Phytomedicine Plus 1 (2021) 100054

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Phytomedicine Plus
journal homepage: www.elsevier.com/locate/phyplu

Investigating wound healing potential of Typha angustata L. inflorescence

in albino Wistar rats
Shraddha Saha, Vibha Sonar, Bansari Soni, Shreya Koladiya, Sumitra Chakraborty,
Meonis Pithawala∗
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Maliba Campus, Bardoli, Surat 394350, Gujarat, India

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

Keywords: Background: Typha angustata L. is a common aquatic weed. Although the folkloric use of Typha in wound healing
Albino Wistar rats has been reported, scientific evidences are still lacking.
Typha angustata L
Wound healing Purpose: The present study aimed to explore the wound healing potentials of T. angustata L.
Method: The ointment was prepared from the aqueous extract of Typha inflorescence and applied on the induced
wounds (viz., Excision, Incision and Burn) in albino Wistar rats. Continuous monitoring of percentage of wound
contraction, epithelization duration, content of hydroxyproline and protein (from the excised wound tissues col-
lected on 14th and 28th day of treatment) were used as parameters to know wound healing effects. In addition,
catalase activity (antioxidant marker) and histopathological studies were also carried out.
Results: Topical application of the test ointment showed significant (p<0.05) wound contraction, fast epithe-
lization, increased hydroxyproline content and increased catalase activity in comparison to the reference drug
treated groups [(treated with (10%w/w) povidone iodine and (10%w/w) burnol ointment)] as well as control
groups (treated with ointment base) in all wound types. Similarly, the histopathological study also revealed better
reparative changes in terms of neo-vascularization and collagen tissue deposition in treated animals as compared
to reference drug treated and control group animals.
Conclusion: The results of the present study suggest the potentials of Typha plant in formulating wound healing
agents.

Introduction
Cutaneous wound healing is a dynamic process consisting of four
stages, namely hemostasis, inflammation, proliferation or repair and
remodelling or resolution (Guo and Dipietro, 2010). Once a wound is
formed, immediately vasoconstriction and fibrin clot formation take
place. The chemotactic growth factors and pro-inflammatory cytokines
are released to facilitate the sequential influx of leukocytes (neutrophils,
macrophages and lymphocytes), fibroblast migration and proliferation
in the wound area (Guo and Dipietro, 2010).
The proliferative phase which follows and overlaps the inflamma-
tory phase is characterized by the presence of endothelial and fibroblast
cells, fibroplasias, granulation epithelization and neo-vascularization.
The stem cells of epidermis and bone marrow give rise to keratinocytes
that migrate to the site of the wound to create re-epithelization and thus
contribute to angiogenesis (Morton and Philips, 2016). The final phase
of the wound healing process is characterized by regression of several of
the newly formed blood vessels, remodelling of the extracellular matrix,
the physical contraction of the wound and conversion of type III colla-
gen fibres into a strong network of type I collagen fibres, thus resulting
into scar tissue formation (Guo and Dipietro, 2010).
Different treatment options are available for the wound management
(such as analgesics, antibiotics and non- steroidal anti-inflammatory
drugs), but majority of these therapeutics produce numerous adverse
side effects. Wound dressings consisting of antibiotic are valuable in the
management of local infections. However, high concentrations of antibi-
otics are needed to treat local infections (Ramasubbu et al., 2017). Addi-
tionally, high concentrations of antibiotics can lead to systemic toxicity
(Everts, 2016). Over last years, the development of new antibiotics has

Abbreviations: ANOVA, Analysis of Variance; CAT, Catalase; CPCSEA, The Committee for the Purpose of Control and Supervision of Experiments on Animals; IAEC,
Institutional Animal Ethics Committee; MPC, Maliba Pharmacy College; OECD, Organization for Economic Co-operation and Development; OD, Optical Density; SD,
Standard Deviation; SOD, Superoxide Dismutase; TIO, Typha angustata L. Inflorescence Ointment.
∗
Corresponding author.
E-mail addresses: meonis_pithawala@yahoo.com, commpithawala@utu.ac.in (M. Pithawala).

https://doi.org/10.1016/j.phyplu.2021.100054
Received 2 December 2020; Received in revised form 12 February 2021; Accepted 2 March 2021
2667-0313/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
S. Saha, V. Sonar, B. Soni et al.
decreased, only small numbers of companies are active in these domains.
In addition, resistant micro-organisms have considerably increased due
to over use and misuse of antibiotics (Das and Horton, 2016). Thus, an-
tibiotic crisis is still in progress and affects treatments (e.g. diabetic foot,
pressure ulcers and venous ulcers) that are dependent on antibiotics.
Usually, Non-steroidal anti-inflammatory drugs (NSAIDs) or Cyclo-
oxygenase-2 (COX-2) inhibitors are reported to inhibit Prostaglandin
E2 (PGE2) production and act as an effective pain killer, as they
are able to reduce inflammation (Park et al., 2006; Megle-Gaw and
Schwartz, 2002). Additionally, NSAIDs are reasonably priced, read-
ily available and familiar; they are often prescribed and used post-
operatively for controlling pain (Reish and Eriksson, 2008). However,
the impact of COX-2 inhibitors or NSAIDs on wound healing is very
controversial as they may have a negative effect on wound healing.
The inflammatory process has direct effect on normal and abnormal
wound healing. Clinical understanding suggests that hypertrophic scar
formation is an aberrant form of wound healing (Aarabi et al., 2007),
that involves an exaggerated function of fibroblasts and excess accumu-
lation of extracellular matrix during wound healing (Butler et al., 2008).
Inflammation process is a driving force behind diverse disorders such
as wounds, trauma, arthritis, neuropathy, atherosclerosis, cancer, dia-
betes, neurodegenerative disease and chronic pain (Medzhitov, 2008;
Couzin-frankel, 2010; Mantovani et al., 2008). Notably, inflammation
is a natural reaction to injury and essential for stimulation of tissue re-
pair. Thus, therapeutic inhibition of inflammation is only logical when it
becomes unregulated, inappropriate or recurrent. Standard treatments
which mainly include steroids, disease modifying drugs (e.g. methotrex-
ate) and non-steroidal anti-inflammatory drugs have severe side effects
(thromboembolic events, gastrointestinal ulcers and bleeding, kidney
and liver toxicity). The anti-inflammatory treatment that uses TNF-alpha
inhibitors can only be used in limited number of selected patients due
to their unaffordable cost, parenteral formulation, tumor induction and
risk for infection (Frank et al., 2009). Thus, plant based therapeutic ap-
proach will be a better alternative.
Traditionally, plants have been used to prepare wide range of formu-
lations for treating skin diseases, burns, wounds and cuts (Kokane et al.,
2009). Western pharmacopoeias comprise of only 1-3% of drugs which
are generally used for dermal wound healing; out of which one third is
based on traditional herbal remedies (Hayouni et al., 2011). Thus, plants
which are already in use for wound healing after proper scientific eval-
uation can be a better option in comparison to the existing approaches.
Recently, several studies have been carried out on herbal drugs to
explicate their potential in wound management. These natural reme-
dies proved their effectiveness as an alternative therapeutic approach to
the available synthetic drugs for the treatment of wound (Thakur et al.,
2011; Shuid et al., 2005; Jalalpure et al., 2008). Many developing
countries, especially in rural areas, rely heavily on traditional healer
and medicinal plant to meet their primary health care. This is mainly
attributed to easy availability of herbal medicine and their low cost
(Shaw, 1998).
The plant derived isolates induce tissue regeneration and heal-
ing through multiple mechanisms, which often have a synergistic ef-
fect on overall healing (Maver et al., 2015). Medicinal plants rich in
polyphenols are reported to possess remarkable anti-oxidant activity
(Ovais et al., 2018b; Shah et al., 2015; Zohra et al., 2019a). These phy-
tochemicals have great wound healing potentials and also used for the
treatments of various skin damages (Dzialo et al., 2016). Several plants
contain therapeutic amount of flavonoids and are also used in traditional
medicine as anti-inflammatory, treating wounds and anti-allergic agents
(Hughes et al., 2017; Salaritabar et al., 2017). Flavonoids promote ex-
cellent wound healing by means of anti-oxidative and anti-microbial
properties (Getie et al., 2002; Shetty et al., 2008). Many plants derived
essential oils have tremendous potential to be used in the treatment of
wound (Woollard et al., 2007).
Typha, commonly called as “Cattail” is a cosmopolitan monocotyle-
donous plant belonging to family Typhaceae, which consists of eleven
2
Phytomedicine Plus 1 (2021) 100054
species (Long and Lakela, 1976). Literatures from India, China and
Turkey, have already reported the medicinal use and medicinal prop-
erties of T. angustata L. This plant is widely used for treating bleed-
ing noses, haematemesis, haematuria, uterine bleeding, dysmenor-
rheal, gastralgia, scrofula and abscesses (Yeng Him-Che, 1985). Further,
Bensky et al., (1993) reported the traditional use of Typha sp. pollens
for stopping bleeding due to external traumatic injuries and invigorate
blood loss. Previous studies reported that pollen grains of Typha mainly
contain sterols, terpenoids, flavonoids, glycosides (Tao et al., 2011).
Till date to the best of our knowledge, the pharmacological wound
healing potentials of T. angustata L. inflorescence has not been reported.
Therefore, the present study was conducted to evaluate the in vivo
wound healing properties of the aqueous extracts of T. angustata L. in-
florescence.
Materials and methods
Collection of material: Inflorescence of T. angustata L. growing
as a natural inhabitant in the campus of Uka Tarsadia Univer-
sity, Bardoli, Gujarat, India was collected and authenticated (voucher
no. SAHA/CGBIBT/01/19). The plant name has been checked with
http://www.theplantlist.org. The plant along with inflorescence was
collected and shade dried for 1 week at 25-30ᵒC temperature. The in-
florescences were placed in desiccators that contained silica gel (con-
taining cobalt chloride). Temperature in desiccators was maintained at
25ᵒC. Drying process was terminated when 70% silica gel changed its
colour from blue to pink (Fischler, 1994). The dried inflorescences were
stored at 4ᵒC temperature for further use.
Extraction process: 100g dried T. angustata L. inflorescence was added
to 200ml of distilled water, mixed and extracted using a soxhlet appa-
ratus. The extraction assembly was set up as described by (Handa et al.,
2008). Continuous extraction was carried out after 5-6 refluxing cycles;
drop wise collection of solvent from the siphon tube was carried out
and observed for residue mark after evaporation. The refluxing cycle
was terminated, when no residue was found from the evaporated drop
so collected. This aqueous extract was then filtered using N1 Whattmann
filter paper, concentrated at 40-60ᵒC temperature in a rota-evaporator
(R-300) and preserved at 4ᵒC temperature until further use.
Preparation of Ointment: The ointment was prepared by mixing 35g
of cetostearyl alcohol, 17.5g of PEG 600, 270g of petroleum jelly, 35g
of liquid paraffin and 0.65g/400g methyl paraben in a water bath with
constant stirring at 60ᵒC temperature so as to maintain a fluid like con-
sistency. The preserved extract was added to this mixture and homoge-
nized to achieve 10%w/w concentration.
Control drugs: Povidone iodine USP 10%w/w (Batodine), Nivca Life-
care, Ahemdabad was used as the reference drug for excision and inci-
sion wounds. Whereas, [Aminacrine HCL & Cetrimide Cream (Burnol)
Dr. Morepen Ltd. New Delhi) ointment (10%w/w) was used as the ref-
erence drug for burn wound.
Experimental Animals: Healthy, adult Wistar albino rats of either sex
weighing approximately 200-400g were used in this study. The study
protocol complying to (CPCSEA, 1960) guidelines were approved by the
IAEC (MPC/IAEC/2019). All experimental animals were acclimatized to
standard laboratory conditions at 25±2ᵒC temperature with a relative
humidity of 45-56% and 12 hours of the photoperiodic cycle for a time
period of 7 days prior to experimentation. These animals were fed with
standard diet and water ad libitum during the entire study.
Animal grouping and drug administration for wound healing activity: In
order to assess the wound healing potentials of T. angustata L. inflores-
cence extracts; excision, incision and burn wound models were created.
The animals were divided into nine groups, each consisting of ten an-
imals (Table 1). The grouping of animals was done on random basis.
All ten animals of each group were of either sex and healthy. The above
grouped animals were treated with the respective ointments (50mg oint-
ment were applied over 1 centimeter area of skin) once daily until the
wounds healed completely.
S. Saha, V. Sonar, B. Soni et al. Phytomedicine Plus 1 (2021) 100054

Table 1
Grouping of animals.

Wound Type Group Group Type Treatment Mean Weight (gm) Sex of animal Number of animals

Excision I Control Base of ointment (10%w/w) 432 4 male + 6 female 10

II Reference drug Povidone iodine ointment (10%w/w) 452 5 male+ 5 female 10
III TIO treated Typha angustata L. inflorescence extract ointment (10%w/w) 436 6 male+4 female 10
Incision IV Control Base of ointment (10%w/w) 431 4 male + 6 female 10
V Reference drug Povidone iodine ointment (10%w/w) 433 5 male+ 5 female 10
VI TIO treated Typha angustata L. inflorescence extract ointment (10%w/w) 451 7 male+3 female 10
Burn VII Control Base of ointment (10%w/w) 446 5 male+ 5 female 10
VIII Reference drug Burnol ointment (10%w/w) 432 3 male+ 7 female 10
IX TIO treated Typha angustata L. inflorescence extract ointment (10%w/w) 444 6 male+ 4 female 10
Total 90

Acute skin irritation test: The ointment was prepared by using in-
florescence extracts as per OECD guideline no.402 (OECD guide-
lines, 1987). The ointment was formulated using different concentra-
tions viz., 0%w/w, 5%w/w, 10%w/w, 15%w/w, 20%w/w, 25%w/w,
30%w/w. The prepared ointments were applied on the depilated dorsal
region of the rats and observed daily for the next 10 days for any of
the adverse dermal reactions such as erythema, inflammation, irritation
and itching. Based on the results the dose was kept constant throughout
the study.
Excision Wound Model: For wound creation, approximately 300mm2
circular area on the dorsal surface of all the rats were depilated using an
epilator. This region was outlined with a marker pen and a full, thick ex-
cision wound was created (Yadav et al., 2018). The wound was cleaned
and topically applied with the aforesaid (Table-1) respective drug treat-
ment protocols and noted as day 0.
Incision Wound Model: Under sterilized condition with all surgical in-
terventions, a longitudinal patch of 1-1.5cm of the dorsal surface of the
animals were depilated and marked with a marker pen. Under anesthe-
sia, using a surgical scalpel, an incision of 2mm thickness was created
and noted as day 0 (Kuwano, 1994). After attaining homeostasis, the
incised skin was sutured with a no.11 curved needle and black no.000
silk surgical thread. The wound was applied with respective ointment
daily till the wound healed completely.
Burn Wound Model: The rats were anesthetized under sterilized con-
ditions and all surgical interventions, their dorsal region was depilated,
marked and burn wound was created by pressing a burner heated
red hot aluminum wire loop for 14 seconds following the method of
Katakura et al., (2005) with slight modifications. A second-degree burn
was created and noted as day 0. This wound was applied with the
aforesaid ointment daily until the wound healed completely which was
marked by scab falling.
Measurement of wound contraction area: Percentage rate of wound
closure and epithelization was measured in the animals belonging to
the excision, incision and burn wound model groups.
Photographs of the wounds were also captured for visual observation
of wound healing process, on alternate days, until complete healing of
the wounds.
Percentage wound closure rate: Percentage rate of wound healing in
the excision, incision and burn wound induced groups was calculated
by using the formula of Jridi et al. (2017).
Percentage wound closure rate =
Wound area on day 0th − Wound area on day nth
X The collected tissues were separately stored in 10% formalin buffer so-
Where,
n = Observation days i.e. 4th , 8th , - - -, 40th .
X = Wound area on 0th day multiplied by 100
Epithelization period was measured by considering the days on
which scabs started falling off from the wound leaving raw wound marks
(Fikru et al., 2012) in the excision, incision and burn wound model an-
imal groups.
Parameters for estimating wound healing process
Hydroxyproline determination: Hydroxyproline content of granulation
tissues were estimated by weighing approximately 250mg of wet tissue
then drying it for 24 hours at 50°C temperature. After 24 hours the dried
tissue was hydrolysed by adding 10ml of 6N HCL and then heated at
130°C temperature for 4 hours in an oven. To this 10ml of 10N NaOH
was added to the hydrolysate tissue to neutralize it and then the solution
was allowed to rest for 20-25 minutes at room temperature. Thereafter,
0.2ml of 2.5N NaOH, 0.2ml of 0.01M CuSO4 and 0.2ml of 6% H2 O2
was added to neutralize tissue hydrolysate and then incubated in a wa-
ter bath for 15 minutes at 80°C temperature. After incubation, the solu-
tion was placed for 5 minutes in cold water. To this cooled solution, 4
ml of 3N H2 SO4 was added cautiously with repeated whirling followed
by addition of 2ml of freshly prepared 5% solution of para-dimethyl
amino-benzaldehyde in n-propanol. The solution was again incubated
at 750 C temperature for 15 minutes and then allowed to stand for 5
minutes. Spectrophotometric readings of the solutions were measured
at 540nm against the blank. Standard curve was prepared by using 50-
1000mg/0.3ml L- hydroxyproline (Newman and Logan, 1950).
Estimation of Protein by Biuret Method: The granulation tissue ho-
mogenates were used for estimation of protein content by Biuret method
(Gornall, 1949).
Assay of Catalase Activity: Bergmeyer’s assay (1974) was used for
measuring catalase activity based on the ability of the enzyme to ox-
idize H2 O2. In a cuvette 450𝜇L of 0.1M phosphate buffer (pH 7.4) was
added to 50𝜇l of granulation tissue homogenate along with 500𝜇L of
20mM H2 O2. Change in the rate of spectrophotometric absorbance at
240nm was monitored at 30s interval over a period of 3 minutes.
One unit of catalase activity is defined as 1mM of H2 O2 degraded per
minute and expressed as units/mg of protein using the below mentioned
formula.
U∕mg = (A0 − A180 ) × Vt ∕ ε240 × d × Ct × 0.001
Where, Vt is total volume of reaction (3mL) Ɛ240 is the molar extinc-
tion coefficient for H2 O2 at OD240 (39.4mol−1 cm−1 ) d is optical path
length of cuvette (1cm) Vs is volume of sample (1mL) Ct is the total
protein concentration in the sample. 0.001 is absorbance change that
caused by one unit of enzyme per min at 240nm OD.
Histological studies: Histological changes were studied from healed
skin tissues collected from each group of rats after respective treatments.
lution, followed by alcoholic grade dehydration and paraffin wax blocks
were prepared, sections of 5𝜇m were cut, processed, stained and counter
stained (Yadav, 2017). A light microscope Magnus-MLX-Plus was used
for observing the stained skin sections for every group models.
Statistical analysis: The data of all parameters were expressed as
Mean±SD and the results were analyzed using SPSS software 22.0. One
Way ANOVA and Turkey’s Test comparison were performed. Mean val-
ues significant at p<0.05 were considered as statistically significant.

3
S. Saha, V. Sonar, B. Soni et al. Phytomedicine Plus 1 (2021) 100054

Table 2
Skin Irritation Test score after applications of Typha angustata L. inflorescence crude extract ointments at varying concentrations.

Erythema Inflammation Irritation Itching

Dose
Day 0 Day 3 Day 6 Day 9 Day 0 Day 3 Day 6 Day 9 Day 0 Day 3 Day 6 Day 9 Day 0 Day 3 Day 6 Day 9

0%w/w 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5%w/w 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10%w/w 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
15%w/w 0 0 0 0 0 0 1 1 0 0 0 0 0 2 2 2
20%w/w 0 1 1 1 0 2 2 2 0 2 2 2 0 0 0 0
25%w/w 0 1 2 3 0 3 3 3 0 2 2 2 0 1 1 1
30%w/w 0 3 3 3 0 3 3 3 0 2 2 2 2 2 2 2

Where,
0- No reaction
1- Mild reaction
2- Moderate reaction
3- Excessive reaction.

Results and discussion

Safety evaluation (Skin irritation test): Application of the ointments

on the depilated areas of the albino rats at different concentrations were
carried out to assess for the signs of redness, inflammation, irritation and
itching (Table 2). The rats were monitored for the time duration of ten
days. Out of all prepared formulations, 10%w/w ointment formulation
showed no signs of erythema, inflammation, irritation and itching. Thus,
the primary skin irritation of the ointment was calculated as 0.00 which
indicated that the ointment was safe for use. As the extract remain in
direct contact with the wounded skin, irritant compounds in the extract
may cause recurrent inflammation on the wounded site and may inter-
fere with the healing processes (Robert et al., 2004). Moreover, in the
presence of persistent inflammation, wounds may fail to heal normally,
which requires treatment to resolve. Additionally, the agents that have
potential to resolve the inflammatory process in wound also promote
or accelerate the healing process (Houghton et al., 2005; Agyare et al.,
2013).
Macroscopic study (Excision/ Incision/ Burn wound models): The heal-
ing time reduces with contraction. Because it helps to decrease the size
of the wound and reduces the amount of extracellular matrix needed to
repair the defect. Additionally, it facilitates re-epithelization by short-
ening the distance of migrating keratinocytes (Strodtbeck, 2001). More-
over, the wound heals at faster rate, if the medication is more efficient
(Prasad and Dorle, 2006). There was statistically significant (p<0.05)
increase in the percentage of wound contraction (Table 3, Figure 1,2,
3, 4A, 4B, 4C) among all group of rats treated with T. angustata L. in-
florescence ointment (TIO) as compared to the control group of rats. Figure 1. Photographic representation of wound contraction in excision wound
The inflorescence ointment facilitated wound contraction from day 8 model.
onwards i.e. 25.78±0.76, 22.08±1.33 and 22.28±0.79 were observed
in excision, incision and burn wound models respectively as compare to
control groups were percentage wound contraction were 11.01±1.00, wound area, skin biochemical parameters play crucial role. These pa-
11.61±1.52 and 12.22±1.38 on day 8 in excision, incision and burn rameters are an important evidence of sub-dermal organization with
wound models respectively. Interestingly, as compared to reference drug respect to the newly synthesized collagen fibrils (Biswas et al., 2017).
treatments, TIO treated rat groups showed slower wound healing abil- The hydroxyproline concentration has been used as an estimate of colla-
ity in the first week of experiments; but not during second or the third gen content. The enhanced wound healing activity has been accredited
weeks. to increased collagen formation (Trabucchi et al., 1986; Shukla et al.,
Furthermore, epithelization involves proliferation and migration of 1999). The protein content of granulation tissue is an indication of the
epithelial cells across the wound bed (Esimone et al., 2008). Table 4 in- level of protein synthesis and cell proliferation. Increase protein con-
dicates period of epithelization. Again, as compared to control group of tent is mainly due to increase in collagen synthesis (Bourguignon et al.,
rats among all three types of wounds (32, 28 and 31 days respectively), 1987; Rasik et al., 1999). In the present study the biochemical results
the TIO treated animals showed significant (p<0.05) reduction in the showed that the ointment prepared from T. angustata L. inflorescence
number of days taken for epithelization i.e. 23, 19, 22 days in case of ex- aqueous extract (10%w/w) was found to be effective as reference drugs
cision, incision and burn wound respectively. Moreover, better healing viz., (10%w/w) povidone iodine and (10%w/w) burnol in treating ex-
activity is distinguished with short epithelization period (Suntar et al., cision, incision and burn wound types. Rapid wound healing of tissue is
2010). Furthermore, epithelization involves proliferation and migration marked by an increase in the hydroxyproline and protein content, esti-
of epithelial cells across the wound bed (Esimone et al., 2008). mated from excised granulation tissue on day 14th and 28th day, which
Determination/ Estimation of biochemical markers on the excision, in- is an acceptable method for predicting the amount of collagen synthe-
cision and burn wound models: To access the quality of the skin in the sized in the wounded tissue (Lodhi et al., 2013).

4
 S. Saha, V. Sonar, B. Soni et al.
Table 3
Percentage of wound contraction from excision, incision and burn wounds in control, reference drug treated and Typha angustata L. inflorescence extract ointment treated (TIO) group of rats (Data pooled from 10
rats/group).

Excision Wound Model Incision Wound Model Burn Wound Model

No. of Days
Group IIReference Group IIITIO Group VReference Group VITIO Group VIIIReference Group IXTIO
Group IControl drug (Povidone iodine) treated Group IVControl drug (Povidone iodine) treated Group VIIControl drug (Burnol) treated

0 Day 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 0.00±0.00 0.0±0.00
4 Day 6.01±1.00 11.10±0.79∗ 10.41±1.79∗ 2.77±0.69 22.36±0.38∗ 14.17±0.97∗ 6.18±0.50 10.65±0.25∗ 10.99±1.16
8 Day 11.01±1.00 29.42±0.51∗ 25.78±0.76∗ 11.61±1.52 41.93±1.94∗ 22.08±1.33∗ 12.22±1.38 20.03±0.31∗ 22.28±0.79∗
12 Day 17.92±0.66 35.84±0.25∗ 35.77±0.78∗ 20.92±1.25 67.12±1.82∗ 32.33±1.31∗ 21.94±1.72 58.19±0.23∗ 46.96±0.36∗
16 Day 40.97±2.32 64.52±0.57∗ 66.75±0.71∗ 32.31±0.822 100±0.00∗ 67.2±0.94∗ 66.09±0.50 73.67±0.51∗ 76.86±1.44∗
20 Day 49.95±1.33 72.41±0.57∗ 74.21±0.766∗ 37.43±1.13 - 100±0.00∗ 73.44±1.19 100±0.00∗ 88±0.50∗
24 Day 70.65±0.95 100±0.00∗ 100±.00∗ 70.01±1.28 - - 84.83±1.18 - 100±0.00∗
28 Day 80.73±0.00 - - 90.86±0.04 - - 90.1±1.02 - -
32 Day 95.12±0.01 - - 97.34±0.02 - - 95.12±0.03 - -
36 Day 98.12±0.03 - - 100±0.00 - - 98.52±0.04 - -
40 Day 100±0.00 - - - - 100±0.00 - -

Values are expressed in Mean ± SD, ∗ p < 0.05, as compared to control group of rats, one way ANOVA followed by Turkey’s test.
5

be lower. The content of hydroxyproline, protein as well as catalase ac-

of control group animals where collagen tissue deposition was found to
fibrils and collagen tissue deposition. This was compared with the group
in the level of hydroxyproline content marked the formation of collagen
drug, povidone iodine and burnol on days 14th and day 28th an increase
and collagen deposition (Hayouni et al., 2011). After application of test
tributed to enhancement of the proliferation and migration of fibroblasts

model.
Figure 3. Photographic representation of wound contraction in burn wound

model.
Figure 2. Photographic representation of wound contraction in incision wound
Higher hydroxyproline content in the treated group might be at-

Phytomedicine Plus 1 (2021) 100054

S. Saha, V. Sonar, B. Soni et al.
tivity was estimated from the excised skin of healed wounds on 14th
and 28th day of treatment. Results represented in (Table 5) revealed
that the TIO treatment groups (Group III, VI, IX) showed statistically
significant increase (p<0.05) in the content of hydroxyproline, protein
as well as catalase activity in comparison to control groups. However,
the contents of hydroxyproline, protein as well as catalase activity after
treatment with TIO were not significantly higher than those from the
reference drug treated groups.
6
Phytomedicine Plus 1 (2021) 100054
Figure 4. Percentage of wound contraction in treated and un-
treated animal groups; 4A- Excision, 4B- Incision and 4C- Burn
wounds (Data pooled from 10 rats / group).
Fibroblast cells stimulate the synthesis of collagen, which is a major
component of the extracellular matrix. Additionally, collagen also facili-
tates the migration of endothelial cells to form new blood vessels that in
turn enhance granulation tissue formation and consequently improves
the wound healing process. However, to estimate the amount of collagen
in the wound bed, the hydroxyproline content of wound tissue is always
assessed. As hydroxyproline is an aminoacid, which is exclusively con-
fined to collagen (Gao et al., 2006). Agyare et al., (2011) reported that
S. Saha, V. Sonar, B. Soni et al. Phytomedicine Plus 1 (2021) 100054

Table 4

Biochemical profile of rat wound tissues on 14th and 28th day post treatment of 10% w/w Typha angustata L. inflorescence extract ointment (TIO); Values are expressed in Mean±SD (Data pooled from 10 rats/ groups).

U/mg of tissue
Period of epithelisation from excision, Incision and burn wounds in control,

19.34±0.026∗

28.55±0.051∗
21.14±0.026∗

27.85±0.035∗
22.16±0.026∗
15.13±0.025

15.54±0.026

15.66±0.032
21.15±0.1∗
reference drug treated and Typha angustata L. inflorescence extract ointment
treated (TIO) group of rats (Data pooled from 10 rats/group).

28th D
Groups Epithelization Periods in Days
Excision Wound Incision Wound Burn Wound

Control 32±0.001 28±0.006 31±0.004

Reference drug treated 19±0.001∗ 18±0.002∗ 21±0.001∗

Catalase activity

U/mg of tissue

10.16±0.015∗

11.16±0.020∗
TIO treated 23±0.003∗ 19±0.003∗ 22±0.005∗

7.83±0.020∗

8.14±0.020∗
4.37±0.106

5.54±0.032

5.54±0.015
9.95±0.02∗

7.78±0.01∗
Where,

14th D
Reference drug in excision wound group is (10%w/w Povidone iodine) and
burn wound group is (10%w/w Burnol); TIO- 10%w/w Typha angustata L. in-
florescence extract ointment.
Values are expressed in Mean ± SD,
∗
p < 0.05, as compared to control group of rats; one way ANOVA followed by

mg/100mg of tissue
Turkey’s test.

51.67±0.118∗
53.07±0.056∗

53.72±0.094∗
32.72±0.081

33.06±0.019
56.70±0.18∗
49.00±0.82∗

51.8±0.041∗
31.55±0.22
geraniin stimulates the collagen biosynthesis from normal human der-

28th D
mal fibroblast. The finding of this study is in agreement with aforesaid
report.
Additionally, increased deposition of collagen and intermolecular

TIO- 10% w/w Typha angustata L. inflorescence extract ointment. ∗ p < 0.05, as compared to control one way ANOVA followed by Turkey’s test.
cross-linking facilitate the remodeling phase of wound healing process,

mg/100mg of tissue
since the tensile strength of wound is mainly due to the amount of col-
lagen organization in wound bed (Diegelmann, 2001).

31.47±0.087∗
24.55±0.041∗
24.52±0.16∗
17.75±0.17∗

31.7±0.026∗
25.62±0.51∗
12.54±0.19

19.97±0.04
19.80±0.10
Johansen et al., (2005) demonstrated that natural cellular oxidants
i.e. CAT and SOD participates in oxygen defence metabolic reactions in

Protein

14th D
tissue matrix by the formation of water and molecular di-oxygen from
superoxide. In the present study, tissues samples excised on 14th and
28th day from all types of wound models showed significantly (p>0.05)
higher level of CAT. This finding provides good evidence of faster wound
healing in treatment groups with test drug and standard reference drugs
μg/100mg of tissue

as compared to non-treated animal groups.

88.125±0.464∗
92.55±0.966∗

94.87±0.267∗
88.90±0.193∗
44.57±0.267

39.65±0.069
44.62±0.070

91.2±0.069∗
84.39±0.69∗
High levels of reactive oxygen species (ROS), like superoxide an-
ion (O2 − ), hydroxyl radicals (OH− ), hydrogen peroxide (H2 O2 ) and
28th D

singlet oxygen (1 O2 ) at wound site promotes breakdown of collagen

and hence destruction of extracellular matrix (ECM). When the ECM
is destroyed, the processes such as angiogenesis and re-epithelization
Excision Wound Model

Incision Wound Model

which are essential for wounds to heal get reduced (Siwik et al.,
2001; Rodriguez et al., 2008). Boakye et al. (2016c) reported that Burn Wound Model
μg/100mg of tissue

geraniin stimulates the production of catalase superoxide dismutase and

Hydroxyproline

48.45±0.267∗
37.25±0.464∗

52.62±0.070∗
37.92±0.202∗

32.82±0.094∗
16.77±0.255

16.91±0.069
20.30±0.564

49.9±0.078∗
ascorbate peroxidase but reduces the activity of malondialdehyde and
myeloperoxidase levels in the excised wounds.
14th D

Histopathology: Histopathological examination was carried out on

day 14th from the skin tissue excised from all group so as to evalu-
ate the wound healing efficacy of T. angustata L. inflorescence crude
extract. Among all three wound types, findings (Table 6, Figure 5) re-
vealed better reparative changes (in terms of re-epithelization, necro-
Reference drug (10 % w/w povidone iodine)

Reference drug (10% w/w povidone iodine)

sis, Inflammatory foci, neo- vascularization, collagen tissue and spindle

cells deposition) from the groups treated with T. angustata L. inflores-
cence crude extract ointment (10% w/w) as compared to control groups.
Reference drug (10 % w/w burnol)

These reparative changes were similar in reference drug treated groups

as well as TIO treated groups.
Histopathological findings also revealed that after 14 days of post
wound, fibroblast cells and collagen deposition appeared in all groups;
notably, 10%w/w Typha angustata L. inflorescence extract ointment
(TIO) treated wounds showed the highest density of collagen and a more
organized fibrous structure, as compared to that of other groups treated
TIO treated

TIO treated

TIO treated
Treatment

with reference drug and non-treated groups. Moreover, collagen is the

Control

Control

Control

main component of extracellular matrix that plays crucial role in wound

healing process. Also, collagen synthesis occurs due to massive prolifer-
ation of fibroblasts (Gil et al., 2013).
A better granulation tissue formation with more accumulated extra-
Table 5

cellular matrix, fibroblasts (spindle cells) and other cells are beneficial to
Group

Where,
VIII
VII

wound healing process. As shown in (Table 6; Figure 5) the granulation

III

IX
IV

VI
II

V
I

tissues in both 10%w/w Typha angustata L. inflorescence extract oint-

7
S. Saha, V. Sonar, B. Soni et al. Phytomedicine Plus 1 (2021) 100054

Table- 6
Histological examination showing comparison of wound healing in control, reference drug treated and Typha angustata L. inflorescence extract ointment treated
(TIO) group of rats groups on the 14th post wound day.

Excision Wound Incision Wound Burn Wound

Histopathological
Group I Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX
observations
Control Reference drug treated TIO Control Reference drug treated TIO Control Reference drug treated TIO

Reepithelization AB. PR. PR. PR. PR PR. PR. PR. PR.

Necrosis AB. AB. AB. AB. + AB. AB. PR. AB.
Infl.Foci AB. AB AB. + ++ + + ++ PR.
Neovascularization + ++ ++ + ++ +++ + + +++
Collegen Tissue + ++ +++ + ++ +++ + + +++
Spindle Cells AB. ++ + + + + + + +

Where, reference drug in excision / Incision wound group is (10% w/w Povidone iodine) and burn wound group is (10% w/w Burnol); TIO- 10% w/w Typha angustata
L. inflorescence extract ointment.
Skin sections stained with hematoxylin/Eosin were classified as (AB.) Absent, (PR.) Present (+) Good (++) Better (+++) Best.

Figure 5. Histopathological changes in granulation tissue on day 14 post-treatment. Median section from tissue samples of dermal wounds or the corresponding
intact dermis were stained with hematoxylin and eosin (H&E). Where, Black arrow indicates epithelization, red arrow indicates spindle cells, yellow arrow indicates
RBC; orange arrow indicates angiogenesis.

ment (TIO) and reference drug treated groups (10%w/w povidone io-
dine and 10%w/w burnol ointment) treated wounds were much thicker
than those of untreated groups.
Angiogenesis is critical phase that occurs during wound repair,
newly formed blood vessels participate in granulation tissue for-
mation. The blood vessels thus formed provides nutrition and oxy-
gen to growing tissues, which are required for wound healing. In
this study, the angiogenesis of the wounds was evaluated by mi-
crovessels formation from excised tissue section at wound site on
day 14. The result showed enhanced microvessel formation in both
10%w/w Typha angustata L. inflorescence extract ointment (TIO) and
reference drug treated groups (10%w/w povidone iodine and 10%
w/w burnol ointment) as compared to the other groups (Table 6;
Figure 5).

8
S. Saha, V. Sonar, B. Soni et al.
Wound healing process helps to restore the damaged tissue layers
and cellular structure to its normal state. With the initiation of fibroblast
stage, the wound area undergoes shrinkage (Chitra et al., 2009). This
complex process consists of contraction, granulation, epithelization and
collagenation (Ayyanar and Ignacimuthu, 2009; Wild et al., 2010).
The multifactorial process of wound healing can be delayed and/or
disrupted by microbial infections, presence of free radicals as well
as due to metabolic disturbances (Frangez et al., 2017). Ozay et al.,
(2018) pointed that wound closure, wound contraction and functional
barrier re-organization are important markers in the process of wound
healing. The type of damage plays an important role in wound heal-
ing, therefore healing stages of wound models such as excision, incision,
burn wound model and dead space are affected differently during this
process (Ananth et al., 2010). The main objective of wound manage-
ment is to heal the injury in shortest possible time with minimal pain
and discomfort to the patient.
In the present study, the effect of T. angustata L. inflorescence extract
was observed on tissue regeneration using experimental animal models
(Wistar rats). After creation of various types of wounds, rats were con-
tinuously monitored and treated topically with prepared ointment as
well as reference drug. The results revealed that treatment groups and
the reference drug treated group (10%w/w povidone iodine) in case of
excision and incision; (10%w/w burnol) in case of burn wound models
showed significant (p<0.05) reduction in wound area as well as rapid
epithelization and rapid rate of wound closure when compared to the
control group animals.
The histopathological findings provide an additional evidence for
the experimental wound healing as observed by wound area contraction
values and rapid epithelization. Wound re-epithelization is a hallmark
of successful wound care (Stadelmann et al., 1998). Granulation, col-
lagen maturation and scar formation are some of the many phases of
wound healing which run concurrently, but independent of each other
(Udupa et al., 2006).
Reports of Gibbs et al., (1983); Qin and Sun (2005); Tao et al.,
(2010) on phytochemical analysis of Typha sp. pollens revealed presence
of sterols, terpenoids, flavonoids and long chain hydrocarbons. Thus, it
could be also predicted that the presence of these compounds in the
inflorescence of T. angustata L. may have acted synergistically in the
process of wound healing process.
The study revealed the potential of aqueous extract of T. angustata
L. inflorescence in wound healing. The prepared topical ointment from
aqueous extract of T. angustata L. inflorescence possess multifaceted
properties in healing incision, excision and burn wound types. The find-
ings showed remarkable improvement in wound contraction, rapid ep-
ithelization, collagen deposition, hydroxyproline and protein content in
rats treated with TIO ointment similar to that reference drugs treated
animals.
Funding
This research did not receive any specific grant from funding agency
in public, commercial or not for profit sector.
Declaration of Competing Interest
The authors declare no conflicts of interest in relation to this article.
Acknowledgements
The authors would like to thank the management of Uka Tarsadia
University for constant support and providing necessary facility to carry
out the work and we are also grateful in particular to Dr Bhavin Vyas,
and Dr Shrikant Joshi from Maliba Pharmacy College to provide guid-
ance as well as animal house facility and animals for study.
9
Phytomedicine Plus 1 (2021) 100054
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