Comparative efficacy of andrographolide and hordenine in the treatment of methicillin resistant Staphylococcus aureus infected wounds: a multifaceted approach

SJ Vidyavarsha; Bibu John Kariyil; Varsha Unni; R Anoopraj

doi:10.1016/j.jaim.2025.101291

. 2026 Feb 12;17(1):101291. doi: 10.1016/j.jaim.2025.101291

Comparative efficacy of andrographolide and hordenine in the treatment of methicillin resistant Staphylococcus aureus infected wounds: a multifaceted approach

SJ Vidyavarsha ^a, Bibu John Kariyil ^a,^⁎, Varsha Unni ^b, R Anoopraj ^c

PMCID: PMC12924907 PMID: 41687516

Abstract

Background

Wound healing is a multifaceted biological process hindered by infections, especially the methicillin-resistant Staphylococcus aureus (MRSA), which resists antibiotics. Natural compounds such as andrographolide and hordenine are being explored as alternative therapeutics.

Objective

To compare the efficacy of andrographolide and hordenine in promoting wound healing in MRSA-infected infectious wounds using in vitro and in vivo methods.

Methods

L929 fibroblast-based MTT and scratch assays assessed cytotoxicity and wound closure potential. CAM assay evaluated angiogenic activity. Wistar rats with MRSA-infected excision wounds were treated topically, followed by biochemical analysis (hydroxyproline and hexosamine levels) and histopathological evaluation.

Results

The study revealed that andrographolide showed significantly (P < 0.001) higher cellular viability than hordenine and significantly reduced the cellular gap in L929 cells by day 3. Andrographolide increased blood vessels significantly (P < 0.001) more than hordenine. Clinical observations indicated reduced pus in andrographolide and hordenine-treated groups than in the control group, with the ceftriaxone-sulbactam group showing the lowest pus formation. Hexosamine levels were comparable in both treatments, while hydroxyproline was higher in the hordenine group. Histopathology showed hordenine-treated animals had faster wound healing and enhanced epithelialisation compared to andrographolide-treated animals.

Conclusion

Andrographolide and hordenine exhibited wound healing activity in MRSA-infected wounds, promotes fibroblast proliferation and migration, as well as fostering angiogenesis.

Keywords: MRSA-infected wound, Fibroblast migration, Angiogenesis, Andrographolide, Hordenine, ECM remodeling

1. Introduction

Wound healing is a multidimensional process that involves four overlapping phases, viz. hemostasis, inflammation, proliferation, and remodeling, that collectively works to restore the damaged or wounded tissue. Despite advances in treatment, wounds remain still a challenging problem that necessitates proper care and management to avoid negative outcomes in both humans and animals. Infections and inflammatory responses are among the most common and unavoidable impediments to wound healing, particularly in chronic wounds. The bacterial pathogens most frequently associated with wound infections include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Proteus species, Streptococcus species, and Enterococcus species. Another critical factor in wound repair is angiogenesis, which involves capillary sprouting and the formation of a microvascular network regulated by vascular endothelial growth factor (VEGF), angiopoietins, fibroblast growth factor, and transforming growth factor-β. However, poorly healing wounds often exhibit impaired or abnormal angiogenesis.

According to recent estimates, multidrug-resistant bacteria are responsible for 67.1 % of infected wounds. Multidrug resistance has been observed in 68.8 % of Gram-positive bacteria and 66.0 % of Gram-negative bacteria [1]. Staphylococcus aureus that exhibits multidrug resistance is one of the most challenging bacterial infections to treat. Methicillin-resistant S. aureus (MRSA) is the most common multidrug-resistant S. aureus in India, with prevalence rates ranging from 25 % in the western region to 50 % in the southern region [2]. High resistance rates in S. aureus highlight the urgency for early detection and evidence-based treatment to prevent infections [3].

The indiscriminate use of antibiotics in both humans and animals has led to the emergence of MRSA strains resistant to nearly all commercially available antibacterial agents. Moreover, the systemic toxicity associated with the administration of high doses of antibiotics remains a major concern. The lack of development of newer antibiotics in recent years further exacerbates the problem. In this context, exploring natural materials or compounds with antibacterial and anti-inflammatory properties—and incorporating them into wound dressings—may offer promising therapeutic benefits, particularly for wounds infected with MRSA.

Andrographolide, a labdane diterpenoid isolated from Andrographis paniculata, a plant widely used in Ayurvedic medicine for treating fever, infections, and skin disorders. Its wound healing, antibacterial, and anti-inflammatory properties have been documented in both traditional and modern pharmacology. Studies have demonstrated its anti-angiogenic effects in the context of wound healing [4]. Hordenine, a phenethylamine alkaloid found in the seeds of Hordeum vulgare, a cereal crop with traditional medicinal applications in Unani and folk systems for treating inflammation and infection. Research indicates that hordenine can inhibit biofilm formation, reduce the production of acylated homoserine lactones (AHLs), and suppress motility and virulence factors of Pseudomonas aeruginosa, suggesting its effectiveness against this pathogen [5]. Additionally, hordenine has been shown to inhibit the secretion and activation of inflammatory cytokines in wounds, which may contribute to its wound healing potential [6]. However, there is no documented evidence to date indicating a significant influence of hordenine on angiogenesis during wound healing.

While both compounds have demonstrated bioactivity in various in vitro and in vivo settings, conflicting data exist regarding their antimicrobial effects against MRSA. A recent in vitro study [7] reported that andrographolide failed to inhibit MRSA, and hordenine exhibited a minimum inhibitory concentration (MIC) ≥ 1000 μg/mL. This highlights the need to investigate their wound-healing effects in MRSA-infected models through host-mediated mechanisms, rather than direct antimicrobial activity alone.

Hence, the present study is envisaged to evaluate the efficacy of hordenine and andrographolide in various models, highlighting the need for alternative treatments in the context of escalating antibiotic resistance and impaired wound healing.

2. Materials and methods

2.1. Reagents and chemicals

Andrographolide 98 per cent and Hordenine ≥97.0 per cent were procured from M/s Sigma-Aldrich India Ltd., Bangalore, India. Brain heart infusion agar base, Muller-Hinton agar base, Muller-Hinton broth no.2 control cations, resazurin sodium extra pure AR dye, thaizolyl blue, sodium carbonate anhydrous, l-hydroxyproline extrapure, 99 per cent glucosamine hydrochloride extrapure, 99 per cent and acetylacetone were purchased from M/s Sisco Research Laboratories Pvt. Ltd., Chennai, India. HiCrome™ MeReSa Agar Base, MeReSa selective supplement (FD229), cefoxitin supplement (FD259), paraformaldehyde solution, gentamicin solution, Dulbecco's phosphate buffered saline and diluent for DNA extraction were procured from M/s Himedia Laboratories Pvt. Ltd., Mumbai, India. Haematoxylin and eosin stain, Weigert's iron haematoxylin, Beibrich scarlet acid fuchsin, aniline blue, phosphomolybdic and phosphotungstic acid were procured from M/s Nice chemicals, Pvt. Ltd., Ernakulam, India. Ketamine (Zokent®), Xylazine (Xylaxin®) and Buprenorphine hydrochloride (Fragesic 1®) injections were purchased from M/s Green Pharmaceuticals, Thrissur, India. Perchloric acid, hydrochloric acid and dimethyl sulfoxide (DMSO) were purchased from M/s Merck Life Science Pvt. Ltd., Mumbai, India.

2.2. Cell culture

The cell line used for the study, L929 mouse skin fibroblasts, was procured from National Centre for Cell Science (NCCS), Pune, India. This cell line was maintained in a humidified incubator at 37 °C with 5 % CO 2. After attaining 70 % confluency, L929 cells were subcultured by enzymatic digestion with 0.25 % trypsin-1mM EDTA solution and these trypsinised cells were used for the study.

2.3. Antibacterial activity assay

The antibacterial activity of andrographolide and hordenine against the MRSA strain used for infection in the current study was previously evaluated and published by the same research group (Vidyavarsha et al., 2022). The study employed both the Kirby-Bauer agar disc diffusion method and the modified resazurin microtiter plate assay.

In the disc diffusion assay, neither andrographolide nor hordenine produced any inhibition zones against Staphylococcus aureus or MRSA isolates at concentrations ranging from 62.5 to 1000 μg/mL. However, the modified resazurin microtiter plate assay revealed that hordenine exhibited a minimum inhibitory concentration (MIC) of 1000 μg/mL, while andrographolide did not demonstrate inhibitory activity at the tested concentrations.

These findings confirm the MRSA strain's resistance to andrographolide and partial sensitivity to hordenine in vitro. The results support the hypothesis that their wound healing effects observed in vivo may be due to non-antibiotic mechanisms, such as anti-inflammatory, immunomodulatory or pro-regenerative pathways.

Full details of this assay are available in the published report [7].

2.4. MTT assay

The preparation of stock stock solutions used for the study is described in Supplementary File S1. The cell viability of andrographolide and hordenine was studied using MTT assay [8]. Trypsinised L929 mouse skin fibroblast cells were seeded at a density of 5 × 10³ cells per well in 200 μL medium and were allowed to attach overnight in a CO₂ incubator at 37 °C. Cells were treated with positive control (three per cent boric acid), andrographolide and hordenine at concentrations of 5, 10, 20, 40, 80, 100 and 160 μg/mL for a period of 24 h. After the treatment, 20 μL of MTT (5 mg/mL) in 150 μL medium was added and incubated at 37 °C for 4 h after removing the medium containing andrographolide, hordenine and three per cent boric acid. Then, the media with MTT was removed and 200 μL of DMSO was added and read at 570 nm in an microtitre plate reader (Varioskan flash, Thermo Fischer Scientific, Finland) The percent inhibition was calculated using the formula:

Per cent viability = (Average absorbance of control cells − average absorbance of treated cells/average absorbance of untreated cells) × 100. The experiment was repeated six times and the effective concentration 50 (EC₅₀) values of andrographolide and hordenine were calculated using Graph Pad Prism 5.

2.5. Scratch assay

The L929 mouse skin fibroblast cells were seeded in 12-well cell culture plate and cells were allowed to attach overnight in a CO₂ incubator at 37 °C. Linear scratch was made in confluent cell monolayer using 200 μL pipette tip. Cell debris were washed out with RPMI. Andrographolide at EC₅₀ of 13.16 μg/mL, hordenine at EC₅₀ of 7.17 μg/mL and three per cent boric acid (positive control) were used for the study. After addition of test substances and three per cent boric acid, images of cellular gap were captured daily for 5 days on inverted microscope (Carl Zeiss Primovert, Germany). Images were captured with calibrated scale bars using ImageJ software [9]. The assay protocol is detailed in Supplementary File S1.

2.6. Chicken embryo chorioallantoic membrane (CAM) assay

CAM assay performed as per method [9] with modifications. The detailed assay protocol used for the study is provided in Supplementary File S2. Ten-day-old fertilized chicken embryos were treated with andrographolide and hordenine at concentrations of 1000, 500, 250 μg/mL. The control groups received 5 % and 3 % DMSO as vehicles. Eggs were incubated at 37 °C for 72 h. After incubation, the membrane was fixed with 4 % paraformaldehyde-PBS which was further incubated at 4 °C overnight. The CAMs were excised, refixed and the images were captured. The number of primary, secondary, tertiary and quaternary blood vessels in control-treated and test substances treated embryos were carried out. The blood vessel branching was counted using ImageJ software by a blinded assessor. Reproducibility was confirmed by repeating the assay in triplicate.

2.7. Animal experiments

The study was approved by the Institutional Animal Ethics Committee (sanction number: IAEC/COVAS/PKD/11/2021) of College of Veterinary and Animal Sciences, Pookode, Kerala, India. The study was carried out in male Wistar rats (160–190 g) using an excision wound model inoculated with a characterised MRSA isolate. Animals were housed individually in polypropylene cages with standard diet and water ad libitum and acclimatised for seven days prior to experimentation. After 7 days of acclimatisation, animals were randomly allocated to treatment groups using Research Randomizer Software. No animals were excluded during the study.

Full-thickness wounds (500 mm², 2 mm depth) were created on the dorsal thoracic region under anaesthesia with ketamine (70 mg/kg) and xylazine (5 mg/kg). Wounds were inoculated with 5 × 10⁷ CFU/mL of MRSA [10], and the animals were housed individually. To confirm infection, wound swabs were collected on days 1, 7 and 15 and streaked on HiCrome MeReSa agar for colony forming unit (CFU) quantification. Colonies were counted after 24 h to assess bacterial burden. On day 2, animals were randomly assigned to four groups (n = 6): Group I received soft paraffin, while Groups II, III, and IV were treated topically with standard drug, ceftriaxone:sulbactam (2:1), 1 % andrographolide and 1 % hordenine respectively for 15 days. Wound healing was monitored by tracing wound areas on days 0, 3, 6, 9, 12, and 15. Percentage wound contraction was calculated as: Reduction (%) = (Wound area on day 0 − Wound area on day n)/Wound area on day 0) × 100. Wound area measurements and scoring were performed by blinded assessors. On day 15, animals were euthanised, and the wound tissues were collected for evaluating hydroxyproline and hexosamine levels. Histopathological and special staining techniques were also performed. The detailed protocol used for performing the animal experiments and preparation of ointments are provided as Supplementary Files S3 and S4, respectively.

2.8. Hydroxyproline estimation

Hydroxyproline estimation followed Woessner's (1961) method with minor modifications [11]. Skin from the granulated wound area was collected, dried and hydrolyzed. After hydrolysis, the samples were evaporated, dissolved in 2 mL distilled water. Standard hydroxyproline solutions were prepared and the samples and standard solutions were treated with chloramine T and perchloric acid and spectrophotometrically analyzed at 557 nm. Hydroxyproline content in the tissue sample was calculated as described by Carlson (2014) [12]. The detailed assay protocol followed is provided as Supplementary File S5.

2.9. Hexosamine estimation

Hexosamine estimation followed Eisele and Eichelberger's (1945) method with modifications [13]. Skin from the granulated wound area collected, dried and hydrolyzed. The obtained residue was dissolved in 2 mL distilled water after evaporation. Standard glucosamine hydrochloride solutions (0.187–96 μg/mL) were prepared. The samples and standard solutions were treated with acetyl acetone-sodium carbonate solution, ethanol, and Ehrlich's reagent. The formation of purple-red color was read spectrophotometrically at 530 nm. Hexosamine content in the tissue sample was calculated as described by Carlson (2014) [12]. The detailed assay protocol is provided as Supplementary File S6.

2.10. Histopathological examinations

Tissue samples were collected in 10 % neutral buffer formalin and subjected to fixation for minimum of 48 h. The tissues were then washed overnight under running tap water, dehydrated through ascending grades of alcohol and cleared in xylene before being embedded in paraffin wax blocks for sectioning. Five-micron sections were taken and mounted on clean glass slides coated with Mayer's egg albumin and air dried. The dried sections were cleared in xylene before staining by routine haematoxylin and eosin stain and modified Masson's trichrome staining to assess the formation of granulation tissue, inflammatory cell infiltration, collagen formation and reepithelialisation [14]. Histopathological evaluation was carried out by Dr. Anoopraj R, Veterinary Pathologist who was blinded to the treatment groups. A semi-quantitative scoring system was used for parameters including epithelial thickness (+ to +++), fibroblast activity, angiogenesis, and inflammatory infiltration.

2.11. Statistical analysis

Statistical analysis was performed using involved SPSS v24.0 (Armonk, NY: IBM Corp). For cell viability and number of blood vessels, statistical analysis between group and between concentrations was done using two-way ANOVA followed by least significant difference test on least square means as pair wise comparison. For cellular gap, tissue hydroxyproline and tissue hexosamine, statistical analysis between groups was conducted using one way ANOVA followed by Duncan multiple range tests. For cellular gap between days within each group and percentage of wound contraction between days within each group, statistical analysis was done using repeated measures ANOVA followed by least significant difference test on least square means.

3. Results

3.1. MTT assay

Andrographolide exhibited significantly (P < 0.001) higher mean cell viability at concentrations from 20 to 160 μg/mL than the concentrations at 10, 5 μg/mL and the positive control. Notably, cell viability was lower at 10 μg/mL, while at 5 μg/mL, it was substantially lower than at 20 μg/mL but higher than at 10 μg/mL. No significant difference was observed between the positive control and andrographolide-treated cells at 5 μg/mL. The mean cell viability for various concentration of andrographolide and hordenine is presented in Supplementary file S7. Table 1 and Fig. 1.

Fig. 1 — Per cent cell viability of 3 % boric acid (positive control), andrographolide, and hordenine in L929 fibroblast cells assessed using MTT assay.

Andrographolide and hordenine exhibited concentration-dependent cytotoxicity. Andrographolide showed significantly higher cell viability (P < 0.001) than hordenine at all concentrations tested, with EC₅₀ values of 13.16 μg/mL and 7.17 μg/mL, respectively. The mean cell viability of hordenine was significantly (P < 0.001) higher at concentrations of 20–160 μg/mL than the concentrations at 10 and 5 μg/mL. Positive control treated L929 cells had significantly (P < 0.001) higher cell viability than hordenine at all concentrations. There was no significant difference between the mean cell viability at 10 and 5 μg/mL of hordenine treated L929 cells.

3.2. Scratch assay

The reduction in the cellular gap between days viz. day one and day two was not significant. On day three, a highly significant (P = 0.010) reduction in the cellular gap in the scratch treated with andrographolide was observed. Similarly, hordenine (P = 0.002), control (P < 0.001) and positive control (P < 0.001) treated scratches showed the significant reduction in the cellular gap on day three (Fig. 2a and b.).

Fig. 2 — (a) Scratch assay images on day 1 and 2 showing fibroblast migration under different treatments. (b) Scratch assay images on day 3 and 4. (c) Quantitative comparison of cellular gap reduction between groups and days.

By day 3, the cellular gap in andrographolide-treated cells was significantly reduced (P = 0.002) compared to hordenine. Boric acid alone showed a marginal effect. By day 3, the cellular gap in andrographolide-treated cells was significantly reduced (P = 0.002) compared to hordenine. Boric acid alone showed a marginal effect.

On day one, between groups, there was no significant reduction in the cellular gap in any of the four groups. On day two, positive control showed a highly significant (P = 0.008) reduction in the cellular gap than hordenine and andrographolide. Among the four groups, andrographolide showed the least significant reduction in the cellular gap. There was no significant difference in the reduction of the cellular gap between control and positive control. Hordenine had a significantly lower reduction than positive control but significantly higher reduction in the cellular gap than andrographolide. On day three, hordenine showed a significantly (P < 0.001) higher reduction in the cellular gap. The reduction of the cellular gap between hordenine, control and positive control was comparable. Among the four groups, andrographolide had the lowest significant reduction in the cellular gap (Table 1 and Fig. 2c). The results of the present study revealed that andrographolide and hordenine treated L929 cells reduced the cellular gap significantly (P < 0.001) at day 3. Among the two treatments, the reduction in cellular gap was significant (P = 0.002) for hordenine treated L929 cells (Fig. 2c).

Table 1.

In vitro evaluation of wound healing activity of andrographolide and hordenine using scratch assay in L929 mouse skin fibroblast cells: comparison of cellular gap between groups and between days, mm.

Days	Control	Positive control	Andrographolide	Hordenine	F-value (P-value)
Day 1	351.13 ± 17.4^a	283.87 ± 4.89^a	321.79 ± 20.46^a	335.96 ± 22.2^a	2.684 (0.117)
Day 2	126.71 ± 4.99^bBC	119.83 ± 12.57^bC	225.48 ± 29.78^abA	181.21 ± 11.09^bAB	8.291^a (0.008)
Day 3	64.15 ± 3.26^cB	46.33 ± 2.28^cB	128.53 ± 5.76^bA	69.09 ± 12.65^cB	24.397^a (<0.001)
F-value (P-value)	150.338^a (<0.001)	190.18^a (<0.001)	17.635^b (0.010)	46.685^a (0.002)

Open in a new tab

NS = non-significant; mm millimetre; Means having different small letter as superscript differ significantly within a column; Means having different capital letter as superscript differ significantly within a row.

Significant at 0.01 level.

Significant at 0.05 level.

3.3. Chicken embryo chorioallantoic membrane (CAM) assay

In the present study at 1000 μg/mL, the number of primary, secondary, tertiary, and quaternary blood vessels rose considerably in the andrographolide and hordenine treated groups compared to other concentrations and controls (Supplementary file S.8. Table 3., S.9. Table 4., S.10. Table 5. and S.11. Table 6.). Andrographolide treatment at 500 μg/mL had a lower significant (P < 0.001) number of primary, secondary, tertiary and quaternary blood vessels than at 1000 μg/mL and at 250 μg/mL, it showed the least significant (P < 0.001) number of primary and secondary blood vessels. The number of primary and secondary blood vessels was not significantly different between the 250 μg/mL treated and control-treated CAM's. In terms of the number of tertiary and quaternary blood vessels, there was a substantial difference between the 250 μg/mL treated and control-treated CAM's. Blood vessels showing the angiogenic potential of control (5 % DMSO) and andrographolide in chicken embryo chorioallantoic membrane are depicted in Fig. 3 (a.1.) and 3 (a.2.), respectively. Hordenine treatment at 500 μg/mL had a lower significant (P < 0.001) number of primary, secondary, tertiary and quaternary blood vessels than at 1000 μg/mL. Between the 250, 500 μg/mL hordenine treated and the control-treated CAM's, there was no significant change in the number of primary and secondary blood vessels. Between the 250 μg/mL hordenine-treated and the control-treated CAM's, there was no significant change in the number of tertiary blood vessels, but there was a substantial difference in the number of quaternary blood vessels. Blood vessels showing the angiogenic potential of control (3 % DMSO) and hordenine in chicken embryo chorioallantoic membrane are depicted in Fig. 3 (b.1.) and 3 (b.2.), respectively. Andrographolide treatment showed significantly higher number of primary (P = 0.001), secondary (P < 0.001), tertiary (P = 0.001) and quaternary (P < 0.001) blood vessels at 1000 and 500 μg/mL compared with hordenine treatment. At 250 μg/mL, significant increase in tertiary (P = 0.001) and quaternary (P < 0.001) blood vessels were observed in andrographolide treatment than in hordenine. In both andrographolide and hordenine treated groups, the number of primary and secondary blood vessels at 250 μg/mL and control were non-significant. The number of tertiary and quaternary blood vessels of control was also observed to be non-significant (Fig. 3 a.2. and 3b.2.). At higher concentrations (250 and 500 μg/mL), andrographolide significantly increased blood vessel branching (P < 0.001) compared to hordenine and control. ImageJ-based quantification revealed reproducible angiogenic activity across three biological replicates.

Fig. 3 — CAM assay: (3a.1.) Blood vessels showing the angiogenic potential of (a) five per cent DMSO and (b) andrographolide in chicken embryo chorioallantoic membrane: P- Primary vessels, S- Secondary vessels, T- Tertiary vessels, Q- Quaternary vessels (3a.2 Blood vessel quantification graphs for andrographolide: number of primary, secondary, tertiary and quaternary blood vessels between various concentrations of andrographolide (3b.1.) Blood vessels showing the angiogenic potential of (a) three per cent DMSO and (b) hordenine in chicken embryo chorioallantoic membrane: P- Primary vessels, S- Secondary vessels, T- Tertiary vessels, Q- Quaternary vessels (3b.2.) Blood vessel quantification graphs for hordenine: number of primary, secondary, tertiary and quaternary blood vessels between various concentrations of hordenine.

3.4. Animal experiments

The per cent reduction in the wound on day 3 was significantly (P = 0.034) greater in control group than in the other groups. There was no significant difference between the ceftriaxone-sulbactam, andrographolide and hordenine treated groups. The per cent reduction in the wound on days 6, 9, 12, and 15 was comparable in all the four groups.

CFU counts confirmed active MRSA infection on day 1 (∼10⁶ CFU/mL), which decreased by day 7 and was undetectable by day 15 in all treatment groups. Andrographolide and hordenine both showed significantly greater wound contraction (P < 0.001) than the infected control on day 15.

In the control group, the per cent reduction in the wound was non-significant on all days. In the ceftriaxone-sulbactam treated group, the per cent reduction in the wound was highly significant (P = 0.014) on day 15 compared to other days. There was no significant difference between days 6 and 9 or, days 3 and 12. In the andrographolide treated group, the per cent reduction in the wound was highly significant (P = 0.017) on days 6, 9 and 15 compared with other days. There was no significant difference on per cent reduction in wound between days 3 and 12. In the hordenine treated group, per cent reduction in wound was highly significant on days 6, 9 and 15 compared with other days (P = 0.003). There was no significant difference on per cent reduction in wound between days 6 and 12; day 3 had the least significant reduction in wound per cent (Supplementary file. S.12. Table 7).

The results of the present study revealed that andrographolide and hordenine showed significant (P = 0.017, P = 0.003 respectively) reduction in the per cent reduction in wound from day 6 onwards. The per cent reduction in the wound was comparable for both the treatments on day 15.

The establishment of wound infection on day 1, and swabs taken on days 7 and 15 was confirmed by reading the swabs streaked on Hi Chrome MeReSa agar. The presence of infection with MRSA was indicated by bluish green colour growth as shown in Fig. 4. According to the clinical observations, the presence of pus was higher in the control group than in the andrographolide and hordenine-treated groups. There was a reduction in pus formation in the ceftriaxone-sulbactam treated group. On swab sample analysis, the presence of MRSA infection on Hi Chrome MeReSa agar was confirmed in all the four groups. MRSA Infected wound healing activity of ceftriaxone-sulbatam, andrographolide, and hordenine are depicted in Fig. 5a and Fig. 5b.

Fig. 4 — Swab samples on HiChrome MeReSa agar confirming MRSA presence through bluish green colonies across experimental days.

Fig. 5 — (a) Representative wound images from excision wound model in different treatment groups. (b) Graph showing percentage reduction in wound area over time.

3.5. Hydroxyproline estimation

The results revealed that the ceftriaxone-sulbactam treated group indicated higher hydroxyproline level than the control, hordenine and andrographolide. The control treated groups had the least amount of hydroxyproline level. However, the level of hydroxyproline in hordenine treated group was found to be greater than andrographolide treated group (Table 2 and Fig. 6a.). Statistical analysis revealed that the hydroxyproline content was significantly higher (P < 0.001) in the hordenine-treated group compared to the andrographolide and infected control groups.

Table 2.

Biochemical estimation of hydroxyproline in granulated wound tissue.

Groups	Tissue hydroxyproline (μg/g)
Control	73.32 ± 3.22^d
Ceftriaxone-sulbactam	196.55 ± 3.22^a
Andrographolide	121.09 ± 4.19^c
Hordenine	145.18 ± 3.75^b
F- value (P- value)	213.61^a (<0.001)

Open in a new tab

Significant at 0.01 level; ns non-significant; Means having different small letter as superscript differ significantly within a column; Means having different capital letter as superscript differ significantly within a row.

Fig. 6 — (a) Hydroxyproline levels in granulated wound tissues. (b) Hexosamine levels in granulated wound tissues.

3.6. Hexosamine estimation

The ceftriaxone/sulbactam treated group exhibited significantly higher hexosamine content in wound tissue compared to other groups. The control group had the lowest hexosamine content. No significant difference was observed in hexosamine content between the ceftriaxone/sulbactam and andrographolide treated groups, or between andrographolide and hordenine treated groups (Table 3 and Fig. 6b.). The hexosamine concentrations in both the andrographolide and hordenine treated groups were found to be comparable in this study. Hexosamine content did not show a statistically significant difference (P > 0.05) between the andrographolide and hordenine-treated groups, but both treatments showed significantly higher values (P < 0.01) compared to the infected control.

Table 3.

Biochemical estimation of hexosamine in granulated wound tissue.

Groups	Tissue hexosamine (mg/g)
Control	6.56 ± 0.30^c
Ceftriaxone-sulbactam	11.60 ± 0.37^a
Andrographolide	10.43 ± 0.73^ab
Hordenine	9.25 ± 0.26^b
F- value (P- value)	27.00^a (<0.001)

Open in a new tab

3.7. Histopathological examination

Histopathological evaluation of wound healing focused on epithelial regeneration, neovascularisation, fibroblast proliferation, collagen deposition, tissue necrosis, inflammation, and the formation of skin appendages, particularly hair follicles on a scale of + (mild) to +++ (extensive) were evaluated by the blinded pathologist using a semi-quantitative scoring system. In control group animals, a single epithelial layer covered granulation tissue near the wound edges was observed (Fig. 7ia). Mild to moderate neovascularisation and moderate fibroblast proliferation were noted. Inflammation was marked, predominantly involving macrophages and neutrophils, with few eosinophils and mast cells. Moderate necrosis was evident. Masson's trichrome staining showed faint, immature fibrillar collagen without bundle formation (Fig. 7ib). In ceftriaxone-sulbactam treated animals, multilayered epithelium extended from the wound edges but did not cover the central areas (Fig. 7iia.). Angiogenesis and fibroblast proliferation were marked. Inflammation was moderate to marked, mainly involving macrophages and mast cells, with minimal superficial neutrophilic infiltration. Necrosis was minimal. Collagen appeared thicker and formed small bundles (Fig. 7iib.). Andrographolide treated animals showed two to three epithelial layers extending from wound margins, without central epithelialisation. Angiogenesis was moderate and fibroblast proliferation was marked (Fig. 7iiia.). Inflammation resembled that in the ceftriaxone-sulbactam group, with predominant macrophages followed by mast cells and minimal neutrophilic infiltration. Necrosis was mild, higher than in ceftriaxone-sulbactam but lower than controls. Collagen was organised in small bundles (Fig. 7iiib.). These findings suggest a faster healing rate than in controls, comparable to ceftriaxone-sulbactam except for epithelialisation, angiogenesis, and necrosis. Hordenine treated animals displayed two to multiple epithelial layers extending further than in the andrographolide group, though central areas lacked epithelialisation (Fig. 7iva). Neovascularisation and fibroblast proliferation matched ceftriaxone-sulbactam levels. Inflammation was marked, with macrophages followed by neutrophils. Necrosis resembled that in the andrographolide group. Collagen appeared as deeply stained small to medium bundles (Fig. 7ivb), indicating enhanced healing over controls. Although all treatment groups showed accelerated wound healing compared to controls, there were no significant differences among them. Notably, skin appendages were absent in all groups. The control group remained in the inflammatory phase, while treated groups had progressed to the proliferative phase. Among the treatments, the hordenine-treated group showed significantly enhanced epithelialisation (P < 0.001), fibroblast proliferation and collagen bundle formation compared to the andrographolide and infected control groups. Inflammatory infiltration was mild to moderate in the treated groups but severe in the control.

(i)Showing the microphotograph of paraffin treated wound tissue section (a) Single layer of epithelium, moderate neovascularisation and fibroblast proliferation (H&E, x100) and (b) Immature faintly stained fibrillary collagen (Masson's trichrome, x400) Fig. 7. (ii) Showing the microphotograph of ceftriaxone-sulbactam treated wound tissue section (a) Multi-layered epithelium over the granulation tissue and marked fibroblast proliferation (H&E, x40) and (b) Deeply stained collagen fibres arranged in small bundles (Masson's trichrome, x400). Fig. 7 (iii) Showing the microphotograph of andrographolide treated wound tissue section (a) Two to three layered epithelium, moderate angiogenesis and marked fibroblast proliferation (H&E, x100) and (b) Deeply stained collagen fibres arranged in small bundles (Masson's trichrome, x100). Fig. 7. (iv) Showing the microphotograph of hordenine treated wound tissue section (a) Multiple layers of epithelium extending from the wound edges (H&E, x100) and (b) Deeply stained collagen fibres arranged in small bundles (Masson's trichrome, x400).

4. Discussion

Wound healing is a complex biological process involving a well-orchestrated interplay between cellular proliferation, extracellular matrix remodeling, angiogenesis, and inflammatory resolution. In the present study, the wound healing efficacy of two phytochemicals—andrographolide and hordenine—was systematically evaluated using in vitro assays, including MTT, scratch, and CAM assays, alongside in vivo excisional wound model, biochemical markers, and histopathological assessment. Collectively, the results elucidate the multifaceted therapeutic potential of these compounds in promoting tissue repair.

The MTT assay demonstrated that both andrographolide and hordenine enhanced the viability of L929 mouse skin fibroblast cells, indicating their biocompatibility and ability to support fibroblast proliferation. Andrographolide-treated cells exhibited higher viability than those treated with hordenine, suggesting comparatively stronger proliferative or protective effects on fibroblasts. This observation corroborates earlier reports where andrographolide enhanced viability in dermal fibroblasts and protected against oxidative and inflammatory damage [15]. In contrast, hordenine has been shown to induce significant cytotoxic effects in human cancer cell lines, such as A549 and H1299, while sparing normal cells like HEK293 [16]. Similarly, Hordeum vulgare seed extracts reduced viability in MDA-MB-231 cells [16,17], and andrographolide has been shown to reduce proliferation in MCF-7 and MDA-MB-231 breast cancer cells through apoptosis induction [18]. These findings suggest a potential dual role of both phytochemicals—promoting proliferation in normal cells while exerting cytostatic or cytotoxic effects on malignant cells—highlighting their therapeutic selectivity.

To further evaluate the migratory and regenerative capabilities of the fibroblasts, a scratch assay was conducted using the EC₅₀ values of each compound (13.16 μg/mL for andrographolide and 7.17 μg/mL for hordenine). The selected concentrations reflect pharmacologically relevant concentrations that promote fibroblast viability without inducing cytotoxicity, aligning with EC50-based therapeutic windows. Both compounds significantly enhanced wound closure by day 3, with hordenine exhibiting a slightly more pronounced reduction in the cellular gap. This enhanced migratory capacity is indicative of active cytoskeletal reorganization and increased extracellular matrix production, essential steps for re-epithelialisation during wound healing. Previous studies have established the role of polyherbal formulations and plant bioactives in stimulating fibroblast migration and gap closure in similar cell line models [9,19]. Moreover, hordenine's superior performance in the scratch assay could be attributed to its capacity to modulate cellular adhesion and signaling cascades that influence fibroblast motility. The observation that extracellular matrix proteins such as fibronectin, laminin, and collagen support cell migration [20] reinforces the hypothesis that hordenine may activate or upregulate such matrix components.

The angiogenic potential of both compounds was evaluated through the CAM assay, a well-established in vivo model for studying neovascularisation. Angiogenesis is pivotal in wound healing, as it ensures sustained delivery of oxygen and nutrients while supporting cell proliferation and matrix remodeling. Treatment with both andrographolide and hordenine significantly increased the number of newly formed tertiary and quaternary blood vessels. This enhancement suggests a pro-angiogenic mechanism likely involving the activation of VEGF-dependent signaling. The angiogenic activity of phytocompounds has previously been observed in models using dinitrosyl iron complexes (DNICs) and oxygen-releasing electrospun mats [21,22]. Likewise, chitosan-collagen matrices infused with amino acids have also been reported to stimulate vascular growth [23]. Notably, andrographolide has been shown to upregulate VEGF expression in endothelial cells under hyperglycemic conditions, indicating its ability to overcome oxidative stress-induced vascular damage [24,25]. The observed angiogenesis in this study aligns with such findings and underscores the role of both test compounds in enhancing capillary sprouting and vascular remodeling, critical elements in granulation tissue maturation.

Although andrographolide showed a stronger angiogenic response in the CAM assay, this did not translate to enhanced wound healing in vivo. This divergence may reflect the differential effects of the two agents on distinct healing stages—angiogenesis versus matrix remodeling. Hence, the CAM and wound healing data are not contradictory but complementary.

The in vivo wound healing analysis further validated the results of in vitro studies. From day 6 onwards, a significant reduction in wound area was observed in both andrographolide and hordenine treated groups. By day 15, the wound closure in both groups was comparable, indicating a steady and sustained healing response. These findings are consistent with studies demonstrating the efficacy of bioactive compounds in MRSA-infected excisional wound models [[26], [27], [28], [29]], wherein the application of phytochemical-based therapies resulted in accelerated wound contraction, decreased bacterial load, and improved histological parameters. Importantly, the histological evaluation in this study revealed that the treated groups had transitioned to the proliferative phase of wound healing, in contrast to the control group, which largely remained in the inflammatory phase. The presence of multilayered epithelial cells, enhanced fibroblast activity, and organized collagen deposition in the treated animals reflects active tissue regeneration and structural repair.

Biochemical quantification of hydroxyproline provided further insights into collagen synthesis. As hydroxyproline is a major constituent of collagen, its levels directly reflect the extent of collagen deposition during wound repair. Statistical analysis revealed that the hydroxyproline content was significantly higher (P < 0.001) in the hordenine-treated group compared to the andrographolide and infected control groups. These findings suggest that both compounds stimulate fibroblast-mediated collagen synthesis, essential for restoring the tensile strength and architectural integrity of the wounded tissue. This aligns with previous studies that recorded elevated hydroxyproline levels following treatment with plant-derived wound healing agents [[30], [31], [32], [33], [34], [35]].

Similarly, hexosamine levels, indicative of glycosaminoglycan synthesis and extracellular matrix expansion, were significantly elevated in both treatment groups. Hexosamine contributes to the stabilization of collagen fibers, influences fibril alignment, and facilitates matrix hydration through electrostatic interactions [36]. Hexosamine content did not show a statistically significant difference (P > 0.05) between the andrographolide and hordenine-treated groups, but both treatments showed significantly higher values (P < 0.01) compared to the infected control. The increase in hexosamine content observed in this study suggests enhanced matrix remodeling and provides a biochemical basis for the histological findings of organized collagen bundles in the treated groups.

Histopathological analysis performed in a blinded manner using a semi-quantitative scoring system based on epithelial thickness, fibroblast activity, angiogenesis, collagen deposition, and inflammatory infiltration further illuminated the cellular events underpinning wound repair. In control animals, histological sections showed marked inflammation, moderate necrosis, and immature collagen, and limited epithelialisation— hallmarks of an unresolved inflammatory phase. In contrast, the treated groups—especially those administered andrographolide and hordenine—exhibited reduced inflammatory cell infiltration, re-epithelialisation extending from wound margins, abundant fibroblasts, neovascularisation, and collagen arranged in small to medium bundles. These findings reflect a shift from inflammation to active tissue proliferation and matrix remodeling. The presence of collagen bundles, visualized through Masson's trichrome staining, also corroborates the biochemical evidence of increased hydroxyproline and hexosamine content. The observed differences in necrosis levels—moderate in controls, minimal in ceftriaxone-sulbactam, and mild in the phytochemical-treated groups—further highlight the cytoprotective effects of the treatments in limiting tissue damage and promoting faster resolution of inflammation [37,38].

Furthermore, collagen not only serves a structural function but also influences key cellular events such as angiogenesis, chemotaxis, and cell differentiation [39,40]. The in vitro scratch assay results, particularly those showing enhanced fibroblast migration in hordenine-treated cells, support these histological observations, suggesting a positive feedback loop between collagen deposition and fibroblast motility.

Previous in vitro data by the same authors [7] confirmed that neither andrographolide nor hordenine produced any zone of inhibition in disc diffusion assays against MRSA. The MIC of hordenine was 1000 μg/mL in the resazurin-based microdilution assay, whereas andrographolide failed to inhibit MRSA at similar concentrations. These published results align with the current findings, suggesting that the in vivo healing was likely mediated by host-directed effects rather than direct antibacterial activity.

In summary, both andrographolide and hordenine exerted pronounced wound healing effects through multiple complementary mechanisms, including enhanced fibroblast proliferation, accelerated cell migration, stimulation of angiogenesis, and effective extracellular matrix deposition. While andrographolide showed higher fibroblast viability in vitro, hordenine appeared more potent in promoting migration and collagen synthesis. Nevertheless, both agents were comparable in terms of overall wound closure efficacy by day 15. The absence of skin appendage formation in all groups suggests that complete skin regeneration may require extended treatment durations or adjunctive therapies. Nonetheless, the current findings provide strong preclinical evidence supporting the therapeutic application of these phytoconstituents in wound management.

5. Conclusion

To conclude, andrographolide and hordenine by enhancing fibroblast proliferation and migration, alongside fostering angiogenesis, underscore a promising therapeutic avenue against MRSA infections. The exploration of their mechanisms offers a beacon of hope in the ongoing battle against microbial resistance, emphasizing the need for continued investigation into natural compounds as viable alternatives in wound management strategies.

Ethical approval

The study was approved by the Institutional Animal Ethics Committee of College of Veterinary and Animal Sciences, Pookode with sanction number IAEC/COVAS/PKD/11/2021.

Author contributions

VSJ carried out the experiment. BJK conceptualized and designed the experiment. VU assisted VSJ in the conduct of experiment. AR interpreted and reported the results of histopathological examinations.

Declaration of generative AI in scientific writing

The authors have not used any generative AI and AI-assisted technologies in the writing process.

Funding sources

The research work was supported with by Kerala Veterinary and Animal Sciences University as MVSc research grant for the first author with grant no. KVASU/2019/088/MVP/VPT. A part of the research work was financially supported by Kerala State Council for Science, Technology and Environment through the KSCSTE student project with grant no. 00218/SPS 65/2021/KSCSTE. The research work was also supported by Kerala State Plan Fund 17–18 granted to the corresponding author with grant no. RSP/17-18/VI entitled “Screening of medicinal plants for wound healing properties”

Data availability statement

The data of the study is available in the institutional repository of Indian National Agricultural Research System - KrishiKosh in the following link:

https://krishikosh.egranth.ac.in/items/fe22ba07-5322-47e1-a605-0acdc47fa510.

Conflict of 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.

Acknowledgements

The authors acknowledge the Dean and Implementing officer of COHEART, College of Veterinary and Animal Sciences, Pookode for providing the facilities for the conduct of research. The authors also acknowledge Dr. Jess Vergis in providing MRSA as gift samples.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jaim.2025.101291.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1

mmc1.docx^{(270.9KB, docx)}

References

1.Alam M.M., Islam M.N., Hawlader M.D.H., Ahmed S., Wahab A., Islam M., et al. Prevalence of multidrug-resistant bacterial isolates from infected wound patients in Dhaka, Bangladesh: a cross-sectional study. Int J Surg. 2021;28:56–62. [Google Scholar]
2.Joshi S., Ray P., Manchanda V., Bajaj J., Chitnis D.S., Gautam V., et al. Methicillin resistant Staphylococcus aureus (MRSA) in India: prevalence and susceptibility pattern. Indian J Med Res. 2013;137:363–369. [PMC free article] [PubMed] [Google Scholar]
3.Nobel F.A., Islam S., Babu G., Akter S., Jebin R.A., Sarker T.C., et al. Isolation of multidrug resistance bacteria from the patients with wound infection and their antibiotics susceptibility patterns: a cross-sectional study. Ann Med Surg. 2022;84 doi: 10.1016/j.amsu.2022.104895. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Lin Y.L., Liao J.W., Wang S., Sridharan B., Lee H.J., Li A., et al. Andrographolide relieves post-operative wound pain but affects local angiogenesis. Pharmaceuticals. 2022;15(12):1586. doi: 10.3390/ph15121586. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Guzzo F., Scognamiglio M., Fiorentino A., Buommino E., D'Abrosca B. Plant derived natural products against Pseudomonas aeruginosa and Staphylococcus aureus: antibiofilm activity and molecular mechanisms. Molecules. 2020;25(21):5024. doi: 10.3390/molecules25215024. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Zhang X., Du L., Zhang J., Li C., Zhang J., Lv X. Hordenine protects against lipopolysaccharide-induced acute lung injury by inhibiting inflammation. Front Pharmacol. 2021;12 doi: 10.3389/fphar.2021.712232. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Vidyavarshha S.J., Kariyil B.J., Unni V., Vergis J., Juliet S., John P., et al. In vitro antibacterial activity of andrographolide and hordenine against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus. J Vet Anim Sci. 2022;53(3):407–412. [Google Scholar]
8.Gopalakrishnan A., Kariyil B.J., John R., Usha P.T. Phytochemical evaluation and cytotoxic potential of chloroform soluble fraction of methanol extract of Thespesia populnea in human breast cancer cell lines. Pharmacogn Mag. 2019;15(62):150–154. [Google Scholar]
9.Talekar Y.P., Apte K.G., Paygude S.V., Tondare P.R., Parab P.B. Studies on wound healing potential of polyherbal formulation using in vitro and in vivo assays. J Ayurveda Integr Med. 2017;8(2):73–81. doi: 10.1016/j.jaim.2016.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Medeiros V.F.L.P., Azevedo I.M., Rêgo A.C.M., Egito E.S.T., Araújo-Filho I., Medeiros A.C. Antibacterial properties and healing effects of Melipona scutellaris honey in MRSA-infected wounds of rats. Acta Cir Bras. 2016;31(5):327–332. doi: 10.1590/S0102-865020160050000006. [DOI] [PubMed] [Google Scholar]
11.Woessner J. Catabolism of collagen and non-collagen protein in the rat uterus during post-partum involution. Biochem J. 1962;83(2):304–314. doi: 10.1042/bj0830304. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Carlson C.G. Determination of hydroxyproline content as a measure of fibrosis in nondystrophic and dystrophic skeletal muscle. Neuromuscul Netw. 2014;1:1–10. [Google Scholar]
13.Eisele C.W., Eichelberger L. Water, electrolyte and nitrogen content of human skin. Exp Biol Med. 1945;58(1):97–100. [Google Scholar]
14.Suvarna K.S., Layton C., Bancroft J.D. seventh ed. Elsevier Health Sciences; London: 2012. Bancroft's theory and practice of histological techniques; p. 654. [Google Scholar]
15.Mussard E., Jousselin S., Cesaro A., Legrain B., Lespessailles E., Esteve E., et al. Andrographis paniculata and its bioactive diterpenoids protect dermal fibroblasts against inflammation and oxidative stress. Antioxidants. 2020;9(5):432. doi: 10.3390/antiox9050432. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Kariyil B., Akhil G., Akshatha G., Bhatt S.V., Dhanusha G., John R. Germinated seeds of Hordeum vulgare target extrinsic pathway of apoptosis in triple-negative breast cancer cells. Pharmacogn Mag. 2020;16(5):531. [Google Scholar]
17.Anwar S., Mohammad T., Shamsi A., Queen A., Parveen S., Luqman S., et al. Discovery of hordenine as a potential inhibitor of pyruvate dehydrogenase kinase 3: implication in lung cancer therapy. Biomedicines. 2020;8(5):119. doi: 10.3390/biomedicines8050119. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Tohkayomatee R., Reabroi S., Tungmunnithum D., Parichatikanond W., Pinthong D. Andrographolide exhibits anticancer activity against breast cancer cells through suppressing cell proliferation and inducing apoptosis via inactivation of ER-α and PI3K/AKT/mTOR signaling. Molecules. 2022;27(11):3544. doi: 10.3390/molecules27113544. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Dandannavar V., Balakrishna J., Dhawale L., Joseph J., Gollapalli S., Pillai A., et al. Wound-healing potential of methanolic extract of Rhaphidophora korthalsii leaves possibly mediated by collagen and fibronectin expression in L929 cells. Natl J Physiol Pharm Pharmacol. 2019;9(0):1. [Google Scholar]
20.Velnar T., Bailey T., Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res. 2009;37(5):1528–1542. doi: 10.1177/147323000903700531. [DOI] [PubMed] [Google Scholar]
21.Chen Y.J., Wu S.C., Wang H.C., Wu T.H., Lu T.T., Liaw W.F., et al. Activation of angiogenesis and wound healing in diabetic mice using NO-delivery dinitrosyl iron complexes. Mol Pharm. 2019;16(10):4241–4251. doi: 10.1021/acs.molpharmaceut.9b00586. [DOI] [PubMed] [Google Scholar]
22.Zehra M., Zubairi W., Hasan A., Butt H., Ramzan A., Azam M., et al. Oxygen-generating polymeric nanofibers that stimulate angiogenesis and show efficient wound healing in a diabetic model. Int J Nanomed. 2020;15:3511–3522. doi: 10.2147/IJN.S248911. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Aleem A.R., Shahzadi L., Tehseen S., Alvi F., Chaudhry A.A., Rehman I.U., et al. Amino acids loaded chitosan/collagen-based membranes stimulate angiogenesis in chorioallantoic membrane assay. Int J Biol Macromol. 2019;140:401–406. doi: 10.1016/j.ijbiomac.2019.08.095. [DOI] [PubMed] [Google Scholar]
24.Johnson K.E., Wilgus T.A. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care. 2014;3(10):647–661. doi: 10.1089/wound.2013.0517. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Duan M., Zhou H., Wu Q., Liu C., Yang X., Deng W., et al. Andrographolide protects against HG-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs. Mediat Inflamm. 2019;2019:1–15. doi: 10.1155/2019/6168340. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Sewall G.K., Robertson K.M., Connor N.P., Heisey D.M., Hartig G.K. Effect of topical mitomycin on skin wound contraction. Arch Facial Plast Surg. 2003;5(1):59–62. doi: 10.1001/archfaci.5.1.59. [DOI] [PubMed] [Google Scholar]
27.Hoffmann J.J., Friedman J., Wang Y., McLachlan J.B., Sammarco M.C., Morici L.A., et al. In situ treatment with novel microbiocide inhibits methicillin-resistant Staphylococcus aureus in a murine wound infection model. Front Microbiol. 2020;10:1234. doi: 10.3389/fmicb.2019.03106. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Bolatchiev A., Baturin V., Bazikov I., Maltsev A., Kunitsina E. Effect of antimicrobial peptides HNP‐1 and hBD‐1 on Staphylococcus aureus strains in vitro and in vivo. Future Microbiol. 2019;34(1):102–108. doi: 10.1111/fcp.12499. [DOI] [PubMed] [Google Scholar]
29.Shetru M.N., Karched M., Agsar D. Locally isolated broad host-range bacteriophage kills methicillin-resistant Staphylococcus aureus in an in vivo skin excisional wound model in mice. Microb Pathog. 2021;152 doi: 10.1016/j.micpath.2021.104744. [DOI] [PubMed] [Google Scholar]
30.Dwivedi D., Dwivedi M., Malviya S., Singh V. Evaluation of wound healing, antimicrobial and antioxidant potential of Pongamia pinnata in Wistar rats. J Tradit Complement Med. 2017;7(1):79–85. doi: 10.1016/j.jtcme.2015.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Ibrahim A.M., Nagarajan A., Majeed M. Evaluation of in vivo wound healing activity of the extract and oleuropein from Morinda tinctoria Roxb. (Rubiaceae)—a traditional medicinal plant. Int J Adv Res. 2017;5(9):889–895. [Google Scholar]
32.Boakye Y.D., Agyare C., Ayande G.P., Titiloye N., Asiamah E.A., Danquah K.O. Assessment of wound-healing properties of medicinal plants: the case of Phyllanthus muellerianus. Front Pharmacol. 2018;9:945. doi: 10.3389/fphar.2018.00945. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Goorani S., Zangeneh M.M., Koohi M.K., Seydi N., Zangeneh A., Souri N., et al. Assessment of antioxidant and cutaneous wound healing effects of Falcaria vulgaris aqueous extract in Wistar rats. Comp Clin Pathol. 2018;28(2):435–445. [Google Scholar]
34.Zangeneh M.M., Zangeneh A., Seydi N., Moradi R. Evaluation of cutaneous wound healing activity of Ocimum basilicum aqueous extract ointment in rats. Comp Clin Pathol. 2019;28(5):1447–1454. [Google Scholar]
35.Kumar R., Gupta R., Kumar B., Kapoor B. Wound healing potential of polyherbal formulation in rats. Res J Pharm Technol. 2021;14(4):2195–2199. [Google Scholar]
36.S M., Mk G., S G., V P., H S., Rk G. Evaluation of in vivo wound healing activity of Bacopa monniera on different wound model in rats. BioMed Res Int. 2013;2013:1–8. doi: 10.1155/2013/972028. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Bekara F., Vitse J., Fluieraru S., Masson R., Runz A.D., Georgescu V., et al. New techniques for wound management: a systematic review of their role in the management of chronic wounds. Arch Plast Surg. 2018;45(2):102–110. doi: 10.5999/aps.2016.02019. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Biranje S.S., Madiwale P.V., Patankar K.C., Chhabra R., Dandekar-Jain P., Adivarekar R.V. Hemostasis and anti-necrotic activity of wound-healing dressing containing chitosan nanoparticles. Int J Biol Macromol. 2019;121:936–946. doi: 10.1016/j.ijbiomac.2018.10.125. [DOI] [PubMed] [Google Scholar]
39.Boyar V. Collagen: providing a key to the wound healing kingdom [Internet] HMP Global Learning Network. 2022 https://www.hmpgloballearningnetwork.com/site/wmp/article/collagen-providing-key-wound-healing-kingdom [cited 2025 Aug 5]. Available from: [Google Scholar]
40.Mathew-Steiner S.S., Roy S., Sen C.K. Collagen in wound healing. Bioengineering. 2021;8(5):63. doi: 10.3390/bioengineering8050063. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1

mmc1.docx^{(270.9KB, docx)}

[bib1] 1.Alam M.M., Islam M.N., Hawlader M.D.H., Ahmed S., Wahab A., Islam M., et al. Prevalence of multidrug-resistant bacterial isolates from infected wound patients in Dhaka, Bangladesh: a cross-sectional study. Int J Surg. 2021;28:56–62. [Google Scholar]

[bib2] 2.Joshi S., Ray P., Manchanda V., Bajaj J., Chitnis D.S., Gautam V., et al. Methicillin resistant Staphylococcus aureus (MRSA) in India: prevalence and susceptibility pattern. Indian J Med Res. 2013;137:363–369. [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Nobel F.A., Islam S., Babu G., Akter S., Jebin R.A., Sarker T.C., et al. Isolation of multidrug resistance bacteria from the patients with wound infection and their antibiotics susceptibility patterns: a cross-sectional study. Ann Med Surg. 2022;84 doi: 10.1016/j.amsu.2022.104895. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Lin Y.L., Liao J.W., Wang S., Sridharan B., Lee H.J., Li A., et al. Andrographolide relieves post-operative wound pain but affects local angiogenesis. Pharmaceuticals. 2022;15(12):1586. doi: 10.3390/ph15121586. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Guzzo F., Scognamiglio M., Fiorentino A., Buommino E., D'Abrosca B. Plant derived natural products against Pseudomonas aeruginosa and Staphylococcus aureus: antibiofilm activity and molecular mechanisms. Molecules. 2020;25(21):5024. doi: 10.3390/molecules25215024. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Zhang X., Du L., Zhang J., Li C., Zhang J., Lv X. Hordenine protects against lipopolysaccharide-induced acute lung injury by inhibiting inflammation. Front Pharmacol. 2021;12 doi: 10.3389/fphar.2021.712232. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7.Vidyavarshha S.J., Kariyil B.J., Unni V., Vergis J., Juliet S., John P., et al. In vitro antibacterial activity of andrographolide and hordenine against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus. J Vet Anim Sci. 2022;53(3):407–412. [Google Scholar]

[bib8] 8.Gopalakrishnan A., Kariyil B.J., John R., Usha P.T. Phytochemical evaluation and cytotoxic potential of chloroform soluble fraction of methanol extract of Thespesia populnea in human breast cancer cell lines. Pharmacogn Mag. 2019;15(62):150–154. [Google Scholar]

[bib9] 9.Talekar Y.P., Apte K.G., Paygude S.V., Tondare P.R., Parab P.B. Studies on wound healing potential of polyherbal formulation using in vitro and in vivo assays. J Ayurveda Integr Med. 2017;8(2):73–81. doi: 10.1016/j.jaim.2016.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Medeiros V.F.L.P., Azevedo I.M., Rêgo A.C.M., Egito E.S.T., Araújo-Filho I., Medeiros A.C. Antibacterial properties and healing effects of Melipona scutellaris honey in MRSA-infected wounds of rats. Acta Cir Bras. 2016;31(5):327–332. doi: 10.1590/S0102-865020160050000006. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Woessner J. Catabolism of collagen and non-collagen protein in the rat uterus during post-partum involution. Biochem J. 1962;83(2):304–314. doi: 10.1042/bj0830304. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 12.Carlson C.G. Determination of hydroxyproline content as a measure of fibrosis in nondystrophic and dystrophic skeletal muscle. Neuromuscul Netw. 2014;1:1–10. [Google Scholar]

[bib12] 13.Eisele C.W., Eichelberger L. Water, electrolyte and nitrogen content of human skin. Exp Biol Med. 1945;58(1):97–100. [Google Scholar]

[bib13] 14.Suvarna K.S., Layton C., Bancroft J.D. seventh ed. Elsevier Health Sciences; London: 2012. Bancroft's theory and practice of histological techniques; p. 654. [Google Scholar]

[bib15] 15.Mussard E., Jousselin S., Cesaro A., Legrain B., Lespessailles E., Esteve E., et al. Andrographis paniculata and its bioactive diterpenoids protect dermal fibroblasts against inflammation and oxidative stress. Antioxidants. 2020;9(5):432. doi: 10.3390/antiox9050432. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.Kariyil B., Akhil G., Akshatha G., Bhatt S.V., Dhanusha G., John R. Germinated seeds of Hordeum vulgare target extrinsic pathway of apoptosis in triple-negative breast cancer cells. Pharmacogn Mag. 2020;16(5):531. [Google Scholar]

[bib17] 17.Anwar S., Mohammad T., Shamsi A., Queen A., Parveen S., Luqman S., et al. Discovery of hordenine as a potential inhibitor of pyruvate dehydrogenase kinase 3: implication in lung cancer therapy. Biomedicines. 2020;8(5):119. doi: 10.3390/biomedicines8050119. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Tohkayomatee R., Reabroi S., Tungmunnithum D., Parichatikanond W., Pinthong D. Andrographolide exhibits anticancer activity against breast cancer cells through suppressing cell proliferation and inducing apoptosis via inactivation of ER-α and PI3K/AKT/mTOR signaling. Molecules. 2022;27(11):3544. doi: 10.3390/molecules27113544. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19.Dandannavar V., Balakrishna J., Dhawale L., Joseph J., Gollapalli S., Pillai A., et al. Wound-healing potential of methanolic extract of Rhaphidophora korthalsii leaves possibly mediated by collagen and fibronectin expression in L929 cells. Natl J Physiol Pharm Pharmacol. 2019;9(0):1. [Google Scholar]

[bib20] 20.Velnar T., Bailey T., Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res. 2009;37(5):1528–1542. doi: 10.1177/147323000903700531. [DOI] [PubMed] [Google Scholar]

[bib21] 21.Chen Y.J., Wu S.C., Wang H.C., Wu T.H., Lu T.T., Liaw W.F., et al. Activation of angiogenesis and wound healing in diabetic mice using NO-delivery dinitrosyl iron complexes. Mol Pharm. 2019;16(10):4241–4251. doi: 10.1021/acs.molpharmaceut.9b00586. [DOI] [PubMed] [Google Scholar]

[bib23] 22.Zehra M., Zubairi W., Hasan A., Butt H., Ramzan A., Azam M., et al. Oxygen-generating polymeric nanofibers that stimulate angiogenesis and show efficient wound healing in a diabetic model. Int J Nanomed. 2020;15:3511–3522. doi: 10.2147/IJN.S248911. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 23.Aleem A.R., Shahzadi L., Tehseen S., Alvi F., Chaudhry A.A., Rehman I.U., et al. Amino acids loaded chitosan/collagen-based membranes stimulate angiogenesis in chorioallantoic membrane assay. Int J Biol Macromol. 2019;140:401–406. doi: 10.1016/j.ijbiomac.2019.08.095. [DOI] [PubMed] [Google Scholar]

[bib24] 24.Johnson K.E., Wilgus T.A. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care. 2014;3(10):647–661. doi: 10.1089/wound.2013.0517. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25.Duan M., Zhou H., Wu Q., Liu C., Yang X., Deng W., et al. Andrographolide protects against HG-induced inflammation, apoptosis, migration, and impairment of angiogenesis via PI3K/AKT-eNOS signalling in HUVECs. Mediat Inflamm. 2019;2019:1–15. doi: 10.1155/2019/6168340. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib26] 26.Sewall G.K., Robertson K.M., Connor N.P., Heisey D.M., Hartig G.K. Effect of topical mitomycin on skin wound contraction. Arch Facial Plast Surg. 2003;5(1):59–62. doi: 10.1001/archfaci.5.1.59. [DOI] [PubMed] [Google Scholar]

[bib27] 27.Hoffmann J.J., Friedman J., Wang Y., McLachlan J.B., Sammarco M.C., Morici L.A., et al. In situ treatment with novel microbiocide inhibits methicillin-resistant Staphylococcus aureus in a murine wound infection model. Front Microbiol. 2020;10:1234. doi: 10.3389/fmicb.2019.03106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 28.Bolatchiev A., Baturin V., Bazikov I., Maltsev A., Kunitsina E. Effect of antimicrobial peptides HNP‐1 and hBD‐1 on Staphylococcus aureus strains in vitro and in vivo. Future Microbiol. 2019;34(1):102–108. doi: 10.1111/fcp.12499. [DOI] [PubMed] [Google Scholar]

[bib29] 29.Shetru M.N., Karched M., Agsar D. Locally isolated broad host-range bacteriophage kills methicillin-resistant Staphylococcus aureus in an in vivo skin excisional wound model in mice. Microb Pathog. 2021;152 doi: 10.1016/j.micpath.2021.104744. [DOI] [PubMed] [Google Scholar]

[bib30] 30.Dwivedi D., Dwivedi M., Malviya S., Singh V. Evaluation of wound healing, antimicrobial and antioxidant potential of Pongamia pinnata in Wistar rats. J Tradit Complement Med. 2017;7(1):79–85. doi: 10.1016/j.jtcme.2015.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Ibrahim A.M., Nagarajan A., Majeed M. Evaluation of in vivo wound healing activity of the extract and oleuropein from Morinda tinctoria Roxb. (Rubiaceae)—a traditional medicinal plant. Int J Adv Res. 2017;5(9):889–895. [Google Scholar]

[bib32] 32.Boakye Y.D., Agyare C., Ayande G.P., Titiloye N., Asiamah E.A., Danquah K.O. Assessment of wound-healing properties of medicinal plants: the case of Phyllanthus muellerianus. Front Pharmacol. 2018;9:945. doi: 10.3389/fphar.2018.00945. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33.Goorani S., Zangeneh M.M., Koohi M.K., Seydi N., Zangeneh A., Souri N., et al. Assessment of antioxidant and cutaneous wound healing effects of Falcaria vulgaris aqueous extract in Wistar rats. Comp Clin Pathol. 2018;28(2):435–445. [Google Scholar]

[bib34] 34.Zangeneh M.M., Zangeneh A., Seydi N., Moradi R. Evaluation of cutaneous wound healing activity of Ocimum basilicum aqueous extract ointment in rats. Comp Clin Pathol. 2019;28(5):1447–1454. [Google Scholar]

[bib35] 35.Kumar R., Gupta R., Kumar B., Kapoor B. Wound healing potential of polyherbal formulation in rats. Res J Pharm Technol. 2021;14(4):2195–2199. [Google Scholar]

[bib36] 36.S M., Mk G., S G., V P., H S., Rk G. Evaluation of in vivo wound healing activity of Bacopa monniera on different wound model in rats. BioMed Res Int. 2013;2013:1–8. doi: 10.1155/2013/972028. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37.Bekara F., Vitse J., Fluieraru S., Masson R., Runz A.D., Georgescu V., et al. New techniques for wound management: a systematic review of their role in the management of chronic wounds. Arch Plast Surg. 2018;45(2):102–110. doi: 10.5999/aps.2016.02019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38.Biranje S.S., Madiwale P.V., Patankar K.C., Chhabra R., Dandekar-Jain P., Adivarekar R.V. Hemostasis and anti-necrotic activity of wound-healing dressing containing chitosan nanoparticles. Int J Biol Macromol. 2019;121:936–946. doi: 10.1016/j.ijbiomac.2018.10.125. [DOI] [PubMed] [Google Scholar]

[bib39] 39.Boyar V. Collagen: providing a key to the wound healing kingdom [Internet] HMP Global Learning Network. 2022 https://www.hmpgloballearningnetwork.com/site/wmp/article/collagen-providing-key-wound-healing-kingdom [cited 2025 Aug 5]. Available from: [Google Scholar]

[bib40] 40.Mathew-Steiner S.S., Roy S., Sen C.K. Collagen in wound healing. Bioengineering. 2021;8(5):63. doi: 10.3390/bioengineering8050063. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Comparative efficacy of andrographolide and hordenine in the treatment of methicillin resistant Staphylococcus aureus infected wounds: a multifaceted approach

SJ Vidyavarsha

Bibu John Kariyil

Varsha Unni

R Anoopraj

Abstract

Background

Objective

Methods

Results

Conclusion

1. Introduction

2. Materials and methods

2.1. Reagents and chemicals

2.2. Cell culture

2.3. Antibacterial activity assay

2.4. MTT assay

2.5. Scratch assay

2.6. Chicken embryo chorioallantoic membrane (CAM) assay

2.7. Animal experiments

2.8. Hydroxyproline estimation

2.9. Hexosamine estimation

2.10. Histopathological examinations

2.11. Statistical analysis

3. Results

3.1. MTT assay

Fig. 1.

3.2. Scratch assay

Fig. 2.

Table 1.

3.3. Chicken embryo chorioallantoic membrane (CAM) assay

Fig. 3.

3.4. Animal experiments

Fig. 4.

Fig. 5.

3.5. Hydroxyproline estimation

Table 2.

Fig. 6.

3.6. Hexosamine estimation

Table 3.

3.7. Histopathological examination

Fig. 7.

4. Discussion

5. Conclusion

Ethical approval

Author contributions

Declaration of generative AI in scientific writing

Funding sources

Conflict of interest

Acknowledgements

Footnotes

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases