1. Introduction

Man has used diverse portions of plants in the prevention and treatment of many illnesses since the dawn of time [1]. The concept of generating medications from plants used in indigenous medical systems is far older. Although there are some direct links between a local and biomedical use, the relationship is much more complex in other cases [2]. According to the Wound Healing Society, wounds are defined as physical damages that disrupt the normal structure of the skin. Chronic wounds, in particular, present a serious challenge for both patients and healthcare providers, as they affect a large population and significantly reduce the quality of life [3]. Recent estimates show that approximately six million individuals worldwide suffer from chronic wounds. These wounds occur at a rate of about 4.5 per 1000 people, while acute wounds have been reported to occur at nearly twice that frequency, approximately 10.5 per 1000 individuals [4]. Medicinal plants play an important role in both traditional and contemporary medicine. Plant-derived compounds with suspected antimicrobial properties require evaluation using appropriate microbial models in order to validate their activity and establish relevant parameters. Consequently, a vast number of researchers from many regions of the world have investigated the effects of plant extracts on bacteria [5]. Despite advances in modern medicine, traditional medicines are still used by over 80 % of the global population to treat various skin ailments [6]. In order to support the creation of a proper environment for natural healing, herbal remedies in wound management include cleaning, debridement, and maintaining a moist atmosphere [6].

Monodora is a plant genus belonging to the Annonaceae family and comprises approximately thirty-five species distributed across tropical Africa. M. myristica (Dunal)is one of the most commonly utilised species and is widely used as a spice [7]. Itis a perennial plant predominantly found in evergreen forests of West Africa and is commonly known as African nutmeg, calabash nutmeg, and ehuru while some common names in Nigeria include: ariwo, ehiri, and airama [7]. The seeds possess a nutmeg-like aroma, and widely incorporated into West African cuisine. Countries such as Liberia, Nigeria, Cameroon, Angola, Uganda, and western Kenya are among the regions where the M. myristica tree grows naturally in evergreen woods. This tropical shrub belongs to the flowering plant family and produces a berry of about 20 cm in diameter. It is smooth, green, and spherical when young, which becomes woody upon maturation. The fruit is attached to a long stalk that may reach up to 60 cm in length and contains numerous oblong pale brown seeds, each averaging about 1.5 cm in length, embedded in a white, aromatic pulp. Studies show that nearly all parts of the M. myristica tree are commercially significant [8]. The wood is durable and commonly used for carpentry, household fittings, and joinery, while the seeds are used in the production of jewellery and other crafts. The seeds, which are encased in a white, sweet-smelling pulp of the sub-spherical fruit, are the most economically valued portions. On average, a single fruit contains between 119 and 122 seeds [7]. Prior to storage or consumption, the seeds undergo processing methods such as fermentation, washing, drying, and cracking [8]. Numerous studies have documented the therapeutic properties of M. myristica. In the parts of Central Africa, the stem bark is traditionally used to manage haemorrhoids, gastrointestinal discomfort, fever, and eye conditions, while the seeds are used to treat headaches and hypertension, also as a condiment and postpartum tonic in Eastern Nigeria [9]. Anti-sickling properties have been discovered in M. myristica, and the powdered kernel is sometimes added to soup to relieve constipation and manage passive postpartum uterine bleeding. Additionally, the fruit is also utilized for its diuretic qualities [9]. Previous studies have reported the use of 5 % and 10 % methanolic seed extracts of M. myristica and M. tenufolia in wound-healing of albino rats [10]. Building on some of this evidence, this present study investigated the wound activity of 100 % methanolic and ethanolic extracts of M. myristica seeds.

• MATERIALS AND METHOD

2.1       Plant material

The seeds of Monodora myristica were obtained from Bodija market in Ibadan, Nigeria. The plant material was identified and authenticated at the Herbarium Unit, University of Ibadan, Nigeria.

2.2       Preparation of seed extract

The seed coats were carefully removed, after which the seeds were air-dried and milled into a coarse powder using an electronic blender. A total of 300 g each of the dried powdered sample was divided equally into two separate aspirator containers. One litre of methanol was added to the first container, and also one litre of ethanol to the second container. Both mixtures were allowed to stand at room temperature for four days with constant agitation to enhance extraction. The resulting mixtures were filtered, and the filtrates were concentrated using a rotatory evaporator at 53 °C for methanol and 65 °C for ethanol. The concentrated extracts were then designated as M. myristica methanolic seed extract (MMMSE) and M. myristica ethanolic seed extract (MMESE), respectively.

2.3       Phytochemical screening

Qualitative phytochemical analyses were conducted to identify the presence of secondary metabolites in both methanolic and ethanolic seed extracts. The compounds screened included alkaloids, steroids, carbohydrates, flavonoids, saponins, glycosides, terpenoids, quinones, and anthraquinones, using standard procedures as described by Balakrishnan et al.  [11].

2.4       Physical Properties

Some of the properties of the purchased M. myristica seed were determined. The weight of some randomly selected seeds was determined with and without the hull. The percentage of hull or husk was then calculated. M. myristica seed length with hull and without hull was also measured in millimeters and the average seed length was determined.

2.5       Test Microorganisms

The microorganisms used in this study were Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Salmonella typhi. Pure bacterial and fungal strains were obtained from the Department of Pharmacy, University of Ibadan, Nigeria. The bacterial isolates were cultured on nutrient agar (Oxoid, Hampshire, UK) and incubated at 37 °C for 24 hours.

2.5.1    Susceptibility tests

The antibacterial activity of MMMSE and MMESE against the test organisms was assessed using the disk diffusion method. Gentamycin served as the positive control. The test microorganisms were grown overnight (24 h at 37 °C) in nutrient agar, after which the inoculum was prepared by diluting cultures in Muller-Hinton Broth to achieve approximately 10⁶ CFU/ml.  Sterile paper disks (6 mm diameter) were impregnated with 10 µl of the extracts at concentrations of 1.00 mg/ml, 0.875 mg/ml, 0.75 mg/ml, 0.5 mg/ml and 0.25 mg/ml. The disks were placed on inoculated agar plates (90 mm) and incubated at 37 °C for 24 hours. Zones of inhibition were measured and compared with those produced by Gentamycin (10 µg/ml). All experiments were conducted in duplicate.

2.5.2    Minimum Inhibitory Concentrations (MIC) of seed extract

The Minimum Inhibitory Concentration (MIC) is defined as the extract concentration capable of preventing visible microbial growth on agar media or turbidity in broth cultures. In this study, the susceptibility of bacterial and fungal strains to the extracts was evaluated using the microdilution technique to establish MIC values. This procedure was applied extensively to extracts that demonstrated inhibition zones of at least 8mm. The extracts were dissolved in 10 % of ethanol 99 % v/v. The assay was conducted using a 96-well microplate following established protocols. The wells of Column 2–11 was filled with 100 µl of Muller Hinton Broth (MHB) and served as solvent control for sterility. The first tubes contained only the pure extracts and were used as sterility controls. Gentamycin, prepared in MHB at concentrations ranging from 1 mg/mL, was used as the standard drug for positive control. The agar plates incubated at 37 °C for 24 h, and MIC was recorded as the lowest concentration at which no visible colony growth was observed. All experiments were carried out in triplicate [12, 13, 14].

2.6       FTIR Analysis

Fourier Transform Infrared (FTIR) spectroscopy was employed as the preferred infrared analytical technique. When the infrared radiation passes through a sample, certain wavelengths are absorbed while others are transmitted. The transmitted radiation generates a signal detected as an infrared spectrum, which represents a molecular “fingerprint” unique to the sample. The effectiveness of FTIR spectroscopy lies in its ability to differentiate chemical structures based on their distinct spectral patterns.

2.7       Experimental Animals

Healthy Wistar rats of both sexes, with body weights ranging between 150 and 250 g, were obtained from Laniba Farm, located near Leyket Gas Station in Ibadan. They were given a conventional unlimited food and water and kept in polypropylene cages under conventional laboratory conditions (25-30 ⁰C, 35-60 % relative humidity). All experimental procedures adhered to the ethical guidelines approved by the University of Ibadan Ethics Committee on Research in Animals, as well as internationally accepted standards for the use and care of laboratory animals.

2.8       Evaluation of Wound Healing Activity

The wound-healing potential of M. myristica seed extracts was evaluated using an excision wound model. The animals were randomly divided into four groups, each comprising six rats. Ointments were applied topically once daily to the rats in groups 1-3. Group 1 animals received MMMSE ointment, while Group 2 was treated with ointment of MMESE. Group 3 served as the untreated control, whereas Group 4 functioned as the positive control and was treated with a standard wound-healing ointment containing 5% povidone-iodine USP. Prior to and during wound induction, the rats were anesthetized with ketamine hydrochloride (100 mg/kg, intraperitoneally) following standard procedures. All the animals were monitored closely for signs of illness, and any diseased animals were excluded from the study [15].

2.8.1 Excision wound model

Excision wounds were created on the animals using the method described by Ajayi and Omolere [16]. An electrical clipper was employed to shave the dorsal fur of the dorsolateral flank area. After preparing the wound region with 70 % alcohol, the skin from the planned shaved area was excised to its full thickness with forceps, a surgical blade, and a scissor to obtain a wound area of roughly 300 mm². Excision incisions were made 2.0 cm from the spinal column on both sides of the dorsal thoracic region.The wound was left open, and all of the animals were treated with the prepared ointments. The wound healing was tracked by tracing the wound on the 3rd, 11th, 15th, 19th, and 23rd days after wounding. Percentage wound closure and epithelialisation time, which is an indicator of new epithelial tissue formation over the wound surface, were calculated based on wound closure measurements recorded at regular intervals [17].

2.8.2    Wound healing activity study

The wound-healing effectiveness of the formulated extracts was assessed using parameters such as wound surface area (mm²), rate of wound contraction, epithelialisation time in the excision wound model, and histological examination of the regenerated tissues.

2.8.3    Rate of wound contraction

Wound contraction was evaluated every four days and expressed as the percentage reduction in size. Wound areas were measured graphically to monitor the percentage of wound closure, which reflects the formation of new epithelial tissue. Transparent paper and a marker were used to trace wound margins and measurements were carried out. The percentage wound healing was calculated using the formula prescribed by Ajayi et al. [10].

Where:

Ao = Initial area of wound on day “0’’ of the experiment.

At = Area of wound at day “t’’ of experiment.

The epithelialisation time was determined by the number of days required for complete scar formation without any remaining raw wound surface.

2.9       Histological Study

On the 25th day of the experiment, skin tissue samples were collected from all the five experimental groups of animals for histological evaluation of collagen deposition and tissue regeneration. Skin samples from the treated animals were fixed in 10 % buffered formalin, sectioned in 6 m thickness slices, and stained with hematoxylin and eosin. The prepared slides were examined under a light microscope to identify histological alterations.

2.10     Statistical analysis

Each experimental group consisted of six animals (n=6). Data analysis was performed using one-way analysis of variance (ANOVA) with IBM SPSS statistical software version 20.0. The Duncan T test was applied to determine significant differences among group means, with statistical significance set at P<0.05. The results were expressed as mean values±standard deviations obtained from six duplicated tests.

• Results and Discussion

3.1    Some determined physical properties of M. myristica seeds

The mean weight and length of hulled and dehulled seeds are presented in Tables 1 and 2 respectively. Ajayi and Ifedi [18] reports kernel percentage of 89.90 % for P. longifolia seeds.

3.2      Phytochemical Analysis

Table 3 shows the phytochemical screening results of MMMSE and MMESE. The analysis confirmed the presence of tannins, alkaloids, terpenoids, steroids, quinones, anthraquinones, and reducing sugars, along with other bioactive constituents. Tannins can bind to proteins, form complexes that prevent blood flow to wound area thereby reducing loss of blood, and thus cause early healing [19]. Saponins are known to a play a role in stimulating the activity of fibroblasts, increasing collagen production, and helping to repair damaged tissue structure [20].  Lestari et al. [21] reports that Azadirachta indica leaf extract contains secondary metabolites with potential as therapeutic agents for wounds, in particular, diabetic wounds.

3.3       Antimicrobial activities assay

The differences observed in the antimicrobial activity of M. myristica seed extracts can be attributed to variations in their chemical composition (Table 4). [22] reported that B. subtilis exhibited the highest sensitivity to ethanol extracts at concentrations of 50-400 mg/ml, whereas E. coli and Proteus mirabilis showed greater resistance. In contrast, findings from the present study identified S. aureus and B. subtilis as the most resistant organisms. Previous studies have shown that the minimum inhibitory concentration for S. aureus and S. typhi was 40 mg/ml. The antibacterial effect of both MMMSE and MMESE the against S. aureus in this study aligns with results reported by [23], who recorded inhibition zones of 9 mm. Overall, the presence of both antibacterial and antifungal activities in MMESE confirms that the plant contains biologically active compounds with potential for multiple therapeutic applications.

3.3         FTIR Analysis of methanol and ethanol M. myristica extract

The FTIRspectra ofmethanol and ethanol extracts of M. myristica seeds demonstrated notable similarities (Figures 1a and 1b). Both extracts have C-H bonds, C-O bonds, and they also have the O-H bonds; the O-H absorption is a broad one in both molecules. There are also C-H and C-O absorptions. The ethanol molecule has more peaks than the methanol molecule because of the presence of C-C bonds in it. The peaks below 1500 cm^-1 also indicate the fingerprint region, as shown in Fig. 1 and 2^. O-H shows a broad absorption at about 3300 cm^-1, C-O has one at near 1200 cm^-1, C-H bonds absorbed at about 3000 cm^-1, while the C-C bond is unique to only ethanol.

3.4      Weekly weight gain of rats

Table 5 presents the weekly weight changes observed in both the test and control animals during the study period. It was noticed that there was a decline in weight after the wound was inflicted (after week 1). This may be due to loss of appetite for about a week due to pain from the wound incision. The weights increased steadily after some days as wound healing progressed until week 4.

3.5       Wound contraction and epithelialisation time

The progressive reduction in wound size among the various experimental over the 23-day treatment period using M. myristica seeds is presented in Table 6 and 7, Figure 2. Results from this study revealed that topical application of M. myristica seed extracts significantly enhanced wound healing in rats. This effect may be linked to the extracts’ ability to neutralize free radicals, suppress inflammatory pathway mediators, and inhibit bacteria growth at the wound site [24]. Group 1, treated with MMMSE, exhibited the fastest wound healing response, achieving nearly complete healing (almost 100 % wound contraction) within 18 days, compared to Group 2, which received MMESE. By the 19th day post-wounding, wound of Group 1 rats was markedly faster than those of the rest of the groups. Group 3 animals showed the slowest wound healing rate, with an epithelialisation period of 24.83±0.14 days. Animals treated with povidone iodine (Group 4) demonstrated epithelialisation times of 20.33±1.86 days, while Group 2 ones recorded 19.83±1.47 days. Complete epithelialisation across all treated groups was observed between the 18^th and 20th post-wounding days. The untreated group (Group 3) showed the poorest healing outcome, characterized by prolonged epithelialisation time, and delayed wound closure. MMMSE ointment healed the wound of the rats faster (epithelialisation time of 18.00±1.09) than MMESE ointment (epithelialisation time of 19.83±1.47).

3.6       Histopathology Analysis

Histological examination of the skin tissues obtained from the wound sites of the rats treated for 21 days with MMMSE and MMESEointments revealed the presence of well-developed granulation tissue extending across most layers of the dermis in the excision wound model. Histological features observed in the healed wounds included reformation of the epidermal skin layer, presence of myofibroblasts and fibroblasts, collagen deposition, angiogenesis and infiltration of inflammatory cells. These regenerative characteristics were evident in both the treated and control groups across all experimental sets (Table 8, Figure 3) indicating active tissue repair and remodelling.

4.1       Conclusion

The findings of this study demonstrate that M. myristica seed extracts significantly enhanced wound healing in rats. This effect is likely associated with the activity of bioactive compounds present in the extracts. The wound healing impact of M. myristica species was also demonstrated by the histological results, as well as the wound contraction rate and duration of epithelialisation. These results suggest that M. myristica seeds possess strong ability for development as a topical wound-healing formulation and many have valuable applications within the pharmaceutical industry.

4.2     Conflict of interest

This study does not have any conflicts of interest.

4.3     Research funding

Research funding was not available for this study.

4.4    Acknowledgement

The Departments of Chemistry, Microbiology and Veterinary Pathology of Faculties of Science and Veterinary, respectively, are acknowledged by the authors for making their facilities available for this research.

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