2Ahmed Maher Teaching Hospital, Cairo, Egypt.
3Botany and Microbiology Dept, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt.
*Corresponding Author E-mail: amrelwaseif@azhar.edu.eg
ABSTRACT:
To repair and regenerate tissue and avoid infections and problems, wound healing is an essential biological process. In recent years, there has been an increasing interest in investigating sustainable wound healing techniques. The innate and adaptive immune systems' natural reaction to infection is inflammation. Low cytotoxicity natural molecules with anti-inflammatory and wound healing properties can target the key players of inflammation, wound healing and exert beneficial skin health effects. In this study, Glycoplex raw material from polygon Life science Company assayed to determine anti-inflammatory properties using red blood cells hemolysis and membrane stability method. Scratch Assay was performed to evaluate the migratory capacity of HFB4 human skin fibroblasts cells in response to treatment with Glycoplex for wound healing application. Human skin fibroblasts cells HFB4 treated with Glycoplex for cytotoxicity assay using MTT method. Anti-inflammatory results indicated that IC₅₀ of Glycoplex at 4.19µg/mL reflects potent bioactivity even at relatively low concentrations if compared with standard indomethacin recorded IC₅₀ at 4.28µg/mL. The wound healing results demonstrate that Glycoplex treatment significantly accelerates fibroblast migration and wound closure compared to the untreated control. The Glycoplex -treated group achieved nearly 90% closure within 48 hours, compared to 66% in the control group, indicating a clear enhancement of the wound healing process. The IC₅₀ value of 196.34µg/mL obtained for the Glycoplex indicates low cytotoxic activity against human dermal fibroblasts. In conclusion, Glycoplex, a novel postbiotic-based bioactive, demonstrates potent anti-inflammatory activity comparable to indomethacin, alongside significant wound-healing benefits through accelerated fibroblast migration and low cytotoxicity toward human dermal fibroblasts. Its proven efficacy in managing mild-to-moderate inflammation, combined with its safety, photo stability, and sustainable origin, positions it as a promising alternative to conventional agents and a valuable innovation in advanced wound care strategies.
KEYWORDS: Glycoplex, Postbiotic, Probiotic, Anti-inflammatory, Scratch Assay, Wound healing.
INTRODUCTION:
The largest organ in the body, the skin, serves as a dynamic barrier that shields inside tissues from viruses, environmental irritants, and physical harm.
It is crucial for immunological surveillance, excretion, hydration, thermoregulation, and vitamin D production. The complex ecology of resident bacteria supports the health and homeostasis of the skin. However, wounds like burns, cuts, surgical incisions, or chronic inflammatory diseases commonly upset this equilibrium 1.
Heamostasis, inflammation, proliferation, and tissue remodeling are the four overlapping stages of the wound healing process, which is usually effective in healthy people. However, wound healing is frequently compromised in people with diabetes mellitus, leading to chronic, non-healing wounds that are extremely prone to infection. These infections raise morbidity and the burden on healthcare systems by aggravating tissue damage and delaying the healing process considerably2.
Generally considered as safe (GRAS), probiotic microorganisms like Lactobacillus, Bifidobacterium, and Lactococcus may tolerate severe gastrointestinal disorders3. They have the ability to modify host immunity4, generate antimicrobial chemicals, and stick to mucosal surfaces. Strain selection and fermentation conditions can be adjusted to further enhance the biological usefulness of the structurally unique EPS produced by each strain5. Crucially, probiotic-derived EPS has shown promise in gastrointestinal and cutaneous wound healing models, provided prebiotic benefits, and encouraged the development of beneficial microbiota6.
Exopolysaccharides (EPS), which are high molecular weight polymers released by microorganisms such as bacteria and fungus, have become one of the most promising options among these bioactive substances. EPS can reside as capsular structures or be secreted into the extracellular environment7. Depending on their monosaccharide content, they can be either homopolysaccharides or heteropolysaccharides. Because of their low toxicity, biodegradability, biocompatibility, and adjustable bioactivity, bacterial EPS particularly those generated by probiotic strains have drawn a lot of attention8. These characteristics make them perfect for a range of biological uses, such as immunological regulation, wound healing, and antimicrobial protection 9,10.
According to recent studies, postbiotic compounds—non-viable bacterial metabolites or metabolic byproducts like EPS provide a secure and efficient substitute for live probiotics in medicinal settings. This is particularly important in wound treatment, where it can be difficult to keep probiotic cells viable. Notably, because of its anti-inflammatory, antimicrobial, and tissue regenerative qualities, probiotic derived EPS has demonstrated encouraging wound healing potential, including in diabetic models3–10.
This study aims to examine anti-inflammatory and wound-healing properties of Glycoplex. The goal includes assaying the effect of Glycoplex on cell viability of HFB4 human skin fibroblasts.
Glycoplex product:
Glycoplex raw material provided from polygon Life science Company, Polygon Technologies (Cairo, Egypt). The nature of Glycoplex is an extracellular polysaccharides metabolite called posbiotic extracted and purified from local probiotic bacterial strain isolated and identified.
HRBC Membrane Stabilization assay:
According to Shinde et al. (1989), anti-inflammatory effects on Glycoplex membrane integrity and HRBC hemolysis have been shown11. Fresh whole blood was drawn from healthy people, placed in heparinized tubes, and centrifuged for 10minutes at 3000rpm to produce the erythrocyte suspension. The red blood pellets and the supernatant were dissolved in an equivalent volume of ordinary saline. Prior to being reconstituted as a 40% v/v suspension (pH 7.4, 10mM sodium phosphate buffer), red blood pellets were dissolved in an isotonic buffer solution and their volume was determined.
One liter of distilled water, 1.15g of Na2HPO4, 0.2g of NaH2PO4, and 9g of NaCl make up the buffer solution. Extract samples were dissolved in distilled water (a hypotonic solution) for this test. Duplicate pairs of centrifuge tubes were filled with the extracts at the specified doses (100, 200, 400, 600, 800, and 1000 µg/ml) in the hypotonic solution (5ml) (per dose).
Furthermore, duplicate pairs (per dose) of centrifuge tubes were filled with isotonic solution (5ml) that contained graded doses of the extracts (100–1000 µg/ml). The control tubes contained five milliliters of the drug (indomethacin 200g/ml) and five milliliters of the vehicle (distilled water). Each tube was filled with 0.1ml of erythrocyte suspension, which was carefully mixed. The solutions were incubated for an hour at 37 °C before being centrifuged for three minutes at 1500 rpm. A Spectronic (Milton Roy) spectrophotometer was used to quantify the hemoglobin conc. supernatant's absorbance (OD) at 540nm12,13.
To calculate the percentage hemolysis, hemolysis in the presence of distilled water was taken to be 100%. The extract's percentage of hemolysis inhibition was estimated to be as follows:
Inhibition of hemolysis (%) =1-(OD2-OD1)/(OD3-OD1) X100
Where OD1 indicates the test sample's absorbance in an isotonic solution, OD2 indicates the test sample's absorbance in a hypotonic solution, and OD3 indicates the control sample's absorbance in a hypotonic solution.
Scratch Assay:
To assess the migratory potential of HFB4 human skin fibroblast cells in response to Glycoplex therapy, the wound healing experiment was carried out in accordance with Cory (2011)14. To enable the creation of a confluent monolayer, HFB4 cells were seeded in 6-well culture plates at a density of 3 × 105 cells/well and incubated for the entire night at 37°C in a humidified environment with 5% CO2.
Using a sterile 200µL (yellow) pipette tip maintained at an angle of roughly 30 degrees to ensure a consistent and narrow scratch width, a uniform linear scratch was inserted into the cell monolayer once confluence was reached. The wells were twice washed with sterile phosphate-buffered saline (PBS) to gently remove detached cells and debris.
The control wells were then filled with fresh medium, and the treatment group was given medium that had been supplemented with Glycoplex. Using an inverted phase-contrast microscope set to 10× magnification, images of the scratch (wound) area were taken at 48 hours, making sure that both wound’s borders were visible.
MII Image View 3.7 software was used to quantify the wound area at each time point. The following formula was used to determine wound closure:
Wound Closure (%) = (A0−At/ A0 ) ×100
Where A0 is the wound area at 0 h and At is the wound area at time (48 h). Experiments were conducted in triplicate, and results were expressed as mean ± standard deviation (SD).
MTT assay:
The HFB4 human skin fibroblasts used for MEM-E media with 10% fetal bovine serum from Thermo Fisher Scientific, Waltham, Massachusetts, USA, were used to cultivate cells at 37°C in a humidified 5% CO2 atmosphere (Jouan SA, Saint-herblain, Pays de la Loire, France). Cells were kept in accordance with the manufacturing procedure, which included decanting the growing media and washing the cells with phosphate buffer saline (Adwia Pharmaceuticals, Sharqia, Egypt).
For five minutes at 37°C, cells were subjected to 0.05% (v/v) EDTA and 0.25% trypsin enzyme (GIBCO). Spent out were separated cells according to necessity. Senthilraja and Kathiresan (2015) examined the cytotoxicity of OEC cell lines grown in a 75cm cell culture (TPP-Swiss)15–17. Cells were plated in 96-well cell culture plates at a density of 105cells/ml and incubated for 24hours at 37°C prior to confluence. After the growth medium was decanted, pre-cultured plates were filled with fresh medium that contained serially diluted Glycoplex samples.
50L of MTT stock solution (0.5mg/ml) was melted in phosphate buffer saline (PBS, pH = 7.2±0.2 (Adwia)) and added to each well twenty-four hours after the dead cells were extracted. After 4hours at 37°C, the supernatant was withdrawn, and 50L of di-methyl sulfoxide (DMSO) was added to each well to make the formazan precipitate soluble. After 30minutes of dark incubation at 37°C, absorbance at 570nm was measured using a micro plate reader (ELx-800, Bio-Tek Instruments, Inc., Winooski, VT, USA).
The ratio of viable cells was calculated using the following formula:
Viability percentage (%) = Mean OD of test dilution × 100/Mean OD of control wells.
Evaluation of Anti-inflammatory Activity of Glycoplex:
The HRBC membrane stabilization analysis's results, which are shown in table 1, demonstrate that the Glycoplex has strong anti-inflammatory properties due to its high hemolysis inhibition. In hypotonic conditions, the sample successfully prevented red blood cells from lysing, indicating cellular membrane stabilization, which is comparable to the stabilization of lysosomal membranes in inflammatory tissues.
When compared to the conventional indomethacin reported IC50 at 4.28µg/mL, the observed IC50 of Glycoplex at 4.19µg/mL indicates strong bioactivity even at relatively low concentrations, supporting the therapeutic potential of Glycoplex in inflammation-related applications. The dose-dependent pattern suggests that some chemical elements that can interact with or strengthen membrane integrity are probably responsible for Glycoplex function.
These results are in line with earlier studies on microbial exopolysaccharides that have cytoprotective and anti-inflammatory qualities, potentially through receptor-modulating or antioxidant mechanisms. The low IC50 and significant hemolysis inhibition make EPS a viable option for additional pharmacological and cosmetic uses that target inflammation18–20.
Table 1: Anti-inflammatory activity of Glycoplex in comparison with standard Indo
|
Concentrations µg /ml |
Glycoplex |
Indomethacin |
||||
|
Mean Hypotonic Ab. |
SD |
Hemolysis Inhibition % |
Mean Hypotonic Ab. |
SD |
Hemolysis Inhibition % |
|
|
1000 |
0.017 |
0.002 |
99.5 |
0.019 |
0.002 |
99.2 |
|
500 |
0.027 |
0.003 |
98.1 |
0.033 |
0.003 |
97.3 |
|
250 |
0.068 |
0.002 |
92.8 |
0.065 |
0.005 |
93.1 |
|
125 |
0.095 |
0.004 |
89.2 |
0.099 |
0.003 |
88.7 |
|
62.5 |
0.117 |
0.002 |
86.3 |
0.135 |
0.003 |
84.1 |
|
31.25 |
0.190 |
0.004 |
77.5 |
0.192 |
0.003 |
77.3 |
|
15.62 |
0.291 |
0.003 |
65.3 |
0.275 |
0.003 |
67.3 |
|
7.8 |
0.394 |
0.004 |
52.9 |
0.392 |
0.004 |
53.2 |
|
3.9 |
0.488 |
0.009 |
41.7 |
0.501 |
0.002 |
40.1 |
|
IC50 µg /ml |
4.19 |
4.28 |
||||
Glycoplex Wound Healing Activity in Vitro:
A scratch assay on human skin fibroblasts (HFB4) was used to assess the Glycoplex's capacity to heal wounds (figure 1). Figure 2 shows the quantification of wound areas using image analysis software after images were taken at 0 and 48hours.
The control group experienced a 65.77% wound closure, with an average wound area that dropped from 1, 067, 205.58µm² at 0hours to 365, 273.0µm² at 48hours. The Glycoplex-treated group, on the other hand, had a much-improved closure, with the wound area decreasing from 1, 067, 205.58µm² to 111, 246.2µm², yielding an 89.58% wound closure.
Furthermore, the area difference, which indicates the degree of closure, was substantially larger in the EPS-treated group (955, 959.4µm²) than in the control group (701, 923.6µm²), and the mean remaining wound width at 48hours decreased from 338.48µm (control) to 103.08 µm (Glycoplex-treated).
When compared to the untreated control, the results of the wound healing (scratch) assay show that Glycoplex treatment dramatically speeds up fibroblast migration and wound closure. The Glycoplex-treated group clearly improved the wound healing process, achieving approximately 90% closure in 48 hours as opposed to 66% in the control group.
The bioactive elements in the EPS, which may encourage cell migration, proliferation, or matrix remodeling, may be responsible for this increase in closure efficiency. The significant decrease in wound area and residual breadth indicates that EPS may be used to promote wound healing, most likely by modifying fibroblast activity and extracellular matrix interaction. These results are consistent with earlier research on microbial exopolysaccharides, many of which have been shown to support epithelial repair and dermal regeneration. To evaluate effectiveness in in vivo wound models and to clarify the molecular mechanisms behind the reported pro-healing actions, more research is necessary21–22.
Figure 1: Wound-healing activity of Glycoplex at concentration 250 µg/mL compared with control
A
B
Figure 2: A: control and B: treated with Glycoplex, Cellular migration in HFB4 human skin fibroblasts cells analyzed through scratch assay for wound-healing properties of Glycoplex at concentration 250 µg/mL
Cytotoxicity effect of Glycoplex:
The MTT assay was used to evaluate the cytotoxicity of the Glycoplex in human skin fibroblasts (HFB4). With an estimated IC50 value of 196.34±1.13µg/mL, which represents the concentration at which 50% cell viability was suppressed, Glycoplex demonstrated a dose-dependent cytotoxic action. Higher doses (1000 and 500 µg/mL) caused a considerable decline in viability, whilst those below 125µg/mL had no cytotoxic effects (figures 3-4).
Figure 3: Effect of Glycoplex on cell viability of HFB4 human skin fibroblasts
Figure 4: Morphology changes in HFB4 skin cells after exposure to Glycoplex at concentrations of 1000 – 31.25 μg/ml
The Glycoplex's IC50 value of 196.34µg/mL suggests that it has little cytotoxic effect against human dermal fibroblasts. This indicates that over 98% of cell viability was maintained at lesser concentrations (<125µg/mL), indicating that the Glycoplex is comparatively biocompatible. Osmotic stress or bioactive elements in the Glycoplex could be the cause of the cytotoxicity seen at greater concentrations. These findings are in line with earlier research on natural polysaccharides, which, depending on their source, molecular weight, and structure, frequently show mild to moderate cytotoxicity.
The excellent cell viability at lower EPS concentrations supports its safety and promise for further development from the standpoint of biomedical applications, especially in topical or wound-healing formulations. To demonstrate biocompatibility, more in vivo and long-term exposure studies are advised1,7,8,21,22.
CONCLUSION:
This study suggests that Glycoplex is a postbiotic that is a safe anti-inflammatory formulation with comparable to the standard indomethacin. Glycoplex can be equivalent to indomethacin in the treatment of mild-to-moderate Inflammation. The study's main conclusion summarizes important findings and provides a synthesized viewpoint on Glycoplex's potential to transform existing understanding of novel strategies for sustainable wound healing techniques. Our findings might be supported by additional research using a bigger sample size and across ethnic groups.
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Received on 11.08.2025 Revised on 04.12.2025 Accepted on 07.02.2026 Published on 20.05.2026 Available online from May 25, 2026 Research J. Pharmacy and Technology. 2026;19(5):2139-2144. DOI: 10.52711/0974-360X.2026.00308 © RJPT All right reserved
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