Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) 

Volume 14, Number 2, October 2025 | Pages: 951-955 | DOI: 10.14421/biomedich.2025.142.951-955 ISSN 2540-9328 (online) 
 

 

 

 

Evaluation of Forest Honey on the Proliferation and Migration of 

Dermal Fibroblasts under Hyperglycemic Conditions: An In Vitro Study 

 
Januar Rizqi1*, Denny Agustiningsih2, Dwi Aris Agung Nugrahaningsih3 

1Departement of Nursing, Faculty of Medicine, Universitas Respati Yogyakarta, Indonesia. 
2Department of Physiology, Faculty of Medicine, Universitas Gadjah Mada, Indonesia. 

3Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Gadjah Mada, Indonesia. 

 

 

Corresponding author* 
arizqi.januar@respati.ac.id 

 

 
Manuscript received: 01 September, 2025. Revision accepted: 27 October, 2025. Published: 28 October, 2025. 

 

 

Abstract 

 

This study aims to evaluate the effect of forest honey on fibroblast proliferation and migration under hyperglycemic conditions in vitro. 

The research method involved culturing primary fibroblasts in high-glucose DMEM (25 mM) and dividing them into five groups: 

standard control (standard medium), hyperglycemia control (high-glucose medium without treatment), 3% honey, 1.5% honey, and 0.75% 

honey. Proliferation was assessed by counting live cells (Trypan Blue staining/hemocytometer) at 24, 48, and 72 hours. Migration was 

measured using a scratch assay (0, 24, 48, 72 hours) and analyzed with ImageJ. The Shapiro–Wilk test was used for normality; data were 

analyzed with ANOVA followed by LSD or Kruskal–Wallis (p<0.05). Results showed that hyperglycemia decreased proliferation 

compared to the standard control. Treatment with 1.5% honey and 0.75% honey consistently increased proliferation compared to the 

hyperglycemic control at all time points (p<0.05), while 3% honey showed a relatively lower increase. The standard control group 

achieved 100% closure at 72 hours in the migration variable. The honey groups (0.75–3%) showed an increase compared to the 

hyperglycemic control, but the difference was not significant at 24–48 hours and approached significance at 72 hours (p≈0.057). In 

conclusion, forest honey at a concentration of 1.5% can increase fibroblast proliferation in a hyperglycemic environment. The effect on 

migration requires confirmation with osmolality controls and more sensitive endpoints. These findings provide a biological basis for 

developing honey-based diabetic wound care adjuvants focusing on dose optimization and formulation standardization. 

 

Keywords: forest honey; in vitro hyperglycemia (25 mM); dermal fibroblast proliferation; scratch migration assay. 

 

 

INTRODUCTION 
 

Diabetes mellitus (DM) continues to increase and has 

become one of the most significant burdens on the global 

health system, with a significant economic impact on 

individuals (IDF, 2025). One of the complications of DM 

is diabetic foot ulcer (DFU), which can lead to disability, 

amputation, and mortality, and requires high healthcare 

resources (Fuentes-Peñaranda et al., 2025). Wound 

healing is a coordinated process that includes the phases 

of hemostasis, inflammation, proliferation, and 

remodeling, with complex intercellular orchestration and 

mediators, one of which is the fibroblast (Mamun et al., 

2024).  

Fibroblasts play a central role in matrix deposition, 

wound contraction, and the secretion of growth factors 

that support angiogenesis and tissue repair (Boraldi et al., 

2024). Reactive oxygen species (ROS) support healing 

signals under controlled conditions, while in excessive 
conditions, they promote chronicity of wounds (Hunt et 

al., 2024). Redox imbalance disrupts proliferation and 

migration by disrupting the cytoskeleton, adhesion–de-

adhesion, and signal transduction. Hyperglycemia 

exacerbates the condition through the AGE–RAGE 

pathway, mitochondrial load, and persistent 

inflammation, thereby delaying the proliferation phase 

(Hunt et al., 2024). In human dermal fibroblasts, high 

glucose exposure is associated with cellular aging and 

dysfunction that impedes healing (Ma et al., 2023). 

Therefore, restoring redox homeostasis and fibroblast 

function is a biological key to accelerating tissue repair 

(Mamun et al., 2024). 

Hyperglycemia consistently reduces fibroblast 

proliferation and migration. Impeded cell-type-specific 

migration is recognized as a key mechanism delaying 

diabetic wound healing (Song et al., 2025). Current 

wound therapies such as nanotechnology, stem cells, and 

innovative dressings are promising, but cost and 

complexity constraints limit their widespread application 

(Mamun et al., 2024). Some high-glucose studies have 

not included iso-osmotic controls, making it challenging 

to distinguish biological effects from osmolality or 
viscosity (Sharma et al., 2018). From a methodological 

perspective, further evidence is needed to strengthen in 

vitro evidence and enable translation (Mamun et al., 

https://doi.org/10.14421/biomedich.2025.142.951-955


 

 
 

952 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 951-955 

 

 

2024). Thus, scientific, biocompatible adjuvant 

interventions that can be integrated into wound care 

practices are a fundamental need (Fuentes-Peñaranda et 

al., 2025). 

Honey is a candidate adjuvant due to its polyphenol 

or flavonoid content and its ability to produce low doses 

of H₂O₂ via glucose oxidase, making it relevant as a 

redox modulator and antimicrobial (Tashkandi, 2021). 

Honey can also modulate inflammation, support the 

proliferation phase, increase collagen synthesis, and 

improve cell migration (Iosageanu et al., 2024). Previous 

studies have assessed the effectiveness of honey on 

various chronic wounds with a good safety profile, 

although the heterogeneity of studies calls for further 

standardization. Variability in sources and phenolic 

composition underscores the importance of precise 

dosing to ensure efficacy without causing osmotic stress 

(Saad, 2025). Therefore, conducting safe concentration 

range trials for fibroblasts under hyperglycemic 

conditions is crucial. 

Although chronic wound therapy rapidly evolves to 

include nanotherapeutics, exosomes, and innovative 

dressings, safe, affordable, and accessible adjuvants 

remain highly needed (Mamun et al., 2024). A significant 

gap in this research is the limited number of studies 

evaluating honey on fibroblast proliferation and 

migration under hyperglycemic conditions. The research 

of this article lies in its focus on forest honey across a 

range of doses and the simultaneous evaluation of two 

key fibroblast functions in a hyperglycemic environment. 

Thus, this study aimed to assess the effect of forest honey 

on fibroblast proliferation and migration under 

hyperglycemic conditions in vitro. 

 

 

MATERIALS AND METHODS 
 

Study Area 

This study was conducted at the Pharmacology 

Laboratory, Physiology Laboratory, and Anatomy 

Laboratory of the Faculty of Medicine, Gadjah Mada 

University, Yogyakarta, and has obtained ethical 

approval from the UGM LPPT Research Ethics 

Committee (No. 00010/04/LPPT/2016). 

 

Cell Culture  

Primary fibroblasts were obtained from the dermal tissue 

of male Wistar strain white rats (aged 3–4 months) 

induced with diabetes mellitus using streptozotocin 

(STZ, 60 mg/kg BW) and nicotinamide (NA, 120 mg/kg 

BW) via intraperitoneal injection. Mice with fasting 

blood glucose levels ≥126 mg/dL were categorized as 

successful diabetes models. Fibroblast cells were isolated 

enzymatically, then cultured in Dulbecco's Modified 

Eagle's Medium (DMEM) containing 10% fetal bovine 

serum (FBS) and antibiotics-antimycotics. The cultures 

were maintained in a 5% CO₂ incubator at 37 °C until 

they reached 80–90% confluence. Hyperglycemia was 

modeled using high-glucose DMEM medium (25 mM). 

 

Experimental Groups and Treatments 

The fibroblast cultures were divided into five groups, 

namely: (1) fibroblasts in standard medium (standard 

control), (2) fibroblasts in high-glucose medium without 

treatment (hyperglycemia control), (3) 3% honey 

fibroblasts, (4) 1.5% honey, and (5) 0.75% honey. Each 

treatment was performed in triplicate (n = 3) and 

observed after 24, 48, and 72 hours of incubation. 
 

Proliferation Assay 

Cell proliferation was assessed by counting the number 

of cells using a hemocytometer after Trypan Blue 

staining. The number of live cells was counted 24-, 48-, 

and 72-hours post-treatment, then expressed as the mean 

± standard deviation. 
 

Migration Assay 

Fibroblast migration activity was tested using the scratch 

assay method. A linear scratch was made using a sterile 

pipette tip in confluent monolayer cultures. The scratch 

area was photographed with an inverted microscope at 0, 

24, 48, and 72 hours. Wound closure was calculated 

using ImageJ software, and the results were expressed as 

the percentage of cell migration relative to the baseline. 
 

Statistical Analysis 

Data normality were tested using the Shapiro–Wilk test. 

Normally distributed data were analyzed using One-Way 

ANOVA followed by LSD post hoc testing. The 

Kruskal–Wallis’s test was used if the data were not 

normally distributed. The significance level was set at p 

< 0.05. 
 
 

RESULTS AND DISCUSSION 
 

Fibroblast Proliferation 

Fibroblast proliferation differed between groups at all 

observation time points (24, 48, 72 hours). Standard 

control (standard medium) showed the highest number of 

cells, while hyperglycemia control (high glucose medium 

without treatment) showed the lowest. Administration of 

1.5% honey and 0.75% honey consistently increased 

proliferation compared to the hyperglycemia control at 

24, 48, and 72 hours (all p<0.05; LSD post hoc test), 

while 3% honey increased but was relatively lower than 

1.5% and 0.75% honey. The Shapiro–Wilk test met the 

normality assumption, and One-Way ANOVA showed 

significant differences between groups (p<0.001). The 

mean and standard deviation for each group is shown in 
Table 1. 

 

 

 

 



 

 
 

 Rizqi et al. – Evaluation of Forest Honey on the Proliferation and Migration of … 953 
 

 
Table 1. Mean number of fibroblasts (mean ± SD) at 24, 48, and 72 hours. 

 

Group 24 h (mean ± SD) 48 h (mean ± SD) 72 h (mean ± SD) 

Standard control 40.68 ± 4.62 49.32 ± 4.46 51.63 ± 1.36 

Hyperglycemic control 16.82 ± 2.86 21.14 ± 4.36 19.19 ± 3.86 

Honey 3% 23.51 ± 2.47 30.06 ± 7.55 22.29 ± 3.54 

Honey 1.5% 30.08 ± 2.70 36.01 ± 5.87 29.33 ± 2.65 

Honey 0.75% 28.61 ± 1.71 31.18 ± 3.90 27.79 ± 1.75 

Note: ANOVA p<0.001; LSD follow-up test shows that 1.5% honey (24 hours p=0.019; 48 hours p=0.011; 72 hours p=0.006) and 0.75% 

honey (24 hours p=0.035; 48 hours p=0.046; 72 hours p=0.012) were higher than the hyperglycemia control. 

 

 

Fibroblast Migration 

Migration activity increased over time in all groups. The 

standard control showed the highest migration 

percentage and reached 100% closure at 72 hours, while 

the hyperglycemia control was the lowest. Treatment 

with 3% honey, 1.5% honey, and 0.75% honey showed 

an increasing trend compared to the hyperglycemia 

control; the differences between groups were not 

significant at 24 hours (Kruskal–Wallis p=0.629) and 48 

hours (p=0.094), and showed a trend at 72 hours 

(ANOVA p=0.057). Detailed values are presented in 

Table 2. 
 

 

Table 2. Percentage of fibroblast migration in various treatment groups. 

 

Group 24 h (median; IQR) 48 h (median; IQR) 72 h (mean ± SD) 

Standard control 78.93 (69.73–79.02) 100.00 (100.00) ᵃ 100.00 ± 0.00ᵃ 

Hyperglycemic control 78.59 (66.50–78.64) 81.23 (68.84–82.29) 79.23 ± 7.69 

Honey 3% 78.40 (65.02–78.47) 82.70 (68.66–83.65) 79.92 ± 8.21 

Honey 1.5% 77.07 (66.02–77.07) 83.16 (70.12–85.38) 81.20 ± 7.45 

Honey 0.75% 77.79 (65.18–79.34) 81.71 (68.93–82.44) 80.35 ± 7.61 

ᵃThe difference is significant compared to other groups (p<0.05). 

 

Figure 1. Fibroblast migration activity at 24, 48, and 72 hours (inverted microscope 100×). Straight lines mark the initial scratch boundaries. Groups: 

standard control, hyperglycemia control, 3% honey, 1.5% honey, 0.75% honey. 

Standar control 

Honey 0.75% 

Honey 1.5% 

Honey 3% 

Hyperglycemic control 

24h Jam 48h 72h 



 

 
 

954 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 951-955 

 

 

Discussion 

This study shows that hyperglycemia suppresses 

fibroblast function, particularly proliferation, compared 

to normoglycemic controls; conversely, honey at 0.75–

1.5% consistently increased proliferation compared to 

hyperglycemic controls, while honey at 3% did not 

increase proliferation. Meanwhile, fibroblast migration in 

the honey group showed an increasing trend compared to 

the hyperglycemic control, but did not reach statistical 

significance; the normoglycemic control achieved 100% 

wound closure at 72 hours. These results are consistent 

with the knowledge that a high-glucose environment 

disrupts redox homeostasis and tissue repair dynamics, 

while the bioactive components of honey have the 

potential to moderate these disturbances (Berry et al., 

2024; Pasupuleti et al., 2020).  

The finding of reduced fibroblast performance in 

high-glucose media is consistent with reports that 

hyperglycemia increases oxidative stress and maintains 

inflammation, thereby delaying the proliferation phase 

until remodeling (González et al., 2023; Nagy et al., 

2019). In human dermal cell models, high glucose 

exposure is associated with cellular aging and functional 

abnormalities that slow healing (Zhang et al., 2021). 

Research results show that honey, especially honey rich 

in phenolics, can suppress inflammation, support 

proliferation, and improve cell migration. 

Hyperglycemia increases ROS production through the 

AGE–RAGE pathway, mitochondrial load, and glucose 

autooxidation; excessive ROS exposure disrupts 

cytoskeletal organization, adhesion–de-adhesion, and 

signaling required for proliferation and migration 

(Comino-Sanz et al., 2021; Hunt et al., 2024). Honey 

contains polyphenols or flavonoids that produce low 

levels of H₂O₂ via glucose oxidase; both factors 

contribute to redox buffering (antiradical, pro-

inflammatory resolution) and support cell cycle and 

matrix survival (Iosageanu et al., 2024; Wilczyńska & 

Żak, 2024). The dose-response correlation indicates that 

honey at 0.75–1.5% is more effective than honey at 3%.  

Scientifically, these results reinforce the hypothesis 

that honey can maintain and enhance fibroblast 

proliferation in a hyperglycemic environment, which is a 

key mechanism for initiating the tissue repair phase 

(Pasupuleti et al., 2020; Scepankova et al., 2021). To 

strengthen the evidence, further studies need to measure 

and control osmolality, add orthogonal endpoints, and 

verify the redox pathway through quantification of ROS 

and antioxidant enzymes (SOD, CAT, GPx) (Gayathri et 

al., 2023). Testing in 3D/explant models and in vivo 

validation in diabetic wounds will increase biological 

relevance toward early clinical trials (Mamun et al., 

2024). 

 

 

 

 

CONCLUSIONS 
 

Hyperglycemia reduces fibroblast function, most notably 

in proliferation, compared to normoglycemic controls. 

Administration of forest honey at concentrations of 0.75–

1.5% consistently increased fibroblast proliferation 

compared to hyperglycemic controls at 24, 48, and 72 

hours, while 3% was relatively less optimal. In 

migration, the honey group showed an increasing trend 

compared to the hyperglycemic control, but it was not 

statistically significant; the standard control achieved 

100% wound closure at 72 hours. Overall, these findings 

confirm that sub-osmotic honey concentrations (≤1.5%) 

are more conducive to supporting fibroblast function in a 

hyperglycemic environment, consistent with the study 

objective of evaluating the effects of honey on fibroblast 

proliferation and migration. 

 

 

Authors' Contributions: Januar rizqi conceptualization; 

methodology; investigation; data curation; formal 

analysis; visualization; writing original draft. Denny 

Agustiningsih provides validation, supervision, 

resources, writing, review & editing, and project 

administration. Dwi Aris' supervision; writing, review 

and editing. All authors contributed to interpreting the 

results, reviewed and approved the final manuscript for 

publication, and agreed to be accountable for all aspects 

of the work. 

 

Competing Interests: The authors declare that there are 

no competing interests. 

 

Funding: The authors declare funding if any funding. 

 

 

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