Assessment of the Clinical, Cellular and Molecular Effects of Royalactin-Royal Jelly on the Healing of Wagner Grad II Diabetic Foot Ulcers.
Phase: PHASE2
Conditions: Diabetic Foot Ulcer
Sponsor: Universidad Autonoma de Nuevo Leon

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Assessment of the clinical, cellular, and molecular effects of Royalactin-Royal Jelly on 
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the healing of Wagner Grade II diabetic foot ulcers: A phase II, double-blind, 
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randomized controlled trial. 
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Valoración de los efectos clínicos, celulares y moleculares de la Royalactina-Jalea Real en la 
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cicatrización de úlceras de Pie diabético Wagner II. Ensayo clínico controlado doble ciego 
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fase II. 
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Completed September 2025  
 
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Abstract 
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Objective: To assess the clinical, cellular, and molecular effects of Royalactin on the healing 
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of Wagner Grade II diabetic foot ulcers compared to a placebo. 
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Materials and Methods: A total of 20 observations were derived from dividing 10 wounds 
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in diabetic patients recruited from a chronic wound and diabetic foot management center at 
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a hospital in Northeastern Mexico for this double-blind randomized controlled trial. Each 
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wound was divided, treated with standard techniques three times per week, and randomized 
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to receive either placebo or Royalactin gel on each side for 4 weeks. At baseline and study 
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completion, wound area (cm²) was measured, and biopsies were taken, stained, and labeled 
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to classify epithelial characteristics, inflammatory response, fibroblast presence, 
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angiogenesis, collagen content, and the expression and intensity of VEGF and EGFR. 
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Results: In the clinical evaluation, both groups showed a statistically significant reduction in 
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wound area; however, this reduction was greater in the Royalactin group (from 6.3 to 3.4 
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cm², p = 0.003) compared to the control group (from 7.8 to 3.9 cm², p = 0.036). Regarding 
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cellular activity, no statistical significance was reached, but the Royalactin group showed an 
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increase in cases with marked infiltration in the granulation tissue (10% to 50%), an increase 
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in fibroblasts with moderate distribution (20% to 60%), an increase in cases with moderate 
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angiogenesis (30–40%), and an increase in collagen content. At the molecular level, both 
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groups showed an increase in VEGF expression, with a higher proportion in the Royalactin 
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group (18% increase: from 32% to 50%) than in the control group (4.5% increase: from 39% 
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to 43.5%), approaching statistical significance (p = 0.072), with an increase in expression 
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intensity from mild to moderate (10% to 30%). No EGFR expression was detected in either 
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group at baseline or after 4 weeks. 
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Conclusions: A significant reduction in average wound area was observed, accompanied by 
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favorable modifications in key histological parameters and a trend toward increased VEGF 
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expression, particularly in the Royalactin-treated group. EGFR expression was not detected 
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(0 in all evaluated cases), suggesting that this pathway was not activated under the study 
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conditions. 
 
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Introduction.  
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The prevalence of diabetic foot ulcers (DFU) ranges from 6% to 15% and accounts for nearly 
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20% of hospital admissions among diabetic patients, who face a 15-fold higher risk of 
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amputation compared to non-diabetics (1,2). Risk factors for DFU include male gender, 
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disease duration exceeding 10 years, high BMI, and comorbidities such as neuropathy, 
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chronic arterial occlusive disease, and infection. Other factors include elevated glycated 
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hemoglobin levels (3-6), foot deformities and instability (7,8), high plantar pressures, and 
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inappropriate foot self-care habits (9-11). Management is aimed at achieving ulcer closure as 
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quickly as possible, based on four principles: optimizing perfusion, adequate debridement, 
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infection control, and balanced pressure redistribution on the affected foot. To these ends, 
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various treatments are used, including the application of topical agents and dressings made 
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of diverse materials; honey with Royal Jelly (RJ) is one of the oldest among them. 
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The re-epithelializing effects of RJ have been reported, such as the stimulation of fibroblast 
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migration to the injury site through a dose-dependent mechanism that accelerates the healing 
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process (12), as well as the stimulation of keratinocyte production and migration (13). Among 
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the components of RJ, Royalactin stands out as the most significant peptide (14,15). This 
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protein activates the signaling pathway for the expression of the Epidermal Growth Factor 
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Receptor (EGFR) in bee larvae, leading to epigenetic changes and the development of larger 
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body size compared to worker bees (approximately double), accelerated growth, and life 
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extension (up to three years compared to an average of three weeks in worker bees) (16). 
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Additionally, a study demonstrated that this molecule also extends lifespan in the nematode 
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Caenorhabditis elegans through the expression of epidermal growth factor and its receptor; 
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another described property is antimicrobial action (17). The activation of EGFR is one of 
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the most critical mechanisms in healing processes and is a key point in cellular signaling to 
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induce the migration and division of fibroblasts, keratinocytes, and endothelial cells in 
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wounds. Furthermore, it activates epithelial-mesenchymal transition processes at the wound 
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periphery, participates in cellular homeostasis, and stimulates angiogenesis. The synergistic 
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activity of these effects induces accelerated granulation, re-epithelialization, wound 
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contraction, and dermal maturation, achieving a healing rate three times faster than 
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conventional methods, especially in diabetic foot ulcers. Controlled clinical trials have 
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demonstrated that its application in wounds promotes the speed of healing (18,19). 
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Objective 
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The objective of this study is to evaluate the clinical, cellular, and molecular effects of 
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Royalactin as an adjunct to standard treatment, compared to standard treatment alone, in the 
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management of Wagner Grade II diabetic foot ulcers by assessing wound dimensions, 
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cellular behavior, and growth factor expression. 
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Study Design 
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This is a randomized, double-blind, prospective, experimental, longitudinal, and comparative 
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controlled clinical trial. 
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Materials and methods 
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Ethical Considerations The study was conducted in compliance with Good Clinical Practice 
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(GCP) guidelines. All procedures adhered to the ethical standards of the Research and Bioethics 
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Committee of the XXXXXXXXXX. the Declaration of Helsinki, and the General Health Law. 
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Protocols were strictly followed, data were handled with rigorous confidentiality, and informed 
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consent was obtained from all participants. 
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Inclusion Criteria Adult patients with Diabetes Mellitus undergoing treatment for foot and ankle 
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wounds were included. Eligibility criteria required wounds larger than 4 cm², with more than 80% 
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granulation tissue, involving skin, fat, or ligaments without bone involvement, and non-infected 
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(Wagner Grade II) (20). Additional requirements included a duration of more than 2 months, an 
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ankle-brachial index (ABI) greater than 0.5, the signing of the informed consent form, and the 
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ability to attend dressing changes three times per week for 4 weeks. 
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Experimental Agents and Randomization The FEMSA Biotechnology Center laboratory 
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produced 20 tubes (50 ml) containing gold nanoparticles and emulsifiers. Ten tubes were 
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formulated with Royalactin at 0.2 g/L, while the other 10 served as the control without the peptide. 
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The laboratory randomized, numbered, and paired the tubes, providing a list for paired application. 
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Treatment Protocol Standardized dressing changes were performed three times per week for 4 
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weeks at the Chronic Wound, Diabetic Foot, and Hyperbaric Medicine Center of XXXXX. Each 
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wound was first irrigated with sterile water and dried with sterile gauze. The wound was then 
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divided (or the division was remarked) using black ink on the skin, and a 0.5 cm strip of Hypafix 
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(BSN medical GmbH, ESSITY) was applied. A circular label was placed on the perilesional skin 
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to identify the tube number (bottom), patient ID (top-right), and dressing change number (top-left). 
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Standard photographs were taken (Camera+ App, iPhone 15 Pro Max). The area was gently 
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debrided three times with sterile gauze moistened with 0.12% Chlorhexidine (Clorhexi-Clean®, 
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PISA). Ketanserin 2% gel (Sufrexal®, Janssen) was applied to both sides, followed by the 
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application of the corresponding gel (Royalactin or placebo) to each designated wound section. 
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Finally, a primary dressing of sodium carboxymethylcellulose hydrofiber and regenerated 
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cellulose fiber (Aquacell extra®, Convatec) was applied, protected by a secondary adhesive border 
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gauze dressing (Border gauze®, Medline). 
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Data Collection and Histological Analysis During the first and last dressing changes (sessions 1 
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and 13), photographs were taken to measure the area of each side in cm² (IMITO measure App, 
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iPhone 15 Pro Max). Additionally, 3 mm punch biopsies of the granulation tissue were obtained 
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from the distal edge of each side. 
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Biopsy samples were sectioned, labeled, and stained using Hematoxylin and Eosin (H&E), 
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Masson’s Trichrome, and immunohistochemistry (IHC) for the expression of: 
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• 
CD31 (Roche) 
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• 
Vascular Endothelial Growth Factor (VEGF) (Biocare) 
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• 
Epidermal Growth Factor Receptor (EGFR) (Cell Marque) (Figure 1 A-F). 
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Statistical Analysis and Evaluation Results from sessions 1 and 13 were evaluated. Wound size 
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was recorded in cm². Epithelialization, polymorphonuclear neutrophils (PMN), fibroblasts, 
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angiogenesis, and collagen were scored according to a semi-quantitative histological scale for 
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wound assessment (Table 1) (21). The percentage of tissue expressing VEGF and EGFR, as well 
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as the intensity of expression (scored 0 to 3+), were reported separately (Figure 1 G-I). Data were 
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compared at baseline and at four weeks, both as a total cohort and by comparing the Control vs. 
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Royalactin groups; individual variables were also analyzed. 
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Results 
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A total of 20 clinical and histological observations were included (2 per wound), corresponding 
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to Wagner Grade II DFUs treated over a 4-week follow-up period. The study population 
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consisted of 3 women and 7 men, with a mean age of 55.1 years, a mean duration of Diabetes 
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Mellitus of 15.5 years, 3 patients with arterial hypertension, and 4 with mild peripheral arterial 
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disease. Wounds had a mean evolution time of 6.7 months and a mean length of 8.6 cm. At 
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baseline, 95% of the wounds showed granulation tissue, all presented mild serosanguinous 
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exudate (staining), and most reported low-intensity pain and localized edema. All wounds 
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presented erythema (half < 2 cm and the other half > 2 cm). 
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All 20 observations were compared between baseline parameters and those obtained at the end of 
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treatment (Table 2). A significant reduction in the mean wound area was observed, decreasing 
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from 7.0 cm² (SD: 6.2) to 3.7 cm² (SD: 3.9) (p = .034). Regarding epithelialization, the lowest 
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value predominated; thickness of cut edges started at 16 cases (80%) and decreased to 13 (70%), 
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cell migration ≥50% remained unchanged (5%), while excision union increased from 5% to 10% 
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and keratinization from 10% to 15% (p = .865). The PMNL analysis showed a trend toward 
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increased evaluation scores after 4 weeks (p = .524), with a decrease in PMNL cases within the 
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dermis/granulation tissue (D/GT) from 45% to 30%. Conversely, moderate infiltrate increased 
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from 25% to 40%, and marked infiltrate in the same area rose from 10% to 35%, consistent with 
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an inflammatory peak associated with the active repair phase. Fibroblast presence showed 
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qualitative improvement, with an increase in moderate infiltrate cases in granulation tissue (GT) 
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from 25% to 45%, with no marked infiltrate observed in any phase (p = .520). Angiogenesis 
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evolved favorably: cases without vascular evidence disappeared (10% to 0%), moderate 
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angiogenesis in GT increased (20% to 30%), and marked angiogenesis appeared in 5% of cases 
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(p = .549). Regarding collagen synthesis, a statistically significant transition toward increased 
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synthesis was observed (p = .008), moving from absence (15% to 0%) and minimal collagen 
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(45% to 40%) toward mild (40% to 25%) and moderate (0% to 35%) presence in GT by the end 
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of treatment. At the molecular level, an increase in VEGF expression was identified, rising from 
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35.5% (SD: 20.8) to 46.75% (SD: 18.22) (p = .344). Furthermore, VEGF 1+ expression intensity 
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increased from 50% to 65%, while 2+ expression remained constant (35%), and extreme values 
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(0 and 3+) disappeared, indicating a more uniform response ($p = .396$). In contrast, no EGFR 
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expression was detected (0 in all evaluated cases), suggesting this pathway was not activated 
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under study conditions. 
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In the initial assessment of the 10 lesions selected as controls and the 10 selected for Royalactin 
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application, results showed that clinical, histological, and molecular characteristics were 
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generally comparable, with no statistically significant differences in most variables, indicating 
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adequate randomization (Table 3). Mean wound area was slightly larger (7.8 cm², SD: 7.1) in 
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Without Royalactin lesions compared to those selected for Royalactin (6.3 cm², SD: 5.4), with no 
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statistically significant difference (p = .684). Regarding epithelialization, thickness in cut edges 
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predominated in both groups (70% vs. 90%), being higher in the Royalactin group, which also 
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showed an exclusive presence of cell migration ≥50%. Keratinization and excision union were 
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observed in the Without Royalactin sections (p = .334). Regarding the PMN inflammatory 
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response, a higher proportion of mild infiltrate in D/GT was seen in Royalactin sections (70% vs. 
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20%), while control sections presented a higher proportion of moderate infiltrate in GT (40% vs. 
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10%) (p = .125). Fibroblast distribution was nearly identical, predominantly mild or moderate in 
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GT (p = .848). Angiogenesis was mainly mild in GT, with higher values in the Royalactin group 
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(80% vs. 50%), while moderate angiogenesis was more frequent in the control (30% vs. 10%) (p 
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= .628). For collagen, sections with minimal and mild presence in GT predominated (70% and 
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20% in control vs. 30% and 50% in Royalactin); both showed similar absence values (p = .484). 
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Molecularly, VEGF expression was similar (Mean: 39% SD: 24 vs. 32% SD: 16, p = .470), with 
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a slight predominance (7%) in control sections. However, expression intensity showed 
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significant differences (p = .014); 1+ intensity was observed in 80% of Royalactin sections 
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versus 20% in the control group, while 2+ expression was more frequent in the latter (60%). No 
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cases of maximum expression (3+) were identified. No EGFR expression was detected at 
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baseline. 
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After four weeks of treatment, clinical, histological, and molecular effects were compared 
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between groups (Table 4). Mean wound area was slightly smaller with Royalactin (3.4 cm², SD: 
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4.2) versus control (3.9 cm², SD: 3.8) (p = .677). Histologically, the initial biopsy site (GT at the 
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wound edge) had epithelialized in all observations by week 4; thus, end-of-study biopsies were 
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taken from the new wound edge. For epithelialization, more cases of thickness in cut edges were 
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seen in the Royalactin group (80% vs. 50%), with the latter showing a single case of cell 
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migration ≥50% and another with keratinization. Keratinization and excision union were slightly 
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more frequent (20%) in control sections (p = .471). In the PMNL infiltrate assessment, both 
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groups showed similar patterns, predominantly mild, moderate, and marked in D/GT ($ = .727), 
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with one case of absent infiltrate in the Royalactin group. Fibroblasts were similarly distributed 
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(mild and moderate in GT, p = .727), with the highest moderate distribution seen in Royalactin 
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(50%). Angiogenesis was very similar (p = .999), predominantly mild in GT (50% vs. 60%); 
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marked angiogenesis was only observed in one control section. Higher collagen percentage was 
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found in the minimal assessment of control sections (50%) compared to Royalactin (30%); 
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however, Royalactin showed higher mild (30% vs. 20%) and moderate (40% vs. 30%) ratings. 
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Molecularly, VEGF expression was higher in Royalactin (Mean: 50%, SD: 17) versus control 
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(Mean: 43.5%, SD: 19.7) (p = .440). VEGF intensity was similar in all sections, predominantly 
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1+ (60% vs. 70%) and 2+ (40% vs. 30%) (p > .999). No EGFR expression was detected. 
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When comparing baseline versus 4-week effects separately for both groups (Table 5), it was 
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observed that both groups showed a decrease in wound area, which was more pronounced in the 
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Royalactin group (from 6.3 SD: 5.4 to 3.4 cm² SD: 4.2, p = .089) compared to the control group 
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(from 7.8 SD: 7.1 to 3.9 cm² SD: 3.8, p = .185) (Graph 1). Histological results followed a similar 
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comparison pattern (Graph 2). In epithelialization, the lowest rating predominated and decreased 
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by week 4 in both control (70% to 60%) and Royalactin (90% to 80%) groups (p = .820 and p = 
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.999 respectively). PMN distribution in control sections showed an increase in mild infiltrate in 
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D/GT (20% to 40%, p = .836). In Royalactin sections, mild D/GT infiltrate decreased (70% to 
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20%), while moderate (10% to 40%) and marked (10% to 30%) infiltrate increased, showing a 
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trend (p = .168). Fibroblasts showed a discrete progression in controls (p = .999), while 
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Royalactin sections highlighted a trend (p = .187) toward moderate GT distribution (20% to 
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50%). Angiogenesis was very similar in controls (p = .999), whereas Royalactin sections showed 
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a progression from mild GT (80% to 60%) to moderate (10% to 30%) (p = .714). Collagen 
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increased in both groups, reaching 0% absence at week 4 and progressing to moderate presence 
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in controls (0% to 30%, p = .276) and further in Royalactin (0% to 40%, p = .071). Molecular 
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assessment showed a significant increase (p = .089) in VEGF expression percentage in 
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Royalactin sections (32% SD: 16 to 50% SD: 17), higher than in controls (39% SD: 24 to 43.5 
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SD: 19.7, p = .795) (Graph 3). VEGF 1+ intensity increased in controls and decreased in 
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Royalactin, while 2+ increased in Royalactin (p = .170 for controls vs. p = .582 for Royalactin). 
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No EGFR expression was detected. 
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Finally, a linear regression between wound area (cm²) and VEGF expression (%) at baseline 
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found a negative correlation in both control (R² = 0.38, p = .057) and Royalactin (R² = 0.16, p = 
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.253) groups. This suggests that higher VEGF levels may be associated with smaller lesions and 
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a more active tissue environment. At 4 weeks, the correlation remained negative in the control 
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group (R² = 0.24, p = .153), but was completely lost in the Royalactin group (R² = 0.04, p = .558) 
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(Graph 4). 
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Discussion 
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The baseline evaluation of both control and Royalactin-treated wound areas demonstrated that, 
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overall, the clinical, histological, and molecular characteristics were comparable, with no 
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statistically significant differences across most variables. This indicates successful randomization, 
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with the sole exception of a higher baseline intensity of VEGF expression in the control sections. 
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Our standard wound care management effectively promoted healing, as evidenced by a statistically 
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significant reduction in all wound areas (p = .034). This reduction was more pronounced in the 
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Royalactin group (p = .089), a finding that aligns with previous studies describing the re-
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epithelializing and pro-angiogenic properties of Royal Jelly (12–14). This result is clinically 
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significant, as an increased rate of wound closure is associated with lower rates of infectious 
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complications and a reduced risk of amputation in patients with diabetic foot ulcers. 
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In our study, the wound edges where initial biopsies were taken achieved complete re-
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epithelialization in all cases. These changes may reflect a more favorable tissue environment for 
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keratinocyte migration, consistent with the findings of Lin et al., who documented the mitogenic 
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effect of royal jelly-derived proteins in human skin models (13). 
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In the Royalactin-treated sections, unlike the control group, a trend toward an increase in moderate 
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infiltrate within the granulation tissue was observed (p = .168 vs. p = .836). This can be interpreted 
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as an intensification of the local inflammatory response a migratory effect previously reported (14) 
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and associated with an active regeneration phase in the absence of clinical infection. Additionally, 
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an increase in fibroblast presence to moderate levels in the granulation tissue was observed (p = 
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.187 vs. p = .999). Linked to a significant increase in collagen (p = .071 vs. p = .276), this likely 
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suggests the stimulation of cellular migration. Angiogenesis also exhibited a discreetly favorable 
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pattern (p = .714 vs. p = .999), with an increase in cases showing moderate angiogenesis. 
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At the molecular level, Royalactin-treated sections showed a statistically significant higher 
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percentage of VEGF expression (p = .0379 vs. p = .759). While the intensity of VEGF expression 
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decreased in the control group, it increased in the Royalactin-treated sections (p = .582 vs. p = 
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.170). 
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This finding is coherent with literature linking Royalactin to enhanced cell survival and growth in 
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Caenorhabditis elegans and tissues exposed to hypoxia via the modulation of EGFR and VEGF 
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(17,18). In our study, immunohistochemical activation of EGFR was not detected in human tissue, 
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possibly due to differences in methodological sensitivity or distinct transcriptional activation 
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requirements in humans. 
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Conclusiones 
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The present study explored the clinical, histological, and molecular effects of Royalactin, a 
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bioactive protein derived from royal jelly, on the healing process of Wagner Grade II diabetic foot 
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ulcers. Our findings demonstrate that, after four weeks of treatment, a reduction in the mean wound 
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area was achieved, accompanied by favorable modifications in key histological parameters and an 
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increase in VEGF expression, particularly in the Royalactin-treated group. These results justify 
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the development of Phase III clinical trials with greater statistical power, as well as complementary 
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studies to explore additional molecular pathways. 
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EGFR expresión was not detected (0 in all evaluated cases), suggesting that this pathway was not 
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activated under the study conditions.  
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FIGURE 1.  A Jars and labels; B Wound division; C Marking and biopsies; D Applied gel; E Primary 
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hydrofiber dressing; F Secondary absorbent dressing; G Patient 2, jar 3, treatments 1 and 13; H Patient 2, 
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jar 5, treatments 1 and 13; I Semiquantitative table of cellular and molecular findings.
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Graph 1  
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Wound area in the control group decreased from 7.8cm2 (SD: 7.1) to 3.9cm2 (SD: 3.8) (p = 0.185), and in 
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the Royalactin group from 6.3cm2 (SD: 5.4) to 3.4cm2 (SD: 4.2) (p = 0.089). 
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Graph 2 – Results of the semiquantitative histological wound assessment scale, comparing control groups 
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at baseline (O-I) and study endpoint (O-T) versus Royalactin-treated groups at baseline (X-I) and study 
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endpoint (X-T). 
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Graph 3: Percentage of vascular endothelial growth factor (VEGF) expression in control sections (39% SD: 
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24 to 43.5% SD: 19.7) (p = 0.795) and in Royalactin-treated sections (32% SD: 16 to 50% SD: 17) (p = 
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0.088). 
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Figure 4  Correlation between wound area and VEGF expression. Linear regression analysis 
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comparing wound area and VEGF percentage at baseline (left) and after 4 weeks of treatment (right) for 
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both the control and Royalactin-treated groups.
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