a
balance in tissue regeneration and scar formation1.
Traditional remedies based on plant sources too
remarkably promote the wound healing process at one
or more stages2. Sage (Salvia spp.) is a popular herb
in the mint family. It is the largest genus of the
Lamiaceae family, with approximately 900 species,
including annual, biennial or perennial herbs along
with woody subshrubswith immense medicinal
potential3,4. In Turkish folk medicine, some Salvia
species have been used as herbal tea4,5 and also to
cure wounds, inflammation and skin ailments6,7.
Extract of dried roots and rhizomes of Salvia
miltiorrhiza Bunge has been reported to have
protective effect on myocardial ischemia-reperfusion
injury8. Pharmacological and phytochemical potential
of Salvia gesneriiflora Lindl. and Salvia hispanica L.
have been demonstrated by Aberto et al.9. Earlier
studies have shown the antibacterial10, antidiarrheal5,
anti-inflammatory6,11 antinociceptive11 antioxidant10,12,
antiproliferative11 diuretic6 and immunomodulatory13
activities of Salvia officinalis. Further, its usage in
traditional phytotherapy for bronchitis, cold, dental
care, fever, liver, kidney and stomach ailments,
midgrade depression, throat ache, women
reproductive system and wounds and ulcers is also
known5,6. S. officinalis is one of the medicinal herbs
used in healthcare products such as band aid14. In
Morocco, women during pregnancy take sage flowers
infusion for getting back in shape15. Süntar et al.7
reported that Salvia cryptantha had a significant effect
on wound contraction and tensile strength.
To our knowledge, only few studies deal wtih the
wound healing activity of various Salvia species.
Thus, in this study, we tried to evaluate the wound
healing activity of Salvia officinalis in topical
application. We explored the effects of different dose
of hydroethanolic extract of S. officinalis leaves on
excision wound model (contraction ratio, period of
re-epithelialization and histopathological changes),
linear incision and dead space wound models in a rat
model.
Materials and Methods
Plant material and extract preparation
Salvia officinalis was collected from the central
district of the region of Urmia, West Azerbaijan
——————
*Correspondence:
Phone: +98 44 332722043; Fax: +98 44 332722043
E-mail: mrf78s@gmail.com

KARIMZADEH & FARAHPOUR: SALVIA OFFICINALIS LEAF EXTRACT IMPROVES WOUND HEALING
99
province, Iran in July 2013 (latitude: 37 34',
longitude: 44 58'). The plant was authenticated by
Agricultural and Natural Resources Research Center,
Hamadan, Iran. Around 600 g of fresh plant material
(leaves) was dried naturally on laboratory benches at
room temperature (23-24ºC) for six days until crisp
and powdered in an electric blender. Then, 150 g of
the plant powder was suspended in 600 mL of
hydroethanolic solution for 96 h at room temperature.
The mixture was filtered using a fine muslin cloth
followed by filter paper (Whatman No 1). The filtrate
was placed in an oven to dry at 40ºC. The clear
residue obtained was used for the study. The obtained
extracts were kept at −15°C until further use2.
Antioxidant activity
DPPH radical scavenging assay
Diphenylpicrylhydrazyl (DPPH) free radical
inhibition was assessed using previous method2.
Microtiter plates of 96-wells were used and five
different concentrations of each sample assessed. A
100 mg/mL of DPPH solution in methanol was used
and to minimize experimental error, all experiments
were done in triplicates. Solutions incubated at 25ºC
for 45 min (heidolph titramax 1000 and incubator
1000, Germany) and absorbance recorded at 517 nm
using power wave XS Micro plate spectrophotometer
(Bio-Tek Instruments, Inc.). The percent of free
radical inhibition (In %) calculated using the formula:
100
blank
A
)
sample
A
blank
(A
In% 


where Ablank: absorbance of control reaction
(containing all reagents except the test compound)
and Asample: absorbance of the test compound. Finally,
concentration of solution in 50 % inhibition (IC50)
was calculated from inhibition percentage of plotted
graph against each sample concentration.
FRAP radical scavenging assay
Ferric reducing ability of plasma (FRAP) assay is
an antioxidant power test based on reduction of ferric
tripyridyltriazine (Fe+3-TPTZ) to ferrous
tripyridyltriazine (Fe+2-TPTZ). Production of Fe+3-
TPTZ generates blue color at 593 nm absorbance2.
FRAP reagent was prepared just before each
experiment by mixing solutions A, B and C in the
ratio of 10:1:1 (A: Acetate buffer 300 mM pH 3.6, B:
10 mM TPTZ [2, 4, 6-tripyridyl-s-triazine] in 40 mM
HCl, C: 20 mM FeCl3. 6 H2O). A 20 µL of sample
mixed with 200 µL FRAP reagent, held for 10 min at
room temperature and recorded at 593 nm absorbance
using Power wave XS Microplate spectrophotometer
(Bio-Tek Instruments, Inc.). Different concentrations
of FeSO4.7H2O (200, 400, 800, 1200 and 1600 μM)
were used as standard solution where reacted with
TPTZ reagent and absorbance plotted against various
ferrous ion concentrations. The results expressed as
µM of Fe2+ equivalents per mg of dried extract.
L-ascorbic acid was used as standard antioxidant.
Determination of the total flavonoid content
Total flavonoid content was assessed using
previous method. The aluminum chloride test applied
to determination total flavonoid content of extracts.
The flavonoid content was expressed as mg of
quercetin equivalents per gram of dried extract2.
Determination of total phenolic content
Total phenolic constituents were assessed using
previous method. Total phenolic constituents of
sample extract were determined by modified methods
using Folin-Ciocalteu reagent and Gallic acid
(ranging from 0-1000 mg/L) as standard phenolic
compound2.
Biological activity test
Animals and study design
Healthy white Wistar male rats weighing
approximately 200 g and 9 wk of age were used in the
present study. Two weeks before and during the entire
experiments, the animals were housed in individual
plastic cages (50×40×20 cm) with an ambient
temperature of 23±3ºC, stable air humidity and a
natural day/night cycle. Animals were handled on a
regular daily basis for 2 wk prior to the study in order
to acclimatize them with testing area and
experimental condition. Rats had free access to chew
food and freshwater. The procedures were carried out
based on the guidelines of the Ethics Committee of
the International Association for the Study of pain2
and the current laws of the Iranian government for
laboratory animal care. The University Research
Council approved all experiments.
Formulation of topical wound application forms
Four variants of the topical application ointment
were prepared. All the variants consisted base
formulation comprising commercial Eucerin (25%)
and Vaseline (75%) in about 1:3 proportions. All rats
randomly were labeled by none toxic color and
divided into five groups. After surgical wound
creation, Group I served as control: had no received
any administration. Group II rats received Placebo
(base formulation). Groups III-V were applied with

INDIAN J EXP BIOL, FEBRUARY 2017
100
1, 3 and 5% of Salvia officinalis hydroethanolic
extract mixed with base formulation (S. officinalis),
respectively2. The ointments were topically applied
once a day, starting from the day of operation, on the
wound area until the wound healed completely. All
rats were monitored for any wound fluid or any
evidence of infection or other abnormalities, until
complete epithelialization.
Acute skin irritation test
The test was carried out based on Farahpour et al.2.
About 500 mm2 areas on the dorsal fur of each rat was
shaved and prepared aseptically. The S. officinalis
formulations were applied. After 4 hr, the skin of each
animal observed for inflammation and or any signs.
Wound healing models
Circular Excision Wound Model
In this wound model, 75 healthy white Wistar rats
used for wound contraction ratio, period of re-
epithelialization and histopathological change studies.
Animals were anesthetized by intraperitoneal (IP)
administration of ketamine 5%, 90 mg/kg (Ketaset 5%;
Alfasan, Woerden, The Netherlands) and xylazine
hydrochloride 2%, 5 mg/kg (Rompun 2%, Bayer,
Leverkusen, Germany). The fur was prepared
aseptically and the predetermined area was marked on
the back of animals. Each rat was fixed on the surgery
table in ventral posture. Following surgical preparation,
a circular surgical full thickness wound was made, 314
mm2 diameters, on the anterior-dorsal side of each rat.
Wound contraction percentage and wound closure
time were used to assess wound-healing property2.
The wound area was measured by immediate
placing of a transparent paper over the wound and
tracing area of this impression was calculated using
the graph sheet. The wound healing percentage was
calculated by Walker formula after measuring the
wound size2. The percentage of wound healing was
computed at the beginning of experiments on days 3,
6, 9, 12, 15, 18, and 21 days postoperative.
100
zero dayon area wound
Xday on area wound-zeroday on area wound
closure wound of% 
X= number of days.
Incision wound model
In this wound model, 30 animals were randomly
divided into five experimental groups with six rats in
each: control (Group I), placebo (Group II), 1 (Group
III), (Group IV)3 and 5% (Group V) S. officinalis-
treated groups. All animals were anesthetized with the
method mentioned above. A 4-cm length incision was
made through the skin and cutaneous muscle at a
distance about 1.5 cm from the middle on the right
side of the depilated back. The wound was closed at
0.5 cm intervals using 3/0 nylon (Dafilon, B/Braun,
Germany). Ointments were applied once daily for
9 days. On day 9, sutures were removed and the
tensile strength of healed wounds was measured on
day10 using Strongraph mechanical test frame
(Toyoseiky Tensile Testing Unit, Model R3, Japan)2.
Tensile strength was calculated using the following
formula:
Tensile strength = breaking strength (g) / cross
sectional area of skin (mm2)
Dead space wound model and hydroxyproline content
estimation
Animals were randomly divided into five
experimental groups (n=6 in each). Group I was
considered as control. Group II drenched phosphate
buffered saline as placebo. Groups III-V treated with
1, 3 and 5% of S. officinalis leave extract ointment,
respectively. The dead space wound was created
using subcutaneous implanting of polypropylene
tubes (2.50.5 cm) in the lumbar region on the dorsal
side. All animals were gavage the extracts for 9 days
post-injury. On day 10 post-wound induction,
granuloma tissue created on the implanted tube was
carefully dissected and used to determine breaking
strength and estimation of hydroxyproline content2.
Histopathological Study
Animals were anesthetized with the same way
mentioned above and specimens from skin were taken
on 3, 6, 9, 12, 15, 18 and 21 days after surgery.
Sample tissues, excised along with 1 to 2 mm
surrounding normal skin in a depth of approximately
3 mm, were pinned on a flat cork surface and fixed in
neutral-buffered formalin 10%. Then the sample
tissues were routinely processed, paraffin wax
embedded, sectioned at 5 µm and stained with
hematoxylin and eosin (H&E) and Masson’s
Trichrome stains for further examination using
microscopy (Olympus CX31RBSF attached
cameraman) to assess the predominant stage of wound
healing. Three parallel sections were obtained from
each specimen. The number of MNC, PMN,
fibroblastic aggregation and angiogenesis (the number
of blood vessels and capillary buds) were
quantitatively evaluated in 5 per high power fields
(HPFs) (400). Acute hemorrhage, congestion,
edema, epithelialization, collagen production and

KARIMZADEH & FARAHPOUR: SALVIA OFFICINALIS LEAF EXTRACT IMPROVES WOUND HEALING
101
density were also evaluated qualitatively and
calculated manually. They were analyzed in 5 per
high power fields (HPFs) (100)2.
Statistical analysis
Experimental results were expressed as means ±
SEM. Statistical analyses were performed using
PASW 18.0 (SPSS Inc., Chicago, IL, USA). Model
assumptions were evaluated by examining the
residual plot. Results were analyzed using one-way
ANOVA. Dunnett's test for pair-wise comparisons
was used to examine the effect of time and treatments.
P <0.05 was considered significant differences.
Results
Antioxidant activity, total phenol and flavonoid contents
The results of IC50 in DPPH, Eq+ Fe2++, total
phenol and total flavonoid contents of hydroethanolic
Salvia officinalis leave extract is presented in Table 1.
A seen, IC50 in DPPH of Salvia officinalis extract
was 7.12 compared to BHT (107.04). Also, Eq+ Fe2++
(m per mg extract) was 18318.0 ± 83.6 in comparsion
to ascorbic acid 7740.2 ± 64.9. Furthermore, total
flavonoid and phenolic compouns of Salvia officinalis
extract was 116.71 ± 2.34 and 298.8 ± 4.3, respectevly.
Acute skin irritation test
The results obtained from acute skin irritation test,
4 h after applying ointment on the skin, there was no
sign of inflammation in all animals during the test.
Wound contraction developed and re-epithelization period in
S. officinalis-treated animals
According to the data (Table 2), there was no
significant difference in wound contraction
percentage between S. officinalis-treated group and
non-treated group on days 3, 6 and 9 after wound
induction (P >0.05). Interestingly, wound contraction
percentage significantly increased in the rat treated
with 3 and 5% of S. officinalis-treated group
compared to the other groups on days 12 and 15
(P <0.05). As noticed, wound contraction percentage
significantly increased in S. officinalis-treated (1, 3
and 5%) animals compared to the control group on
days 18 and 21 after wound creation (P <0.05). The
positive effect of 1% S. officinalis is seen from day 18
which indicates for low therapeutic properties of 1%
S. officinalis-treated group. Likewise, re-epithelialization
period significantly increased in S. officinalis-treated
rats compared to non-treated group (P <0.05), but
there was no significant effect between 1 and 3% of S.
officinalis administrated rat (P >0.05).
Linear incision wound model and dead space wound model
changed depending on dose
Effects of hydroethanolic S. officinalis leaf extract
ointment on the linear wound incision is presented in
Table 3. According to the obtained data, a significant
effect observed in S. officinalis administrated groups
compared to control and placebo groups. As observed,
Table 1—Antioxidant properties, total phenol and total flavonoid
contents of of hydroethanolic Salvia officinalis Leaf extracts
IC50 in
DPPH
inhibition
assay
(µg/mL)
Eq+ Fe2++
(m per mg
extract)
Total
flavonoid
(µg eq
Rutin/mg
dried extract)
Total
phenols
(µg/mg
dried
extract)
7.12 18318.0±83.6
116.71±2.34
298.8±4.3
Salvia
officinalis
BHT 107.04 - - -
Ascorbic
acid - 7740.2±64.9 - -
Table 3—Effect of Salvia officinalis on the linear
wound incision model in rat
Groups Mean±SEM
Control
Placebo
S. officinalis 1%
S. officinalis 3%
S. officinalis 5%
280.2±15.6
289.96±17.7
386.03±12.67a
484.03±10.11a
527.51±37.11b
n= 6 animals in each group. Data are presented as the mean±SEM.
There are significant differences between groups with different codes
in a column (superscript letters a,b; P <0.05 vs. control).
Table 2—Effect of Salvia officinalis on wound area and period of re-epithelialization in rat model in rat
Groups Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21 re-epithelialization
Control 4.12±3.28 20.45±4.28
41.30±2.38
56.90±2.03 64.55±1.10
78.40±0.62 86.65±0.11
22.87±1.31
Placebo 7.75±2.01 20.74±3.84
44.55±4.84
63.75±1.83 77.60±1.05
81.45±0.29 89.25±0.12
21.83±1.24
S. officinalis 1% 7.75±3.90 30.35±3.37
51.80±5.57
87.85±3.86 93.10± 0.29
98.87±0.30a 100±0.00b 18.16±1.42a
S. officinalis 3% 8.59±4.57 33.35±6.05
67.40±5.07
92.70±1.03a 96.81±0.28a
100±0.00b 100±0.00b 17.16±1.53a
S. officinalis 5% 10.49±5.48
38.55±5.47
78.35±0.79
96.00±0.65b
100±0.00b 100±0.00b 100±0.00b 15.65±1.57b
n= 6 animals in each group. Data are presented as the mean±SEM. There are significant differences between groups with different codes
in a column (superscript letters a, b; P < 0.05 vs. control).

INDIAN J EXP BIOL, FEBRUARY 2017
102
S. officinalis-treated group significantly increased
wound incision compared to non-treated group
(P <0.05). Additionally, there was no significant
effect between 1 and 3% of S. officinalis-treated
groups (III and IV) (P >0.05) while 5% of
S. officinalis-treated group significantly increased
wound incision compared to the other groups
(P <0.05).
Dead space wound model and hydroxyproline content
estimation
The effect of S. officinalis on wound healing of
dead space wound is shown in Table 4. As seen,
different levels of S. officinalis-treated groups (3 and
5%) significantly increased hydroxyproline content in
dead space wound compared to control group
(P <0.05). Likewise, 1% of S. officinalis-treated group
had no significant effect on hydroxyproline content in
dead space wound compared to control and placebo
groups (P >0.05). Also, different levels of
S. officinalis-treated group (1, 3 and 5%) significantly
increased wet weight of the granulation tissue
compared to both non-treated and placebo groups
(P <0.05). According to the data, the 3 and 5%
S. officinalis-treated groups (IV and V) significantly
promoted dry weight of the granulation tissue
compared to non-treated group (P <0.05).
Histopathology
The histological results for PMN, MNC, Fib
infiltration and new vascular formation are presented
in Table 5. The S. officinalis-treated groups (III-V)
significantly diminished PMN infiltration compared
to control group on 3 day postopertion (P <0.05).
Following 7 days after wound induction, the PMN
Table 4—Effect of Salvia officinalis on wound healing of dead space wound model in rat
Groups Dead space wound model
Hydroxyproline content
(μg/mL) Wet weight of the granulation tissue
(mg) Dry weight of the granulation tissue
(mg)
Control 11.63±0.4 84.18±5.4 13.21±0.89
Placebo 12.38±0.22 84.88±5.88 13.54±0.76
S. officinalis 1% 13.58±0.4 97.98±2.73* 15.37±0.55
S. officinalis 3% 14.7±0.36* 117.4±4.26* 16. 83±0.57*
S. officinalis 5% 16.22±0.31* 125.83±3.3* 20.38±0.51*
n= 6 animals in each group. P <0.05 vs.
control. Data are presented as the mean±SEM. There are significant differences between groups
with different codes in a column (superscript letters a, b; P <0.05).
Table 5—Effects of Salvia officinalis on polymorphonuclear and mononuclear cells, new vessels and fibroblasts formation
on subsequent wound healing in rat
Day Group PMN MNC New vessels Fibroblast
Control 62.4±4.1 a 12.1±0.54 a 2.5±2.1 a 21.5±1.52 a
S. officinalis 1% 42.7±5.1b 19.2±0.95 b 5.2±0.62 b 39.3±1.63 b
S. officinalis 3% 39.8±4.2 b 23.1±0.77 b 6.5±1.01 b 47.8±2.19 b
3
S. officinalis 5% 22.1±2.6 bc 26.1±0.45 bc 7.8±1.29 bc 55.2±2.38 bc
Control 50.2±3.4 a 14.3±0.4 a 3.2±0.28 a 35±1.26 a
S. officinalis 1% 33.2±1.4 b 15.8±0.71 a 4.1±0.71 b 58±2.12 b
S. officinalis 3% 29.91±0.4 b 16.5±1.6 a 4.7±0.29 b 60± 2.41 b
7
S. officinalis 5% 15.12±1.8 c 19.9±0.81 b 5.6±0.68 bc 89±2.25 bc
Control 32.1±1.7 a 9.12±0.67 a 2.8±0.81 a 57±2.62 a
S. officinalis 1% 18.4±1.4 b 12.4±0.58 a 1.3±0.56 b 79±3.91 b
S. officinalis 3% 15.81±0.3 b 12.8±1.41 a 2.5±0.88 a 85±4.33 b
14
S. officinalis 5% 8.92±0.6 c 14.32±1.09 a 3.6±1.03 a 99±4.81 bc
Control 12.9±0.9 a 3.3±0.1 a 1.1±0.29 a 28±1.87 a
S. officinalis 1% 5.11±0.3 b 3.8±0.81 a 0.98±0.32 a 35±2.72 b
S. officinalis 3% 3.99±0.5 b 3.1±0.88 a 1.9±0.68 b 42±3. 42 b
21
S. officinalis 5% 1.1±0.11 c 4.3±0.79 a 2.2±1.09 b 49±3.55 bc
n= 6 animals in each group. PMN: polymorp
ho nuclear cells, MNC: mononuclear cells. Data are presented as the mean±SEM. There are
significant differences between groups with different codes in a column (superscript letters a, b, c; P <0.05 vs. control).

KARIMZADEH & FARAHPOUR: SALVIA OFFICINALIS LEAF EXTRACT IMPROVES WOUND HEALING
103
distribution was significantly lower in S. officinalis-
treated group versus to those in non-treated animals.
Comparing different doses of S. officinalis showed that
high dose-administrated S. officinalis exerted
significantly (P <0.05) better results compared to lower
doses (1% and 3%) on day 14 after operation (Fig. 1).
In this study, administrating 5% from S. officinalis
significantly diminished MNCs infiltration compared
to the other groups on day 3 postperation (P <0.05).
Whereas no significant differences were observed
between control group and 1 or 3% S. officinalis-
treated rats (P >0.05). Compared to control group, the
S. officinalis-treated animals exhibited significantly
(P <0.05) lower MNCs on day 7 after wound induction.
However, no significant differences were observed
between treated and non-treated animals for MNCs on
days 14 and 21 days after wound induction (P >0.05).
In contrary, administration of different levels of
S. officinalis-treated group significantly increased NV
formation compared to control group during to the
postoperative days (P <0.05). As noticed, the 5%
S. officinalis-treated group (V) had more potential but
not significantly on NV formation compared to the
other S. officinalis-treated groups (III and IV) on days
3 and 7 post wound creation injury (Fig. 1). Also,
dose of 1% S. officinalis had no significant effect on
NV (P >0.05), whereas 3 and 5% of S. officinalis-
treated groups significantly improved NV compared
to non-treated group on day 21 (P >0.05).
Based on our data, a significant (P <0.05)
distribution of fibroblasts was observed in the
S. officinalis-treated animals compared to control
group (Fig. 2). Additionally, group V (5% of
S. officinalis-treated) had a slight better effect, but not
Fig. 1—
Cross section from the wound area after 7 days. (A, B)
Control group; (C, D) 3% Salvia officinalis-
treated group; (E, F)
5% S. officinalis-
treated group. The necrotic is shown by arrow.
Note, the cross sections from higher magnification of the marked
region with faint granulation tissue formation, impressive
leukocytes infi
ltration and edema in control group. At the same
time, see well-
formed granulation tissue formation with
remarkable reduction in the leukocytes infiltration and edema,
along with remarkable fibroblast migration in S. officinalis-
treated
groups (Masson trichrome staining).
Fig. 2—
Cross section from the wound area after 14 days. (A, B)
Control group ; (C, D) 3% Salvia officinalis-
treated group ; (E, F)
5% S. officinalis-treated group. The necr
otic is shown by arrow.
Note, the cross sections from granulation tissue with higher
magnification of the marked region with less fibroblast
distribution and
collagen formation in control group. At the same
time, see well-formed granulation tissue formatio
n with
remarkable in collagen formation in S. officinalis-
treated groups,
specially in 3% S. officinalis-treated group (
Masson trichrome
staining).

INDIAN J EXP BIOL, FEBRUARY 2017
104
more significant than the other two treated group
levels (1 and 3%) (P >0.05).
Discussion
In this study, different wound models are used to
evaluate the healing activity of hydroethanolic
extracts of Salvia officinalis as a traditional remedy.
For this purpose, the preliminary phytochemical
assessment of hydroethanolic extract of S. officinalis
were performed. Biochemical analysis results showed
that the S. officinalis hydroethanolic extract contains
high levels of phytochemicals such as phenolic and
flavonoid compounds. It is reported that flavonoids
and phenols16,17 have antioxidant activity18-21 and
accelerate wound healing activity2,22-25, by their free
radical scavenging25-28, astringent, anti-microbial
properties2,25,29,. Also, flavonoids are responsible for
activating anti-inflammatory system which acts
against lipid peroxidation,28,30-32.
In contrast, it is revealed that poly morphonuclear
cells (PMN) is one of the primery sources of reactive
oxygen species (ROS)2,33. Administration of S.
officinalis significantly decreased in PMN cells
infiltration and subsequently inhibited RNA damage.
Therefore, it seems the flavonoid content of the
S. officinalis extract could considerably decrease the
inflammation-induced oxidative stress and RNA
damage in epidermal and dermal cells.
Mononuclear cells (MNC), specially Macrophage
cells are important phagocytosis cells in the
inflammatory phase of wound healing process1,2,34.
The presence of macrophage cells in the wound area
is an indicator for the beginning of the second phase
of the healing process35. On day 3 post-operative, the
number MNC significantly increased in 3 and 5%
S. officinalis-treated groups, compared with non-
treated group.
Vascular endothelial cells attracted to the wound
site by macrophage cells1,2,34. In histological
assessment of on day 3 after exision wound creation,
significant increase was observed innumber of new
blood capillaries formation in 3 and 5% S. officinalis-
treated groups compared to the untreated group. On the
other hand, macrophage cells release PDGF, TGF-β
and collagen which atracts fibroblast and smooth
muscle cells cells into the wound site2,34. In histological
assessment on days 7 and 14 post wound creation, the
number of fibroblasts significantly increased in 3 and
5% S. officinalis-treated groups compared to the
untreated group. Our observations demonstrated
that administration of S. officinalis diminished
inflammatory phase and promoted proliferative stage
by increased in fibroblast distribution.
There is a positive correlation on collagen
synthesis and fibroblasts distribution1,36,37. Increased
collagen content, cross-linkages between collagen
fibers and extracellular matrix secreted by fibroblasts
to granulation tissue38. This phenomenon increases
tensile ratio and onset wound contraction2,39,40. Our
analysis showed that wound contraction and breaking
tensile strength ratio significantly accelerate in
S. officinalis-treated animals, especially 3 and 5%
treated animals.
Hydroxyproline is a direct estimation marker for
collagen synthesis41,42. Therefore, increase in the
collagen content of granulation tissue, collagen cross
linking and subsequent maturation in collagen,
increases hydroxyproline, wet and dry granulation
tissue weight43,44. Our results revealed that
hydroxyproline content significantly amplified in 5%
S. officinalis-treated animals which sunsequently
increased wet and dry granulation tissue weight.
Neovascularization is an essential factor for
migration of epithelial cells from the margins to the
central of wound1,45. Also, re-epithelialization
decreases distance wound size2,35,46. Rapid re-
epithelialization in the wound healing process is
considered as a hallmark for well wound
treatment35,46. In this study, the epithelization time
significantly reduced in 5% S. officinalis-treated
group. The surveillance of epithelial cells in groups
treated with S. officinalis could be attribued to its
antioxidant characteristics which prevented cellular
damage mediated by oxidative stress and accelerated
process of their migration.
In conclusion, our data suggested that different
topical doses of the Salvia officinalis hydroethanolic
leaves extract increased tensile strength, facilitated
wound contraction, decreased healing time as well as
increased collagen deposition by up-regulating
macrophage and fibroblast cell distribution followed
by promoted proliferative stage of the healing
process. Findings of the current study suggest that
hydroethanolic extract of Salvia officinalis leaves
support wound healing activity, and thereby
indicating the potential of this plant in reducing the
healing time.
Acknowledgement
Authors acknowledge the Indian Council of
Agricultural Research networking project for financial
support.

KARIMZADEH & FARAHPOUR: SALVIA OFFICINALIS LEAF EXTRACT IMPROVES WOUND HEALING
105
References
1 Harper D, Young A, & McNaught CE, The physiology of
wound healing. Surgery (Oxford), 32 (2014) 445.
2 Farahpour MR, Mirzakhani N, Doostmohammadi J, &
Ebrahimzadeh M, Hydroethanolic Pistacia atlantica hulls
extract improved wound healing process; evidence for mast
cells infiltration, angiogenesis and RNA stability. Inter
J Surgery, 17 (2015) 88.
3 Royal Botanic Garden E, 28 (1967) 11.
4 Polat R, Cakilcioglu U, Ulusan MD & Paksoy MY, Survey
of wild food plants for human consumption in Elazığ
(Turkey). Inidan J Tradit Knowl, 14 (2015) 69.
5 Popović Z, Smiljanić M, Kostić M, Nikić P & Janković S,
Wild flora and its usage in traditional phytotherapy
(Deliblato Sands, Serbia, South East Europe). Inidan
J Tradit Knowl, 13 (2014) 9.
6 Korkmaz M, Karakuş S, Özçelik H & Selvi S, An
ethnobotanical study on medicinal plants in Erzincan,
Turkey. Inidan J Tradit Knowl, 15 (2016) 192.
7 Süntar İ, Akkol EK, Şenol FS, Keles H & Orhan IE,
Investigating wound healing, tyrosinase inhibitory and
antioxidant activities of the ethanol extracts of Salvia
cryptantha and Salvia cyanescens using in vivo and
in vitro experimental models. J Ethnopharmacol, 135 (2011) 71.
8 Zhou C, Hu T, Li X, Jiao S & Wu Z, Determination of
tanshinone ⅡA in Salvia miltiorrhiza Bunge extract and its
effect on ischemia-reperfusion injury. Inidan J Tradit Knowl,
14 (2015) 387.
9 Alberto MEJ, Imelda PM, Clarenc AR, Susana PG, Edgar SP &
Bernarda GO, Pharmacological and phytochemical potential
study of plants collected in Amecameca, State of Mexico,
Mexico. Inidan J Tradit Knowl, 15 (2016) 62.
10 Kontogianni VG, Tomic G, Nikolic I, Nerantzaki AA,
Sayyad N, Stosic-Grujicic S, Stojanovic I, Gerothanassis IP &
Tzakos AG, Phytochemical profile of Rosmarinus officinalis
and Salvia officinalis extracts and correlation to their
antioxidant and anti-proliferative activity. Food Chem, 136
(2013) 120.
11 Kozics K, Klusová V, Srančíková A, Mučaji P, Slameňová D,
Hunáková Ľ & Horváthová E, Effects of Salvia officinalis
and Thymus vulgaris on oxidant-induced DNA damage and
antioxidant status in HepG2 cells. Food chem., 141 (2013)
2198.
12 Farhat MB, Chaouch-Hamada R, Sotomayor JA, Landoulsi A
& Jordán MJ, Antioxidant potential of Salvia officinalis L.
residues as affected by the harvesting time. Indust Crops
Prod, 54 (2014) 78.
13 Mukherjee PK, Nema NK, Bhadra S, Mukherjee D,
Braga FC & Matsabisa M, Immunomodulatory leads from
medicinal plant. Inidan J Tradit Knowl, 13 (2014) 235.
14 Chandrasekaran K, Anbumani N & Priyal VV, Analysis of
eco-friendly medicinal herb extracts and essential oil
applications on textile products for healthcare applications.
Inidan J Tradit Knowl, 14 (2015) 481.
15 Elkhoudri N, Baali A & Amor H, Maternal morbidity and the
use of medicinal herbs in the city of Marrakech, Morocco.
Inidan J Tradit Knowl, 15 (2016) 79
16 Saha B, Subramanian M, Gupta P, Patro BS, Ray J,
Bandyopadhyay SK & Chattopadhyay S, trans-4,4'-
Dihydroxystilbene (DHS) protects PC12 cells from oxidative
damage but induces reactive oxygen species-mediated
apoptosis in SHSY-5Y cell line. Indian J Exp Biol, 54 (2016)
719.
17 Nile SH & Park SW, Determination of polyphenols and
antioxidant activity of Vitis labrusca cv. baile berries. Indian
J Exp Biol, 53 (2015) 671.
18 Bhatt A & Patel V, Antioxidant potential of banana: Study
using simulated gastrointestinal model and conventional
extraction. Indian J Exp Biol, 53 (2015) 457.
19 Sreejith G, Jayasree M, Latha PG, Suja SR, Shyamal S,
Shine VJ, Anuja GI, Sini S, Shikha P, Krishnakumar NM,
Vilash V & Shoumya S, Hepatoprotective activity of Oxalis
corniculata L. ethanolic extract against paracetamol induced
hepatotoxicity in Wistar rats and its in vitro antioxidant
effects. Indian J Exp Biol, 52 (2014) 147.
20 Singha I & Das SK, Scavenging and Antioxidant properties
of different grape cultivars against ionizing radiation-induced
liver damage ex vivo. Indian J Exp Biol, 54 (2016) 280.
21 Bouaziz-Ketata H, Zouari N, Salah HB, Rafrafi M & Zeghal N,
Flavonoid profile and antioxidant activities of methanolic
extract of Hyparrhenia hirta (L.) Stapf. Indian J Exp Biol, 53
(2015) 208.
22 Agar OT, Dikmen M, Ozturk N, Yilmaz MA, Temel H, &
Turkmenoglu FP, Comparative Studies on Phenolic
Composition, Antioxidant, Wound Healing and Cytotoxic
Activities of Selected Achillea L. Species Growing in
Turkey. Molecules, 20, (2015) 17976.
23 Gopalakrishnan A, Ram M, Kumawat S, Tandan SK &
Kumar D, Quercetin accelerated cutaneous wound healing in
rats by increasing levels of VEGF and TGF-. Indian J Exp
Biol, 54 (2016) 187.
24 Khan A, Manna K, Chinchubose, Das DK, Sinha M,
Kesh SB, Das U, Dey RS, Banerji A & Dey S, Seabuckthron
(Hippophae rhamnoides L.) leaf extract ameliorates the
gamma radiation mediated DNA damage and hepatic
alterations. Indian J Exp Biol, 52 (2014) 952.
25 Yadav DK, Bharitkar YP, Chatterjee K, Ghosh M, Mondal
NB & Swarnakar S, Importance of Neem Leaf: An insight
into its role in combating diseases. Indian J Exp Biol, 54
(2016) 708.
26 Satapathy R, Jadhav RS & Swamy PL, Antioxidant activity
of Wattakaka volubilis (Linn. f.) leaf extract in carbon
tetrachloride induced mice. Indian J Exp Biol, 54 (2016) 467.
27 Saha MR, Dey P, Chaudhuri TK, Goyal AK, Sarker DD &
Sen A, Assessment of haemolytic, cytotoxic and free radical
scavenging activities of an underutilized fruit, Baccaurea
ramiflora Lour. (Roxb.) Muell. Arg. Indian J Exp Biol, 54
(2016) 115.
28 Effect of flavonoid rich fraction of Tephrosia purpurea (L.)
Pers. on complications associated with streptozotocin-
induced type I diabetes mellitus. Indian J Exp Biol, 54 (2016)
457.
29 Mamta, Mehrotra S, Amitabh, Kirar V, Vats P, Nandi SP,
Negi PS & Misra K, Phytochemical and antimicrobial
activities of Himalayan Cordyceps sinensis (Berk.) Sacc.
Indian J Exp Biol, 53 (2015) 36.
30 Adebayo SA, Dzoyem JP, Shai LJ, & Eloff JN, The anti-
inflammatory and antioxidant activity of 25 plant species
used traditionally to treat pain in southern African. BMC
Complement Altern Med, 15 (2015) 159.
31 Lingaraju MC, Anand S, Begum J, Balaganur V, Kumari RR,
Bhat RA, More AS, Kumar D, Bhadoria BK & Tandan SK,

INDIAN J EXP BIOL, FEBRUARY 2017
106
Anti-inflammatory effect of dikaempferol
rhamnopyranoside, a diflavonoid from Eugenia jambolana
Lam. Leaves. Indian J Exp Biol, 54 (2016) 801.
32 Sikder K, Das N, Kesh SB & Dey S, Quercetin and β-
sitosterol prevent high fat diet induced dyslipidemia and
hepatotoxicity in Swiss albino mice. Indian J Exp Biol, 52
(2014) 60.
33 Verbeke J, Boulougouris X, Rogiers C, Burvenich C,
Peelman L, De Spiegeleer B, & De Vliegher S, Reactive
oxygen species generation by bovine blood neutrophils with
different CXCR1 (IL8RA) genotype following Interleukin-8
incubation. BMC Vet Res, 11(1), (2015) 104.
34 Duque GA, & Descoteaux A, Macrophage cytokines:
involvement in immunity and infectious diseases. Front
Immunol, 5 (2014) 491.
35 Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG,
Sawaya A & Tomic-Canic M, Epithelialization in wound
healing: a comprehensive review. Adv Wound Care, 3 (2014)
445.
36 Liu J, Pampillo M, Guo F, Liu S, Cooperman BS, Farrell I &
Leask A, Monitoring collagen synthesis in fibroblasts using
fluorescently labeled tRNA pairs. J Cell Physiol, 229 (2014)
1121.
37 Arbi S, Eksteen EC, Oberholzer HM, Taute H, & Bester MJ,
Premature collagen fibril formation, fibroblast-mast cell
interactions and mast cell-mediated phagocytosis of collagen
in keloids. Ultrastruct Pathol, 39 (2015) 95.
38 Zhang Y, Lin Z, Foolen J, Schoen I, Santoro A, Zenobi-
Wong M & Vogel V, Disentangling the multifactorial
contributions of fibronectin, collagen and cyclic strain on
MMP expression and extracellular matrix remodeling by
fibroblasts. Matrix Biol, 40 (2014) 62.
39 Darby IA, Laverdet B, Bonté F & Desmoulière A,
Fibroblasts and myofibroblasts in wound healing. Clin
Cosmet Investig Dermatol, 7 (2014) 301.
40 Werner S & Antsiferova M, Wound Healing: An
Orchestrated Process of Cell Cycle, Adhesion, and Signaling.
Encyc Cell Biol, 3 (2016) 216.
41 Qiu B, Wei F, Sun X, Wang X, Duan B, Shi C & Mu W,
Measurement of hydroxyproline in collagen with three
different methods. Mol Med Rep, 10, (2014) 1157.
42 Wang T, Gu Q, Zhao J, Mei J, Shao M, Pan Y & Liu F,
Calcium alginate enhances wound healing by up-regulating
the ratio of collagen types I/III in diabetic rats. Int J Clin Exp
Path, 8 (2015) 6636.
43 Nayak BS, Ramlogan S, Rao AC & Maharaj S, Neurolaena
lobata L. promotes wound healing in Sprague Dawley rats.
Int J Appl Basic Med Res, 4 (2014) 106.
44 Honnegowda TM, Kumar P, Udupa P, Rao P, Bhandary S,
Mahato KK & Mayya SS, Effect of limited access dressing
on hydroxyproline and enzymatic antioxidant status in
nonhealing chronic ulcers. Indian J Plast Surg, 47 (2014)
216.
45 Cerqueira MT, Pirraco RP, Martins AR, Santos TC,
Reis RL & Marques AP, Cell sheet technology-driven re-
epithelialization and neovascularization of skin wounds. Acta
biomater, 10 (2014) 3145.
46 Amar MB & Wu M, Re-epithelialization: advancing
epithelium frontier during wound healing. J R Soc Interface,
11 (2014) 1013.