Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

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Pharmacological Research - Modern Chinese Medicine

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Ameliorative effect of apple cider vinegar and p-coumaric acid combination

in Ex ovo antimicrobial and in vivo wound healing models
Jegadheeswari Venkadakrishnan 1, Amrita Chatterjee 1, Rajdeep Saha , Kaberi Chatterjee ,
Prashanta Kumar Deb , Biswatrish Sarkar , Papiya Mitra Mazumder *
Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India

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

Keywords: Background: Apple cider vinegar (ACV) is most commonly used in sweet and sour Chinese cuisine. This study
Apple cider vinegar aimed to overcome the side effects of raw ACV, by increasing the concentration of p-Coumaric acid (p-CA), a
p-Coumaric acid polyphenolic component of ACV. The combination (diluted ACV with p-CA) has also been compared with in­
Antioxidant
dividual raw ACV and p-CA to confirm if the overall dosage of the ACV can be reduced to avoid side effects and if
Ex-ovo
Antimicrobial
the combination therapy had any better effect than the individual component itself.
Wound healing Objective: To evaluate and compare antioxidant, antimicrobial, and wound healing effects of ACV and p-CA
combination with individual components ACV and p-CA.
Methods: The antimicrobial properties of the samples were assessed by determining the Minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) using the broth microdilution method, and
zone of inhibition (ZOI) and an ex ovo study was also done to evaluate the antimicrobial effect of the samples in
live embryo. For the evaluation of the test samples excision wound was created in Swiss male albino mice
weighing 25–30 g of either sex to estimate parameters like wound contraction rate, WBC and platelet count, SOD
and GSH levels. Histopathological analysis of the skin was also done.
Result: In DPPH and ABTS antioxidant assays, the combined sample (ACV + p-CA) had lower IC50. The combi­
nation therapy also showed the best antimicrobial potential against P. aeruginosa and B. subtilis. In this context,
ex-ovo antimicrobial study results showed that diluted ACV + p-CA treated live embryo samples had the least
bacterial growth after 48 h, in comparison with non-treatment group as well as individually ACV and p-CA
treated samples. In vivo study depicted that the highest dose of the combination test sample had the best wound
contraction rate and antioxidant marker enzymes elevation compared to diseased control proving the potency to
restore the wound healing progression.
Conclusion: ACV and p-CA combination can be used with daily intake as this combination can prevent microbial
contamination and oxidative stress additionally can repair wounds more safely than raw ACV.

ROS Reactive oxygen species.

Abbreviations
1. Introduction
ACV Apple cider vinegar.
p-CA p-Coumaric acid.
The wound is a rupture in the cellular, anatomical, or functional
MIC Minimum inhibitory concentration.
continuity of living tissues [1]. Healing is a complicated process which
MBC Minimum bactericidal concentration.
requires a proper approach to restoring anatomical continuity and
ZOI Zone of inhibition.
functional status of the skin. Healing is delayed when there is microbial
WBC White blood cells.
contamination and increased oxidative stress [2]. The common causa­
SOD Superoxide dismutase.
tive organisms of wound infections are Streptococcus pyogene,
GSH Reduced glutathione.

* Corresponding author.
E-mail address: pmitramazumder@bitmesra.ac.in (P.M. Mazumder).
1
The first two authors contributed equally.

https://doi.org/10.1016/j.prmcm.2024.100364
Received 1 December 2023; Received in revised form 11 January 2024; Accepted 15 January 2024
Available online 15 January 2024
2667-1425/© 2024 The Authors. 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/).
J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Table 1 for its chronic wound healing property, where the inflammatory phase
IC50 Values of various samples by DPPH assay and ABTS assay. was reduced by p-CA, which improved angiogenesis, tissue repair, and
Samples IC50 (µg/mL) in DPPH assay IC50 (µg/mL) in ABTS assay collagen generation throughout healing [23]. Another study suggested
that ε-caprolactone-p-CA copolymers regenerated skin [24]. As dis­
Ascorbic acid 44.91 ± 0.35 34.89 ± 0.61
ACV 112.51 ± 0.56b 93.37 ± 0.35b cussed earlier although having a lot of therapeutic benefits, chronic and
p-CA 82.84 ± 0.35b 81.43 ± 0.64b excessive usage of ACV can cause side effects. This study aimed to
ACVþ p-CA (1:1) 73.29 ± 1.00b 69.43 ± 0.46b overcome this issue, by increasing the concentration of p-CA, the
Values are expressed as mean ± SD (n = 3), ap < 0.01, bp < 0.001 when phenolic compound already present in ACV. ACV p-CA combination was
compared to Ascorbic acid, One-way ANOVA followed by Tukey’s multiple assessed for its antioxidant, antimicrobial, and wound-healing proper­
comparison test. ties in two different doses. This combination was compared with indi­
vidual ACV and p-CA to confirm if the overall dosage of the ACV could
be reduced to avoid side effects and if the combination therapy had any
Table 2 better effect than the individual component itself.
Zone of Inhibition of various samples.
Bacteria Zone of inhibition (cm) [Including 0.6 cm diameter disc] 2. Methods
ACV p-CA ACV + p- Streptomycin
CA (Standard) 2.1. Chemical and reagents
S. aureus 1.15± 1.48± 0.08 1.78 ± 1.95 ± 0.05
0.13a 0.02ns Apple cider vinegar was procured from Dabur Company Ltd. (An ISO
B. subtilis 1.17± 1.31 ± 1.9 ± 0.08a 2.32 ± 0.08 27001:2013 certified company). p- Coumaric acid, DPPH (2, 2-diphenyl-
0.13c 0.08c 1-picryl-hydrazyl-hydrate), ABTS (2, 2′-azino-bis (3-ethylbenzothiazo­
P. aeruginosa 1.2 ± 1.45± 1.65 ± 1.78 ± 0.04
line-6-sulfonic acid)) were procured from Sigma Aldrich, hematoxylin
0.05c 0.03ns 0.03ns
E. coli 1.05± 1.42±0.02c 1.38 ± 2.13 ± 0.32 from leica biosystems and eosin from ACS Chemicals.
0.98b 0.02c
2.2. Microorganisms
Values are expressed as Mean ± SD (n = 3).
a
p < 0.05.
b
p < 0.01. Two Gram-positive bacteria including Staphylococcus aureus MTCC
c
p < 0.001, ns; non-significant when compared to standard. One way ANOVA 96 (S. aureus), Bacillus subtilis MTCC 441 (B. subtilis), and two Gram-
followed by Tukey’s multiple comparison test. negative bacteria including Escherichia coli MTCC 443 (E. coli) and
Pseudomonas aeruginosa MTCC 741 (P. aeruginosa) were procured from
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella species, the Institute of Microbial Technology Chandigarh, India. On Luria-
Escherichia coli, etc. [3]. Excessive and unregulated oxidative stress plays Bertani medium (LB media) purchased from HIMEDIA, all bacteria
a critical role in the pathophysiology of chronic non-healing wounds by were grown at 37 ◦ C. As a standard, HIMEDIA-purchased streptomycin
maintaining and deregulating inflammatory processes [4]. Alternative sulphate was utilized.
experimental approaches such as ex-ovo culture of avian embryos can be
used in a variety of domains of fundamental research [5]. This type of 2.3. Fertilized eggs
culture of infection is used to assess the effect of drugs in the infected
embryos for their antimicrobial potential [6]. This model is simple, The fertilized eggs were purchased from the Ranchi College of Vet­
rapid and low-cost, and it more closely resembles the in vivo condition erinary Sciences and Animal Husbandry, Kanke, Ranchi, Jharkhand,
than typical antimicrobial testing based on agar plates and dilution India.
experiments.
Apple cider vinegar (ACV) is popular in Chinese food preparation for 2.4. Animal care and handling
its distinct fruity tone which is balanced by its acidity and can be pro­
duced by fermenting apple juice containing different organic acids and Swiss male albino mice weighing 25–30 g of either sex were used in
compounds [7,8]. ACV so far has been proven for its effectiveness in the study. Animals were procured from Laboratory Animal House of
antibacterial, antifungal, antiviral, cytotoxic properties, neuro­ Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India (1968/
protective, anti-obesity and anti-diabetic properties. It has been claimed PO/Re/S/17/CPCSEA) and were maintained in polyacrylic cages with
to provide a variety of health advantages, including a reduction in the standard housing conditions of temperature (24–27 ◦ C), 12:12 light:
risk of cardiac disorders and weight loss [9–13]. The wound healing dark cycles and humidity (60–65%). They underwent seven days of
property of ACV has been explored by Ali et al. that it had the potential acclimatization. Dry pellets were available as food, and water was
to decrease the bacterial load in the infected wound similar to the extent available at all times. The experimental study (Protocol No. 1972/PH/
of the standard drug cefotaxime [14]. ACV is popular in traditional BIT/136/21/IAEC) was approved by the Institutional Animal Ethics
herbal Chinese medicine effecting in lowering blood pressure, and also Committee of Birla Institute of Technology, Mesra, Ranchi, Jharkhand,
as an alternate treatment method to treat fungal infections including India.
vaginal Candidiasis infection and all tineas infections [15–17]. ACV has
been found to have side effects in large doses or chronic usage, especially 2.5. Treatment schedule
in an undiluted form. Due to its acidic nature, apple cider vinegar may
dissolve tooth enamel and cause cavities, delay stomach emptying, and The total 20 animals were randomly divided into 5 groups (n = 4
irritate the oesophagus when consumed in excess and undiluted each). A wound was created and the doses of test samples were given for
[18–20]. 14 days to animals via the oral route once per day before administering
p-coumaric acid (p-CA) or 4-Hydroxycinnamic acid, a phenolic food. The wound was created and normal saline was administered each
compound present in ACV is a major component present in different day in the diseased control group animals [25].
fruits, vegetables and coffee [8]. p-CA was proven for its anti-oxidant,
antimicrobial, anti-inflammatory, hypolipidemic and immunomodula­ 2.6. Animal groups
tory effects [21,22]. In a very recent study, p-CA was also been studied
The treatment in different groups was as follows: [26–27]

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J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Fig. 1. Zone of inhibition of various samples along with standard amongst different bacterial strains. The combination test drug (ACV + p-CA) had the best zone of
inhibition in bacterial strains. This depicts that combined drug has antimicrobial potential against gram +ve and gram -ve bacteria. Samples were taken at a
concentration of ACV (Neat solution), p-CA (2.5 mg/mL), the combination of ACV + p-CA (neat: 2.5 mg/mL) and standard drug streptomycin (STRP) (1 mg/mL).

Table 3
The minimum inhibitory concentration and minimum bactericidal concentration of various samples.
Bacteria Minimum Inhibitory Concentration Minimum Bactericidal Concentration

ACV (µL/ p-CA (µg/ ACV (µL/mL) + p-CA STD (µg/ ACV (µL/ p-CA (µg/ ACV (µL/mL) + p-CA STD (µg/
mL) mL) (µg/mL) mL) mL) mL) (µg/mL) mL)

Gram- S. aureus 12.5 62.5 12.5: 31.25 6.25 25 125 25: 62.5 12.5
positive B. subtilis 25 62.5 25: 62.5 12.5 50 250 50: 125 50
Gram- P. aeruginosa 25 125 25:62.5 50 50 250 50:125 100
negative E. coli 12.5 125 25:62.5 25 25 250 50:125 50

Group 1: The disease control or non-treatment group received CA (1:1) were prepared (10, 20, 40, 80, and 160 µg/mL) in water and the
normal saline only (0.9% NaCl solution) DPPH assay was carried out [28]. Briefly, 100 µL of freshly prepared 0.2
Group 2: wounded animals treated with Apple cider vinegar (0.7%, mM DPPH solution in methanol was mixed with 50 µL of different
p.o.) concentrations of sample. The reaction was carried out in triplicate and
Group 3: wounded animals treated with p-Coumaric acid (50 mg/kg the decrease in absorbance with an increase of concentration was
BW, p.o.) measured at 517 nm after 30 min incubation in the dark using a
Group 4: wounded animals treated with ACV + p-CA (0.35% + 25 UV–visible spectrophotometer.
mg/kg BW, p.o.) The% inhibition was calculated using the formula,
Group 5: wounded animals treated with ACV + p-CA (0.7% + 50 mg/
Inhibition% = (Ac − As / Ac) × 100
kg BW, p.o.)
Where, Ac = absorbance of the control, As = absorbance of the sample
2.7. Antioxidant assay IC50 was calculated.

2.7.1. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay

Different concentrations of Ascorbic acid, ACV, p-CA, and ACV +p-

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J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Fig. 2. Ex-ovo study for the evaluation of antimicrobial effect against S. aureus of the test samples in the viable chick embryo. At the time of inoculation, there was no
bacterial growth. After 48 h, there was the least bacterial growth in the combination group than in individual test groups.

2.7.2. 2, 20-azino-bis (3-ethylbenzothiazoline) 6-sulfonic acid (ABTS)

Table 4 radical scavenging assay
Percentage of wound contraction. ABTS radical scavenging assay was performed according to the
Day Percentage of wound contraction (%) method reported by Deb et al. (2021). ABTS reagent was prepared by
adding 7 mM ABTS with 2.45 mM potassium persulfate in methanol and
Non- ACV p-CA (25 ACV+p-CA ACV+p-CA
treatment (0.7%) mg/kg) (0.35%:25 mg/ (0.7%:50 mg/ keeping it in the dark for 12 to 16 h at room temperature. Before initi­
treated treated kg) treated kg) treated ating the assay, methanol was added to dilute the stock of ABTS solution
group group group group to an absorbance of 0.7 at 734 nm. 100 µL of ABTS reagent was mixed
3 12.34 ± 18.48 ± 18.75 ± 19.34 ± 0.99c 18.20 ± 0.32c with a 50 µL sample of five different concentrations (10, 20, 40, 80, and
0.37 0.275c 0.28c 160 µg/mL) and incubated in the dark for 30 min at room temperature
6 25.86 ± 30.79 ± 30.79 ± 31.31 ± 0.80a 36.40 ± 0.64c [28]. Absorbance was measured at 734 nm and IC50 was calculated as
1.48 0.46a 0.46a
above.
9 37.02 ± 56.89 ± 49.27 ± 47.70 ± 1.49c 60.68 ± 1.89c
1.12 0.64c 0.74c
12 53.87 ± 81.53 ± 81.50 ± 73.20 ± 1.089c 81.80 ± 0.32c
0.80 0.27c 0.25c 2.8. Antimicrobial assay
14 64.65 ± 92.84 ± 91.99 ± 88.03 ± 0.33c 93.93 ± 0.10c
0.85 0.09c 1.25c 2.8.1. Zone of inhibition
Values are expressed as Mean ± SEM (n = 4), ap < 0.05, bp < 0.01, cp < 0.001 For the determination of the test samples to possess any antimicro­
when all the other groups compared to the non-treatment group. One way bial activity, a zone of inhibition was performed using the Agar disc
ANOVA followed by Tukey’s multiple comparison test. diffusion technique [29]. To generate a consistent microbial suspension,
strains were typically subcultured to the mid-log phase (5 × 106
CFU/mL) and inoculated to a sterile liquid medium (0.01%). At 37 ◦ C,
the test microorganisms were cultured for 24 h after being inoculated

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J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Fig. 3. Wound contraction of mice on different days of different groups. The animals treated with test drugs were observed to increase the percentage of wound
contraction as compared to the non-treatment group from the 9th to 14th day. The high dose of combination drug therapy showed the best wound contraction rate in
comparison with the non-treatment group till day 14th.

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J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Table 5 did not exhibit visible growth after 24 h of incubation at 37 ◦ C. The

Effect of various test samples on WBC and platelet count on the 0th, 7th and 14th growth of the microorganism was indicated by turbidity. To determine
day after the creation of wound. MBC, on LB agar medium, 100 μL of the previously grown medium up to
Groups WBC Count (103/mm3) Platelet count (105/mm3) MIC concentration were plated and incubated for 24 h at 37 ◦ C. MBC of
Day 0 Day 7 Day 14 Day 0 Day 7 Day 14
the test samples is the lowest concentration at which no microbe could
survive [31]. All the MIC and MBC analyses were carried out in
Non-treatment 6.2 ± 11.95 7.54 ± 5.06 11.65 7.44
triplicate.
0.26 ± 0.18 0.11 ± 0.26 ± 0.15 ± 0.21
ACV (0.7%) 6.37 10.4 ± 7.36 ± 4.18 8.53 ± 5.23
± 0.16 0.46c 0.64ns ± 0.14 0.14c ± 2.8.3. Ex-ovo study for antimicrobial assessment
0.07c The fertilized eggs were taken and an ex-ovo set up for a model of
p-CA (25 mg/ 4.68 9.45 ± 7.36 ± 4.23 7.48 ± 4.47 infection was created to assess its antimicrobial potential in a live em­
kg) ± 0.23 0.23c 0.17ns ± 0.11 0.27c
bryo. After the CAM was infected with S. aureus, test sample-infused
±
0.27c
ACV þ p-CA 6.23 8.18 ± 5.64 ± 4.49 7.46 ± 4.47 discs were placed on CAM near the spot where the infection was
(0.35%: 25 ± 0.27 0.13c 0.10b ± 0.14 0.17c ± created. A solution of 1 mg/mL of ACV, 2.5 mg/mL of p-CA and ACV+ p-
mg/kg) 0.11c CA (1 mg/mL: 2.5 mg/mL) in sterile water was prepared, and 6 mm
ACV þ p-CA 5.15 7.99 ± 4.6 ± 4.63 6.47 ± 4.66
diameter filter paper discs were dipped into it. The survival of the em­
(0.7%: 50 ± 0.27 0.13c 0.21c ± 0.21 0.17c ±
mg/kg) 0.27c bryo in the presence of the test sample was observed at 24 h and 48 h
after the infection was induced. Also, the presence of infection was
Values are expressed as Mean ± SEM (n = 4), ap < 0.05, bp < 0.01, cp < 0.001
evaluated at 24 h and 48 h after infection by examination of allantoic
when compared to the non-treatment group. One way ANOVA followed by
fluid by streaking in a sterile agar plate [6].
Tukey’s multiple comparison test.

2.9. In-vivo wound healing activity

Table 6
Effect on the SOD and GSH levels in the treatment skin tissue post-14th day. 2.9.1. Induction of excision wound
Sl. Groups % Inhibition for SOD GSH/mg
Diethyl ether was used to anaesthetize the mice. Hair was removed
No. determination protein from the dorsal part of the animals after anaesthesia by shaving. Before
forming the wound, EtOH (70%) was used as an antiseptic for the shaved
1. ACV (0.7%) 46.78 ± 0.73c 0.099 ±
0.006b region, and an excision wound was created by taking a thick piece of
2. p-CA (50 mg/kg) 34.45 ± 1.54b 0.094 ± skin out from a 150 mm square circular area from the designated shaved.
0.001a
3. ACV (0.35%) + p-CA (25 41.09 ± 1.21c 0.119 ± 2.9.2. Determination of wound contraction
mg/kg) 0.009b
4. ACV (0.7%) + p-CA (50 53.37 ± 1.76c 0.18 ±
Following the excision, wound edges were drawn on translucent
mg/kg) 0.005c graph paper with a millimetre scale and measured with a calibre with a
5. Non-treatment group 22.6 ± 1.36 0.055 ± 1/20 mm precision at every 3-day intervals. Measurements were carried
0.003 out for a total of 14 days [32]. The healed area was calculated by sub­
Values are expressed as Mean ± SEM (n = 4). tracting the contracted wound area from the initial wound area.
a
p < 0.05.
b Percentage wound contraction = (Healed area / Total area) × 100
p < 0.01.
c
p < 0.001 when compared to the non-treatment group. One way ANOVA
followed by Tukey’s multiple comparison test. 2.10. White blood cells (WBC) and platelet count

into the liquid medium. To achieve confluent growth, 25 µL of bacterial
suspension was typically equally distributed throughout the entire agar
plate (using LB agar medium). The test samples along with standard
streptomycin were taken at a concentration of ACV (Neat solution), p-CA
(2.5 mg/mL), and a combination of ACV + p-CA (neat: 2.5 mg/mL) and
standard drug streptomycin (STRP) (1 mg/mL) were soaked in paper
discs of 8 mm with 10 µL of each sample and were placed on previously
streaked plates with bacteria namely S. aureus, B. subtilis (gram +ve),
E. coli and P. aeruginosa (gram -ve) and was incubated for 24 h and their
zone of inhibition was calculated.
2.8.2. Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC)
The MIC values of ACV, p-CA and their combination against different
strains were evaluated by the broth microdilution method [29,30]. MIC
and MBC of test samples were assessed against S. aureus, B. subtilis (gram
+ve), E. coli and P. aeruginosa (gram -ve) bacteria were evaluated.
Two-fold dilution was carried out in a 96-well plate to obtain the series
dilutions with concentrations ranging from 15.625 to 1000 µg/mL for
p-CA and Streptomycin, for ACV the range was 1.56 to 100 µL/mL. The
diluted medium was added with 100 μL of the inoculum containing
approximately 106 CFU/mL of each strain. The bacterial suspension
containing DMSO (2%) without a sample was used as a control. The MIC
was defined as the lowest concentration of the oil at which the bacteria
Blood samples were collected from all the groups before and after
induction of the wound on the 0th, 7th, and 14th days by retro-orbital
puncture and haematological parameters were studied. The influence
of WBC and platelet count was assessed in diseased and treated groups
[33].
2.10.1. Biochemical antioxidant evaluation
The biochemical antioxidant enzyme assays were carried out on skin
tissue formed after the 14th day of wound formation. The levels of
reduced glutathione (GSH) and Superoxide dismutase (SOD) were
evaluated in homogenized skin tissue [34–35].
2.10.2. Histopathological analysis
Fixed skin specimens from diseased and different test sample-treated
groups were processed for dehydration with isopropyl alcohol, cleaning,
and impregnation with paraffin wax. A microtome was used to cut
sections of 4 µM thickness. The tissue was deparaffinized with xylene
after being mounted on the plates with Mayer’s albumin solution. The
sections were stained with hematoxylin and eosin. Light microscopy was
used to examine the dried and mounted specimens [36].
2.10.3. Statistical analysis
The results were expressed as mean ± SD values (n = 3). All the
statistical analyses were performed using GraphPad Prism (Version 5.0).
One-way ANOVA followed by Tukey’s test was performed to determine

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J. Venkadakrishnan et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364

Fig. 4. The histopathological images of skin tissue of various groups (a) Non-treatment, (b) ACV Treated (0.7%), (c) p-CA Treated (50 mg/kg), (d) ACV (0.35%) + p-
CA (25 mg/kg), (e) ACV (0.7%) + p-CA (50 mg/kg). Where mononuclear cell (m), fibroblast cell (f), and collagen deposition(c) are depicted.

any statistical differences between the groups. (p < 0.001) than the individual components and the standard ascorbic
acid had the highest antioxidant potential (Table 1).
3. Results

3.1. Antioxidant assay 3.2. Antimicrobial property

3.1.1. DPPH and ABTS radical scavenging assay 3.2.1. Determination of zone of inhibition by disc diffusion method
The lower the IC50 value higher the antioxidant activity. In both the The standard drug streptomycin (STRP) had the highest zone of in­
DPPH and ABTS assay, the combination ACV + p-CA had a better effect hibition and the combination test sample had an almost closer extent of
the zone of inhibition to that of STRP for S. aureus and P. aeruginosa it

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J. Venkadakrishnan et al.
was non-significantly different (Table 2). The test samples had the best
zone of inhibition in bacterial strains of P. aeruginosa and B. subtilis
(Fig. 1). This depicts that combined sample has antimicrobial potential
against gram +ve and gram -ve bacteria.
3.2.2. Minimum inhibitory concentration and minimum bactericidal
concentration
The minimum inhibitory concentration and minimum bactericidal
concentration of streptomycin were the lowest followed by ACV + p-CA.
ACV had a lower MIC and MBC value than p-CA. The MIC and MBC
values of the test samples were comparatively lower in the bacterial
strain of S. aureus (Table 3). So, ACV, p-CA and their combination had
greater potency to kill S. aureus in comparison to other bacteria. The
samples had the potency to kill the gram +ve and gram -ve bacteria.
Also, it was observed that ACV had greater potential to kill the bacteria
in the liquid medium than in solid media and p-CA had a greater extent
of bactericidal activity in the solid medium but their combination
potentiated the antimicrobial activity of each other in both conditions.
3.2.3. Ex-ovo study to assess the antimicrobial property
The antimicrobial effect of the test samples in live embryos in an ex
ovo set-up was evaluated. In this study, S. aureus has been chosen as all
the test samples showed potent inhibitory activity against this gram-
positive microbe in both MIC and MBC estimation. At the time of
inoculation, there was no bacterial growth but later after 48 h, there was
lesser growth from test samples treated live embryo samples, in com­
parison with non-treatment group samples. Also, ACV+ p-CA had the
least bacterial growth (Fig. 2).
3.3. In-vivo wound healing activity
3.3.1. Determination of wound contraction
The excision wound model showed that all of the test samples treated
animals exhibited a gradually significant (p < 0.001) increase in the
percentage of wound contraction as compared to the non-treatment
group from the 9th to 14th day (Table 4, Fig. 3). From the results ob­
tained, it was found that both ACV and p-CA treated groups had a sig­
nificant increase in the percentage of wound contraction from non-
treatment group depicting that both of the samples individually have
prominent wound healing potential. However, the high dose of combi­
nation sample therapy that is ACV (0.7%) + p-CA (50 mg/kg) group had
constantly the best wound contraction rate in comparison with the non-
treatment group till day 14th.
3.3.2. WBC and platelet count
The platelet and WBC count of every group on the 7th day after
wound creation was at an elevated level. WBC and platelet count in the
ACV + p-CA (0.7%:50 mg/kg) treated group were low (p < 0.001) when
compared to diseased group animals (Table 5). Even other treatment
groups had lower WBC and platelet counts when compared to non-
treatment. On the 14th day, all the treated groups had normal WBC
and platelet counts and the non-treatment group had elevated levels of
WBC and platelet count even on the 14th day.
3.3.3. Biochemical estimation- SOD and reduced GSH level estimation
A trend of decrease was observed in SOD activity in disease-control
skin (Table 6). Wound tissues from treatment groups were found to
have increased SOD activity (p < 0.001) in comparison with non-
treatment group. Whereas the animals treated with ACV (0.7%) + p-
CA (50 mg/kg) had SOD and GSH enzyme elevation compared to
diseased control proving the potency to restore the wound healing
progression [36–37]. The reduced glutathione (GSH) was found to
remain decreased in disease-control animals and a partial recovery in
their content was observed in treated animals. The ACV (0.7%) + p-CA
(50 mg/kg) was found to have reduced GSH levels (p < 0.001) somewhat
closer to normal control animals indicating nearly complete healing of
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Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
the wound.
3.3.4. Histopathological studies
In the non-treatment group, the granulation tissue showed less ag­
gregation of mononuclear cells poor migration of fibroblast cells, less
formation of the new blood vessel, and less collagen deposition which is
not good, and indicates incomplete healing of the wound in the 10th
post-wounding day in the control group (Fig. 4). Whereas in the case of
the test groups, it is evident that granulation tissues had marked ag­
gregation of the mononuclear cell, increased migration of fibroblast
cells, formation of new blood vessels, and increased collagen deposition
indicating complete healing of wound in 10 post-wounding days in mice
after treatment [38]. The ACV + p-CA (0.7%; 50 mg/kg) group was
found to show better healing parameters than other treated groups [36].
4. Discussion
Acute wound healing requires the coordination of cellular and mo­
lecular reactions. Inflammation, myofibroblast accumulation, extracel­
lular matrix production, angiogenesis, re-epithelialization, and tissue
remodelling are among the overlapping phases that make up the intri­
cate molecular and cellular processes of wound healing [4]. In addition
to eradicating invasive infections and regulating the healing process,
immune cells first move to the wound site. Genes related to wounds are
activated by cut epidermal edges, allowing for mass cell migration. To
create the wound granulation tissue, local and blood-borne fibroblasts
multiply, move, supply structure and signalling cues, and deposit new
extracellular matrix. To help in wound contraction, certain fibroblasts
undergo myofibroblast differentiation. Via newly formed blood vessels
produced by angiogenesis, the wound surface is supplied with nutrients
and oxygen [39]. Wound healing is influenced by various factors out of
which this study mainly concentrated on the oxidative stress and mi­
crobial contamination parameters.
In the present research work, test drugs ACV and p-Coumaric acid
were used, which come under the category of polyphenols and have
been studied for various pharmacological properties [12,39]. In DPPH
and ABTS antioxidant assays both ACV and p-CA scavenged free radi­
cals, but the combined sample (ACV+p-CA) had lower IC50 values. The
addition of p-CA with diluted ACV has increased the polyphenolic con­
tent of ACV; hence it showed more antioxidant potential than in­
dividuals at a lower dose.
In this investigation, the antimicrobial potential of ACV is evident
based on the results of the zone of inhibition, MIC, MBC, and ex-ovo
antimicrobial studies. The presence of organic acids in ACV is believed
to contribute to its antimicrobial properties. These organic acids, being
weak in nature, exert their antibacterial effects primarily in their un­
dissociated forms. They undergo passive diffusion through the bacterial
cell wall, entering the cell at a neutral pH and subsequently dissociating
into anions and protons. The release of protons results in a decrease in
internal pH, exerting inhibitory effects on microorganisms. The study
suggests that upon the addition of ACV, there was a significant decline in
bacterial populations. This decline was associated with damage to the
bacterial nuclear material, structural and metabolic proteins, and
overall cell integrity [40,41].
p-CA, a well-known polyphenolic compound, a potent antioxidant
and a key polyphenol present in ACV had the potency to kill the gram
+ve and gram -ve bacteria. According to the MIC and MBC values, it is
most effective against S. aureus which is 62.5 and 125 µg/mL respec­
tively. This compound binds to the phosphate anion in the DNA double
helix and intercalates the groove in the DNA double helix, which might
affect the replication, transcription, and expression of the microor­
ganism [42]. In this context, ex-ovo antimicrobial study results showed
that ACV (1 mg/mL) + p-CA (2.5 mg/mL) treated live embryo samples
had the least bacterial growth after 48 h, in comparison with
non-treatment group as well as individually ACV and p-CA treated
samples.
J. Venkadakrishnan et al.
The wound contraction rate was better in combination groups than
individual ACV and p-CA (0.7%:50 mg/kg), maybe due to more poly­
phenols present as the study by Denis et al. showed the anti-
inflammatory effect of apple phenols on gastrointestinal cell inflam­
mation, which involved downregulating TNF-α and IL-6 [41,43]. These
are the pro-inflammatory cytokines that aid in the development of
chronic wounds. By reducing inflammatory cell infiltrates and reducing
proinflammatory macrophage activity, TNF-α and IL-6 neutralization at
the site of the incision greatly accelerate chronic wound healing in mice
[44]. These results suggest that p-CA and diluted ACV combination had
the potential to facilitate the movement of fibroblasts in granulation
tissue collagen and pulling forces of granulation tissue myofibroblasts
on the skin edges which also have been supported by histopathological
analysis results. Leukocytes, namely neutrophils, macrophages, and
mast cells, also have a direct involvement in the initial recruitment of
fibroblasts. Platelets are the initial cells to infiltrate the wound region,
contributing to the activation of the coagulation cascade to halt further
blood loss. Simultaneously, they establish a provisional extracellular
matrix, facilitating subsequent cell infiltration. Additionally, platelets
play a pivotal role in recruiting and activating neutrophils and macro­
phages. They stimulate fibroblasts and mesenchymal cells by secreting
transforming growth factor-β1 and platelet-derived growth factors.
Platelet-rich plasma has been shown to boost wound resolution, and
deficiencies in platelets are linked to suboptimal wound healing [44].In
this study, it was found that WBC and platelet content was elevated on
the 7th day after the wound was created but in comparison with other
groups the WBC and platelet count was lower in the combination
sample-treated group depicting the wound healing rate was better, and
also on 14th day the count came back to normal indicating the complete
healing.
Reactive oxygen species (ROS) play a pivotal role in regulating
various stages of the healing process. Minimal ROS formation is essential
for defence against invasive pathogens and cell survival signalling.
Chronic wounds, often attributed to oxidative damage, result from an
imbalance caused by either excessive ROS generation or inadequate ROS
detoxification [4]. The skin tissue’s biochemical antioxidant potential
was assessed by examining levels of superoxide dismutase (SOD) and
glutathione (GSH). The treated group exhibited higher levels of these
antioxidant marker enzymes, suggesting that the test compounds
contributed to the elevation of these crucial antioxidants. Abdulrauf
et al. reported that phenolic acids and vitamins in ACV possess the
ability to scavenge superoxide anion and free radicals, manifesting sig­
nificant antioxidant effects [45].
Analysis of the study results indicates that combination therapy
demonstrated the highest potential compared to individual components,
possibly due to the additive effect of p-Coumaric acid (p-CA) in diluted
apple cider vinegar (ACV). This combination appears to offer a more
efficacious and safer option for expediting wound healing.
5. Limitation
This study reduced the overall dosage of ACV combined with p-CA
and the combination drug therapy had a better effect than the individual
components itself. Besides these strengths, this study has some limita­
tions. In the preclinical study, the taste of the new form of diluted ACV
could not be tested and its cost may have little increment from raw ACV
due to the addition of p-CA.
6. Conclusion
To reduce the gastric irritation and other side effects of acid in ACV,
p-Coumaric acid, a well-known polyphenolic compound and a key
component of ACV has been used in combination with ACV to improve
the wound healing effect. Vinegar is acidic and can cause some gastric
irritations and other side effects. To reduce the dose of apple cider
vinegar and also to improve the wound healing effect, p-Coumaric acid,
9
Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
a well-known polyphenolic compound and a key component of ACV,
was taken to assess its wound healing potential individually as well as in
combination. Throughout the study, this combination sample showed
additive effects in antioxidant, antimicrobial and wound healing po­
tential in a diluted and safe dose of ACV. So, the combination can be the
better option to heal wounds faster and at a safer dose. It can proceed for
clinical study in future and can further be commercialized for daily use
with food items safely.
Consent for publication
All the authors have provided their consent for publication of this
manuscript.
Ethics approval
All animal experiments were approved by the Institutional Animal
Ethical Committee (Reg. no. 1972/PH/BIT/136/21/IAEC). The experi­
ments complied with the WMA Statement on animal use in biomedical
research.
Funding
The authors declare that no funds or grants were received during the
preparation of this manuscript.
CRediT authorship contribution statement
Jegadheeswari Venkadakrishnan: Conceptualization, Formal
analysis, Methodology, Software. Amrita Chatterjee: Investigation,
Writing – review & editing. Rajdeep Saha: Conceptualization, Meth­
odology. Kaberi Chatterjee: Investigation, Writing – review & editing.
Prashanta Kumar Deb: Conceptualization, Resources. Biswatrish
Sarkar: Supervision. Papiya Mitra Mazumder: Conceptualization,
Methodology, Resources, Supervision, Writing – review & editing.
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.
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