Current Research in Biotechnology 6 (2023) 100151

Contents lists available at ScienceDirect

Current Research in Biotechnology

journal homepage: www.elsevier.com/locate/crbiot

Assessment of wound healing activity and safety profile of Amaranthus

spinosus extract using incision wound healing model
Shravan Kumar Paswan a, b, *, Pritt Verma a, b, Lucy Mohapatra b, Chandana Venkateswara Rao a,
Sajal Srivastava b, Sachin Kumar c
a
Pharmacology Division, CSIR-National Botanical Research Institute, Lucknow 226001, Uttar Pradesh, India
b
Amity Institute of Pharmacy, Amity University Uttar Pradesh, Lucknow Campus, Gomati Nagar, Lucknow 226010, Uttar Pradesh, India
c
Division of Medical Laboratory Technology, School of Allied Health Sciences and Management, Delhi Pharmaceutical Sciences and Research University, Gate no-1, Opp.
Sainik Farm, Mehrauli- Badarpur Road, Sector-3, New Delhi 110017, India

A R T I C L E I N F O A B S T R A C T

Keywords: The objective of the study was to determine efficacy of Amaranthus spinosus extract against inflammation and
Inflammation wound along with assessment of safety profile of drug upon topical application. The collected whole plant was
Wound extracted with ethanol (65 %). In vitro efficacy of extract was performed on HaCaT and MEF cells. Clonogenic and
Plant
scratch assays were carried out to determine the wound healing activity of the extract. Acute oral and dermal
Herb
Medicine
toxicity studies were conducted in accordance with OECD recommendations in order to assess the drug’s profile
for safety. Biological efficacy of A. spinosus was determined using incision wound healing model and measuring
the tensile strength of a healing skin. Results showed that A. spinosus extract significant reduced inflammation
and wound area. The percentage viability of cells were 96.20 and 96.23 %, respectively for HaCaT and MEF cells
after 48 h of treatment; however, surviving factor were 0.71 ad 0.79, respectively. The tensile strength was
574.5 g after treatment with drug as compared to the control group. It can be concluded based on findings that
A. spinosus may be used as an alternative medicine in the healing of wound.

Introduction
Skin is the protective organs of the body that extends to whole body
parts and prevents entry of pathogens. It minimized fluid loss, protect
the body and act as thermal barrier (Kalva et al., 2021). Hence, in the
case of a wound, it is crucial to restore the functionality of this multi­
purpose organ. Maintaining skin integrity and restoring injured tissues.
It is a multistep biochemical process including hemostasis, proliferation,
inflammation, and remodelling (Karaly et al., 2021; Saha et al., 2021).
The associated factors responsible for disruption of wound healing
phases are infection, reduced oxygenation and antibiotic drugs (Won
et al., 2021).
The conventional and synthetic drugs are associated with side effects
(Zhao-fleming et al., 2018). High concentration of antibiotics drugs
cause significant systemic toxicities (Yang et al., 2020). NSAIDs are
readily available, over-the-counter, reasonably priced and most familiar
drugs against inflammation and pain (Drini, 2017). However, they have
several toxicities in which gastric ulcer and hepatotoxicity are major
ones (Ghlichloo and Gerriets, 2021).
The normal and abnormal wound healing is directly linked with
inflammatory process. Inflammation is a natural and protective mech­
anism of the body for stimulation of tissue repair (Rodrigues et al., 2018;
Nourian Dehkordi et al., 2019). Thus, therapeutic use of anti-
inflammatory drugs is only logical when the inflammation becomes
inappropriate, unregulated and recurrent. Due to several toxicities
associated with these drugs, the herbal medicines may be a better
alternative in treating inflammation and wound (Karimi et al., 2015).
Traditionally, herbal medicinal plants have been for treating wounds,
skin diseases, cuts and burns. Plants metabolites possess tissue regen­
eration and healing properties. They contain a high concentration of
polyphenols, which have been shown to have extraordinary antioxidant
activity, and phytochemicals, which can be used to treat a wide range of
skin damages (Mojzer et al., 2016; Forni et al., 2019). Hence, wound-
healing medicinal plants may be preferable to current pharmaceutical
options given adequate scientific study.
Polyphenols, crude fibre, proteins and saccharides are all abundant
in the medicinal herb Amaranthus spinosus, which makes it an effective
treatment for a variety of conditions. It has been used historically to heal

* Corresponding author.
E-mail address: paswanshravan@gmail.com (S.K. Paswan).

https://doi.org/10.1016/j.crbiot.2023.100151
Received 7 July 2023; Received in revised form 13 October 2023; Accepted 21 October 2023
Available online 30 October 2023
2590-2628/© 2023 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.K. Paswan et al. Current Research in Biotechnology 6 (2023) 100151

Table 1 decoction on wound by the ethnic people. In this study, the plant was
Percentage cell viability of HaCaT and MEF cells after treatment with Amar­ extracted macerated with ethanol (65 %). It was applied daily for 10
anthus spinosus plant extract. days on topical wound to evaluate efficacy of the extract. The safety
Culture condition Percentage viability of Percentage viability of evaluation of plant extract via acute and dermal toxicity studies has also
(μg/ml) HaCaT cells MEF cells performed. Mouse embryonic fibroblast (MEF) and Human keratinocyte
AS (5 %) AS (10 %) AS (5 %) AS (10 %) (HaCaT) cells were used in migration, cytotoxicity, and proliferation
experiments for wound healing activity of A. spinosus extract. In vivo
Control 91.35 ± 93.35 ± 93.35 ± 93.35 ±
0.28 0.29 0.30 0.31 activity was performed after positive in vitro results using cell lines. The
30 92.41 ± 94.33 ± 93.40 ± 94.28 ± degree of wound closure was ensured by observing tensile strength of
0.23 0.20 0.42 0.28 the healing skin.
60 93.21 ± 94.04 ± 94.30 ± 95.26 ±
0.21 0.24 0.11 0.42
120 93.21 ± 95.32 ± 94.33 ± 95.47 ±
Materials and methods
0.33 0.30 0.19 0.09
240 94.23 ± 95.23 ± 95.10 ± 96.23 ± Cell lines, chemicals and reagents
0.21 0.27 0.43 0.18
480 95.11 96.20 ± 95.97 ± 96.23 ±
±
MEF and HaCaT cell lines were purchased from NCCS, Pune. The
0.35 0.34 0.26 0.23
standard drug soframycin and diclofenac were purchased from Glax­
Data are represented as mean ± SD (n = 3). Control, Dulbecco’s modified eagle oSmithKline, USA. Other chemicals such as Tween 80, Formalin, Poly­
medium; AS, Amaranthus spinosus. sorbate 60, Soft paraffin, Xylene, Eosin, Butylated hydroxyl anisole and
Cetostearyl alcohol were obtained from Sigma Aldrich, USA.
wounds, reduce inflammation, alleviate pain, treat depression, and even
cure malaria (Paswan et al., 2020a, 2020b). Carbon tetrachloride (CCl4)
causes severe damage to the liver in rats, however A. spinosus has been
Table 2
demonstrated to protect the liver. The stem bark of the plant possesses
Percentage of cell migration and wound closure after treatment with Amaranthus
phenolic acid such as vanillic, ferric, sinapic and syringic acid and other
spinosus plant extract.
flavonoids compounds like kaempferol and quercetin isolated from
sprouts (Rjeibi et al., 2016). The specific compounds found in A. spi­ Time (h) Percentage of cell migration (%)

nosus extract were caffeic acid, ferulic acid, chlorogenic acid, gallic acid, Control Standard AS (5 %) AS (10 %)
protocatechuic acid, and quercetin in our previous work (Paswan et al., 0 0 0 0 0
2020a, 2020b). 12 25.39 35.02 31.11 36.11
Herein, we proposed that the topical application of A. spinosus may 24 31.26 52.39 41.32 57.24
48 40.22 97.06 93.34 96.49
be helpful in reducing inflammation and healing of wounds without
systemic side effects. Traditionally, it has been used to apply leaf Control, untreated; standard, cipladin (5 μg/ml); AS, Amaranthus spinosus.

Fig. 1. Morphology of HaCaT and MEF cells after treatment with A. spinosus plant extract Control, Dulbecco’s modified eagle medium; A. spinosus.

2
S.K. Paswan et al. Current Research in Biotechnology 6 (2023) 100151

Fig. 2. Migration potential of HaCaT cells treated with A. spinosus plant extract Control, Dulbecco’s modified eagle medium; A. spinosus.

Fig. 3. Migration potential of MEF cells treated with A. spinosus plant extract Control, Dulbecco’s modified eagle medium; A. spinosus.

Collection and extraction of A. Spinosus plant authenticated by in the department of botany, Venkateswara University,
Tirupati, India. The specimen of the collected plant was deposited in the
Plant was collected from Andhra Pradesh, India. It was identified and herbarium of the institute via voucher no. 02423. Briefly, extraction of

3
S.K. Paswan et al. Current Research in Biotechnology 6 (2023) 100151

Table 3 after being exposed to extract at 5 % and 10 % concentrations for

Clonogenic assay for HaCaT and MEF cells treated with Amaranthus spinosus twenty-four hours at density of 1 × 107 cells/well. DMEM (200 μl/well)
extract. was added to the culture plates containing cells and incubated for 12 h.
Control AS 5 % AS 10 % The cells were treated with various concentration of plant extract i.e. 30,
Surviving factor HaCaT cells
60, 120, 240, and 480 μg/ml and then incubated for 48 h at 37 ◦ C. Ef­
0.95 0.78 0.71 ficacy of plant extract of viability of cells was determined post-
Surviving factor MEF cells incubation by adding MTT reagent followed by incubation for 2 h. Un­
0.96 0.81 0.79 treated cells were considered as control and showed 100 % viability
(López-García et al. 2014). Using a microplate reader, absorbance was
measured at 565 nm. This was accomplished by dissolving formazan
was performed by grounding the whole plant (100 g) and macerated
crystals in DMSO (100 μl).
with ethanol (65 %, 250 ml) for 48 h. The extractive was contracted over
rotary evaporator (Buchi R-200, USA) to remove extra solvent from the
Clonogenic assay
extract. The percentage yield of the extract was 15.34 %.
Franken et al., 2006 method was used to perform Clonogenic assay.
In this assay, cells were plating after irradiation. Cells were plated
In vitro assay for A. Spinosus extract following irradiation for this test. The flasks of cells were let to expand
until they reached 90 % confluence. The cells were treated with the
MTT assay for evaluation of A. Spinosus extract on cell viability extract in the concentration of 100 μg/mL maintained at 37 ◦ C in air + 5
HaCaT and MEF cells were planted in a microtitre plate (96-well)

Fig. 4. Clonogenicity assay for HaCaT and MEF cells treated with A. spinosus extract Control, Dulbecco’s modified eagle medium; A. spinosus.

Fig. 5. Acute dermal toxicity study of A. spinosus extract A = Control; B = AS1; C = AS2.

4
S.K. Paswan et al. Current Research in Biotechnology 6 (2023) 100151

Fig. 6. Wound healing effect of A. spinosus extract on rats A = Positive Control; B = Negative control, C = Standard, D = AS1; E = AS2.

% CO2 under saturated humidity overnight. To prevent contamination cells continued for 24 h. Using a microscope, researchers could see cells
from radiation passing through air layers, DMEM was used to fill the migrate and undergo morphological changes. There were three sets of
flasks. Cells are re-plated immediately or delayed to examine healing of each determinant. The depth of a scratch and closure of wounds (0, 12,
possibly lethal damage following ionising radiation treatment. Repair of 24 and 48 h) were evaluated using images captured with the Image J
possibly fatal damage is finished after 6 h. Cells should be pipetted onto software.
the test dishes at least twice. (ii) Keep the plates in the incubator until
clones have formed adequately in the control dishes. The following day,
trypsinization was used to collect the extract-treated cells. It was Assessment of A. Spinosus extract for toxicological activity
determined as:
Animals used in experiments
PE = colony forming unit count/cell seeding quantity × 100
The protocol of the study was by the animal ethical committee with
approval number. 1732/GO/RE/CPCSEA. The light cycle was set at 2 h
SF = Count of irradiated colonies relative to the initial seeding of cells × PE
and the dark cycle at 12 h. They were given pelleted food to eat and had
free access to water. Experiments were performed on animals after
Scratch assay
acclimatized to the laboratory.
Scratch assays based on cell migration experiments with HaCaT and
MEF cells evaluated the wound-healing activities of A. spinosus extract
Analysis of acute toxicity
(Liang et al. 2007). The cells are maintained at 37 ◦ C in air + 5 % CO2
The toxicity test was performed in accordance with the OECD’s
under saturated humidity overnight.The cells were grown in 6-well
standard methodology for measuring acute toxicity, OECD 420 (OECD,
plates after being seeded at a density of 2 × 105 cells/well/ml. After
2001). The test subjects consisted of six female Swiss mice weighing
rinsing the cells in Delbucco’s Phosphate Buffered Saline to get rid of any
between 25 and 30 g. Animals were observed for 14 days after a single
remaining cell debris, a sterile tip (200 μl) was used to make the scratch
dose of a plant extract at a dose of 2000 mg/kg for toxicity markers like
on the cells. Standard drug Cipladine (5 μg/ml) was utilised alongside
drooling, abnormal behaviour, excessive salivation, urination, fur
A. spinosus extract (5 % and 10 %) to treat the cells. Incubation of the
colour, disturbed feeding and lethargy.

5
S.K. Paswan et al. Current Research in Biotechnology 6 (2023) 100151

Table 4
Safety profile of Amaranthus spinosus plant extract upon topical application on animals skin.
Time
Response
4 hr 24 hr 1 day 2 days 4 days 6 days 8 days 10 days 12 days 14 days

Alertness + + + + + + + + + +
Irritability – – – – – – – – – –
Fearfulness – – – – – – – – – –
Touch Response + + + + + + + + + +
Restlessness – – – – – – – – – –
Abdominal Tone + + + + + + + + + +
Tremors – – – – – – – – – –
Writhing – – – – – – – – – –
Corneal reflexes + + + + + + + + + +
Diarrhea – – – – – – – – – –
Food and water intake + + + + + + + + + +
Respiration rate + + + + + + + + + +
Pupil reaction to light + + + + + + + + + +
Skin color & texture + + + + + + + + + +
Spontaneous activity + + + + + + + + + +
Heart rate + + + + + + + + + +
Convulsion – – – – – – – – – –
Aggressiveness – – – – – – – – – –
Vomiting – – – – – – – – – –
Sedation – – – – – – – – – –
Locomotion + + + + + + + + + +
Edema – – – – – – – – – –
Mortality – – – – – – – – – –

+, present; -, absent.

Incision wound model

Table 5 An intraperitoneal injection of ketamine hydrochloride at a dose of
Tensile strength activity of Amaranthus spinosus. 50 mg/kg was used to put the animals to anesthetize before they were
Group Dose Tensile strength (g) shaved on the dorsal side. The animals’ shaved backs were marked with
Control Ointment base 239.3 ± 3.7
a series of symbols. With a sharp scalpel, an incision was created on skin
Standard Soframycin 579.1 ± 5.2*** on either side of the spine, measuring 6 cm in length. In order to close
AS1 5 %, w/w 455.4 ± 6.4 * the wound after homeostasis, curved needle was used with silk surgical
AS2 10 %, w/w 574.5 ± 7.2 *** thread (number 000) to create interrupted sutures spaced at 1 cm. The
Data is represented as mean ± SEM (n = 6), significantly different at *p < 0.05, wound was kept open after stitching, and the animals received therapy
**p < 0.01, ***p < 0.001 vs. control. Standard, Soframycin; AS, A. spinosus. once daily for a total of 10 days (Nagar et al. 2016). The animals were
sacrificed after the experiment by giving euthanasia. The animals were
Acute dermal toxicity study euthanized with the dissociative anesthetic ketamine and the adreno­
The acute toxicity study followed OECD recommendation 402 ceptor agonist xylazine at a dose of 300 and 30 mg/kg, respectively.
(OECD, 2004). Twelve female Wistar albino rats were used in the study,
with six in each of the control and treatment groups. In order to apply Tensile strength measurement
the extract, we shaved the dorsal fur off of 5 cm2 of animals. Using a tiny A wound’s recovery might be measured in terms of its tensile
spatula, the A. spinosus extract was rubbed into the animal’s shaved skin. strength as the skin regenerated. The strength of the restored tissue, as
Ointment was made by combining 10 mg of extract with 12 mg of measured by its resistance to tearing, was used to draw this conclusion.
glycerin, 25 mg of soft paraffin, 4 mg of cetostearyl alcohol, 0.02 mg of Animals were put under anaesthesia and their sutures were removed on
butylated hydroxyl anisole, and 5 mg of polysorbate 60. A porous gauze day 10. Tensiometer readings were taken to determine the wound’s
dressing and non-irritating tape were used to increase the amount of tensile strength (Kuwano et al. 1994)
time the ointment had contact with the skin during the 24-hour exposure
period, and the substance was washed off with warm water or sunflower
Statistical analysis
oil after the 24-hour period had passed. Over the course of 14 days, we
kept a close eye on the animals from above the cage and recorded their
Mean ± SD was used to describe the results. Two-way analysis of
weight once a week. After 4 h of ointment application, the skin was
variance (ANOVA) was performed for statistical analysis using Graph­
monitored for 14 days for signs of inflammation, swelling, irritation and
Pad Prism software version 8.0. Results were considered statistically
redness.
significant with p < 0.05*, p < 0.01** and p < 0.001***.

Wound healing activity of A. Spinosus on animals Results

Experimental protocol Effect of A. Spinosus extract on cell viability

Albino Incision wound healing activity was tested on Wistar rats (n
= 24), weighing 180–200 g. There were four sets of six animals each. For
the aim of this experiment, Group 1 was given a placebo ointment,
Group 2 was positive control received no treatment, Group 3 was given
Soframycin (10 %, w/w) as standard group, and Groups 4 and 5 were
given ointments containing 5 % and 10 % A. spinosus plant extract,
respectively as test groups.
Cell viability after treatment with A. spinosus plant extract, as
measured by trypan blue exclusion, is presented in Table 1. The results
showed that at the maximum dose (480 μg/ml), the plant extract
reduced HaCaT cell viability by 95.11 % and 96.20 % at concentrations
of 5 % and 10 %, respectively. Equally, the percentage vitality of MEF
cells was 95.97 and 96.13 when treated with A. spinosus plant extract at

6
S.K. Paswan et al.
doses of 5 % and 10 %, respectively. In other words, the plant extract
was safe to use and could be refined for testing its wound-healing abil­
ities. Fig. 1 shows A. spinosus plant extract-treated HaCaT and MEF cell
morphology. No signs of toxicity were seen in the cells after incubation
for 48 h.
Fibroblast cell migration assay
The ability of biologically active substances to promote wound
healing is measured by means of the scratch assay. In the current
investigation, plant extract from A. spinosus (125 μg/ml) was used to
treat HaCaT and MEF cells for 48 h. Results exhibited that wound gaps
created by A. spinosus extract (10 %) were filled by 96.49 % after 48 h
(Table 2). Micrographs presented in Figs. 2 and 3 for migrating HaCaT
and MEF cells, respectively after treatment with A. spinosus plant
extract. However, standard medication treatment reduced wound gap
closure success to 99.06 % in 48 h.
Clonogenic assay
Clones of HaCaT and MEF cells were cultured in a plate-based clo­
nogenic test, as depicted in Fig. 3. The control HaCaT cells revealed only
70 cells after planting of 200 cells during untreated condition. 200 cells
were seeded, and after radiation therapy, 65 clones emerged (Table 3).
However, after seeding 200 cells, only 66 and 64 cells were visible in
HaCaT cells after treatment with 5 % and 10 % concentrations of
A. spinosus, respectively. Radiation treatment resulted in 55 and 50
clones for 5 % and 10 % concentrations of A. spinosus, respectively, out
of 200 cells (Fig. 4). After planting 200 MEF cells, only 68 cells were
visible. In contrast, 62 clones were generated after radiation therapy
from a seed of only 200 cells (Table 3). However, after seeding 200 cells,
only 63 (5 % ointment) and 61 cells (10 % ointment) were visible in MEF
cells. Out of 200 cells, radiation treatment revealed 54 clones of
A. spinosus at 5 % concentration and 51 clones at 10 % concentration
(Fig. 4).Fig. 5.Fig. 6..
Analysis of A. Spinosus plant extract for acute toxicity
There were no adverse effects seen after 14 days of treatment with
A. spinosus plant extract at a dose of 2000 mg/kg. Neither behaviour nor
diet nor any of the physiological indicators tested showed any signifi­
cant change. No toxicity was found in any organ while analyzed gross
histopathology of the vital organs. We determined that the plant extract
was secure for further testing.
Evaluation of the A. Spinosus plant extract for acute skin toxicity
When tested for toxicity using an extract from the A. spinosus plant, it
was shown to be completely safe for use on animal skin. There was no
evidence of skin irritation, inflammation, or any other change in
appearance. There were no indications of convulsions, tremors, diar­
rhoea, salivation, sleep, lethargy and coma. The results of cage-side
observations of animals given an A. spinosus plant extract are shown in
Table 4.
Incision wound model
Table 5 shows that when an ointment containing an extract from the
A. spinosus plant was applied to a wound, the wound’s tensile strength
reduced. After being treated with either the 5 % or 10 % ointment.
Tensiometer was used to measure tensile strength of wounds. However,
the standard group showed substantial improvement in wound tensile
strength. Both the AS1 (5 %) and AS2 (10 %) groups’ tensile strengths
were substantially higher than those of the control group, at 455.4 and
574.5 μg, respectively. The control group, on the other hand, revealed a
tensile strength of 579.1 μg.
7
Current Research in Biotechnology 6 (2023) 100151
Discussion
The effectiveness of A. spinosus on wound healing was examined in
the present study. Based on A. spinosus historical significance in India,
this procedure was developed. Both animals and people can benefit
greatly from using medicinal plants to address a wide range of condi­
tions (Singh et al. 2016; Jain et al. 2021a, 2021b; Rathore et al. 2014;
Jain, 2015; Kumar et al. 2016; Jain et al. 2021c; Jain et al. 2020a; Kumar
et al. 2017; Jain et al. 2020b; Jain et al. 2020c; Rao et al. 2014; Rao et al.
2016; Jain et al. 2020d; Jain et al. 2020e). Before the herbal medicine
may be used in a clinical setting, however, its toxicity must be deter­
mined (Sharwan et al. 2016; Jain et al. 2016; Jain et al. 2015; Sharwan
et al. 2015; Jain et al. 2014). Using extract topically or ingesting it orally
were both revealed safe in our experiment. It was quite difficult to
produce the required effect while using extract, thus the plant extract
was applied topically in the form of ointment. A. spinosus’ flavonoid
content has been shown to have a wide variety of biological effects (Jain
and Shrivastava, 2020). The majority of medicinal herbs used for
treating wounds include several flavonoids, the effects of which work
together synergistically (Aslam et al. 2018). According to a 2007 study
by Ambiga et al., flavonoids speed up the healing process by contracting
wounds and killing bacteria, respectively (Ambiga et al., 2007). We
observed that the plant extract greatly decreased skin inflammation,
redness, and edoema in our previous study.
Wound healing is another beneficial application of these effects.
Despite the need of their production and early induction for optimal
healing, these cytokines are downregulated during the healing process
(Paswan et al. 2017). Plant extract’s wound-healing properties were
tested in vitro utilising fibroblast cell lines including HaCaT and MEF.
Cytotoxicity of test drugs at various concentrations and their potential
application in future treatments are evaluated using the MTT assay
(Muniandy et al. 2018). After being treated with A. spinosus extract (5 %
and 10 %), the survival rate of both HaCaT and MEF cells in our study
increased to 96 % and 97 %, respectively. After 48 h, the healing rate for
wounds treated with 10 % A. spinosus extract was 97.13 percent,
significantly higher than the rate for the control group. The study was in
agreement with the findings of Nelson et al. (2020) to perform clono­
genic for determining the efficacy of plant extract on HCT-116, PC-3,
MCF-7 and RCC-45. WI-38 cells line. Unlike necrosis, apoptosis is an
important cell death mechanism that does not trigger an inflammatory
response that occasions collateral destruction of normal cells in the
surrounding microenvironment (Elmore, 2007). Thus, apoptosis is a
protective mechanism that maintains tissue homeostasis by removing
ailing cells (Fan et al., 2005).
In the early days of treatment, a high dose of A. spinosus ointment
caused a considerable contraction in wound size, indicating the begin­
ning of the healing process, and by the tenth day of treatment, the
wound had entirely closed. Collagen, the substance that constructs and
maintains extracellular tissue, is largely composed of the amino acid
hydroxyproline (Malcor and Mallein-Gerin, 2022). A. spinosus extract
ointment may have contributed to the high tensile strength of the
treatment group because it increased the number of intramolecular and
intermolecular cross links in mature collagen. Tensile strength was high
in the untreated control group, which might be attributed to either the
proper interaction of glycosaminoglycan with the collagen fibrils or the
existence of a sizable amount of mature collagen (Farooq et al., 2023).
Results revealed that the fibroblast migration prompted by
A. spinosus extract sped up the wound healing process. This finding is
consistent with the research conducted by Bolla et al., 2019. After
applying the A. spinosus ointment, the tensile strength of wounded tis­
sues increased significantly, according to in vivo animal studies. The
wound healing ability of Sida cordifolia ointment was also evaluated in
an animal study by Pawar et al., 2013. They reported a dramatic
reduction in wound size beginning on day 8, an indication of progress
towards full healing. Researchers found that treated incision wounds
had greater tensile strength, which may be attributable to an increase in
S.K. Paswan et al.
collagen concentration and fiber stabilization.
Conclusion
Rapid wound closure has been shown in in vitro and in vivo models
of the disease when A. spinosus is treated with a whole plant ethanolic
extract. There was no sign of skin irritation or toxicity from the plant
extract. It was found that application of A. spinosus on animals skin at
either 5 % or 10 % concentration significantly fasten the healing of
wounds. In an incision wound model, animal skin treated with
A. spinosus ointment demonstrated impressive tensile strength. Thus,
A. spinosus extract may be used as an alternative herbal treatment
therapy for wounds.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
The author was awarded by University Grant Commission-RGNSRF,
Fellowship, (Grant Number F1-17.1/2014-15-RGNF-2014-15-SC-UTT-
60684) from University Grant Commission, New Delhi, India, for pur­
suing his doctoral studies. We are also thankful to the Probecell: Sci­
entific Writing Services (www.probecell.com) for proofreading and
editing of this article.
References
Ambiga, S., Narayanan, R., Gowri, D., Sukumar, D., Madhavan, S., 2007. Evaluation of
wound healing activity of flavonoids from ipomoea carnea. Jacq. Anc Sci Life. 26 (3),
45–51.
Aslam, M., Riaz, H., Raza, S.A., Hussain, S., Qureshi, O., Hamzah, Z., et al., 2018. Role of
flavonoids as wound healing agent. In 95–102.
Bolla, S.R., Mohammed Al-Subaie, A., Yousuf Al-Jindan, R., Papayya Balakrishna, J.,
Kanchi Ravi, P., Veeraraghavan, V.P., 2019. In vitro wound healing potency of
methanolic leaf extract of Aristolochia saccata is possibly mediated by its stimulatory
effect on collagen-1 expression. Heliyon. 5 (5), e01648.
Drini, M., 2017. Peptic ulcer disease and non-steroidal anti-inflammatory drugs. Aust.
Prescr. 40 (3), 91–93.
Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35 (4),
495–516.
Fan, T.-J., Han, L.-H., Cong, R.-S., Liang, J., 2005. Caspase family proteases and
apoptosis. Acta Biochim. Biophy. Sin. 37 (11), 719–727.
Farooq, N., Anwar, F., Saleem, U., Ashfaq, M., Shafi, A., Ismail, T., 2023. The wound
healing potential of Hedychium spicatum Sm. and Zinnia peruviana (L.) ethanolic
extracts against excision wound model in rats. J. Ethnopharmacol. 311, 116404.
Forni, C., Facchiano, F., Bartoli, M., Pieretti, S., Facchiano, A., D’Arcangelo, D., et al.,
2019. Beneficial role of phytochemicals on oxidative stress and age-related diseases.
Biomed. Res. Int. 2019, 8748253.
Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J., van Bree, C., 2006. Clonogenic
assay of cells in vitro. Nat. Protoc. 1 (5), 2315–2319.
Ghlichloo, I., Gerriets, V., 2021. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs).
StatPearls. StatPearls Publishing, Treasure Island (FL).
Jain, P., 2015. Secondary metabolites for antiulcer activity. Nat. Prod. Res. 1–17.
Jain, P., Rao, S.P., Singh, V., Pandey, R., Shukla, S.S., 2014. Acute and sub-acute toxicity
studies of an ancient ayurvedic formulation: agnimukha churna. Columbia J.
Pharma. Sci. 1, 18–22.
Jain, P., Pandey, R., Shukla, S.S., 2015. Acute and subacute toxicity studies of polyherbal
formulation talisadya churna in experimental animal model. MJPMS 1 (1), 7–10.
Jain, P., Pandey, R., Shukla, S.S., 2016. Reproductive and developmental toxicity study
of talisadya churna: an ancient polyherbal formulation. IAJPR 6 (5), 5641–5653.
Jain, P., Satapathy, T., Pandey, R.K., 2020a. A mini review of methods to control ticks
population infesting cattle in Chhattisgarh with special emphasis on herbal
acaricides. IJNPR 11 (12), 217–223.
Jain, P., Satapathy, T., Pandey, R.K., 2020b. Efficacy of arecoline hydrobromide against
cattle tick Rhipicephalus (Boophilus) microplus. Int. J. Acarol. 46 (4), 268–275.
8
Current Research in Biotechnology 6 (2023) 100151
Jain, P., Satapathy, T., Pandey, R.K., 2020c. First report on ticks (Acari: Ixodidae)
controlling activity of cottonseed oil (Gossypium Sp.). Int. J. Acarol. 46 (4),
263–267.
Jain, P., Satapathy, T., Pandey, R.K., 2020d. Rhipicephalus microplus (acari: Ixodidae):
Clinical safety and potential control by topical application of cottonseed oil
(Gossypium sp.) on cattle. Exp. Parasitol. 108017.
Jain, P., Satapathy, T., Pandey, R.K., Rhipicephalus microplus,, 2020e. A parasite
threatening cattle health and consequences of herbal acaricides for upliftment of
livelihood of cattle rearing communities in Chhattisgarh. Biocatal. Agric. Biotechnol.
101611.
Jain, P., Satapathy, T., Kumar, R., 2021a. Acaricidal activity and biochemical analysis of
citrus limetta seed oil for controlling ixodid tick rhipicephalus microplus infesting
cattle. Syst. Appl. Acarol. 26.
Jain, P., Satapathy, T., Kumar, R., 2021b. Veterinary Parasitology Acaricidal activity and
clinical safety of arecoline hydrobromide on calves infested with cattle tick
Rhipicephalus microplus (Acari : Ixodidae). Vet. Parasitol. 298 (May), 109490.
Jain, P., Satapathy, T., Kumar, R., 2021c. Veterinary Parasitology First report on efficacy
of Citrus limetta seed oil in controlling cattle tick Rhipicephalus microplus in red
Sahiwal calves. Vet. Parasitol. 296 (June), 109508.
Jain, A., Shrivastava, S., 2020. Isolation and characterization of bioactive components
derived from whole plant of selaginella bryopteris. J Drug Deliv Ther. 10 (4).
Kalva, S.N., Augustine, R., Al Mamun, A., Dalvi, Y.B., Vijay, N., Hasan, A., 2021. Active
agents loaded extracellular matrix mimetic electrospun membranes for wound
healing applications. J Drug Deliv Sci Technol. 102500.
Karaly, A.H., Sarhan, W.A., El-Sherbiny, I.M., 2021. Development of a silk fibroin-based
multitask aerosolized nanopowder formula for efficient wound healing. Int. J. Biol.
Macromol.
Karimi, A., Majlesi, M., Rafieian-kopaei, M., 2015. Herbal versus synthetic drugs; beliefs
and facts. J. Nephropharmacol. 4 (1), 27–30.
Kumar, V., Jain, P., Rathore, K., Ahmed, Z., 2016. Biological evaluation of pupalia
lappacea for antidiabetic, antiadipogenic, and hypolipidemic activity both in vitro
and in vivo. Scientifica 9.
Kumar, V., Rathore, K., Jain, P., Ahmed, Z., 2017. Biological activity of bauhinia
racemose against diabetes and interlinked disorders like obesity and hyperlipidemia.
Clin. Phytosci. 3, 7.
Kuwano, H., Yano, K., Ohno, S., 1994. Dipyridamole inhibits early wound healing in rat
skin incisions. J. Surg. Res. 56 (3), 267–270.
Liang, C.C., Park, A.Y., Guan, J.L., 2007. In vitro scratch assay: a convenient and
inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2, 329–333.
López-García, J., Lehocký, M., Humpolíček, P., Sáha, P., 2014. HaCaT keratinocytes
response on antimicrobial atelocollagen substrates: extent of cytotoxicity, cell
viability and proliferation. J Funct Biomater. 5 (2), 43–57.
Malcor, J.D., Mallein-Gerin, F., 2022. Biomaterial functionalization with triple-helical
peptides for tissue engineering. Acta Biomatter. 148, 1–21.
Mojzer, E.B., Hrncˇicˇ, M.K., Škerget, M., Knez, Z., Bren, U., 2016. Polyphenols: extraction
methods, antioxidative action, bioavailability and anticarcinogenic effects.
Molecules 21, 1–38.
Muniandy, K., Gothai, S., Tan, W.S., Kumar, S.S., Mohd Esa, N., Chandramohan, G., et al.,
2018. In vitro wound healing potential of stem extract of alternanthera sessilis. Evid.
Based Complement. Alternat. Med.
Nagar, H.K., Srivastava, A.K., Srivastava, R., Kurmi, M.L., Chandel, H.S., Ranawat, M.S.,
2016. Pharmacological investigation of the wound healing activity of Cestrum
nocturnum (L.) ointment in wistar albino rats. J. Pharm. 9249040.
Nelson, V.K., Sahoo, N.K., Sahu, M., Sudhan, H.H., Pullaiah, C.P., Muralikrishna, K.S.,
2020 Nov 23. In vitro anticancer activity of Eclipta alba whole plant extract on colon
cancer cell HCT-116. BMC Complement Med Ther. 20 (1), 355.
Nourian Dehkordi, A., Mirahmadi Babaheydari, F., Chehelgerdi, M., Raeisi, D.S., 2019.
Skin tissue engineering: wound healing based on stem-cell-based therapeutic
strategies. Stem Cell Res Ther 10 (1), 111.
OECD, 2001. OECD Guideline for testing of chemicals. Test Guideline 420: Acute Oral
Toxicity - fixed dose Procedure.
OECD, 2004. OECD Guideline for testing of chemicals. Test Guideline 402: Acute Dermal
Irritation Toxicity – Fixed Dose Procedure.
Paswan, S.K., Srivastava, S., Rao, C.V., 2020. Wound healing, antimicrobial and
antioxidant efficacy of Amaranthus spinosus ethanolic extract on rats. Biocatal.
Agric. Biotechnol. 26, 101624.
Pawar, R.S., Chaurasiya, P.K., Rajak, H., Singour, P.K., Toppo, F.A., Jain, A., 2013.
Wound healing activity of Sida cordifolia Linn. in rats. Indian J Pharmacol. 45 (5),
474–478.
Rao, S.P., Amrit, I., Jain, P., Singh, V., 2014. Antiulcer activity of Agnimukha Churna.
Int. J. Ayur. Pharma Research 2 (2), 40–46.
Rao, S.P., Jain, P., Rathore, P., Singh, V.K., 2016. Larvicidal and knockdown activity of
Citrus limetta Risso oil against dengue virus vector. Indian J. Nat. Prod. Resour. 7
(3), 256–260.
Rathore, K., Singh, V., Jain, P., Rao, S.P., Ahmed, Z., Thakur, V.S., 2014. In- vitro and In-
vivo antiadipogenic, antidiabetic and hypolipidemic activity of Diospyros
melanoxylon (Roxb.). J. Ethnopharmacol. 155, 1171–1176.
Rjeibi, I., Ben Saad, A., Hfaiedh, N., 2016. Oxidative damage and hepatotoxicity
associated with deltamethrin in rats: The protective effects of Amaranthus spinosus
seed extract. Biomed. Pharmacother. 84, 853–860.
Rodrigues, M., Kosaric, N., Bonham, C.A., Gurtner, G.C., 2018. Wound healing: a cellular
perspective. Physiol. Rev. 99 (1), 665–706.
Saha, S., Sonar, V., Soni, B., Koladiya, S., Chakraborty, S., Pithawala, M., 2021.
Investigating wound healing potential of Typha angustata L. inflorescence in albino
Wistar rats. Phytomed. Plus 1 (4), 100054.
S.K. Paswan et al.
Sharwan, G., Jain, P., Pandey, R., Shukla, S.S., 2015. Toxicity profile of traditional herbal
medicine. J. Ayu. Herb. Med. 1 (3), 81–90.
Sharwan, G., Jain, P., Pandey, R., Shukla, SS., 2016. Toxicity and safety profiles of
methanolic extract of pistacia integerrima J. L. Stewart ex Brandis (PI) for Wistar
Rats. J. Pharmacopuncture 19 (3), 253–258.
Singh, P., Jain, P., Pandey, R., Shukla, S.S., 2016 Phytotherapeutic review on diabetes,
Spatula DD, 6(2).
Current Research in Biotechnology 6 (2023) 100151
Won, J.E., Shin, J.H., Kim, J., Kim, W.J., Ryu, J.J., Shim, J.S., 2021. Multi-functional
effects of a nitric oxide-conjugated copolymer for accelerating palatal wound
healing. Mater. Sci. Eng. C 125, 112090.
Yang, C., Song, G., Lim, W., 2020. A review of the toxicity in fish exposed to antibiotics.
Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 237, 108840.
Zhao-fleming, H., Hand, A., Zhang, K., Polak, R., Northcut, A., Jacob, D., et al., 2018.
Effect of Non-Steroidal Anti-Inflammatory Drugs on Post-Surgical Complications
against the Backdrop of the Opioid Crisis. 1–9.