Journal of Ethnopharmacology 191 (2016) 95–106

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Systematic investigation of ethanolic extract from Leea macrophylla:

Implications in wound healing
Apurva Joshi a, Vinod K. Joshi b, Deepali Pandey a, S. Hemalatha a,n
a
Department of Pharmaceutics, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India
b
Department of Dravyaguna, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Leea macrophylla Roxb. ex Hornem. (Leeaceae) commonly known as
Received 8 December 2015 Hastikarnapalasa is mainly distributed throughout the tropical parts of India. Traditionally, the plant is
Received in revised form found to be effective against guinea worm, ringworm and is applied to sores and wounds.
17 April 2016
Aim of the study: The present study aims to validate traditional wound healing claim of Leea macrophylla
Accepted 13 June 2016
scientifically.
Available online 14 June 2016
Material and methods: Box–Behnken design (BBD) was used to optimize the extraction process. The opti-
Keywords: mized root tuber extract of Leea macrophylla was standardized with chlorogenic acid by HPLC for the first
Leea macrophylla time. Both oral and topical routes were selected as administrative means for the wound healing study using
Bioadhesive gel excision and incision wound model. For topical treatment bioadhesive gel was formulated and characterized
Wound healing
for mechanical and physical characteristics by texture profile analysis (TPA) and scanning electron micro-
Proinflammatory cytokines
scopy (SEM). The effect on wound healing was also assessed by evaluating antioxidant enzymes viz. glu-
VEGF
Ki-67
tathione (GSH), superoxide dismutase (SOD) and catalase (CAT), free radicals lipid peroxidation (LPO) and
nitric oxide (NO), inflammatory marker myeloperoxidase (MPO), collagen markers hydroxyproline, hex-
osamine and hexuronic acid along with the histopathological examination. Furthermore, the effect on the
level of the proinflammatory cytokines interleukin-1β (IL-1β), interleukin -6 (IL-6), tumor necrosis factor-α
(TNF-α) and growth factor, vascular endothelial growth factor (VEGF) were determined. The expression of cell
proliferation nuclear marker Ki-67 was also analyzed by Western blot analysis.
Results: With mesh openings Sieve no. 20, semi polar nature of solvent (92.5:7.5 ethanol-water blend) and
extraction time of 18 h, substantially greater extraction efficiency (29%) and phenolic yield (181.54 mg/g)
were obtained. The content of chlorogenic acid in ethanol extracts of Leea macrophylla was obtained as 9.01%
w/w. In incision model, oral treatment with 500 mg/kg ethanolic extract increased wound breaking strength
by 23.41% while bioadhesive gel (5% w/v) showed a higher increase of 44.68%. Topical application produced
complete wound contraction in 20 days against 22 days taken by oral treatment. Topical treatment also
produced a significant (po0.05) increase in antioxidants glutathione, superoxide dismutase and catalase
whereas the level of enzymes lipid peroxidation and nitric oxide and inflammatory markers myeloperoxidase
were reduced. Further advantageous effects were reflected by significantly (po0.05) increased levels of
hydroxyproline, hexosamine and hexuronic acid. Favorable effects on the level of proinflammatory cytokines
interleukin-1β, interleukin-6, tumor necrosis factor – α and growth factor, vascular endothelial growth factor
were also observed. The wound healing potential of Leea macrophylla was further supported by its ability to
promote cell proliferation during wound healing as demonstrated by Western blot analysis of proliferation
marker Ki-67.
Conclusion: The study justified traditional use of Leea macrophylla in wound healing and demonstrated that the
bioadhesive gel of ethanolic extract produced faster and more significant healing as compared to oral treatment.
& 2016 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: BBD, Box–Behnken design; ELM, Ethanolic extract of Leea macrophylla; HPLC, High Performance Liquid Chromatography; CGA, Chlorogenic acid; CMC,
Carboxymethylcellulose; SCMC, Sodium carboxy methyl cellulose; PVP, Polyvinyl pyrrolidone; PC, Polycarbophil; ELMO, Oral treatment with ethanolic extract of Leea
macrophylla; ELMT, Topical treatment with bioadhesive gel of ethanolic extract of Leea macrophylla; WBS, Wound breaking strength; HA, Healed area; TWA, Total wound
area; CAT, Catalase; SOD, Superoxide dismutase; GSH, Reduced glutathione; NO, Nitric oxide; LPO, Lipid peroxidase; HTAB, Hexadecyl trimethyl ammonium bromide; MPO,
Myeloperoxidase; IL-1β, Interleukin-1β; IL-6, Interleukin-6; TNF-α, Tumor necrosis factor-α; VEGF, Vascular endothelial growth factor; ELISA, Enzyme linked immunosorbent
assay; ANOVA, Analysis of variance; TPA, Texture profile analysis; SEM, Scanning Electron Microscopy; ROS, Reactive oxygen species; FRSE, Free radical scavenging enzymes
n
Corresponding author.
E-mail address: shemalatha.phe@itbhu.ac.in (S. Hemalatha).

http://dx.doi.org/10.1016/j.jep.2016.06.034
0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
96 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
1. Introduction
Compromise in congruity of any tissue is classified as a wound
(example skin breaks, muscle tears, burns or a bone fracture). It
may result from a random fall, a bruising accident, surgery, an
infection or a covert etiological ailment. For instance, disruption of
skin integrity leads to cutaneous wounds (Mallefet and Dweck,
2008), whereas abrupt collision with a moving vehicle may cause
visceral, deep lying wounds. The causative factors, signaling in-
termediaries, pre-existent diseases, and type of injury all cumu-
latively determine whether the healing process will be acute or
chronic (Schreml et al., 2010).
Wound healing is a dynamic, complex mechanism aimed to-
wards re-attainment of tissue integrity and homeostasis (Eming
et al., 2007) involving inflammation, re-epithelization, granulation
tissue formation, neovascularization, wound contraction and re-
modeling of extracellular matrix (Singer and Clark, 1999). It is
coordinated by a complicated signaling system involving various
growth factors, cytokines and chemokines. Cell proliferation is an
imperative step in tissue repair and regeneration in wound healing
process (Xing et al., 2015).
Constrained healing hampers the life of plenty, and conse-
quently, more efforts are being directed towards investigating cost
efficient and accessible therapeutic approaches. Plants and their
phytoconstituents have been long-established to treat wounds
(Wang et al., 2011). Leea macrophylla Roxb. ex Hornem. (Leeaceae),
commonly known as Hastikarnapalasa is a wild edible plant with
high nutritive value in terms of minerals and vitamins content (B1,
B2, C and B12) (Jadhao et al., 2009). The dried powdered root of
Leea macrophylla is taken along with clarified butter in the
morning as age sustainer (Jadhao and Wadekar, 2010). Tradition-
ally, the plant has been reported to be effective against guinea
worm, ringworm and is applied on sores and wounds (Kirtikar and
Basu, 1975; Bhavamishra, 2010). Roots are applied externally to
allay pain and are alexipharmic (Kirtikar and Basu, 1975). In Uttar
Pradesh (India) its local name is Bado hanshia where the local
tribes use the root tubers orally and locally for treating wound
(Anonymous, 1999). Pharmacologically, the plant has been re-
ported to possess anti- urolithiasis (Nizami et al., 2012) and anti-
inflammatory activities (Dewanjee et al., 2013). Recently, we have
successfully evaluated the potential in vitro antioxidant and anti-
bacterial activity of ethanolic extract of Leea macrophylla root tu-
bers (ELM), which is mainly attributed to the presence of phenolic,
tannins, flavonoid, steroids and alkaloid (Joshi et al., 2016). How-
ever, the root tubers have not been scientifically validated for their
wound healing activity.
The present study intends to investigate wound healing po-
tential of Leea macrophylla root tuber extract to scientifically va-
lidate its traditional claims. Apart from employing statistical de-
scriptors to optimize the extractive process, the current manu-
script also entails the development and thorough mechanical
characterization of bioadhesive hydrogel for topical application of
obtained extract and comparison of its wound healing effect with
its oral formulation, to discern the most feasible route of
administration.
2. Material and methods
2.1. Plant material and extraction
The root tubers of Leea macrophylla were collected from a
medicinal plant garden of Department of Dravyaguna, Banaras
Hindu University in the month of September – October 2013 and
were authenticated by Prof. V.K. Joshi, Department of Dravyaguna,
Banaras Hindu University and submitted to Department of
Pharmaceutics, Indian Institute of Technology, Banaras Hindu
University, Varanasi (No. COG/LM/01). The acquired root tubers
were weighed (200 g), and shade dried followed by a cycle of
powdering before extraction. The process of Soxhlet extraction
was optimized using Box–Behnken design (BBD) where three
discerning factors influencing the extraction process were ana-
lyzed including mesh size, extraction solvent and extraction time.
Design Expert (Version 6.0.0 Trial, Stat-Ease Inc., MN) was used to
determine the boundary of the experiment, for evaluating re-
sponse and for finally optimizing the process. Each specific design
factor was constrained at three levels: low, medium and high level.
Following the design, the powdered drug was passed through
the sieve (Sieve no. 10, 20 and 40) and then subjected to extraction
in Soxhlet apparatus using ethanol-water blend at different ratios
(85:15, 92.7:7.5 and 100:0) for a particular time (12 h, 18 h and
24 h) at 70 °C. The solvent was removed by concentrating the
extract in a rotary evaporator (IKA) at 40 °C under reduced pres-
sure. The residue left behind was reddish brown in colour and was
stored in a desiccator.
2.2. Working with BBD towards response surface
The efficiency of Soxhlet extraction depends on factors such as
the degree of sample homogenization, nature of the solvent used
for extraction and length of time invested in the completion of
extraction process (Gullberg et al., 2004). These critical factors
were identified by preliminary experimentation and data mining
as being capable of influencing extraction. Please refer to Supple-
mentary information for detailed optimization of soxhlet extrac-
tion process.
2.3. Confirmation of chlorogenic acid in ethanolic extract by High
Performance Liquid Chromatography (HPLC)
A validated HPLC method (Yuan et al., 2005) was followed for
standardizing ethanolic extract of Leea macrophylla. Commercially
procured chlorogenic acid (CGA, Sigma-Aldrich [purity: 95%]) was
employed as standard. HPLC system (Waters) equipped with gra-
dient pumps was used for analysis. A Cosmosil C18 column
(150 mm
4.6 mm, 5 mm particle) was used for chromatographic
separation of the sample. The injection volume of sample was
10 mL. An aqueous phase consisting of 0.4% acetic acid and 4.5%
tetrahydrofuran in triple distilled water was modified with acet-
onitrile in a variable pattern to form the mobile phase flowing at
1 ml/min. Gradient elution began with an aqueous phase to acet-
onitrile phase ratio of 5:95 and changed up to 25:75 during the
initial 15 min. Further, the ratio of the mobile phase was altered
from 25:75 to 60:40 for next 35 min. An equilibration period of
10 min was allowed using the initial mobile phase composition
before injecting the next sample. The entire operation was carried
out in ambient conditions. The peak area of extracted data was
calculated at 326 nm using class VP series software. The identity of
the peak was affirmed by cross checking retention time of the
standard chlorogenic acid sample.
2.4. Experimental animals
Young albino rats (Charles Foster) of either sex weighing from
150 to 200 g bred in the Institutional animal facility, were utilized
for animal studies. Rats were granted free access to standard feed
and water. Temperature and relative humidity were maintained at
25 °C and 50% respectively. The animals were afforded with suf-
ficient time for acclimatization before experiment initiation. Care
and non-experimental handling of animals were performed by
dedicated animal house staff. All experimental protocols were
conducted after approval from Central Animal Ethical Committee
 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
of Banaras Hindu University (No. Dean/2015/CAEC/980) as per
accepted standard guidelines.
2.5. Oral dose
For oral administration, the ethanolic extract of Leea macro-
phylla was suspended in carboxymethylcellulose (CMC, 0.5%) at
250, 500 and 750 mg/kg p.o. Vitamin E (VTE, Sigma-Aldrich,
[purity 95%]) was selected as oral standard drug to evaluate
wound healing activity in experimental animals as reported earlier
(Joshi et al., 2013). Ketamine 100 mg/kg i.p. (Homayouni-Tabrizi
et al., 2015) was used as the anesthetizing agent.
2.6. Topical formulation and its characterization
Sodium carboxy methyl cellulose (SCMC; 2.5 or 4%, w/v) was
initially dispersed in triple distilled water overnight using a
magnetic stirrer. Following complete solubilization, poly-
vinylpyrrolidone (PVP; 2.5 or 4%, w/v) was added to swelled SCMC
dispersion. To the SCMC/PVP containing gel, polycarbophil (PC; 2%,
w/v) followed by differing concentration of ethanolic solution of
extract (2.5, 5.0 and 7.5 g per ml ethanol) were added and mixed,
which yielded 2.5%, 5% and 7.5% w/v of active extract containing
bioadhesive gel. All samples were tapped vigorously to remove
entrapped air and kept in a desiccator. For comparative reference
blank gels were also prepared by following the above described
steps with the exception of adding extract. Experimental Design
for the manufacture of bioadhesive gel is given in Table 1. The
optimized biogel was lyophilized using lyophilizer (Lypholizer,
Decibel, India) for 36 h at  40 °C for further analysis (Singh et al.,
2015c).
Mechanical behavior of the extract containing bioadhesive gel
was evaluated using a texture analyzer (BrookField, CT3) working
in TPA mode. TPA was calibrated for the probe, force and frame
stiffness using Texture Pro CT 64 bit software package which
drives the CT3 Texture analyzer. The biogels were packed into the
sample holder, and a polymeric probe (20 mm across) was com-
pressed twice into them at a rate of 2.0 mm/s to a defined depth
(30 mm) allowing delay period (10 s) between two consecutive
compression cycles. From the resultant force–time plot obtained
for three replicate samples, hardness, compressibility and adhe-
siveness were calculated. On the basis of these parameters, the
best topical formulation was selected and carried forward for
further biological evaluation (Singh et al., 2015c).
The surface morphology of optimized gel and the blank gel was
captured using scanning electron microscope (EVO-50, ZEISS,
United Kingdom). The samples were prepared by mounting lyo-
philized gels on a double adhesive tape attached to an aluminium
stub and sputter coated with a gold-palladium alloy under an ar-
gon atmosphere using a high vacuum evaporator (SC7640 Polaron
Sputter Coater). Gels were imaged using a 5 kV accelerating vol-
tage, at a working distance of 10 mm by randomly scanning sev-
eral imaging fields at appropriate magnifications (Singh et al.,
2015b).
2.7. Acute oral toxicity study
The study was conducted on fasted regular nulliparous and
non-pregnant female rats by orally administering ethanolic extract
of Leea macrophylla. The treated animals were then observed
vigilantly for 48 h for signs of acute toxicity (OECD, 2008) by no-
ticing their para-sympathetic and communicative changes. More
precisely occurrence of convulsions, excessive salivation, lacrima-
tion, defecation, tremors, shivers and any alteration in sleep
feeding pattern were observed.
97
Table 1
Experimental design for manufacture of bioadhesive gels with their codes.
Batch no. SCMC (%w/v) PVP (%w/v) PC (gm) (%w/v)
BG 1 2.5 2.5 2
BG 2 4 2.5 2
BG 3 4 4 2
BG 4 2.5 4 2
2.8. Acute skin irritation test
The method of Gfeller et al. (1985) was used on rats to de-
termine acute skin irritation of Leea macrophylla extract bioadhe-
sive gel. 500 mm2 area of dorsal hair of animals were shaved and
cleaned. The bioadhesive gel formulations (2.5%, 5% and 7.5% w/v)
were then applied to different groups of animals. The skin of each
animal was checked for any sign and symptom of inflammation
following 4 h of application.
2.9. Wound healing activity
2.9.1. Incision wound model
Each group of animals for this study had six rats. The control
group (Group 1) remained untreated whereas animals in Group
2 were treated with blank gel. The animals in Group 3–5 received
oral treatment with ethanolic extract of Leea macrophylla (ELMO)
at 250, 500 and 750 mg/kg p.o. while Group 6–8 animals were
treated topically with bioadhesive gel of ethanolic extract of Leea
macrophylla (ELMT) at a concentration of 2.5%, 5% and 7.5%. Group
9 served as oral standard and was given Vitamin E (200 mg/kg p.
o.), and Group 10 animals were treated with Aloe vera cream as
topical standard (Chithra et al., 1998). Each animal was kept se-
parately in cages. The rats were anesthetized universally across the
vertebral column, and two paravertebral incisions (6 cm long)
were created along the full thickness of the skin. The wounds were
closed with interrupted sutures at a distance of 1 cm. Oral and
topical treatments were continued up to 10 days with sutures
being removed after the 6th day. The method described by Lee
(1968) was used to determine wound breaking strength (WBS) on
the 10th post-wounding day.
2.9.2. Excision wound model
A total of thirty animals were divided into five groups of six
animals each. The control (Group 1) remained untreated whilst
Group 2 was administered 500 mg/kg p.o. ethanolic extract of Leea
macrophylla (equivalent to optimum dose in incision model) and
Group 3 animals were treated with bioadhesive gel of ethanolic
extract of Leea macrophylla at concentration of 5% (dose optimized
in incision model), Group 4 received standard oral dose of 200 mg/
kg Vitamin E, and Group 5 animals received Aloe vera cream as
topical standard. The excision wounds were created as per the
protocol prescribed by Morton and Malon (1972). The wound was
traced on 1 mm2 graph paper on the wounding day and subse-
quently on the passage of each 4-day interval till the 12th day. The
tracing process was then accelerated and performed on each al-
ternate day until completion of healing to accurately determine
scar area. Following formula was used for calculating the change in
the wound area.
% wound contraction = (HA /TWA) × 100
where healed area (HA) is the difference between the total wound
area (TWA) and current wound area. Epithelization period is the
number of days required for falling off the scar without any re-
sidual raw wound.
98 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
Excision wound was created in a parallel group of animals, and
similar treatment was given as mentioned above. On 10th post-
wounding day, the treated animals from the parallel groups were
sacrificed, and the granulation tissue was removed from the re-
spective wound patches. Biochemical evaluations and histological
studies were performed using the granulation tissue samples ob-
tained from the parallel group.
2.9.3. Biochemical estimations
The granulation tissue obtained from excision model was
evaluated for biochemical parameters. A part of granulation tissue
was subjected to drying for 24 h at 60 °C to determine the con-
nective tissue parameters. The dried tissue was weighed and
treated with 6N HCl on the water bath for a period of 24 h for
hydrolysis. To the cooled hydrolysate, 10N NaOH using phe-
nolphthalein was added for neutralization and its concentration
was made to 20 mg/ml of dried granulation tissue with distilled
water. Hydroxyproline, hexuronic acid and hexosamine were
evaluated by using the final solution as per standard methods. The
remaining part of granulation tissue was homogenized using
phosphate buffered saline (PBS, 10% homogenate) at 4 °C followed
by centrifugation at 40,000
g for 30 min. The supernatant ob-
tained was analyzed for catalase (CAT), superoxide dismutase
(SOD) and reduced glutathione (GSH). Free radical inducing com-
ponents like nitric oxide (NO) and tissue lipid peroxidase (LPO)
were also gauged. Further, acute inflammatory marker myeloper-
oxidase was estimated by homogenizing granulation tissue in 0.5%
hexadecyl trimethyl ammonium bromide (HTAB, Sigma-Aldrich,
Co., St. Louis, MO, USA) with 50 mM potassium phosphate buffer
(pH 6). The homogenate was freeze-thawed three times; sonicated
for 10 s followed by cool centrifugation at 4 °C for 45 min at
14,000
g. The supernatant was then used for evaluation of
Myeloperoxidase (MPO) as per standard procedure (Murthy et al.,
2013).
2.9.4. Estimation of IL-1β, IL-6, TNF-α and VEGF
The pro-inflammatory cytokines interleukin-1β (IL-1β), inter-
leukin  6 (IL-6), tumor necrosis factor – α (TNF-α) and growth
factor, vascular endothelial growth factor (VEGF) were assessed by
enzyme linked immunosorbent assay (ELISA) using IL-1β, IL-6,
TNF-α (Komabiotech) and VEGF (RayBiotech) ELISA kits on gran-
ulation tissue homogenate obtained on 10th post-wounding day
from excision wound model. The ELISA protocol was followed as
per manufacturer guideline.
2.9.5. Western Blot analysis of proliferation marker Ki-67
Effect of Leea macrophylla on cellular proliferation was eval-
uated using Western blot analysis of proliferation marker Ki67. The
rats were divided into three groups including control, ELMO and
ELMT treated groups with 9 animals of either sex in each. An ex-
cision wound was created as described earlier and skin wound
specimens were collected on 1st, 7th and 14th-day post- wound-
ing. The excised wound tissue was subjected to homogenization in
RIPA buffer followed by centrifugation at 14,000
g for 15 min
discarding the supernatant. Separation of protein from aliquots
was processed on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose
membrane followed by incubating the membrane with primary
antibody against ki67 (Sigma, St. Louis, MO). Anti rabbit antibodies
were used as secondary antibody. Visualization of protein was
performed by enhanced chemiluminescence employing lumines-
cent image analyzer.
2.9.6. Histological studies
The animals from excision wound model were anesthetized on
the 10th post-operative day of the experiment, and full cross-
sectional thickness of the wound tissue was collected followed by
immediate blotting, drying and fixing in 10% formalin. The treated
tissues were parched in dehydrant acetone and embedded in
paraffin wax blocks only to be cut into thin sections (5 μm) using a
microtome cutter. Haematoxylin-eosin stained sections were then
examined with the help of a microscope (Nikon Trinocular Mi-
croscope, Model E-200, Japan) (Piskin et al., 2014).
2.10. Statistical analysis
The results of the experiment are indicated as mean 7S.E.M.,
with six animals in each group followed by one-way analysis of
variance (ANOVA). Newman–Keuls multiple comparison test was
selected to compare different groups and to evaluate the statistical
significance between them. Also, two-way ANOVA followed by
Bonferroni post hoc test was used to find the significant level in
excision model and cellular proliferation study. The statistical
analysis was performed using GraphPad Prism (version 4; San
Diego, CA, USA) software. Po 0.05 was deemed as significant.
3. Results
3.1. Working with BBD towards response surface
Ethanolic extract of Leea macrophylla was obtained by using
Soxhlet extraction of powdered root tuber. BBD was utilized to
optimize the extractive process so as to maximize the extraction
efficiency and the phenolic content obtained. Three input factors,
solvent blend, mesh size and duration of extractive processing
were varied over three levels to arrive at the best response model
which was used to predict the conditions for optimized extraction
cycle. With mesh openings Sieve no. 20, semi polar nature of
solvent (92.5:7.5 ethanol-water blend) and extraction time of 18 h,
substantially greater extraction efficiency (29%) and phenolic yield
(181.54 mg/g) were obtained. See Supplementary information for
detailed results.
3.2. Quantification of chlorogenic acid by HPLC
The phytochemical study revealed that the extract was highly
rich in phenolics. As per literature review, it was found that the
family Leeaceae to which plant belongs had shown the presence of
CGA (Kubitzki, 2007) which has been implicated with wound
healing potential in several studies (Chen et al., 2013). Considering
the above probability TLC was performed to substantiate the lead.
TLC confirmed the presence of CGA, hence, it was the molecule
which we selected out of all the possible active constituents for
quantification with the help of HPLC. From calibration plot of
chlorogenic acid, the content of chlorogenic acid in ethanol ex-
tracts of Leea macrophylla was obtained as 9.01% w/w (Fig. S2).
3.3. Topical formulation and its texture profile analysis (TPA)
The mechanical properties of the extract containing topical
bioadhesive gel were obtained using texture profile analysis, as
presented in Fig. 1A. Fig. 1B is a force time plot of BG2. The
hardness of gels was: BG1 (1.022 7 0.03 N), BG2 (2.7307 0.04 N),
BG3 (3.15070.12 N), BG4 (2.400 70.08 N). The compressibility
was: BG1 (14.78 72.10 N-mm), BG2 (25.26 7 0.83 N-mm), BG3
(29.66 71.80 N-mm), BG4 (24.6571.60 N-mm). The adhesiveness
was: BG1 (14.79 70.31), BG2 (21.80 70.67 N-mm), BG3
(22.09 71.19 N-mm), BG4 (15.807 0.54 N-mm). As it may be dis-
cernible from the figure, increasing the concentration of SCMC
from 2.5% to 4% (w/v) and PVP 2.5–4% (w/v) in BG1 to BG3 sig-
nificantly increased formulation hardness, compressibility and
 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
Fig. 1. Physicochemical characteristics of bioadhesive gel of ethanolic extract of Leea macrophylla. (A) Hardness, compressibility and adhesiveness of the batches of
bioadhesive gel formulated. Considering the highest adhesiveness, moderate hardness and compressibility BG2 was selected as the best topical formulation (B) Force time
plot of best selected topical formulation BG2. Surface morphology of (C) blank gel and (D) bioadhesive gel BG2 captured by scanning electron microscope.
Fig. 2. Effect of oral and topical treatment of ethanolic extract of Leea macrophylla
on breaking strength in rats. Value are mean 7SEM (n¼ 6). Statistical comparison
determined by one way ANOVA followed by the Newman–Keuls multiple com-
parison test, where ap o 0.05 vs. control, bp o0.05 vs. gel base, cpo 0.05 vs. ELM
250, dp o 0.05 vs. ELM 500, epo 0.05 vs. ELM 750, fp o 0.05 vs. ELM 2.5, gp o0.05 vs.
ELM 5, hp o 0.05 vs. ELM 7.5, ip o0.05 vs. VTE. A significant rise in WBS was found
in animals treated topically with ELM gel 7.5% w/v (461.677 10.78 g) followed by
ELM gel 5% w/v (453.337 9.19 g) which were comparable to topical standard Aloe
(481.677 9.80 g) while increase in WBS with topical treatment with ELM gel 2.5%
w/v (398.337 10.14 g) and oral administration with ELM 750 (400.007 8.56 g) and
ELM 500 (386.6776.15 g)were comparable with oral standard VTE
(410.007 10.65 g). Blank gel (321.677 7.03 g) didn’t exhibit any significant increase
in WBS.
adhesiveness. Considering the highest adhesiveness, moderate
hardness and compressibility of BG2 was selected as the best to-
pical formulation and used for further studies. BG2 and an
equivalent blank gel were lyophilized and observed under Scan-
ning Electron Microscopy (SEM).
SEM micrographs for blank gel (Fig. 1C) revealed a smooth
undulating texture. The presence of distorted irregular entities in
case of lyophilized BG2 (Fig. 1D) is probably evidence of pre-
cipitated micro particles of Leea macrophylla extract upon drying.
99
3.4. Acute oral toxicity study
The results of acute oral toxicity study found the safety limit of
extract to be 5 g/kg when administered via p.o. route. The extract
did not exhibit any behavioral modulation or symptoms of toxicity
or morbidity in the experimental animals.
3.5. Acute skin irritation test
Acute dermal toxicity study showed that formulated bioadhe-
sive gel containing ethanolic extract of Leea macrophylla did not
induce any noticeable swelling, inflammation, irritation or any
others abnormalities on the skin of the treated animals.
3.6. Wound healing activity
3.6.1. Incision wound model
Fig. 2 represents the effect of orally administered and topically
applied ethanolic extract of Leea macrophylla on the tensile
strength of wounds. The wound breaking strength (WBS) after oral
administration and topical application was increased significantly
when incision study was performed. However, the increase in
results was more pronounced when the extract was applied to-
pically. The blank gel alone didn't produce any significant rise in
wound breaking effect. Highly significant increment in WBS was
obtained in treated animals, with percentage increase of 23.41%,
27.66% and 27.13% when treated with ELM orally at 500, 750 mg/kg
and topically at 2.5% w/v respectively which were comparable
with oral standard Vitamin E (30.85%). A percentage increase of
44.68% and 47.34% were observed with topical treatment at 5% w/v
and 7.5% w/v ELM bioadhesive gel respectively which were com-
parable to topical standard Aloe vera cream (53.73%). However, the
WBS of rats plateaued at doses exceeding 500 mg/kg p.o.
100 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
Interestingly 5% w/v and 7.5% w/v bioadhesive gel of Leea macro-
phylla extract exerted almost parallel effects on the tensile
strength of wounded tissue. Therefore, 500 mg/kg orally was fixed
as effective dose of ELM and 5% w/v for topical application for
further investigation.
3.6.2. Excision wound model
In excision wound model the untreated control rats exhibited
chronologically retracting wound contraction, with complete
wound contraction attained on the 24th day. Healing rate of ex-
cised wound was faster in rats treated orally or topically with
ethanolic extract compared to the control group. A statistically
significant difference, within the treatment modules (oral and
topical), was also observed. Topical treatment with ELM showed
optimum wound contraction on 16th day (96.8%), which was faster
as compared to oral treatment (91.7%), also evident with complete
wound contraction which was found to be on 20th day with to-
pical treatment while that with oral treatment was observed on
22nd day (Figs. 3 and 7).
The results also depicted that topically treated animals showed
faster healing as compared to oral treatment and the results were
comparable to standard Vitamin E as well as Aloe vera cream.
Mean epithelization period and scar area justified significant
healing effect of topically treated animals in comparison to oral
treatment (Fig. S3A and B).
3.6.3. Biochemical estimations
Animals which underwent topical treatment showed a greater
increase in weight of granulation tissue and protein in comparison
to those receiving oral treatment (Fig. S4). The increment in dry
and wet weight of granulation tissue and protein content was
highly significant in the topically treated group when compared to
control group. Increase in wet tissue and protein was found to be
37.94% and 28.09% respectively, and dry tissue and protein was
found to be 36.90% and 37.01% respectively when treated topically
with ELM bio gel. Oral treatment also afforded significant rise in
wet granulation tissue (17.68%), protein content (12.77%) and dry
granulation tissue weight (22.04%). However, the increase in dry
tissue protein was not significant.
Fig. 4 represents the effect of the extract on antioxidants, free
radicals, inflammatory markers and collagen determinants. ELM
extract exhibited a significant increase in the value of antioxidants
GSH, SOD and CAT whereas the level of free radical generating
enzymes LPO and NO, and inflammatory markers, MPO was
reduced.
Fig. 3. Effect of oral and topical treatment of ethanolic extract of Leea macrophylla
on wound area. Value are mean 7SEM (n¼ 6). Statistical comparison was per-
formed by two way ANOVA followed by Bonferroni post hoc test. Complete wound
contraction in animals treated topically with ELM gel 5% w/v were achieved in 20
days while oral treatment with ELM 500 mg/kg exhibited complete wound con-
traction in 22 days. Control animals showed delayed wound healing with complete
wound contraction in 24 days.
Further advantageous effects on wound healing were reflected
by significantly increased levels of hydroxyproline, hexosamine
and hexuronic acid levels per unit of dried granulation tissue (g) as
well protein (mg). However, the effect was more pronounced and
significant in the case of topical application in comparison to oral
administration.
3.6.4. Estimation of IL-1β, IL-6, TNF-α and VEGF
Fig. 5 represents the effect of ethanolic extract of Leea macro-
phylla on proinflammatory cytokines IL-1β, IL-6, TNF-α and growth
factor VEGF. On 10th post-wounding day in excision model level of
IL-1β were significantly reduced (1232.89 79.26 pg/ml) in orally
treated animals whereas the effect were more pronounced in to-
pically treated animals (646.67710.73 pg/ml) and were compar-
able to topical standard drug Aloe (601.38 78.18) while untreated
control animals showed higher levels (2061.2477.01 pg/ml). The
results also indicated a significant decrease in IL-6 when treated
with ethanolic extract of Leea macrophylla. Control animals de-
monstrated a higher level of IL-6 (3328.13 73.84 pg/ml) and the
decrease was more in topically treated animals (1468.6778.67 pg/
ml) than orally treated (2932.44 74.75 pg/ml). The data from
ELISA analysis further showed a higher reduction in TNF-α levels
when animals were treated topically (2040.067 13.25 pg/ml) as
compared to orally administered (4563 78.61 pg/ml) while levels
were higher in control animals (6528.44 710.20 pg/ml). The re-
sults depicted significant augmentation in VEGF production in
treated groups as compared to control group which showed a low
level of VEGF (418.35 74.71 pg/ml). Topical application
(907.217 7.48 pg/ml) illustrated effect comparable to topical
standard Aloe (1112.31 79.48 pg/ml) in comparison to oral treat-
ment (714.35 75.41 pg/ml).
3.6.5. Western Blot analysis for proliferation marker Ki-67
Cell proliferation in wounded skin investigated by Western blot
analysis of proliferation marker Ki67 indicated that the expression
of Ki67 was evident from day 1 post-wounding in control and
treated groups (orally and topically treated with ethanolic extract
of Leea macrophylla) however there was no significant difference
in its level between the groups. On 7th day rise in the level of Ki67
was highly significantly in orally and topically treated animals as
compared to control however the increase in Ki67 level was
greater in topically treated. On 14th day decline in Ki67 was evi-
dent in all groups however the decline rate was considerably
higher in topically treated groups followed by orally treated and
control groups. The study illustrated that ethanolic extract of Leea
macrophylla treatment triggered cell proliferation during wound
healing while the effect was more prominent with topical treat-
ment as compared to oral administration (Fig. 6).
3.6.6. Histological studies
To gain mechanistic insight into the stages of wound healing
(initial inflammation, proliferation and skin remodeling) which
results in re-epithelization; histology of excised skin-wound from
experimental animals was observed on ‘day 10’ after treatment
initiation. Accelerated healing was observed in topically treated
group with greater collagen content, angiogenesis, keratinization,
fibroblast proliferation and epithelialisation compared to orally
treated group. The repaired architecture of skin was observed with
orderly epithelization, along with the renewal of fibroplasia, neo-
vascularization and adnexa in treated groups whereas wound
healing rate was protracted in the control group (Fig. 7).
4. Discussion
Design of experiments (DoE) was used to determine the re-
levant influential factors and their admissible limit range on
 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106 101

Fig. 4. Effect of oral and topical treatment of ethanolic extract of Leea macrophylla on (A) Reduced glutathione (B) Superoxide dismutase (C) Catalase (D) Lipid peroxidation
(E) Nitric oxide (F) Myeloperoxidase(G) Hydroxyproline (H) Hexosamine and (I) Hexuronic Acid. Value are mean 7SEM (n¼ 6). Statistical comparison was determined by one
way ANOVA followed by Newman–Keuls multiple comparison test where ap o 0.05 vs. control, bp o 0.05 vs. ELM oral, cpo 0.05 vs. ELM topical, dp o 0.05 vs. VTE. Effect of
ELM treatment on level of antioxidants, free radicals and collagen determinants were evaluated by oral administration and topical application.

Soxhlet extraction, which was further utilized to develop a re-
sponse surface model that in turn aided estimation of a descriptive
mathematical model (Singh et al., 2015a). For detailed discussion
please refer to Supplementary information.
Biogels fabricated for topical delivery should have low hardness
and compressibility yet high adhesiveness. Low gel hardness will
reduce the mechanical input required to take out the gel from a
storage container, whereas low compressibility will improve the
flow property of gel (relatable to spreadability) and application
onto the topical biologic membrane. High gel adhesiveness will
ensure prolonged contact of the biogel onto the wounded area.
BG2 aptly combined these three properties with a low magnitude
of compressibility, hardness and adequate adhesiveness prompted
us to select it for further preclinical evaluation. The presence of
distorted irregular entities in case of lyophilized BG2 (Fig. 1D) is
probably the evidence of precipitated microparticles of Leea mac-
rophylla extract. This is the most critical distinction, between blank
gel and BG2, suggesting proper incorporation of extract in bioad-
hesive gel network.
The results obtained from excision and incision model as well
as from biochemical estimations demonstrated potential wound
healing effect of Leea macrophylla via both oral and topical route.
But significant improvement was considerably more when the
ethanolic extract was applied topically by means of a bioadhesive
gel. In incision model, the wound breaking strength was assessed
after oral and topical application. During initial stages of wound
processing, only the formed blood clot holds edges of the wound
together, and consequently, little breaking strength is required to
disrupt or reopen the wound (Kumara Swamy et al., 2007). With
the progression of time, breaking strength increases via re-
arrangement of collagen and production of stable intra and in-
termolecular crosslinked fibre (Mallefet and Dweck, 2008). From
the study, it was evident that topical treatment was decisively
more effective than oral administration.
Excision wound model showed progressive lessening of
wounded area amongst the treated groups. The fastest and most
complete wound healing (100% wound contraction) was observed
with ELMT (5% w/v) within 20 days whereas orally treated rats
showed complete healing within 22 days as compared to the
control group where complete healing took more than 22 days.
Hence, topical treatment explicates faster rate of wound con-
traction, epithelization period and reduced scar area. The success
of any medicament professing wound healing hinges on its ability
to cause immediate wound closure without scar formation (Clark,
102 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106

Fig. 5. Effect of oral and topical treatment of ethanolic extract of Leea macrophylla on (A) IL-1β (B) IL-6 (C) TNF α and (D) VEGF. Value are mean 7 SEM (n¼ 6). Statistical
comparison was determined by one way ANOVA followed by the Newman–Keuls multiple comparison test where ap o 0.05 vs. control, bpo 0.05 vs. ELM oral, cp o 0.05 vs.
ELM topical, dpo 0.05 vs. VTE. Levels of IL-1β, IL-6, TNF α and VEGF were determined using ELISA kits on granulation tissue homogenate obtained on 10th post-wounding day
from excision wound model. ELM treatment lowered the level of pro-inflammatory cytokines while level of VEGF was augmented. The results were more pronounced with
topical treatment.

1996). Contraction is characterized by a reduction in the wound,
thus accelerating healing without new tissue formation; and
centripetal movement of the contracting wound edges to expedite
closure of wound opening determines the period of epithelializa-
tion (Upadhyay et al., 2013).
Regular immunological representatives (monocytes) and con-
nective cells like fibroblasts are crucial for normal wound closure
(Kant et al., 2013). To validate their role, the data from excision
model was substantiated by histological finding which showed re-
appearance of skin structure with distinct layers of dermis and
epidermis in treated groups (Fig. 7). On comparison: the control
group had lesser intact tissue in dermal layer than the treated
group animals. Formation, alignment and contraction of extra-
cellular matrix molecules determine the intactness of tissue. Re-
establishment is critically dependent on the efficiency of re-
organization of extra cellular matrix molecules (Yariswamy et al.,
2013). The rise in granulation tissue mass and protein amount
signifies increased cellular proliferation resulting in increased
synthesis of collagen which is the principal extracellular protein in
the granulation tissue (Fikru et al., 2012). Increase in collagen in
orally and topically treated drug is also evident through histolo-
gical studies.
Our studies on SOD, CAT, GSH, LPO, NO, and MPO status in
granulation tissue revealed significant anti-oxidant activity which
would reduce free radicals stress and nominal MPO level which
would prevent oxidative damage and thereby accelerate the
healing process. Inflammatory actions instigated by phagocytic
cells during healing often lead to the generation of reactive oxygen
species (ROS). Balance between reactive oxygen species (ROS) and
free radical scavenging enzymes (FRSE) is essential for completion
of wound healing (Joshi et al., 2013). Although originally unin-
tended, these responses thrust an oxidative stress on the damaged
tissue and delay wound healing as a result of an imbalance be-
tween free radical generation and antioxidants. Such is the di-
lemma with the ubiquitousness of inflammatory responses that
both their over expression and under expression lead to deleter-
ious consequences like susceptibility to innocuous environmental
factors, severe damage and even tumorogenesis. Therefore, cur-
tailing the influence of ROS on wound openings could be an im-
portant alternative strategy (White and Heckler, 1990; Mikhal’chik
et al., 2006). ROS can be eliminated by enhancing expression of
antioxidant enzymes like SOD, GSH and CAT or by scavenging pre-
existing free radicals such as LPO products and NO. SOD carries out
disproportionation of superoxide radicals and quenches genera-
tion of any new free radicals. MPO, on the other hand, generates
free radicals. Its elevated status in any milieu (for instance gran-
ulation tissue or infiltrating neutrophils) is indicative of bur-
geoning inflammation. MPO activity and neutrophil derived oxi-
dants subject tissue to significant oxidative stress and contribute
significantly towards wound recovery induced chronic inflamma-
tion (Zhang et al., 2011). GSH assists in detoxification of radicals by
acting as a helper participant in the glutathione peroxidase (GPx)-
instigated reduction of H2O2 and lipid peroxides (Roy et al., 2012).
Biochemical estimation elucidated increased levels of hydro-
xyproline, hyaluronic acid and hexosamine in topically treated
animals as compared to oral treatment. The implications of these
 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106 103

Fig. 6. Effect of oral and topical treatment of ethanolic extract of Leea macrophylla on cellular proliferation. Ki67 in rat wound was determined by Western blot at indicated
days. The IOD (integration optical density) of Ki67 protein band was calibrated using β-actin. Value are mean 7 SEM (n¼ 3). Statistical comparison was performed by two way
ANOVA followed by Bonferroni post hoc test. *po 0.05 and ***po 0.001 compared to control group. Treated groups showed a significant increase in expression of proliferation
marker Ki-67 this suggested treatment with ethanolic extract of Leea macrophylla promotes cellular proliferation and hence wound healing.

Fig. 7. Wound images obtained from excision wound model with oral and topical treatment of ethanolic extract of Leea macrophylla. (A) Day 0 of the experiment (B) Day 20
of the experiment (C) Histopathological view of excision wound on 10th post-operative day. Arrow indicates the epithelization zone.
104 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
finding can be discerned in the following explanation. Collagen is
made up of hydroxyproline, and its relative concentration is an
indicator of collagen production (Joshi et al., 2013). The enhanced
production of collagen is stabilized by hexosamine content which
provides collagen sites for electrostatic bonding. Since, collagen
critically controls the healing process by making up a major por-
tion of connective tissue and also manages its construction, de-
position and ensuing evolution; a higher concentration of hydro-
xyproline and hexosamine, therefore, promote faster wound
healing. Collagen is also vital for re-epithelialisation of cellular-
matrix and inter-cellular interactions thereby strengthening and
integrating the wound matrix (Pather and Kramer, 2012). Collagen
not only provides the tissue matrix with strength and integrity,
but it also caters to the homeostatic demands in the latter portion
of wound healing timeline (Roy et al., 2012). The above stated
functions of collagen are complimented by hyaluronic acid, which
does not participate in its synthesis or maintenance but instead it
is involved in water retention, nutrient exchange, cell differentia-
tion and cell mobilization. Hyaluronic acid plays an active role in a
variety of physiologic and pathophysiologic developments in-
cluding embryo implantation, cutaneous wound healing and os-
teoarthritis. Historically, dermatologists and cosmetic practitioners
have been investigated hyaluronic acid for its contributory roles
which help in regaining elasticity, turgor and moisture of skin
(Chen et al., 2014).
The results supported the finding as treatment with ethanolic
extract of Leea macrophylla reduces the level of proinflammatory
cytokines including IL-1β, IL-6 and TNF-α which are involved in the
process of inflammation, trauma and wound healing regulated by
activity released by macrophages, endothelial and keratinocytes
(Grellner, 2002). VEGF levels were enhanced by treatment. VEGF is
another imperative growth factor involved in wound healing
produced from keratinocytes smooth muscle cells, fibroblasts,
thrombocytes, macrophages and neutrophils and endothelial cells
during wound healing which plays a key role in angiogenesis
(Muthukumar et al., 2014).
Proliferation during wound healing is indicative of multifaceted
events occurring in the time of course. Proliferation occurs during
different phases including epithelialization, angiogenesis, granu-
lation tissue formation, and collagen deposition in order to restore
barrier function, protection against fluid loss and bacterial intru-
sion. Impaired proliferation, migration and contraction during
wound healing advances towards the inefficient repair of the
wound, reluctant epithelization, reduced tensile strength and in-
creased susceptibility towards bacterial infection (Broughton et al.,
2006; Janis and Harrison, 2014). Ki67 is a commonly used pro-
liferation marker. The expression of Ki67 protein is thought to be
an indicator of growing cells within the overall cell population
(Choi et al., 2012).
The study demonstrated that treatment with ethanolic extract
of Leea macrophylla causes an increase in cellular proliferation as
indicated by the level of Ki67, which was found to be highest on
day 7 post wounding while a decline was observed on the 14th
day. However, the effect was more pronounced with topical
treatment as compared to oral administration. Increased cellular
proliferation is an essential aspect of wound healing (Lee et al.,
2012). Increased cellular proliferation in the study demonstrated
early wound healing in treated animals as compared to control
while decline in Ki67 expression may due to the fact that cellular
proliferation might have been subsided as a result of transit from
proliferation phase to remodeling phase (Xian et al., 2015) which
was evident from the study as topical treatment exhibited highly
significant decrease in cellular proliferation on 14th day produced
earlier wound healing on 20th day as compared to oral treatment
where complete wound healing was achieved on 22nd day.
Significant amount of tannins, phenols, flavonoids,
carbohydrate and saponins were present in the extract. Phenolics
(flavonoids and phenolic acids) have been reported for their redox
potential, which allows them to chip in significantly as oxygen
quenchers and reducing agents and in some cases even metal
chelators (Pattanayaka and Sunita, 2008) imparting them potential
in tackling inflammation (Sengar et al., 2015) and thereby wound
healing (Akkol et al., 2012). Moreover, phenolic compounds have
the ability to facilitate wound healing at different stages of wound
healing either by stimulating collagen synthesis, cell proliferation
and angiogenic effect (Agar et al., 2015). Chlorogenic acid quanti-
fied in the present study is a phenolic acid representing the class
of hydroxycinnamic acid polyphenolic compounds and is a dimer
of quinic acid and caffeic acid connected with an ester bond (Sato
et al., 2011). It is a natural antioxidant found abundantly amongst
plant species reportedly possessing antimicrobial, antimutagenic,
anti-inflammatory activity and wound healing properties (Chen
et al., 2013; Xiang and Ning, 2008). Wound healing potential of
chlorogenic acid is accredited to its ability to increase cellular
proliferation and its antioxidant potential accelerating the process
(Chen et al., 2013). Earlier studies have also reported that honey
which exhibits presence of chlorogenic acid also illustrated wound
healing potential through free radical scavenging activity pro-
moting cell proliferation in the acquisition of concurrence ap-
proaching rapid wound healing (Chaudhary et al., 2015). Further-
more, tannins act by chelating free radicals, facilitating wound
contraction, accelerating capillary vessel and fibroblast formation
and by instigating keratinocyte proliferation thus hastening the
process of wound healing (Prasad et al., 2010). Hence, all these
class of phytochemicals has a significant role in wound healing
processes. Therefore, the observed potent wound healing activity
of the extract may be attributed to the presence of chlorogenic
acid along with the cumulative effect of other phytochemicals
present in the extract. Probably such effect is the result of a sy-
nergy with all the molecules contained in the extract.
The study demonstrated that topical application of ethanolic
extract of Leea macrophylla showed faster and more significant
healing as compared to oral treatment. This can be explained due
to the fact that topical delivery is mainly used for achieving the
local effect. It is expected that systemic administration would re-
quire high doses in order to be secreted substantially at a per-
ipheral location. Skin being a large organ, switching to alternative
routes of administration, such as the topical route, can, in principle
provides predicable, effective and reliable drug delivery. Topical
application allows local drug targeting with minimal systemic ef-
fects and is mainly used for delivery of anti-inflammatory, anti-
histaminic, antibiotics, wound healing and analgesic drugs (Liu
et al., 2014).
5. Conclusion
The present study corroborates scientifically the traditional
claims of Leea macrophylla in wound healing. This effect may be
attributed to increased collagen synthesis and reduced in-
flammation through effect on proinflammatory cytokines includ-
ing IL-1β, IL-6 and TNF-α and VEGF growth factor, enhanced cel-
lular proliferation as well as potential antioxidant and free radical
scavenging effect which may be mediated due to the presence of
polyphenols mainly chlorogenic acid in the extract. The study also
showed that the topical formulation hastens wound healing in
comparison to the orally treated animals.
Conflict of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
 A. Joshi et al. / Journal of Ethnopharmacology 191 (2016) 95–106
Acknowledgment
The authors wish to acknowledge Indian Institute of Technol-
ogy (Banaras Hindu University), Varanasi (TA/12621EN002) for
providing financial support to Miss. Apurva Joshi for the present
research work. Authors would also like to express their gratitude
towards Central Instrument Facility Centre IIT-BHU for facilitating
SEM of samples.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2016.06.034.
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