Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
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/).
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 Keywords:
Apple cider vinegar p-Coumaric acid Antioxidant Ex-ovo Antimicrobial Wound healing
A B S T R A C T
Background: Apple cider vinegar (ACV) is most commonly used in sweet and sour Chinese cuisine. This study aimed to overcome the side effects of raw ACV, by increasing the concentration of p-Coumaric acid (p-CA), a polyphenolic component of ACV. The combination (diluted ACV with p-CA) has also been compared with in- dividual raw ACV and p-CA to confirm if the overall dosage of the ACV can be reduced to avoid side effects and if the combination therapy had any better effect than the individual component itself.
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.
Abbreviations
ACV Apple cider vinegar.
p-CA p-Coumaric acid.
MIC Minimum inhibitory concentration.
MBC Minimum bactericidal concentration.
ZOI Zone of inhibition.
WBC White blood cells.
SOD Superoxide dismutase.
GSH Reduced glutathione.
ROS Reactive oxygen species.
1. Introduction
The wound is a rupture in the cellular, anatomical, or functional continuity of living tissues [1]. Healing is a complicated process which requires a proper approach to restoring anatomical continuity and functional status of the skin. Healing is delayed when there is microbial contamination and increased oxidative stress [2]. The common causa- tive organisms of wound infections are Streptococcus pyogene,
* Corresponding author.
E-mail address: [email protected] (P.M. Mazumder).
1 The first two authors contributed equally.
Contents lists available at ScienceDirect
Pharmacological Research - Modern Chinese Medicine
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https://doi.org/10.1016/j.prmcm.2024.100364
Received 1 December 2023; Received in revised form 11 January 2024; Accepted 15 January 2024
Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, etc. [3]. Excessive and unregulated oxidative stress plays a critical role in the pathophysiology of chronic non-healing wounds by maintaining and deregulating inflammatory processes [4]. Alternative 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 culture of infection is used to assess the effect of drugs in the infected embryos for their antimicrobial potential [6]. This model is simple, rapid and low-cost, and it more closely resembles the in vivo condition than typical antimicrobial testing based on agar plates and dilution experiments.
Apple cider vinegar (ACV) is popular in Chinese food preparation for its distinct fruity tone which is balanced by its acidity and can be pro- duced by fermenting apple juice containing different organic acids and compounds [7,8]. ACV so far has been proven for its effectiveness in antibacterial, antifungal, antiviral, cytotoxic properties, neuro- protective, anti-obesity and anti-diabetic properties. It has been claimed to provide a variety of health advantages, including a reduction in the risk of cardiac disorders and weight loss [9–13]. The wound healing property of ACV has been explored by Ali et al. that it had the potential to decrease the bacterial load in the infected wound similar to the extent of the standard drug cefotaxime [14]. ACV is popular in traditional herbal Chinese medicine effecting in lowering blood pressure, and also as an alternate treatment method to treat fungal infections including vaginal Candidiasis infection and all tineas infections [15–17]. ACV has been found to have side effects in large doses or chronic usage, especially in an undiluted form. Due to its acidic nature, apple cider vinegar may dissolve tooth enamel and cause cavities, delay stomach emptying, and irritate the oesophagus when consumed in excess and undiluted [18–20].
p-coumaric acid (p-CA) or 4-Hydroxycinnamic acid, a phenolic compound present in ACV is a major component present in different fruits, vegetables and coffee [8]. p-CA was proven for its anti-oxidant, antimicrobial, anti-inflammatory, hypolipidemic and immunomodula- tory effects [21,22]. In a very recent study, p-CA was also been studied
for its chronic wound healing property, where the inflammatory phase was reduced by p-CA, which improved angiogenesis, tissue repair, and collagen generation throughout healing [23]. Another study suggested that ε-caprolactone-p-CA copolymers regenerated skin [24]. As dis- cussed earlier although having a lot of therapeutic benefits, chronic and excessive usage of ACV can cause side effects. This study aimed to overcome this issue, by increasing the concentration of p-CA, the phenolic compound already present in ACV. ACV p-CA combination was assessed for its antioxidant, antimicrobial, and wound-healing proper- 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 better effect than the individual component itself.
2. Methods
2.1. Chemical and reagents
Apple cider vinegar was procured from Dabur Company Ltd. (An ISO 27001:2013 certified company). p- Coumaric acid, DPPH (2, 2-diphenyl- 1-picryl-hydrazyl-hydrate), ABTS (2, 2
′
-azino-bis (3-ethylbenzothiazo- line-6-sulfonic acid)) were procured from Sigma Aldrich, hematoxylin from leica biosystems and eosin from ACS Chemicals.2.2. Microorganisms
Two Gram-positive bacteria including Staphylococcus aureus MTCC 96 (S. aureus), Bacillus subtilis MTCC 441 (B. subtilis), and two Gram- negative bacteria including Escherichia coli MTCC 443 (E. coli) and Pseudomonas aeruginosa MTCC 741 (P. aeruginosa) were procured from the Institute of Microbial Technology Chandigarh, India. On Luria- Bertani medium (LB media) purchased from HIMEDIA, all bacteria were grown at 37 ◦C. As a standard, HIMEDIA-purchased streptomycin sulphate was utilized.
2.3. Fertilized eggs
The fertilized eggs were purchased from the Ranchi College of Vet- erinary Sciences and Animal Husbandry, Kanke, Ranchi, Jharkhand, India.
2.4. Animal care and handling
Swiss male albino mice weighing 25–30 g of either sex were used in the study. Animals were procured from Laboratory Animal House of Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India (1968/
PO/Re/S/17/CPCSEA) and were maintained in polyacrylic cages with standard housing conditions of temperature (24–27 ◦C), 12:12 light:
dark cycles and humidity (60–65%). They underwent seven days of acclimatization. Dry pellets were available as food, and water was available at all times. The experimental study (Protocol No. 1972/PH/
BIT/136/21/IAEC) was approved by the Institutional Animal Ethics Committee of Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India.
2.5. Treatment schedule
The total 20 animals were randomly divided into 5 groups (n =4 each). A wound was created and the doses of test samples were given for 14 days to animals via the oral route once per day before administering food. The wound was created and normal saline was administered each day in the diseased control group animals [25].
2.6. Animal groups
The treatment in different groups was as follows: [26–27]
Table 1
IC50 Values of various samples by DPPH assay and ABTS assay.
Samples IC50 (µg/mL) in DPPH assay IC50 (µg/mL) in ABTS assay Ascorbic acid 44.91 ±0.35 34.89 ±0.61
ACV 112.51 ±0.56b 93.37 ±0.35b
p-CA 82.84 ±0.35b 81.43 ±0.64b
ACVþp-CA (1:1) 73.29 ±1.00b 69.43 ±0.46b
Values are expressed as mean ±SD (n = 3), ap < 0.01, bp < 0.001 when compared to Ascorbic acid, One-way ANOVA followed by Tukey’s multiple comparison test.
Table 2
Zone of Inhibition of various samples.
Bacteria Zone of inhibition (cm) [Including 0.6 cm diameter disc]
ACV p-CA ACV +p-
CA Streptomycin
(Standard) S. aureus 1.15±
0.13a 1.48±0.08 1.78 ±
0.02ns 1.95 ±0.05 B. subtilis 1.17±
0.13c 1.31 ±
0.08c 1.9 ±0.08a 2.32 ±0.08 P. aeruginosa 1.2 ±
0.05c 1.45±
0.03ns 1.65 ±
0.03ns 1.78 ±0.04
E. coli 1.05±
0.98b 1.42±0.02c 1.38 ±
0.02c 2.13 ±0.32 Values are expressed as Mean ±SD (n =3).
ap <0.05.
b p <0.01.
cp <0.001, ns; non-significant when compared to standard. One way ANOVA followed by Tukey’s multiple comparison test.
J. Venkadakrishnan et al.
Group 1: The disease control or non-treatment group received normal saline only (0.9% NaCl solution)
Group 2: wounded animals treated with Apple cider vinegar (0.7%, p.o.)
Group 3: wounded animals treated with p-Coumaric acid (50 mg/kg BW, p.o.)
Group 4: wounded animals treated with ACV +p-CA (0.35% +25 mg/kg BW, p.o.)
Group 5: wounded animals treated with ACV +p-CA (0.7% +50 mg/
kg BW, p.o.)
2.7. Antioxidant assay
2.7.1. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay Different concentrations of Ascorbic acid, ACV, p-CA, and ACV +p-
CA (1:1) were prepared (10, 20, 40, 80, and 160 µg/mL) in water and the DPPH assay was carried out [28]. Briefly, 100 µL of freshly prepared 0.2 mM DPPH solution in methanol was mixed with 50 µL of different concentrations of sample. The reaction was carried out in triplicate and the decrease in absorbance with an increase of concentration was measured at 517 nm after 30 min incubation in the dark using a UV–visible spectrophotometer.
The% inhibition was calculated using the formula, Inhibition%= (Ac− As/Ac) ×100
Where, Ac =absorbance of the control, As =absorbance of the sample IC50 was calculated.
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/
mL) p-CA (µg/
mL) ACV (µL/mL) +p-CA
(µg/mL) STD (µg/
mL) ACV (µL/
mL) p-CA (µg/
mL) ACV (µL/mL) +p-CA
(µg/mL) STD (µg/
mL) Gram-
positive S. aureus 12.5 62.5 12.5: 31.25 6.25 25 125 25: 62.5 12.5
B. subtilis 25 62.5 25: 62.5 12.5 50 250 50: 125 50
Gram-
negative P. aeruginosa 25 125 25:62.5 50 50 250 50:125 100
E. coli 12.5 125 25:62.5 25 25 250 50:125 50
Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
2.7.2. 2, 20-azino-bis (3-ethylbenzothiazoline) 6-sulfonic acid (ABTS) radical scavenging assay
ABTS radical scavenging assay was performed according to the 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 keeping it in the dark for 12 to 16 h at room temperature. Before initi- ating the assay, methanol was added to dilute the stock of ABTS solution to an absorbance of 0.7 at 734 nm. 100 µL of ABTS reagent was mixed with a 50 µL sample of five different concentrations (10, 20, 40, 80, and 160 µg/mL) and incubated in the dark for 30 min at room temperature [28]. Absorbance was measured at 734 nm and IC50 was calculated as above.
2.8. Antimicrobial assay 2.8.1. Zone of inhibition
For the determination of the test samples to possess any antimicro- bial activity, a zone of inhibition was performed using the Agar disc 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 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.
Table 4
Percentage of wound contraction.
Day Percentage of wound contraction (%) Non-
treatment ACV (0.7%) treated group
p-CA (25 mg/kg) treated group
ACV+p-CA (0.35%:25 mg/
kg) treated group
ACV+p-CA (0.7%:50 mg/
kg) treated group 3 12.34 ±
0.37 18.48 ±
0.275c 18.75 ±
0.28c 19.34 ±0.99c 18.20 ±0.32c 6 25.86 ±
1.48 30.79 ±
0.46a 30.79 ±
0.46a 31.31 ±0.80a 36.40 ±0.64c 9 37.02 ±
1.12 56.89 ±
0.64c 49.27 ±
0.74c 47.70 ±1.49c 60.68 ±1.89c 12 53.87 ±
0.80 81.53 ±
0.27c 81.50 ±
0.25c 73.20 ±1.089c 81.80 ±0.32c 14 64.65 ±
0.85 92.84 ±
0.09c 91.99 ±
1.25c 88.03 ±0.33c 93.93 ±0.10c Values are expressed as Mean ±SEM (n =4), ap <0.05, bp <0.01, cp <0.001 when all the other groups compared to the non-treatment group. One way ANOVA followed by Tukey’s multiple comparison test.
J. Venkadakrishnan et al.
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.
Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364
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
did not exhibit visible growth after 24 h of incubation at 37 ◦C. The growth of the microorganism was indicated by turbidity. To determine MBC, on LB agar medium, 100 μL of the previously grown medium up to MIC concentration were plated and incubated for 24 h at 37 ◦C. MBC of the test samples is the lowest concentration at which no microbe could survive [31]. All the MIC and MBC analyses were carried out in triplicate.
2.8.3. Ex-ovo study for antimicrobial assessment
The fertilized eggs were taken and an ex-ovo set up for a model of infection was created to assess its antimicrobial potential in a live em- bryo. After the CAM was infected with S. aureus, test sample-infused discs were placed on CAM near the spot where the infection was created. A solution of 1 mg/mL of ACV, 2.5 mg/mL of p-CA and ACV+p- CA (1 mg/mL: 2.5 mg/mL) in sterile water was prepared, and 6 mm diameter filter paper discs were dipped into it. The survival of the em- 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 evaluated at 24 h and 48 h after infection by examination of allantoic fluid by streaking in a sterile agar plate [6].
2.9. In-vivo wound healing activity 2.9.1. Induction of excision wound
Diethyl ether was used to anaesthetize the mice. Hair was removed 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 region, and an excision wound was created by taking a thick piece of skin out from a 150 mm square circular area from the designated shaved.
2.9.2. Determination of wound contraction
Following the excision, wound edges were drawn on translucent graph paper with a millimetre scale and measured with a calibre with a 1/20 mm precision at every 3-day intervals. Measurements were carried out for a total of 14 days [32]. The healed area was calculated by sub- tracting the contracted wound area from the initial wound area.
Percentage wound contraction = (Healed area/ Total area) × 100
2.10. White blood cells (WBC) and platelet count
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 Table 5
Effect of various test samples on WBC and platelet count on the 0th, 7th and 14th day after the creation of wound.
Groups WBC Count (103/mm3) Platelet count (105/mm3) Day 0 Day 7 Day 14 Day 0 Day 7 Day 14 Non-treatment 6.2 ±
0.26 11.95
±0.18 7.54 ± 0.11 5.06
±0.26 11.65
±0.15 7.44
±0.21 ACV (0.7%) 6.37
±0.16 10.4 ±
0.46c 7.36 ± 0.64ns 4.18
±0.14 8.53 ± 0.14c 5.23
± 0.07c p-CA (25 mg/
kg) 4.68
±0.23 9.45 ±
0.23c 7.36 ± 0.17ns 4.23
±0.11 7.48 ± 0.27c 4.47
± 0.27c ACV þp-CA
(0.35%: 25 mg/kg)
6.23
±0.27 8.18 ±
0.13c 5.64 ± 0.10b 4.49
±0.14 7.46 ± 0.17c 4.47
± 0.11c ACV þp-CA
(0.7%: 50 mg/kg)
5.15
±0.27 7.99 ± 0.13c 4.6 ±
0.21c 4.63
±0.21 6.47 ± 0.17c 4.66
± 0.27c Values are expressed as Mean ±SEM (n =4), ap <0.05, bp <0.01, cp <0.001 when compared to the non-treatment group. One way ANOVA followed by Tukey’s multiple comparison test.
Table 6
Effect on the SOD and GSH levels in the treatment skin tissue post-14th day.
Sl. No. Groups % Inhibition for SOD
determination GSH/mg
protein
1. ACV (0.7%) 46.78 ±0.73c 0.099 ±
0.006b
2. p-CA (50 mg/kg) 34.45 ±1.54b 0.094 ±
0.001a 3. ACV (0.35%) +p-CA (25
mg/kg) 41.09 ±1.21c 0.119 ±
0.009b 4. ACV (0.7%) +p-CA (50
mg/kg) 53.37 ±1.76c 0.18 ±
0.005c
5. Non-treatment group 22.6 ±1.36 0.055 ±
0.003 Values are expressed as Mean ±SEM (n =4).
ap <0.05.
b p <0.01.
cp <0.001 when compared to the non-treatment group. One way ANOVA followed by Tukey’s multiple comparison test.
J. Venkadakrishnan et al.
any statistical differences between the groups.
3. Results
3.1. Antioxidant assay
3.1.1. DPPH and ABTS radical scavenging assay
The lower the IC50 value higher the antioxidant activity. In both the DPPH and ABTS assay, the combination ACV +p-CA had a better effect
(p <0.001) than the individual components and the standard ascorbic acid had the highest antioxidant potential (Table 1).
3.2. Antimicrobial property
3.2.1. Determination of zone of inhibition by disc diffusion method The standard drug streptomycin (STRP) had the highest zone of in- 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 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.
Pharmacological Research - Modern Chinese Medicine 10 (2024) 100364 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
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,
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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