Evaluation of the antifungal efficacy of Mitracarpus scaber extracts and Ocimum gratissimum oil against clinical isolates of fungi

Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 29 ORIGINAL RESEARCH article Evaluation of the antifungal efficacy of Mitracarpus scaber extracts and Ocimum gratissimum oil against clinical isolates of fungi Miriam G.U. Nwaneri 1 , Chidinma R. Chukwunwejim 1 , Chinelo K. Ezejiegu 1 Chinenye H. Nedum 2 , Roselyn N. Egbuna 3 , and Ijeoma N. Ebenebe 1 * 1 Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, 2 Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, and 3 Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Chukwuemeka Odumegwu Ojukwu University, Igbariam, Anambra State, Nigeria * Author to whom correspondence should be addressed Article number: 228, Received: 02-09-2025, Accepted: 25-10-2025, Published online: 27-10-2025 HOW TO CITE THIS Nwaneri MGU, et al. Evaluation of the antifungal efficacy of Mitracarpus scaber extracts and Ocimum gratissimum oil against clinical isolates of fungi. Mediterr J Pharm Pharm Sci. 2025; 5(4): 29-37. [Article number: 228]. https://doi.org/10.5281/zenodo.17455781 Keywords: Antifungal activity, dermatophytes, Candida albicans, Nigeria Abstract: Dermatophytes and Candida albicans are widespread with increasing prevalence and pose a grave threat to public health globally. Ocimum gratissimum and Mitracarpus scaber have been used in Southeastern Nigeria for several purposes, including antimicrobial effects, and have exhibited inhibition of growth to fungi. This study aimed to evaluate and ascertain the antifungal potential of these extracts against dermatophytes and Candida albicans. A total of 50 samples of dermatophytes and Candida albicans previously isolated from clinical samples at two different Hospitals, in the Southeast of Nigeria were used. The clinical samples were vaginal discharge, sputum, swab samples from the endocervix, urine, groin, mouth thrush, and palm. These organisms were all identified using standard mycological identification and characterization techniques. The sensitivity of selected fungi to the extracts (ethanolic extract of M. scaber and O. gratissimum oil) and ketoconazole was evaluated using a modified cup-agar diffusion plate method. The minimum inhibitory concentration (MIC) of the extracts was determined by the agar dilution method. Their minimum fungicidal concentration (MFC) and killing rates against the isolates were also determined. The organisms remain an important etiological agent in this species, implicated in several kinds of infections. The result showed that the activity of the O. gratissimum oil was comparable with that of the conventional drugs, indicating the potential in this extract. The MIC values for Candida isolates were consistently lower against O. gratisimum compared to ketoconazole. The MFC results indicated that O. gratissimum oil had a greater biocidal effect against most of the test organisms in contrast to ketoconazole. The killing rate study also indicated that the oil has very good activity against the isolates. O. gratissimum oil holds great potential for use in treating a wider spectrum of fungal pathogens. Copyright© 2025. This open-access article is distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 30 Introduction Pathogenic fungi such as dermatophytes and Candida albicans cause superficial and severe systemic infections and are now widely recognized as essential agents of hospital-acquired infection [1]. These two organisms are widespread with increasing prevalence and stance a grave threat to public health globally [2-4]. Dermatophytes are superficial mycoses that cause tinea infections. They are keratinophilic moulds that infect human and animal skin, nails and hair and are typically confined to the superficial keratinized tissue [5]. They are of three genera namely: Trichophyton, Microsporum and Epidermophyton [6]. Infection can be present in children [7], but there is an indication of increase in infection with an increasing age [8, 9]. An increased incidence of dermatophyte infection in men compared to women is well known [10, 11]. As such, the tendency for men to develop dermatophytosis appears to be a widespread phenomenon. Candida albicans on the other hand is an opportunistic fungal pathogen of humans that colonizes the skin and mucosal surfaces of most healthy individuals. It causes superficial infections such as vaginal, oral and cutaneous candidiasis [12, 13] and more severe systemic infections like candidemia, endocarditis, endophthalmitis, pneumonia, septicemia, meningitis, osteomyelitis and fungal arthritis in individuals with compromised immune systems, leading to prolonged hospital stay and high mortality rate [14]. It is responsible for approximately one third to half of all nosocomial candida infections worldwide [15]. The focus of Candida albicans infection is the kidney [16]. For thousands of years now, plants have been seen as a valuable source of medicinal agents with proven potential for treating infectious diseases and with lesser side effects compared to the synthetic drug agents [17]. Medicinal plants have been used for several purposes including antimicrobial effects and have exhibited inhibition of growth to fungi. Some concerns have been expressed about the rising prevalence of pathogenic microorganisms that are resistant to the newer or modern antifungals [18]. There is a problem stood by the high cost and adulteration of these synthetic drugs [19] leading to a rising incidence of failures in the treatment of mycoses in the case of severely immunosuppressed patients, found more especially in the developing countries [20]. Thus, there is a need for an alternative therapy which is safer, cheaper, and more available to those in peculiar countries. Coincidentally, there is an increasing intensive study on extracts and biologically active compounds isolated from plant species used as natural herbal therapies [21]. Based on this, this study was aimed at evaluating and ascertaining the antifungal potentials of herbal extracts from M. Scaber and Oil from O. gratissimum against dermatophytes and Candida albicans. This study therefore, tested the susceptibility of isolated dermatophytes to local herbs used in the area for the treatment of these infections to ascertain their efficacy. Materials and methods Culture media: The culture media used were Sabouraud’s Dextrose Agar (SDA) and Sabouraud Dextrose Broth (Middlesex-UK). These media were prepared according to the manufacturer’s instructions. Plant materials: Mitracarpus scaber and Ocimum gratissimum were collected from Nsukka, Enugu State and authenticated in the Department of Botany, University of Nigeria, Nsukka, Nigeria. Voucher specimens were deposited accordingly. Extraction of plants: 500 g of Sun-dried powdered plant material of Mitracarpus scaber was extracted with 2000 ml of ethanol using the cold maceration method [22]. This filtrate was exposed to air until the solvent evaporated to dryness. The residue seen after drying (which is the extract from the plant) was collected, weighed, and kept in a container for further use. Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 31 Volatile oil extraction: Fresh leaf samples were subjected to steam distillation in a modified Clevenger-type apparatus (Sunbim, India) for a minimum of three hours. The oil was obtained in a yield of 0.3% per 100 g, stored in a sealed glass vial and kept in a refrigerator at 4oC until required. Sample collection and processing: A total of 50 samples each of dermatophytes and Candida albicans previously isolated from clinical samples at University of Nigeria Teaching Hospital, and Federal Medical Centre Owerri, Southeast of Nigeria were used for the study. The clinical samples were scalp and skin for dermatophytes and vaginal discharge, sputum, swab samples from endocervix, urine, groin, mouth thrush, and palm. The isolates were inoculated onto Sabouraud dextrose agar plates containing 0.03% w/v chloramphenicol and incubated for 24 hrs and 7 days for Candida albicans and dermatophytes at 28oC [23]. The colonial growths were stored in Sabouraud dextrose agar (SDA) slants containing 0.03% w/v chloramphenicol at 4oC. Before use, an aliquot of the test isolates was cultured again onto fresh Sabouraud dextrose agar containing 0.03% w/v chloramphenicol and then incubated for 24 hrs and seven days for Candida albicans and dermatophytes at 28oC for reactivation. Reactivated cultures were standardized by growing a 40 mm diameter of the mycelia growth into a 20.0 ml SDA plate and processed [for dermatophytes]. For Candida albicans, overnight (18 hrs) sub-cultures in sabouraud’s dextrose broth were adjusted to 90.0% transmittance at 530 nm using distilled water [23]. Preliminary sensitivity testing of the isolates: The sensitivity of selected fungi to the herbal extracts (ethanolic extract of M. scaber and O. gratissimum oil) and ketoconazole were evaluated using a modified cup-agar diffusion plate method [24]. Evaluation of MIC of the two extracts and ketoconazole against fungi isolates using the agar dilution method: The MIC of the two herbal extracts in addition to ketoconazole were prepared separately in distilled water or DMSO. Each of these solutions was diluted two-fold serially with dilute DMSO up to eight dilutions. Thereafter, 1.0 ml from each dilution was seeded into 19.0 ml of molten sterile SDA and allowed to solidify. The plates were divided into eight segments and eight representative isolates (four isolates each of dermatophytes and Candida albicans selected based on their sources) were streaked in triplicates in each segment. The plates were then incubated for 48 hrs (Candida albicans) and five days (dermatophytes). Signs of growth were checked. Evaluation of MFC of O. gratissimum oil and ketoconazole against fungi isolates: Based on the outstanding antifungal properties O. gratissimum oil extract exhibited, it was further selected for further testing in comparison with ketoconazole as the positive control. For the determination of MFC, the plates were further incubated for double the incubation period (four days for Candida albicans and 10 days for dermatophytes). Killing rate evaluation of O. gratissimum oil and ketoconazole against fungal isolates. Stock solution (10 times the MFC) of the oil and ketoconazole that had a minimum fungicidal concentration was prepared in Sabouraud dextrose broth. One ml of this stock was added into 8.0 ml of Sabouraud dextrose broth and 1.0 ml of representative test organisms was added immediately. At various time intervals of 0.0 min, 20 min, 40 min, 60 min, 90 min, 120 min, 180 min, 6 hrs, and 24 hrs, 0.1 ml was withdrawn from the reaction mixture and diluted 100-fold in sterile normal saline. These various dilutions were plated in sterile SDA plates containing 0.03%w/v chloramphenicol and colonies were counted. Results Table 1 shows the different species of dermatophytes isolated from different sources causing skin infections. Both male and female ratio is shown with age ranging from 9 to 20 years old. Different species of dermatophytes were recovered from the hospital and the spectrum of causing skin infections is wide in this area. Different sources Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 32 of isolates of Candida albicans recovered from palm, high vaginal swab, urine, Endocervical swab, Mouth thrush, sputum and groin. The inhibitory effects of the two extracts and ketoconazole against isolates of dermatophytes and Candida albicans are presented in Table 2. The MIC results of M. scaber, O. gratissimum oil extracts, and ketoconazole against selected fungal isolates are shown in Table 3. Based on the impressive MIC data exhibited by O. gratissimum oil, the MFC was further determined with ketoconazole as the positive control for comparative purposes. The minimum fungicidal concentration (MFC) results of ketoconazole and O. gratissimum oil against the isolates are presented in Table 4. Table 1: Different species of dermatophytes isolated and their sources Gender Age Source(s) Isolates/ isolate number Male 20 Skin/Scalp T. soudanense (TS1) and Cladosporium spp (CS1) Male 17 Skin/Scalp Trichophyton soudanense (TS2) Male 16 Skin/Scalp Cladosporium spp (CS2) Male 12 Skin/Scalp Penicillium spp (PS1) Male 11 Skin/Scalp Curvularia spp (CSS1) Male 11 Scalp Aspergillus niger (AN1) Male 12 Skin/Scalp Fusarium spp (FS1) Male 18 Skin/Scalp Trichophyton soudanense (TS3) Male 11 Skin scrapping Aspergillus niger (AN2) and A. flavus (AF1) Male 14 Skin scrapping Mixed with the growth of T. soundenense (TS4) and Cladosporium spp (CS3) Female 14 Skin Trichophyton soudanense (TS5) Female 11 Skin Aspergillus niger (AN3) Female 16 Skin Fusarium spp (FS2) Female 09 Skin Fusarium spp (FS3) Female 18 Skin Cladosporium spp (CS4) Male 19 Scalp Cladosporium spp (CS5) Male 19 Skin/Scalp Cladosporium spp (CS6) Male 10 Scalp Penicillium lilacinum (PL1) Female 19 Scalp Aspergillus flavus (AF2) mixed with the growth of Trichophyton mentagrophytes (TM1) Female 15 Skin Fusarium solani (FSS1) Table 2: Preliminary test on selected strains of Candida albicans and dermatophytes Isolates A (50 µg/ml) B (50 µg/ml) Keto (50 µg/ml) 1 65 14 20 2 44 15 20 3 64 15 14 10 70 15 - 11 54 - 30 12 60 14 25 13 60 13 20 14 55 10 - 15 53 - 33 18 64 118 Trichophyton soudanense 54 15 35 Trichophyton mentagrophytes 55 15 35 Penicillium lilacinum 66 13 - Fusarium spp 40 13 17 Cladosporium spp 50 14 20 Curvularia spp 40 15 18 Apergillus niger 61 15 18 A: O. gratissimum; B: Mitracarpus scaber; -, no growth; + - growth. Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 33 Table 3: MIC of extracts and ketoconazole against selected isolates of dermatophytes and Candida albicans MIC (µg/ml) Test organisms/Isolates M. scaber Oil O. gratissimum Keto Trichophyton soundanense 40.00 0.625 1.25 Trichophyton mentagrophytes 20.00 0.625 5.00 Apergillus niger 20.00 1.25 2.50 Trichophyton soundanense 40.00 - + 02 40.00 0.16 5.00 11 40.00 0.31 2.50 13 40.00 0.16 2.50 17 20.00 0.31 0.31 Oil - O. gratissimum oil; Keto – ketoconazole; + - growth; -, no growth. Table 4: MFC of ketoconazole and oil against selected isolates of moulds and Candida albicans Test organisms/Isolates MIC (µg/ml) Ketoconazole Oil of O. gratissimum Trichophyton soundanense 2.50 0.625 Trichophyton mentagrophytes 5.00 2.50 Apergillus niger 2.50 1.25 Trichophyton soundanense 2.50 0.156 2 + 0.156 11 2.50 2.50 13 5.00 0.625 17 1.25 0.625 +, growth The results of killing rate of O. gratissimum oil and ketoconazole against the isolates are presented in Tables 5 to 8. This is in line with the results of the MIC and MFC that were obtained earlier in Tables 3 and 4. Table 5: Killing rate (total viable counts) of ketoconazole against dermatophytes Test org./time (mins) 0 20 40 60 90 120 180 360 1440 T. soundanense 7.5 6 4 3 2 1 1 1 - A. niger 8 6 5 3 2 2 2 1 - T. mentagrophytes 9 8.5 3 2 2 1 1 - - T. soundanense 7 6 4 2 1 1 1 1 - Table 6: Killing rate (total viable counts) of ketoconazole against Candida albicans over time Isolates/time (mins) 0 20 40 60 90 120 180 360 1440 2 69 39 34 23 16 14 12 12 - 11 65 40 32 23 16 12 12 - - 13 50 45 32 30 23 22 12 - - 17 50 46 31 30 28 24 22 20 - Table 7: Killing rate (total viable counts) of O. gratissimum oil against dermatophytes over time Test Org./Time (mins) 0 20 40 60 90 120 180 360 1440 T. soundanense 44 34 15 4 - - - - - A. niger 60 34 28 15 6 - - - - T. mentagrophytes 44 42 34 15 3 - - - - T. soundanense 80 62 48 44 53 73 47 30 - Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 34 Table 8: Killing rate (total viable counts) of O. gratissimum oil against Candida albicans over time Test Org./Time (mins) 0 20 40 60 90 120 180 360 1440 T. soundanense 44 34 15 4 - - - - - A. niger 60 34 28 15 6 - - - - T. mentagrophytes 44 42 34 15 3 - - - - T. soundanense 80 62 48 44 53 73 47 30 - Discussion Fungal infections are emerging opportunistic infections that often occur in immunocompromised people and especially those living with HIV/AIDS. In these people, attacks commonly called mycosis are both superficial (skin) and invasive (systemic). These pathogenic fungi cause both superficial and serious systemic diseases and are now widely recognized as important agents of hospital-acquired infection [1]. Fungi implicated in these infections are molds and yeasts [25]. Fungal attacks are recurrent and therapeutic management is complicated by the emergence of multidrug resistant (MDR) fungi. This situation calls for research for new antifungal compounds with a broad spectrum of activity in addition to the existing molecules. One of the solutions is to explore traditional medicine to identify plants with interesting therapeutic properties, since medicinal plants have been used for several purposes and are known to inhibit the growth of several microorganisms including fungi. Among these plants Mitracarpus scaber and Ocimum gratissimum feature prominently. This current research reveals the ease at which fungi isolates can be present in human body parts, as shown in Tables 1 and 2. The preliminary result shows that both antifungal extracts and the drug have activity against the fungal isolates tested. The activity of the O. gratissimum oil was comparable with that of the conventional drugs indicating the potential in this extract. This finding is consistent with the findings of other workers [26, 27]. Other findings revealed that the phenolic compounds isolated from M. scaber extracts are used for the treatment of skin infections caused by Staphylococcus aureus and Candida albicans [28, 29]. It has been previously shown that essential oils as well as compounds derived from O. gratissimum have a wide range of activities with the antimicrobial properties being the most studied [30]. Some work reported that 60% of essential oil derivatives examined to date were inhibitory to fungi while 30.0% inhibited bacteria [31]. Candida albicans still remains an important etiological agent in Southeastern Nigeria with this species implicated in several kinds of infections. As shown in Table 3, the MIC values for Candida isolates (isolates 1-3, 10-15, and 18) were consistently lower (0.16-5.0 ug/ml) against O. gratisimum compared to ketoconazole (1.25-5.0 ug/ml). This work is in contrast with the work done by Anejionu et al. [23], which revealed that in vitro antifungal activity of the ethanol extract of Mitracarpus scaber (50 ug/ml) showed that the clinical isolates were sensitive to the herbal extracts but were more sensitive to O. gratissium oil extract (MIC range of 0.8-1.25 ug/ml) than ketoconazole (MIC range of 0.31-5.00 ug/ml). The data from Table 4 shows that O. gratissimum oil had a greater biocidal effect against most of the test organisms in contrast to ketoconazole. However, the MFC of ketoconazole against the test organisms indicated antagonism. This indicates that the effect of ketoconazole on the isolates is biostatic rather than biocidal; while that of O. gratissimum oil reveals more biocidal activity. This finding is in support with the studies of other researchers [32, 33]. The studies indicated that oil has very good activity against the isolates and kills them faster than ketoconazole especially against Candida albicans. This is an indication that the oil will be good for the treatment of Candida albicans. Notwithstanding the deviation of the total viable count in Table 8, where the total number of viable counts at 90 and 120 min are higher than others, there is still evidence that the oil has strong activity against dermatophytes, confirming the study done by Nwaneri et al. [33]. The inhibitory activities of the extract have been attributed to the presence of hexadecanoic acid (37.21%), oleic acid Mediterranean Journal of Pharmacy & Pharmaceutical Sciences ISSN: 2789-1895 online www.medjpps.com ISSN: 2958-3101 print Nwaneri MGU, et al (2025) Mediterr J Pharm Pharm Sci. 5(4): 29-37. 35 (25.38%), and octadecanoic acid (16.19%) in the extracts of O. gratissimum. [34]. The extracts of O. gratissimum have previously been discovered to have other antifungal properties. A component of O. gratissimum essential oil, called Tymol, was shown to be extremely effective against T. rubrum, T. mentagrophytes, Candida neoformans, Candida albicans, and Malassezia pachydermatis by Chand et al. [35]. Kinetic studies have been consistently used to quantify antimicrobial activity or interactions [36, 37]. 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Evidence-Based Complementary and Alternative Medicine. 2017; 2017: 1-15. doi: 10.1155/2017/ 4534350 37. Alizadeh-Sani M, Hamishehkar H, Khezerlou A, Maleki M, Azizi-Lalabadi M, Bagheri V, et al. Kinetics analysis and susceptibility coefficient of the pathogenic bacteria by titanium dioxide and zinc oxide nanoparticles. Advanced Pharmaceutical Bulletin. 2020; 10(1): 56-64. doi: 10.15171/apb.2020.007 Author contribution: MGUN & INE conceptualized, designed the study and drafted the manuscript. CHN & RNE contributed to data analysis. MGUN, CRC, CKE & INE performed data analysis and interpretation of data. while writing, editing, and proofreading was done by MGUN & UAU. All the authors contributed to data collection and approved the final version of the manuscript. Conflict of interest: The authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical issues: The authors completely observed ethical issues including plagiarism, informed consent, data fabrication or falsification, and double publication or submission. Data availability statement: The raw data that support the findings of this article are available from the corresponding author upon reasonable request. Author declarations: The authors confirm that they have followed all relevant ethical guidelines and obtained any necessary IRB and/or ethics committee approvals.