Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 972028, 9 pages
http://dx.doi.org/10.1155/2013/972028
Research Article
Evaluation of In Vivo Wound Healing Activity of
Bacopa monniera on Different Wound Model in Rats
S. Murthy,1 M. K. Gautam,1 Shalini Goel,2 V. Purohit,1 H. Sharma,1 and R. K. Goel1
1
2
Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
Department of Pathology and Lab Medicine, Medanta-he Medicity, Sector 38, Gurgaon 122001, India
Correspondence should be addressed to R. K. Goel; rkgoelbhu50@gmail.com
Received 29 April 2013; Revised 20 June 2013; Accepted 8 July 2013
Academic Editor: Alethéa Gatto Barschak
Copyright © 2013 S. Murthy et al. his is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Wound healing efects of 50% ethanol extract of dried whole plant of Bacopa monniera (BME) was studied on wound models in
rats. BME (25 mg/kg) was administered orally, once daily for 10 days (incision and dead space wound models) or for 21 days or more
(excision wound model) in rats. BME was studied for its in vitro antimicrobial and in vivo wound breaking strength, WBS (incision
model), rate of contraction, period of epithelization, histology of skin (excision model), granulation tissue free radicals (nitric
oxide and lipid peroxidation), antioxidants (catalase, superoxide dismutase, and reduced glutathione), acute inlammatory marker
(myeloperoxidase), connective tissue markers (hydroxyproline, hexosamine, and hexuronic acid), and deep connective tissue
histology (dead space wound). BME showed antimicrobial activity against skin pathogens, enhanced WBS, rate of contraction,
skin collagen tissue formation, and early epithelization period with low scar area indicating enhanced healing. Healing efect was
further substantiated by decreased free radicals and myeloperoxidase and enhanced antioxidants and connective tissue markers
with histological evidence of more collagen formation in skin and deeper connective tissues. BME decreased myeloperoxidase and
free radical generated tissue damage, promoting antioxidant status, faster collagen deposition, other connective tissue constituent
formation, and antibacterial activity.
1. Introduction
Wounds are physical injuries that result in an opening or
breaking of the skin. Proper healing of wounds is essential
for the restoration of disrupted anatomical stability and disturbed functional status of the skin. Repair of injured tissues
occurs as a sequence of events, which includes inlammation,
proliferation, and migration of diferent cell types [1]. he
inlammation stage begins immediately ater injury, irst
with vasoconstriction that favors homeostasis and releases
inlammation mediators. he proliferative phase is characterized by granulation tissue proliferation formed mainly by
ibroblast and the angiogenesis process. he remodeling stage
is characterized by reformulations and improvement in the
components of the collagen ibre that increases the tensile
strength [2]. Factors that contribute to causation and perpetuation of the chronicity of wounds include repeated trauma,
poor perfusion or oxygenation, and excessive inlammation
[3]. Imbalance in free radical generations and antioxidants
has been observed to induce oxidative stress and tissue
damage and delayed wound healing. herefore, elimination
of ROS could be an important strategy in healing chronic
wounds [4].
Bacopa monniera (BM, Scrophulariaceae) called as water
hyssop is a prostrate herb, commonly found in wet or marshy
habitats and along the stream and river margins throughout India. he Charaka Samhita considers BM (Synonyms:
Herpestis monniera) as medhya rasayana and Ayurvedic
texts advocate the use of BM in ascites, enlarged spleen,
indigestion, inlammation and leprosy, and so forth and, as a
result, researchers have evaluated its sedative and tranquillizing, cognition, antidepressant and antianxiety, antiepileptic,
antioxidant and adaptogenic, antiulcer, and anti-Helicobacter
properties [5–13]. Recently, alcoholic extract of Bacopa monniera and its isolated constituent Bacoside-A were screened
for wound healing activity by excision, incision, and dead
space wound on Swiss albino rats and were found to enhance
2
wound healing in terms of increase in tensile strength, wound
epithelization, and connective tissue formation [14].
he present study was, therefore, undertaken to do an
in-depth study on the wound healing activities of 50%
ethanol extract of whole plant of Bacopa monniera in incision,
excision, and dead space wound models in rats when given by
oral route.
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day 1, 4 hours ater the induction of excision and dead space
wounds. he animals received CMC/BME/VTE orally with
the help of an orogastric tube in the volume of 1 mL/100 g
body weight for 10 days for dead space wound and incision
wound studies and 20 days or till the period of complete
epithelization for excision wound study.
2.5. Wound Healing Studies
2. Materials and Methods
2.1. Animals. Inbred Charles-Foster albino rats (160–180 g)
of either sex were obtained from the central animal house
of Institute of Medical Sciences, Banaras Hindu University,
Varanasi. hey were kept in the departmental animal house at
26±20∘ C and relative humidity 44–56%, light and dark cycles
of 10 and 14 h, respectively, for one week before and during the
experiments. Animals were provided with standard rodent
pellet diet (Pashu Aahar Vihar, Ramnagar, Varanasi) and
water ad libitum. “Principles of laboratory animal care” (NIH
publication no. 82-23, revised 1985) guidelines were followed.
Approval from the Institutional Animal Ethical Committee
was taken prior to the experimental work (Notiication no.
Dean/2010-11/275 dated 13.10.2010).
2.2. Plant Material and Preparation of Extract. he whole
plant of Bacopa monniera (BM) (Ayurvedic Gardens, Banaras
Hindu University) was collected during April–June and identiied with the standard sample preserved in the department
of Dravyaguna, Institute of Medical Sciences, Banaras Hindu
University, Varanasi. 50% ethanolic extract of BM (BME) was
prepared by adding 500 g of dried, crushed, and powdered
whole plant of BM in 1000 mL of 50% ethanol in a round
bottom lask and was kept at room temperature for 3 days
in shade. he extract was iltered and the previous procedure
was repeated twice. he extract iltrate so obtained was
pooled and evaporated on water bath till it dried. he yield
of BME was about 28.16% (w/w).
2.3. Drug and Chemicals. Vitamin E (Merck Ltd., Mumbai,
India) and all the other chemicals and reagents were used of
analytical grade.
2.4. Dose Selection and Treatment Protocol. A preliminary
dose-response efect using BME was irst undertaken to study
the wound breaking strength, in incision wound model,
in rat. Graded doses of BME 12.5, 25, and 50 mg/kg were
administered once daily orally for 10 days in rats following
induction of incision wound. he sutures were removed on
7th day of experiment and wound breaking strength (WBS)
was measured on 10th postwounding day. he result of the
dose response study in incision wound model indicated
that 25 mg/kg of BME had the optimal efect. herefore,
dose of 25 mg/kg of BME was chosen for further study on
various physical, biochemical, and histopathological parameters of wound healing in rat dead space wound models.
BME (25 mg/kg) and the standard drug Vitamin E (VTE;
200 mg/kg), suspended in 0.5% carboxy methyl cellulose
(CMC) in distilled water, were given orally once daily from
2.5.1. Incision Wound Model. Two parallel six cm paravertebral incisions were made through the full thickness of the
skin, 1 cm lateral to the midline of vertebral column ater
giving anaesthesia [15]. Wounds were closed with interrupted
sutures, 1 cm apart, with surgical suture. he sutures were
removed on the 7th postwounding day. Wound breaking
strength (WBS) was measured on the 10th postwounding
day in anaesthetized rats. A line was drawn on either side
of the incision line 3 mm away from the wound. Two Allis
forceps were irmly applied on to the line facing each other.
One of the forceps was ixed, while the other was connected
to a freely suspended lightweight polypropylene graduated
container through a string run over to a pulley. Standard
weights were put slowly and steadily into the container. A
gradual increase in weight was transmitted to the wound site
pulling apart the wound edges. As and when the wound just
opened up, the weight was stopped and noted.
2.5.2. Excision Wound Model. Rats were anesthetized with
ketamine (30 mg/kg, ip) and an area of about ≈500 mm2
was marked on the back of the rat by a standard ring. Full
thickness of the marked skin was then cut carefully. Wounds
were traced on 1 mm2 graph paper on the day of wounding
and subsequently at a gap period of 4 days till 12th day, then
on the alternate days until healing was complete. Changes
in wound area were measured regularly and the rate of
wound contraction calculated as given in the formula below.
Signiicance in wound healing of the test groups is derived by
comparing healed wound area on respective days with healed
wound area of control group. he period of epithelization,
that is, day of fall of eschar and the scar area, was also noted
down [15]:
% wound contraction = [
( ∘ Healed area
Healed area∘
] × 100,
Total wound area
(1)
= original wound area − present wound area) .
2.5.3. Dead Space Wound Model. Rats were anesthetized with
ketamine and 1 cm incision was made on dorsolumbar part
of the back. Two polypropylene tubes (0.5 × 2.5 cm2 each)
were placed in the dead space of lumbar region of rat on
each side, and wounds were closed with a suture material. On
the 10th postwounding day, the animals were sacriiced and
granulation tissue formed on and around the implanted tubes
was carefully dissected out, weighed, and processed for the
estimation of free radicals, antioxidants, and collagen tissue
parameters [15].
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2.6. Estimation of Granulation Tissue Free Radical and Antioxidant. Antioxidants—superoxide dismutase, SOD [16]; catalase, CAT [17] and reduced glutathione, GSH [18]; free
radicals—lipid peroxidation, LPO [19] and nitric oxide,
NO [20] and acute inlammatory marker, myeloperoxidase
(MPO) [21], and protein [22] were estimated in wet granulation tissue homogenates. Briely, the wet granulation tissues
were homogenized in a glass Telon homogenizer (10% w/v)
at 4∘ C in Phosphate bufered saline (PBS, pH 7) used for
the estimation of protein, free radicals, and antioxidants. he
assay of SOD is based on the inhibition of the formation
of NADH-phenazine methosulphate-nitro blue tetrazolium
formazan. One unit of enzyme activity is deined as the
amount of enzyme that gave 50% inhibition of nitro blue
tetrazolium reduction in one minute. CAT measurement was
done based on the ability of catalase to oxidize hydrogen
peroxide. One unit (U) of catalase is the enzyme, which
decomposes one mM of H2 O2 /min at 25∘ C. GSH activity
in the homogenate was estimated by the ability of GSH to
reduce DTNB within 5 min of its addition against blank. LPO
levels were estimated in terms of malondialdehyde (MDA)
released during lipid peroxidation Nitrites and nitrates are
formed as end products of reactive nitrogen products during
NO formation which are measured by using Griess reagent.
For myeloperoxidase (MPO) estimation, granulation tissue (5% w/v) was homogenized in 0.5% hexadecyltrimethylammonium bromide (HTAB, Sigma-Aldrich, Co., St. Louis,
MO, USA) with 50 mM potassium phosphate bufer (pH 6).
he previous homogenate was freeze-thawed three times,
sonicated for 10 seconds, and then centrifuged at 14000 ×g for
45 minutes at 4∘ C and the resulting supernatant was used for
estimation of MPO. A unit of MPO activity is deined as that
converting 1 �mol of H2 O2 to water in 1 min at 25∘ C.
2.7. Estimation of Connective Tissue Parameters. Approximately 250 mg of wet tissue was dried at 50∘ C for 24 h.
It was weighed and kept in glass stoppered test tubes. To
each tube containing 40 mg of the dried granulation tissue,
1 mL of 6 N HCl was added. he tubes were then kept on
boiling water bath for 24 h (12 h each day for two days) for
hydrolysis. he hydrolysate was then cooled and excess of
acid was neutralized by 10 N NaOH using phenolphthalein
as indicator. he volume of neutral hydrolysate was diluted
to a concentration of 20 mg/mL with distilled water. he inal
hydrolysate was used for the estimation of hydroxyproline,
hexosamine, and hexuronic acid following the standard curve
prepared using the proper substrate.
2.7.1. Hydroxyproline (HPR). To the each tube, 0.3 mL each of
hydrolysate, 2.5 N NaOH, 0.01 M CuSO4 , and 6% H2 O2 were
added. Tubes were shaken vigorously and placed immediately
in water bath at 80∘ C. Ater 15 minutes, tubes were removed
and cooled for 5 minutes in cold water. 0.6 mL of freshly
prepared 5% solution of paradimethyl amino-benzaldehyde
in n-Propanol and 1.2 mL of 3 N H2 SO4 was added. he test
tubes were once again placed in a hot water bath at 75∘ C for 15
minutes and then cooled for 5 minutes under running stream
of water. Color intensity was measured at 540 nm against the
3
blank. Hydroxyproline content in the tissue was estimated
as per standard curve prepared with standard 4-HydroxyL-proline (HiMedia Laboratories Pvt. Ltd., Mumbai, India),
from 75 to 900 �g/0.3 mL using 3 mg/mL working solution
[23].
2.7.2. Hexosamine (HXA). 0.05 mL of hydrolyzed fraction
was diluted to 0.5 mL with distilled water. To this was added
0.5 mL of acetyl acetone reagent and heated in boiling water
bath for 20 min then cooled under tap water. To this 1.5 mL of
95% alcohol was added, followed by an addition of 0.5 mL of
Ehrlich’s reagent. he reaction was allowed for 30 minutes to
complete. Color intensity was measured at 530 nm against the
blank. Hexosamine content of the samples was determined
from the standard curve prepared with D (+) glucosamine
hydrochloride (HiMedia Laboratories Pvt. Ltd., Mumbai,
India), from 5 to 50 �g/0.5 mL using 100 �g/mL working
solution [24].
2.7.3. Hexuronic Acid (HUA). 2.5 mL of 0.025 M Borax in
concentrated sulphuric acid is placed in stoppered tubes ixed
in a rack and cooled to 4∘ C. 0.125 mL of hydrolysate was
diluted 0.5 mL by adding distilled water. Now, this 0.5 mL
of hydrolysate is layered carefully on Borax-sulphuric acid
mixture kept in rack at 4∘ C. he tubes were closed with glass
stoppers and then shaken, irst slowly then vigorously, with
constant cooling by placing tubes in ice container. he tubes
were then heated for 10 min in a vigorously boiling water
bath and cooled to room temperature. hereater, 0.1 mL of
0.125% carbazole reagent in absolute alcohol was then added
to each tube, shaken, again heated in the boiling water bath
for further 15 min, and then cooled to room temperature.
Color intensity was measured at 530 nm against the blank.
Hexuronic acid content of the samples was determined from
the standard curve prepared with D (+) Glucurono-6, 3lactone (HiMedia Laboratories Pvt. Ltd., Mumbai, India),
from 5 to 40 �g/0.5 mL using 100 �g/mL working solution
[25].
2.8. Histopathology. he cross-sectional full-thickness skin
specimens and deep granulation tissues from the implanted
tube were collected on the 10th day of the experiment for
the histopathological alterations. Samples were ixed in 10%
bufered formalin, processed, blocked with parain, then
sectioned into 5 �m sections, and stained with hematoxylin
and eosin.
2.9. Antimicrobial Susceptibility and Minimum Inhibitory
Concentration (MIC). In vitro antibacterial susceptibility test
of BME was done using serial concentrations of 50, 100,
150, and 200 mg/mL following the approved standards of
the National Committee for Clinical Laboratory Standards
(NCCL) [26] against common skin bacteria Staphylococcus aureus (ATCC 25323), Staphylococcus epidermidis, and
Pseudomonas aeruginosa obtained from the American Type
Culture Collection (ATCC) and clinical strain preserved at
Department of Microbiology, Institute of Medical Sciences,
BHU, Varanasi, India, following the disk difusion method
4
[27] while minimum inhibitory concentration (MIC) was
performed by microdilution method [28]. Briely, 24 h old
culture of selected microbes was adjusted to 0.5 McFarland
standard in sterile normal saline to achieve concentration of
∼107 (colony forming units) CFU/mL. Standard antibiotics
used as positive control. Dimethylsulfoxide (DMSO) was
used as negative control. MIC was determined by microbroth
dilution method. Speciically 0.1 mL of standardized inoculums of bacteria (1-2 × 107 CFU/mL) was added in each well
of microtiter plate which was incubated aerobically at 37∘ C
for bacterial growth for 18–24 h. he lowest concentration
(highest dilution) of the extract that produced no visible
bacterial growth (no turbidity) when compared with the
control was regarded as MIC.
2.10. Statistical Analysis. Statistical comparison was performed using either unpaired t-test or one-way analysis of
variance (ANOVA) followed by Dunnett’s test for multiple
comparisons” instead of ”for multiple comparisons verses
control group was done by Dunnett’s test. All statistical
analysis was performed using SPSS statistical version 16.0
sotware package (SPSS Inc., USA).
3. Results
3.1. Incision Wound Model. Control rats showed WBS as
283.3±18.6 g on 10th postwound day. BME 12.5, 25, 50 mg/kg
treated rats showed WBS as 353.3 ± 19.3 g, 373.3 ± 13.8 g,
375.0 ± 14.8 g (� < 0.05 to � < 0.01), respectively, while
vitamin E (VTE, 200 mg/kg) treated rats showed WBS as
405.0 ± 21.1 g (� < 0.01).
3.2. Excision Wound Model. Rate of wound contraction in
control rats was 21.6% to 68.3% from day 4 to day 12 and
80.6% to 98.1% from day 14 to day 20, while complete
epithelization and healing were observed on day 24. he
average number of days that took for the shedding of eschar
without leaving any residual raw wound in these rats was
12.7 days and mean of scar area ater completing healing
was 99.8 mm2 . he percent rate of wound contraction in
rats, treated orally with BME (25 mg/kg), was from 32.2%
on day 4 to 85.4% on day 12 and 92.1% to 100% from day
14 to day 20, respectively, while VTE treated rats showed
increase in wound contraction from 32.4% on day 4 to
87.6% on day 12 and 92.2% to 100% from day 14 to day 20,
respectively. he mean epithelization period and scar area
observed with BME were 10.3 days and 75.2 mm2 while the
mean epithelization period and scar area observed with VTE
were 10 days and 74.3 mm2 , respectively. BME treated rats,
thus, showed faster healing which was comparable with VTE
treated group (Table 1 & Figure 1).
Histology of excision biopsy of skin wound at day 10
showed healed skin structures with normal epithelization,
restoration of adnexa and ibrosis within the dermis in
BME and VTE treated groups while the control group lag
behind treated group in formation of the amount of ground
substance in the granulation tissue, as observed in tissue
sections (Figure 2).
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Control
BME
VTE
0 day
12th day
20th day
Figure 1: Photographic representation of contraction rate showing
percent wound contraction area on diferent postexcision days of
control, BME (25 mg/kg), and VTE (200 mg/kg) treated rats.
3.3. Dead Space Wound Model. BME caused an increase in
wet weight mg per 100 g body weight and protein mg/g
granulation tissue by 17.6% (� < 0.05) and 18.7% (� < 0.05),
respectively, compared with control group. BME efect was
comparable with that of VTE on the previous parameters
(Table 2).
3.4. Wet Granulation Tissue Antioxidants, Free Radicals, and
Myeloperoxidase. BME showed signiicant increase in the
level of antioxidants, GSH, SOD, and CAT while free radicals,
LPO and NO, and acute inlammatory marker, MPO were
decreased. he results with BME were comparable with VTE
on the previous sot-tissue parameters (Table 2).
3.5. Dry Connective Tissue HPR, HXA, and HUA. Dry weight
of granulation tissue and protein content were increased by
19.4% and 18.1% (� < 0.05), respectively, in BME treated
groups compared with control group. HPR, HXA, and HUA
were signiicantly increased in BME treated group by 59.8%,
59.1%, and 153%, respectively, compared to control group. he
results with BME were comparable with VTE on the previous
connective tissue parameters (Table 3).
Histology of granulation tissue of deeper structure of
control rat showed mononuclear inlammatory cells, scattered ibroblasts (minimal ibrosis), and few proliferating
vasculature in granulation tissue, while the granulation tissue
of rats treated with BME and VTE showed abundance of
eosinophilic collagen tissue and neovascularisation with few
inlammatory cells indicative of healing by ibrosis (Figure 3).
3.6. Antimicrobial Susceptibility and MIC. BME showed positive susceptibility test against common skin bacteria Staphylococcus aureus (ATCC 25323), Staphylococcus epidermidis,
and Pseudomonas aeruginosa, showing zone of inhibition
≥10 mm, at 200 mg/mL. BME had least minimum inhibitory
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Table 1: Efect of BME and VTE on wound contraction, epithelization period, and scar area in excision wound.
Oral treatment
(mg/kg, od)
Control 0.5%
CMC
BME 25
VTE 200
0 day
532.8 ± 6.93
(0.00)
530.3 ± 7.15
(0.00)
550.0 ± 7.02
(0.00)
4th day
417.7 ± 6.77
(21.6 ± 1.0)
359.2 ± 12.2b
(32.2 ± 2.71)
371.7 ± 12.1b
(32.4 ± 2.03)
8th day
322.3 ± 10.8
(39.5 ± 1.73)
217.8 ± 20.8b
(59.0 ± 3.96)
229.8 ± 14.2c
(58.3 ± 2.48)
Wound area in mm2 /rat (% contraction)
12th day
14th day
16th day
168.5 ± 10.1
103.3 ± 5.79
81.2 ± 2.36
(68.3 ± 2.03) (80.6 ± 1.16) (84.8 ± 0.48)
42.0 ± 3.86c
22.3 ± 2.33c
77.2 ± 5.21c
(85.4 ± 1.08) (92.1 ± 0.72) (95.8 ± 0.46)
68.3 ± 2.55c
42.8 ± 6.13c
19.7 ± 3.02c
(87.6 ± 0.49) (92.2 ± 1.12) (96.4 ± 0.55)
18th day
36.2 ± 1.25
(93.2 ± 0.20)
4.60 ± 0.56c
(99.0 ± 0.09)
3.83 ± 0.98c
(99.3 ± 0.17)
20th day
10.3 ± 0.67
(98.1 ± 0.15)
0.0 ± 0.0c
(100.0)
0.0 ± 0.0c
(100.0)
Epithelization Scar area
period (days) (mm2 )
22nd day
3.17 ± 0.75
12.7 ± 0.67
99.8 ± 4.92
(99.4 ± 0.15)
c
0.0 ± 0.0
10.3 ± 0.49a
75.2 ± 4.04b
(100.0)
0.0 ± 0.0c
10.0 ± 0.58a
74.3 ± 3.98b
(100.0)
Values are mean ± SEM (Percent) of 6 rats in each group. a � < 0.05, b � < 0.01, and c � < 0.001 compared to respective day control group (statistical analysis was done by one-way analysis of variance followed by
Dunnett’s test for multiple comparisons).
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6
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(a)
(b)
(c)
Figure 2: Histopathology of skin at day 10 stained with H&E (100x). (a) Skin of control rat showing ulceration and edema showed by white
arrow, early epithelization showed by yellow arrow, and granulation tissue and abundance of mononuclear inlammatory cells showed by
black arrow. (b) BME treated rats showing large amount of granulation tissue by black arrow, small number of mononuclear inlammatory
cells, and restoration of adnexa and extensive ibrosis. (c) VTE treated rats showing healed skin structures with well-formed, near to normal
epidermis, restoration of adnexa, and extensive ibrosis and collagen tissue within the dermis.
Table 2: Efect of BME and VTE on wet granulation tissue weight, protein, free radicals (LPO and NO), antioxidants (GSH, SOD, and CAT),
and myeloperoxidase (MPO).
Oral treatment Wet tissue
(mg/kg, od × mg/100 g bw
10 day)
Protein
mg/g tissue
359.3 ± 18.3
48.6 ± 2.75
Control 0.5%
CMC
BME 25
VTE 200
422.7 ± 18.1
461.5 ± 11.5b
a
57.7 ± 2.05
59.1 ± 3.40a
a
GSH
nM/mg
protein
20.7 ± 1.17
24.6 ± 1.11
24.5 ± 0.88a
a
Antioxidants
SOD
IU/mg
protein
CAT
mU/mg
protein
0.41 ± 0.07
42.9 ± 1.84
0.72 ± 0.02
0.79 ± 0.04c
b
Free radicals
LPO
NO
nM/mg
nM/mg
protein
protein
151.6 ± 0.68
210.4 ± 0.84c
c
6.33 ± 0.50
3.24 ± 0.26
1.78 ± 0.15c
c
Myeloperoxidase
MPO
mU/mg protein
36.8 ± 4.53
15.3 ± 0.87
15.2 ± 1.53b
c
24.1 ± 0.55
19.3 ± 0.29c
14.6 ± 0.37c
Values are mean ± SEM of 6 rats in each group. a � < 0.05, b � < 0.01, and c � < 0.001 compared to respective control group (statistical analysis was done by
one-way analysis of variance followed by Dunnett’s test for multiple comparisons).
concentration (MIC) of 0.39 mg/mL against Staphylococcus
aureus (ATCC 25323) and 3.125 mg/mL against Pseudomonas
aeruginosa, whereas MIC of BME against other organisms
ranged from 6.25 to 25.0 mg/mL.
4. Discussion
Wound represents a major health problem, both in terms of
morbidity and mortality. he processes involved in wound
healing are epithelization, contraction, and connective tissue
deposition. he healing process depends, to a large extent,
on the regulated biosynthesis and deposition of new collagens and their subsequent maturation [15]. In the tissue
repair process, inlammatory cells promote the migration and
proliferation of endothelial cells, leading to neovascularisation of connective tissue cells which synthesize extracellular
matrices including collagen, and of keratinocytes resulting to
reepithelialization of the wounded tissue [29]. Inlammation,
collagen maturation, and scar formation are some of the
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7
(a)
(b)
(c)
Figure 3: Histopathology of granulation tissue at day 10 stained with H&E (100x). (a) Granulation tissue of control rat showed mononuclear
inlammatory cells by white arrow, scattered abundance of eosinophilic ibroblasts showed by black arrow. (b) BME treated showing large
number of collagen tissue (ibrosis) and neovascularisation with minimal inlammatory cells. (c) VTE treated showing near to normal features,
collagen tissue (ibrosis), and neovascularisation.
many phases of wound healing, which run concurrently but
independent of each other.
In incision wound study, BME showed an increase in
breaking strength which may be due to the increase in
collagen concentration and stabilization of the ibres [30].
he collagen molecules synthesized are laid down at the
wound site and become cross-linked to form ibres. Wound
strength is acquired from both remodeling of collagen and
the formation of stable intra- and intermolecular crosslinks.
BME showed greater breaking strength which may be due
to increased collagen synthesis as found in the dead space
wound study.
In excision wound, BME showed faster healing compared with control group. Further, excision biopsy of skin
wound at day 10 showed healed skin structures with normal
epithelization, restoration of adnexa and ibrosis within the
dermis in BME and VTE treated groups, while the control
group lags behind treated group in formation of the amount
of ground substance in the granulation tissue. he faster
wound contraction by BME may be due to stimulation of
interleukin-8, an inlammatory �-chemokine which afects
the function and recruitment of various inlammatory cells,
ibroblasts and keratinocytes, and may increase the gap
junctional intracellular communication in ibroblasts, and
induces a more rapid maturation of granulation tissue [31].
Collagen is the predominant extracellular protein in
the granulation tissue of a healing wound and there is a
rapid increase in the synthesis of this protein in the wound
area soon ater an injury. Breakdown of collagen liberates
free hydroxyproline and its peptides. Measurement of this
hydroxyproline, therefore, has been used as an index of
collagen turnover. he biochemical data of dead space wound
study showed an increase in wet tissue weight and protein per
g tissue in BME treated groups. he increased hydroxyproline
content in the dead space wounds has indicated faster
collagen turnover leading to rapid healing with concurrent
increase in the breaking strength of the treated wounds.
Hexosamine and hexuronic acids are matrix molecules,
which act as ground substratum for the synthesis of new
extracellular matrix. he glycosaminoglycans are known
to stabilize the collagen ibres by enhancing electrostatic
and ionic interactions with it and possibly control their
ultimate alignment and characteristic size. heir ability to
bind and alter protein-protein interactions has identiied
them as important determinants of cellular responsiveness
in development, homeostasis, and disease [32]. In our study,
hexuronic acid and hexosamine concentrations which are
the components of glycosaminoglycans were signiicantly
increased with BME when compared with control indicating
stabilization of collagen ibres.
8
BioMed Research International
Table 3: Efect of BME and VTE on dry granulation tissue, protein, hydroxyproline, hexosamine, and hexuronic acid content.
Oral treatment
(mg/kg, od × 10
day)
Control 0.5%
CMC
BME 25
VTE 200
Dry tissue
mg/100 g bw
Protein
mg/g dry tissue
71.2 ± 3.80
85.0 ± 3.70
92.2 ± 2.0c
a
244.1 ± 14.4
288.4 ± 10.3
295.5 ± 17.0a
a
Connective tissue parameters
Hydroxyproline
Hexosamine
Hexuronic Acid
�g/mg protein
�g/mg protein
�g/mg protein
145.7 ± 12.6
204.2 ± 9.44
191.1 ± 11.2b
b
86.6 ± 7.02
122.1 ± 7.70
126.2 ± 8.97b
b
20.8 ± 3.94
52.6 ± 2.90c
46.0 ± 2.86c
values are mean ± SEM of 6 rats in each group. a � < 0.05, b � < 0.01, and c � < 0.001 compared to respective control group (statistical analysis was done by
one-way analysis of variance followed by Dunnett’s test for multiple comparisons).
Experimental and clinical lines of evidence suggest that
chronic wound undergoes substantial oxidative stress by
neutrophils-derived oxidants and MPO activity, both of
which contribute markedly to tissue damage during chronic
wound inlammation [33]. Over production of reactive oxygen species (ROS) results in oxidative stress thereby causing
cytotoxicity and delayed wound healing and elimination of
ROS could be an important strategy in healing of chronic
wounds [4]. herefore, estimation of antioxidants like GSH,
SOD, and CAT in granulation tissues is relevant because the
antioxidants have been reported to hasten wound healing
by decreasing the free radicals [34]. Our studies on the
antioxidants, free radicals, and MPO status revealed that
BME had signiicant antioxidant activity, reduced MPO and
free radicals stress, helped to prevent inlammation and
oxidative damage, and promoted the healing process.
he preliminary phytochemical analysis of Bacopa monniera (BM) revealed the presence of saponins, glycosides, and
alkaloids [35]. Animal research has shown that the phytoconstituents present in BM extract modulate the expression
of certain enzymes involved in generation and scavenging
of reactive oxygen species in the brain [7]. BM’s antioxidant
properties and its ability to balance superoxide dismutase
and catalase levels were postulated to account for this efect
[8]. BM by modulating the extent of lipid peroxidation and
enhancing the antioxidant status has DNA protective efects;
hence, it is believed to prevent cell damage [36]. Extracts
of BM have been reported to have a broad spectrum of
antibacterial activity [37], which seemed to have beneicial
efects on wound healing. BME was found to show antibacterial activity against Staphylococcus aureus, Staphylococcus
epidermidis,and Pseudomonas aeruginosa and least MIC was
found with BME against Staphylococcus aureus.
5. Conclusion
hus, in our present study involving three diferent wound
models, which included observation of diferent physical, histological, biochemical parameters, and antimicrobial activity,
indicated the wound healing activity in the 50% ethanolic
extract of dried whole plant of Bacopa monniera. he healing
efects seemed to be due to decreased free radical generated
tissue damage, promoting efects on antioxidant status, faster
collagen deposition, and other connective tissue constituent
formation, and antibacterial activity.
Conflict of Interests
he authors declare that they have no conlict of interests.
Authors’ Contribution
S. Murthy and M. K. Gautam equally contributed to the
paper.
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