IN-SILICO AND IN-VIVO EVALUATION OF A TOPICAL FORMULATION CONTAINING CREATINE MONOHYDRATE FOR THE MANAGEMENT OF CHRONIC DIABETIC WOUNDS IN MALE WISTAR RATS

IN-SILICO AND IN-VIVO EVALUATION OF A TOPICAL FORMULATION CONTAINING CREATINE MONOHYDRATE FOR THE MANAGEMENT OF CHRONIC DIABETIC WOUNDS IN MALE WISTAR RATS

S. L. Bajaj, M. V. Kadwaikar, Malti Salunke, Amol Muthal, Ravindra Kulkarni and V. M. Shinde *

Department of Pharmacognosy, Bharati Vidyapeeth (Deemed to be University), Poona College of Pharmacy, Erandwane, Pune, Maharashtra, India.

ABSTRACT: Chronic diabetic wounds are a common complication in individuals with diabetes and can lead to severe health issues if not treated quickly. Due to its capacity to improve cellular energy metabolism, Creatine monohydrate (CrM) has been suggested to possess potential health benefits. The effectiveness of a topical CrM formulation in healing chronic diabetic wounds in male Wistar rats was investigated in this research along with placebo, marketed formulation. Over the course of 21 days, the reduction of the wound and its fast pace of healing were observed. In contrast to the disease control group, the treatment group lesion size was significantly reduced and its wound closure rate was significantly higher. Increased collagen deposition and angiogenesis were also found by histological analysis in the CrM group, suggesting improved tissue regeneration. In conclusion, the topical CrM formulation showed encouraging benefits on wound healing and may have application in the therapeutic management of chronic diabetic wounds.

Keywords: Diabetic wound healing, Creatine monohydrate, In-silico study, In-vivo validation, Tissue regeneration, Collagen deposition, Histological analysis

INTRODUCTION: Diabetes is a prevalent illness that exposes a considerable number of individuals to the threat of cardiovascular disease, blindness, chronic ulcers, kidney disease and other associated health issues ¹. According to the estimates by the International Diabetes Federation, the global incidence of new diabetes cases exceeds 96,000 annually ².

Diabetic chronic ulcers, typically in the lower limbs, present a major risk with potential for infections, amputations or even death and their healing is affected by factors like impaired inflammation, angiogenesis, glycation and neuropathy ³.

Cutaneous wound healing is complicated process encompassing four distinct phases: a) Hemostasis – to stop bleeding b) Inflammation – where inflammatory cells are recruited and promote healing c) Proliferation – for tissue repair) Remodeling to develop mature scar ^{4, 5, 6, 7}. Growth factors control angiogenesis and matrix re-organization ^{8, 9}. Keratinocytes, fibroblasts ¹⁰ and enzymes like matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs), play key roles ¹¹. Diabetes and wounds can cause oxidative stress, hinder healing and lead to complications ¹². Substances with antioxidant and anti-inflammatory properties may accelerate wound healing in diabetic individuals by reducing oxidative stress and inflammation ¹³.

The connection between nutrition and oxidative stress is well-established, with phytochemicals explored for diabetic wound healing ¹⁴. Nutrition influences allostatic load and tissue repair, requiring adequate energy and protein for proper healing ¹⁵. Current therapeutic options for diabetic wound healing are limited and exploring natural, safer molecules offers an appealing alternative ¹⁶.

Creatine, a natural compound, serves as a vital cellular energy source and can be obtained through diet ¹⁷. It’s known for enhancing muscle mass and has research interest for its potential in preventing neurodegenerative diseases due to its antioxidant properties ¹⁸. Creatine monohydrate (CrM) is the commonly used form in dietary supplements and various food products ¹⁹. Therefore, the goal of this research was to assess Creatine monohydrate’s effects, particularly its antioxidant and anti-inflammatory properties, on wound healing in Streptozotocin induced hyperglycemic rats ²⁰.

MATERIALS AND METHOD:

Materials: Creatine monohydrate (98% purity) with molecular weight 131.14 was purchased from Yucca Enterprises, Mumbai, with appropriate certificate of analysis. Streptozotocin, nicotinamide of Merk and all other reagents were used of analytical grade.

Methods:

In-silico Study: Swiss ADME was used to predict the pharmacokinetic characteristics like absorption, distribution, metabolism and excretion. Established a compound-target network for better understanding of pharmacological process behind the impact of CrM. To create a drug effected target network diagram, information about compound and effective targets was input into Cytoscape 3.9.1 software. The identified targets were then subsequently transferred to a string database, and the species “Homosapiens” was chosen to build a protein-protein interactions.

Design and Preparation of Formulation: The formulation contains Creatine monohydrate (CrM), zinc oxide and arrowroot powder, the process involves several steps to ensure uniformity and quality. First, all the powders are carefully weighed and then they are passed through a 100# mesh size sieve. This ensures that the particles are of consistent size. Next, the ingredients were mixed in geometric proportion, creating a well-blended mixture. The mixture is then sterilized for hour to eliminate any potential contaminants. Once the sterilization process is complete, the powder is mixed thoroughly and transferred in a container. Multiple batches were prepared containing 2.5% (w/w), 5% (w/w) and 10% (w/w) of the drug mixture. Various evaluation parameters are assessed. This comprehensive evaluation ensures that the drug formulation meets the desired quality standards and is suitable for its intended use.

In-vivo Study:

Animals: Adult male Wistar rats of around 8-10 weeks old (180-200 g) were ordered from Global Bioresearch Solutions Pvt. Ltd. Pune, India. The research was conducted in compliance with the guidelines set forth by CPCSEA guidelines and IAEC of Poona College of Pharmacy in Pune, India granted the approval for the experimental protocol (PCP/IAEC/2023/2-5).

Acute Skin Irritation Analysis: Male Wistar rats weight between 180-200 g were utilized for the study. After 24 to 72 hours the skin was evaluated for the sign of erythema, edema and inflammation ta assess any potential skin irritation caused by the formulation ²¹.

Induction of Disease: Diabetes was induced initially by injecting single i.p dose of 110 mg/kg body weight Nicotinamide in saline and after 15 minutes Streptozotocin was injected with dose of 65 mg/kg body weight emulsified in 0.1 M fresh cold citrate buffer (pH 4.5)²². After injecting rats were permitted to utilize 5% glucose solution to prevent hypoglycemic shock. After a period of 72 hours, fasting blood glucose level (BGL) was examined of the animals and BGL > 200 mg/dl were selected as diabetic ^{23, 24}.

Excision Wound Model: When the wounds were created, animals were anesthetized with 35 mg/kg intraperitoneal of thiopentone sodium. The circular wound of about 300 mm² was created with the help of surgical scissor. The rats were kept in separate cages for further experiments ²⁵.

The study consist of five groups with 6 animals in every group and treatment was given topically.

Normal Control (NC): Healthy rat + Wound + Water.

Diabetic Control (DC): Diseased rat + Wound + Water.

Marketed Formulation (NS): Diabetic rat + Wound + Neosporin topical powder.

F (5%): Diabetic rat + Wound + Creatine monohydrate topical 5% w/w formulation.

F (10%): Diabetic rat + Wound + Creatine monohydrate topical 10% w/w formulation.

Measurement of Wound area and Percent Contraction: The changes in the wound area was examined by camera and tracing technique on following days starting from 0 to 4, 8, 12, 16 till 20th. Then percent wound contraction was evaluated with the formula ²⁶:

% Contraction of wound = (Initial area – Specific day area) / Initial area × 100

Determination of Period of Epithelialization and Wound Index: The endpoint for determining complete epithelialization was marked by the absence of any raw wound once the scab had naturally fallen off. The duration of epithelialization was measured as the number of days required for the entire healing process to be completed. The Wound index was assessed using a subjective scoring system, as described earlier ²⁷.

Histopathological Examination: Tissue samples were collected from rat skin after the experiment to assess Histopathological changes. The samples were treated with 10% buffered formalin for fixation, processed and embedded in paraffin blocks. The sections were cut and stained with hematoxylin and eosin (H and E) ²⁸.

Hydroxyproline Content: The segments of skin were weighed and the mixed along with phosphate buffer (0.1M, pH 7.4) and subject to homogenization on the ice for 15 minutes at 10,000 rpm. Supernatant of tissue homogenate was used for determination of hydroxyproline content as described ²⁹.

Statistical Analysis: All the values were stated as Mean±SEM (n=6). Data analysis was conducted using GraphPad Prism 9.0 software. Each statistics was analyzed by one way or two-way ANOVA followed by Dunnett’s or Bonferroni’s test ³⁰.

RESULTS AND DISCUSSION:

In-silico Activity: The network was created between the molecules, its gene targets and disease categories related to diabetes and wound healing. The summary statistics of network shows number of nodes is 20 and number of edges is 22. The network shows creatine monohydrate is linked with 6 genes and disease associated with these genes related to diabetic wound healing are 13 which is depicted in Fig. 1.

FIG. 1: NETWORK PHARMACOLOGY

Evaluation of Formulation: The physicochemical evaluation of the CrM formulation and the commercially available counterpart was conducted, and the results are presented in Table 1.

The pH values of formulations ranged from 5.5 to 6.3, which falls within the normal pH range for human skin. The CrM (10%) formulation closely resembled the properties of the marketed formulation. The angle of repose for the test formulation was found to be 33.69° and its porosity was 2.76%.

In comparison, the marketed formulation exhibited an angle of repose of 33.69° and a porosity of 2.52%. The Moisture content of the 10% formulation was determined to be 3.50%, while the marketed formulation had a moisture content of 2.70% respectively.

TABLE 1: THE PHYSICOCHEMICAL EVALUATION OF DIFFERENT FORMULATIONS

Acute Skin Irritation Study: The study involved testing various doses of the test formulation to assess its acute toxicity and determine the appropriate therapeutic dose. Throughout the study, it was noted that concentrations up to 30% w/w of CrM formulation did not induce any alteration in behavior, itching of the skin, inflammation, swelling, erythema redness of the skin, irritation or instances of mortality. As a result, 5% and 10% concentration of the formulation were chosen for further investigation in subsequent study.

Blood Glucose Level and Body Weight: The Fig. 2 depicts the blood glucose level of diabetic rats over time, showing a consistent increase as diabetes developed. However, there was no considerable change in blood glucose level observed among the various treated groups of diabetic rats (p > 0.05). Conversely, the blood glucose levels of non-diabetic rats differed significantly from those of the diabetic rats (p < 0.05). Additionally, it was found that topical application did not lead to a noteworthy decrease in glucose level in diseased rats (p > 0.05). There was a considerable reduction in body weight of diabetic wound control rats when compared with normal wound control rats as well as treated groups( p < 0.05).

FIG. 2: EFFECT OF CRM FORMULATION ON BLOOD GLUCOSE AND BODY WEIGHT

Wound area and %Wound Contraction: The Wound healing progress was visually documented over 21 days, as shown in the Fig. 3. All experimental groups exhibited a remarkable reduction (p>0.05) in wound area (mm²) and a considerable elevation (p<0.05) in %wound closure related to disease control group, on days 0, 4, 8, 12, 16, 20 as depicted in the Fig. 4. Quantitative measurement of the wound area confirmed that the %wound closure from day 12 in the disease control group was significantly different (p<0.05) from the normal control group. Conversely, all treated groups exhibited a significant difference (p<0.05) in %wound contraction correlated to the disease control group. By the 21^st day post-wounding, the %wound closure in treated groups was 99.3±0.5%, respectively. Remarkably, the F (10%) treated group showed significantly higher wound healing compared to all other treated groups (p < 0.05). These results suggest that the inclusion of CrM in the formulation accelerates the wound recovery process by enhancing wound re-epithelialization.

FIG. 3: PHOTOGRAPHIC DEPICTION OF THE EFFECT OF CRM FORMULATION ON HEALING PHASE IN AN EXCISION MODEL

FIG. 4: EFFECT OF CRM FORMULATION ON WOUND AREA (MM²) AND % CONTRACTION

Epithelialization Period and Wound Index: The Fig. 5 presents the Mean±SEM (n=6) of the period required for epithelialization and wound index. In the group treated with the formulation, the sloughing of the scab took about 20 days and resulted in no residual wound scar. However, in the disease group, the wounds remained partially unhealed. Throughout the experiment, all treated groups showed a superior wound index compared to that of disease control group, indicating better wound healing progress.

FIG. 5: IMPACT OF CRM FORMULATION ON PERIOD OF EPITHELIALIZATION AND WOUND INDEX

Histopathology: Histopathological assessment of wound tissue is of critical importance and outcomes presented in Fig. 6 and Table 2. In the control group the production of fibrosis in the dermis, epithelization of tissues, and adnexa restoration was lagged as compared to Treated groups. Microscopic examination of skin tissue on the 21^st day after injury in the normal control group revealed a high presence of cells showing inflammation. The control group rat skin had an early tissue epithelization and granulation tissue in addition with ulceration and edema and a high concentration of mononuclear inflammatory cells. The diabetic control group showed necrosis, less collagen fibers, pus cells and an average amount of inflammatory cells along with decreased re-epithelialization. The standard treated group/ neosporin revealed repaired structures, including well-formed, nearly regular epidermis, restored adnexa, and a dermis with significant fibrosis and collagen tissue. The formulation 5% and 10% treated exhibited significant fibrosis, a considerable quantity of granulation tissue, a low number of mononuclear inflammatory cells and the repair of adnexa.

FIG. 6: HISTOPATHOLOGICAL EXAMINATION OF SKIN OF MALE WISTAR RATS BY H AND E STAINING

TABLE 2: TISSUE HISTOPATHOLOGICAL DATA OF 21^ST DAY OF POST WOUNDING

Histological section represents (-) absent, (+) Mild, (++) Moderate, (+++) Extensive.

Estimation of Hydroxyproline Content: Table 3 displays the hydroxyproline content in the wound tissues of the rat groups under examination. The group that received the 10% formulation exhibited the highest concentration of L-hydroxyproline. On the 21^st day after injury, the hydroxyproline levels were notably elevated in the groups treated with the commercially available formulation and the 10% formulation when compared to the disease control (p<0.05).

TABLE 3: EFFECT OF CRM FORMULATION ON ESTIMATION OF HRDROXYPROLINE CONTENT

CONCLUSION: Successful wound healing is characterized by the effective closure of wounds without any adverse effects. Our study focused on the wound healing phase in diabetic rats, and we observed promising results with the topical application of Creatine monohydrate (CrM). The use of CrM demonstrated strong wound healing effects, along with notable antimicrobial and antioxidant activities. The results of the wound closure assessments indicated that diabetic wounds treated with the CrM formulation showed superior wound closure capacity. Histological analysis further revealed increased collagen deposition and enhanced re-epithelialization in the treated wounds. Overall, our findings suggest that the topical application of CrM accelerates wound recovery through following key mechanisms. Stimulation of granulation tissue formation through collagen synthesis, facilitation of tissue remodelling via collagen replacement could be reasons for promotion of wound contraction. These positive effects indicate the potential of CrM as a beneficial treatment for enhancing chronic diabetic wound healing.

ACKNOWLEDGEMENT: Authors are thankful to BVDU Poona College of Pharmacy for providing facilities to carry out the research work.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Mieczkowski M, Mrozikiewicz-Rakowska B, Siwko T, Bujalska-Zadrozny M, de Corde-Skurska A, Wolinska R, Gasinska E, Grzela T, Foltynski P, Kowara M, Mieczkowska Z and Czupryniak L: Insulin, but Not Metformin, Supports Wound Healing Process in Rats with Streptozotocin-Induced Diabetes. Diabetes Metabolic Syndrome and Obesity 2021; 14: 1505-17.
2. Mehdinezhad N, Aryaeian N, Vafa M, Saeedpour A, Ebrahimi A, Mobaderi T and Fahimi Ran Sajadi Hezaveh Z: Effect of spirulina and chlorella alone and combined on the healing process of diabetic wounds: an experimental model of diabetic rats. Journal of Diabetes & Metabolic Disorders 2021; 20(1): 161-169.
3. Okonkwo UA and DiPietro LA: Diabetes and Wound Angiogenesis. International Journal of Molecular Science. 2017; 18(7): 1419.
4. Bodas K and Shinde V: Healing of wounds: a detailed review on models, biomarkers, biochemical and other wound assessment parameters. International Journal of All Research Education and Scientific Methods 2021; 9(3): 2069-2085.
5. Varghese R and Shinde V: Novel therapeutics and treatment regimen in wound healing, International Journal of Herbal Medicine 2021; 9(1): 12-18.
6. Varghese R and Shinde V: Therapeutic potential of novel phyto-medicine from natural origin for accelerated wound healing. International Journal of Pharmacognosy 2021; 8(1): 14-24.
7. Shinde V, Shende A and Mahadik K: Evaluation of antioxidant and wound healing potential of pomegranate peel gel formulation. International Journal of Pharmacognosy 2020; 7(1): 23-8.
8. Younis NS, Mohamed ME and El Semary NA: Green Synthesis of Silver Nanoparticles by the Cyanobacteria Synechocystis: Characterization, Antimicrobial and Diabetic Wound-Healing Actions. Marine Drugs 2022 Jan 6; 20(1):56.
9. Chen LY, Huang CN, Liao CK, Chang HM, Kuan YH, Tseng TJ, Yen KJ, Yang KL and Lin HC: Effects of Rutin on Wound Healing in Hyperglycemic Rats. Antioxidants (Basel) 2020; 9(11): 1122.
10. Gourishetti K, Keni R, Nayak PG, Jitta SR, Bhaskaran NA, Kumar L, Kumar N, Krishnadas N and Shenoy RR: Sesamol-Loaded PLGA Nanosuspension for Accelerating Wound Healing in Diabetic Foot Ulcer in Rats. International Journal Nanomedicine 2020; 15: 9265-9282.
11. MacLeod AS and Mansbridge JN: The innate immune system in acute and chronic wounds. Advances in Wound Care (New Rochelle) 2016; 5(2): 65-78.
12. Chen XY, Jiang WW, Liu YL, Ma ZX and Dai JQ: Anti-inflammatory action of geniposide promotes wound healing in diabetic rats. Pharmaceutical Biology 2022; 60(1): 294-9.
13. Conceiçao M, Gushiken LFS, Aldana-Mejia JA, Tanimoto MH, Ferreira MVS, Alves ACM, Miyashita MN, Bastos JK, Beserra FP and Pellizzon CH: Histological, Immunohistochemical and Antioxidant Analysis of Skin Wound Healing Influenced by the Topical Application of Brazilian Red Propolis. Antioxidants (Basel) 2022; 11(11): 2188.
14. Barchitta M, Maugeri A, Favara G, Magnano San Lio R, Evola G, Agodi A and Basile G: Nutrition and wound healing: an overview focusing on the beneficial effects of curcumin. International Journal of Molecular Sciences 2019; 20(5): 1119.
15. Giraldo-Vallejo JE, Cardona-Guzmán MÁ, Rodríguez-Alcivar EJ, Kočí J, Petro JL, Kreider RB, Cannataro R and Bonilla DA: Nutritional Strategies in the Rehabilitation of Musculoskeletal Injuries in Athletes: A Systematic Integrative Review. Nutrients 2023; 15(4): 819.
16. Beserra FP, Vieira AJ, Gushiken LFS, de Souza EO, Hussni MF, Hussni CA, Nóbrega RH, Martinez ERM, Jackson CJ, de Azevedo Maia GL, Rozza AL and Pellizzon CH: Lupeol, a dietary triterpene, enhances wound healing in streptozotocin-induced hyperglycemic rats with modulatory effects on inflammation, oxidative stress, and angiogenesis. Oxidative Medicine and Cellular Longevity 2019; 3182627.
17. Singh S and Dash AK: Creatine Monohydrate. InProfiles of Drug Substances, Excipients and Related Methodology Academic Press 2009; 34: 1-35.
18. Helvacioglu F, Kandemir E, Karabacak B, Karatas I, Pecen A, Ercan I, Sencelikel T and Dagdeviren A: Effect of creatine on rat sciatic nerve injury: a comparative ultrastructural study. Turkish Neurosurger 2018; 28(1): 128-136.
19. Fernández-Landa J, Fernández-Lázaro D, Calleja-González J, Caballero-García A, Córdova A, León-Guereño P and Mielgo-Ayuso J: Long-Term Effect of Combination of Creatine Monohydrate Plus β-Hydroxy β-Methylbutyrate (HMB) on Exercise-Induced Muscle Damage and Anabolic/Catabolic Hormones in Elite Male Endurance Athletes. Biomolecules 2020; 10(1): 140.
20. Farhin A. Sheikh and Manish G. Baheti: Formulation and Evaluation of Anti-microbial Dusting Powder. International Journal of Pharmacy & Life Sciences 2020; 11(7): 6850-57.
21. Yadav J, Patel DK, Dubey NK, Mishra MK, Verma A, Grishina M, Khan MM and Pathak P: Wound healing and antioxidant potential of Neolamarckia cadamba in streptozotocin-nicotinamide induced diabetic rats, Phytomedicine Plus 2022; 2(2): 100274.
22. Dhalwal K, Shinde V, Singh B and Mahadik K: Hypoglycemic and Hypolipidemic Effect of Sida rhombifolia retusa in Diabetic Induced Animals. International Journal of Phytomedicine 2010; 2(2).
23. Rani R, Dahiya S, Dhingra D, Dilbaghi N, Kaushik A, Kim KH and Kumar S: Antidiabetic activity enhancement in streptozotocin + nicotinamide-induced diabetic rats through combinational polymeric nanoformulation. International Journal Nanomedicine 2019; 14: 4383-95.
24. Furman BL: Streptozotocin‐induced diabetic models in mice and rats. Current Protocols in Pharmacology 2015; 70(1): 5-47.
25. Vinayak P. Nakhate, Natasha S. Akojwar, Saurabh K. Sinha, Amarsinh D. Lomte, Mahaveer Dhobi, Prakash R. Itankar and Satyendra K. Prasad: Wound healing potential of Acacia catechu in streptozotocin-induced diabetic mice using in-vivo and in-silico Journal of Traditional and Complementary Medicine 2023; 13(5): 489-99.
26. Tan WS, Arulselvan P, Ng SF, Mat Taib CN, Sarian MN & Fakurazi S: Improvement of diabetic wound healing by topical application of Vicenin-2 hydrocolloid film on Sprague Dawley rats. BMC Complementary and Alternative Medicine 2019; 19: 1-6.
27. Kandhare, A, Patil MV, Mithun, Bhise and Sucheta: Pharmacological evaluation of ameliorative effect of aqueous extract of Cucumis sativus L. fruit formulation on wound healing in Wistar rats. Chronicles of Young Scientists 2011; 2(4): 207-13.
28. Kandhare AD, Alam J, Patil MV, Sinha A and Bodhankar SL: Wound healing potential of naringin ointment formulation via regulating the expression of inflammatory, apoptotic and growth mediators in experimental rats. Pharmaceutical Biology 2016; 54(3): 419-32.
29. Andjic M, Draginic N, Kocovic A, Jeremic J, Vučićevic K, Jeremic N, Krstonošić V, Božin B, Kladar N, Čapo I and Andrijević L: Immortelle essential oil-based ointment improves wound healing in a diabetic rat model. Biomedicine & Pharmacotherapy 2022; 150: 112941.
30. Murkute AB and Shinde VM: Exploratory studies on diabetic wound healing potential of Cipadessa baccifera (ROTH.) MIQ International journal of Pharmacognosy, 2019; 6(8): 277-288.
    

    ---

    How to cite this article:

    Bajaj SL, Kadwaikar MV, Salunke M, Muthal A, Kulkarni R and Shinde VM: In-silico and in-vivo evaluation of a topical formulation containing creatine monohydrate for the management of chronic diabetic wounds in male wistar rats. Int J Pharmacognosy 2024; 11(10): 569-76. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.11(10).569-76.

    ---

    This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.