Iranian Journal of Medical Microbiology

2.1 Collection and Preparation of Plant
The E. japonica seeds were sourced from local markets in Baghdad. Upon acquisition, the fruits underwent incision, facilitating the separation of seeds from their outer coating. Meticulously, the seeds were sifted, rinsed, and subjected to a three-week drying period under shade at room temperature, with regular rotation to ensure uniform drying. Subsequently, they underwent grinding to a fine powder using a grinder. Following established protocols (11), hot ethanolic extracts were meticulously prepared to utilize 100 g of the dried powder obtained from the Soxhlet apparatus, which was carefully packed into a thimble. The resulting extracts were aliquoted into serial tubes and stored at 4°C until further use, ensuring preservation of their bioactive constituents.
2.2 Phytochemical Screening by Gas chromatography-mass spectrometry (GC-MS) Analysis
A total of 5 μL of ethanol extract derived from loquat seeds underwent GC-MS analysis conducted by Agilent Technologies (SHIMADZU Japan) employing a high-temperature column for effective separation. Helium gas served as the carrier gas at a flow rate of 1.0 mL/min, with a split ratio of 1:10, adhering to a meticulously designed temperature program. Both injector and detector temperatures were maintained at 280°C, while the initial column temperature was set at 70°C. A sample volume of 5 μL was injected into the column and subjected to analysis utilizing split (1:10) mode.
Following 2 minutes, the oven temperature underwent incremental elevation to 110°C at a ramp rate of 5°C/min, with a subsequent hold time of 9 minutes. Subsequently, the oven temperature was further increased to 280°C at a ramp rate of 7.5°C/min, with an additional hold time of 9 minutes. Identification of compounds was facilitated by comparing their mass spectra with authentic standards (12), ensuring rigorous characterization of the constituents present in the loquat seed extract.
2.3 Collection of Specimens
Specimens gathered for the investigation of dermatophytic diseases were sourced from individuals afflicted with various manifestations of Tinea across different age groups. Collection methods included skin scraping, hair fragments, and nail clippings. The fungal laboratory at the College of Science, University of Baghdad, oversaw the sample collection. These samples were extracted from patients previously diagnosed with dermatophytes and subsequently cultured on low-pH growth media, ensuring a comprehensive representation of the fungal pathogens under investigation.
2.4 Determination of Eriobotrya japonica Seeds Extract on Dermatophytes
The ethanolic seed extract underwent preparation by dissolution in a DMSO solution to yield three distinct dilution sets (300, 150, and 75 mg/mL), each combined with 200 mL of Sabouraud dextrose agar (SDA). The amalgamation of extracts and SDA was meticulously stirred before being poured into sterile Petri dishes, where it solidified under controlled conditions.
Segments measuring 10 mm, derived from mycelial proliferation in mold cultures cultivated over 14 days, were delicately positioned at the center of each plate. Following inoculation, the plates underwent incubation at a temperature of 28°C for a duration ranging from 7 to 14 days. Subsequent assessment involved measuring. The diameters of fungal colonies using a ruler and comparing them against control samples to determine the antifungal activity of each concentration, as indicated by growth inhibition (13).
2.5 Statistical Analysis
The collected data underwent a one-way analysis of variance (ANOVA) test to enable comparison among groups representing control-negative, control-positive, Nystatin, and Ethanol at concentrations of 300, 150, and 75 mg/mL. Significance levels were determined based on the P-values obtained, with values less than 0.05 being considered statistically significant.

This study was conducted to emulate the effects of crude alcoholic extract of the medical plant E. japonica. The results of the GC/MS analysis identified and focused on the detection of numerous active compounds found in the ethanolic extracts of seeds E. japonica, their retention indexes and percentage composition are summarized in Table 1 and are illustrated in Figure (1). Our findings reveal the presence of 25 peaks in the ethanolic extracts.
The effect of the ethanolic seed extract of E. japonica on the nine types of dermatophytes is shown in Table (2). This table presents the different concentrations of the ethanolic extract of loquat seeds with a positive control (nystatin) and a negative control (naturally grown). The effect of the ethanolic seed extract is calculated by measuring the diameter of the fungal growth. The analysis reveals that at the concentrations of 300, 150, and 75 mg/mL of the ethanolic seed extract, there is a highly significant difference between all tested groups and the negative control. Additionally, there is a significant difference between the 150 and 75 mg/mL concentrations of ethanol and the positive control, but there is no significant difference between the positive control and the 300 mg/mL concentration of ethanol.

Figure 1. GC-MASS analysis chromatogram of E. japonica ethanolic seeds extract.

Table 1. The major compounds that are found in various parts of hot ethanolic seeds extract of E. japonica. by using GC-mass.

Table 2. The percentage of the E. japonica plant's ethanolic seed extract inhibits growth at varying concentrations against dermatophytes.

* The ethanol concentration is provided as mg/mL.
* P value =between tested groups (P≤0.05 were considered significantly different).
* (A, B, and C) LSD represented the levels of significant, highly significant start from the letter (A) and decreasing with the last one.
* NS: Non-significant

The seeds extract containing at least 5 peaks with a percentage area of more than 5% were Propylene glycol monooleate 5.03%, D-Mannitol or DL-Glucitol 5.67%, Beta-sitosterol 5.70%, n-Hexadecanoic acid 14.53% and 9,12-Octadecadienoic acid (Z, Z)- 22.85% (Figure 1). The ethanolic seeds extract of E. japonica has been found to exhibit a range of active compounds, with 9,12-Octadecadienoic acid (Z, Z)- (22.854%) emerging as the most prominent. Conversely, Phthalic acid was noted to have the lowest value (1.162%). The active compound 9,12-octadecadienoic acid (Z, Z)- was the most predominant, which is in agreement with the reports of [14] and [15] on tamarind leaves and seeds. Moreover, 9,12-octadecadienoic acid (Z, Z)- has been shown to possess a range of pharmacological potentials, including antioxidant, anti-inflammatory, hepatoprotective, antibacterial, and antifungal properties [16, 17]. The ethanolic extract of seeds contains a diverse range of secondary metabolites that have been observed to exhibit numerous pharmacological properties, including antifungal activity, such as Hexanal (18), Benzaldehyde (19), benzyl alcohol (20), 4H-Pyran-4-one (21), Benzoic acid (22), Eugenol (23), Sorbitol (24), Dibutyl phthalate (25), Dodecanoyl chloride (26), and Phthalic acid (27).
The results showed that using ethanol as an extraction solvent resulted in the seed extract (Table 1). This is because bioactive compounds with high polarity are more soluble in highly polar solvents like ethanol, enabling ethanol to extract non-glyceride constituents such as phosphatides, sterols, tocopherols, and pigments. These results are consistent with earlier research, which demonstrated that polar solvents yielded more extracts than non-polar solvents (28). Additionally, bio-solvents like ethanol and water pose lower hazards due to their reduced toxicity. Ethanol is frequently utilized as a polar solvent during extraction processes and is also regarded as a bio-renewable substance, as it can be easily acquired at a low cost. Both equipment and solvent types have been shown to significantly impact the quality of extracts (29). Polar solvents such as ethanol exhibit the capacity to extract bioactive constituents, including phenols, alkaloids, glycosides, flavonoids, and tannins. Notably, the total quantity of phytochemical compounds extracted is intrinsically linked to the polarity of solvents employed. Specifically, an increase in the polarity of solvents directly corresponds to a higher concentration of extracted compounds (30). However, these findings are still in the early stages, and future experimental and clinical studies are subsequently needed in this kind of GC-MS investigation. It represents an initial step towards recognizing the dynamic standards in this medicinal field for further detailed study.
As Scheibler et al. indicated (31), Nystatin is the most extensively recommended antifungal medication for treating superficial infections. This study employs nystatin to compare medicinal antifungal drugs for the skin and seed extracts by studying the efficiency and side effects of these plants and drugs. For this purpose, a 2% concentration of nystatin was used with the nine types of dermatophytes: Trichophyton rubrum, Trichophyton mentographytes, Microsporum canis, Trichophyton verrucosum, Trichophyton quinckeanum, Epidermophyton, Trichophyton simii, Trichophyton interdigitale, and Fusarium fungi. The results showed that loquat plant seed extracts significantly inhibited all nine types of dermatophytes.
The effect of the ethanolic seed extract of Eriobotrya japonica on the nine types of dermatophytes is shown in Table (2). This table presents the different concentrations of the ethanolic extract of loquat seeds with a positive control (nystatin) and a negative control (naturally grown). The effect of the ethanolic seed extract is calculated by measuring the diameter of the fungal growth. The analysis reveals that at the concentrations of 300, 150, and 75 mg/mL of the ethanolic seed extract, there is a highly significant difference between all tested groups and the negative control. Additionally, there is a significant difference between the 150 and 75 mg/mL concentrations of ethanol and the positive control, but there is no significant difference between the positive control and the 300 mg/mL concentration of ethanol.
There was a significant difference in the amount of growth inhibition between 150 and 75 mg/mL of ethanol as well as between the positive and negative controls. However, the percentage of growth inhibition was not statistically significant between ethanol concentration of 300 mg/mL and the positive control group. This indicates that using a concentration of 300 mg/mL of the ethanolic extract leads to effectiveness similar to that of the antifungal agent against pathogenic fungi (Nystatin) in most cases.
Increasing the concentration of the ethanolic seed extracts enhanced the efficiency of inhibition, likely attributed to an escalation in the concentration of active compounds within them. Statistical analysis revealed significant differences at the 0.05 level in inhibiting the growth of dermatophytes used in this study. When utilizing ethanol extraction of seeds at 300 mg/mL concentration, it demonstrated extreme efficiency in inhibiting the growth of dermatophytes, with a growth inhibition percentage of 100% observed in almost all dermatophytes examined in this study, except for Fusarium, which exhibited a growth inhibition percentage of 96.56%. At ethanol concentrations of 150 and 75 mg/mL, the percentage of growth inhibition was lower compared to the positive and negative controls, likely due to the diversity and complexity of the natural mixtures of bioactive compounds in crude plant extracts. It is rather challenging to comprehensively analyze and characterize all compounds present in the extract and elucidate their structures in a single study due to the phytochemical results revealing different components of plant extracts with different solvents based on the polarity of each solvent (32).

 

Our results suggest that loquat plant extracts hold promise as environmentally friendly antifungal agents against dermatophytes. Moreover, they encompass a spectrum of phytochemicals exhibiting enhanced biological activity, thereby presenting potential as medications with minimal risk of fungal resistance development and reduced environmental hazards. The present study underscores the superior impact of loquat seed ethanolic extract on inhibiting dermatophyte growth compared to the antifungal nystatin. However, further research is warranted to delve into these effects, especially amidst the ongoing quest for more economical and safer alternatives to prescription drugs.

 

We're highly thankful to the of College of Science, Mustansiriyah University and University of Baghdad to providing us the facilities and also opportunity to achieve our research.
 
 

Conflicts of Interest

The authors declare no conflict of interest.