In silico drug-like properties prediction and in vivo antifungal potentials of Citrullus lanatus seed oil against Candida albicans

2.1 Plant materials

The plant material used in this study was the fresh Citrullus lanatus seeds. The seeds of C. lanatus (watermelon) was procured from the dealers within Zuba in Bwari area council of FCT Abuja, Nigeria. The identity of the plant was confirmed by Mr Felix Nwafor, while the Voucher specimens UNN/PSB/Consult/2019/2721-03 were deposited at the herbarium at the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka,

2.2 Fungal and bacteria strains

Candida albicans strain (SC5314) strain used for this study was obtained from the Department of Microbiology University of Nigeria, Nsukka.

2.3 Collection and processing of Citrullus lanatus seeds

The fruits were washed, cut, and the seeds selected manually from the pulp. Tap water was used to wash off pulp on the seeds before sun-drying for 72 h. Dried samples of watermelon seeds were ground employing a commercial blender (TSK-949, WestPoint, France), preserved in an airtight container, and stored in a desiccator ready for analyses.

2.4 Oil extraction

The pulverized samples weighing 2480 g were extracted for 6 h with n-hexane in a soxhlet extractor equipped with a thimble (chem glass). Rotary evaporator (EYELA, N-N Series; Rikakikai Co Ltd., Tokyo, Japan) was used to concentrate the oil in order to obtain n-hexane-free C. lanatus seed oil. After concentration, crude C. lanatus was immediately placed in a hot air oven maintained at 50 °C for 24 h to ensure complete removal of solvents. The concentrated oil (642 ml) sample was then stored in air-tight bottle in a cool dry place until degumming.

2.5 Degumming of Citrullus lanatus seed oil

The oil was degummed according to the method of Zufarov et al (2008). The crude oil sample was heated to 80 °C, mixed with water (5% vol) and stirred for 15 mins by a magnetic stirrer. Then the mixture was centrifuged (7000 rpm) for 20 mins. Precautions were taken in order to prevent the introduction of air and subsequent oxidation of the oil. The degummed oil supernatant was collected in sterile tubes after centrifuge.

2.6 Susceptibility assay (in-vitro)

Mueller-Hinton agar (Oxoid) plates supplemented with chloramphenicol were prepared for the antifungal activity assay, according to the CLSI agar well diffusion test (CLSI, 2019). Using sterile swab sticks, the standardized fungal cell suspensions (1 × 10⁴ CFU/ml) were inoculated and spread onto the prepared agar surfaces. Within 15 mins of inoculation, wells of 6 mm in diameter were bored into the agar using sterile cork borer and the various oil concentration (63.5, 125.0, 250.0, and 500 mg/ml) were added into the wells using DMSO as a vehicle of choice. The plates were incubated at 37 °C for 24 h (bacteria) and 48 h (fungi).

Antibacterial activities of the oil extract were determined by the agar well diffusion method as described by Kim and Rhee (2016). Different concentrations of 63.5, 125.0, 250.0, and 500 mg/ml of the oil were used for the bioassay. Ciprofloxacin (5 μg) was used as standard antibacterial agent for positive control. After incubation, zones of inhibition formed in the medium were measured in millimeter (mm) diameter.

2.7 Result and interpretations

At the end of incubation, antimicrobial activity of both crude and degummed oil at a particular concentration was indicated by inhibition of growth around each well. The Zone of inhibition was measured with a meter rule in mm and recorded as inhibition zone diameter (IZD) of the oil.

2.8 Docking analysis

The software used includes OpenBabel Program 2.1.1 (downloaded from http://openbabel.org/), AutoDock Vina program (Molecular Graphics Laboratory, The Scripps Research Institute in 2011 downloaded from http://vina.scripps.edu/download. html), Structures of Secreted Aspartic Protein-5 (SAP5) (PDB ID: 2QZX) and lanosterol 14-demethylase (CYP51) (PDB ID: CYP51) enzyme was obtained from protein databank (www.rcsb.org). The protein targets retrieved from protein databank were uploaded to Chimera 1.14 workspace. All non-standard residues including ions, water molecules and co-crystallized ligands were removed. The protein structures were minimized at 200 steepest descent steps, 0.02 steepest descent steps size (Ă), 10 conjugate gradient steps, 0.02 conjugate gradient steps size (Ă), and 5 update intervals, using the structure editing wizard panel of Chimera 1.14. Solvents were removed, hydrogens were added, charges were assigned using AMBER ff14SB force field, histidine was set for the protonation state. All non-standard amino acids in the protein crystal structures were converted to standard amino acids. The output files were converted to PDBQT using software. Structure data file (SDF) structures of palmitic acid and linoleic acid and standard inhibitors were retrieved from the PubChem database.

OpenBabel plugin of Autodock tools was used to convert the proteins and ligand molecules to dockable pdbqt format. Docking of the ligands to various protein targets as well as determination of binding affinities was carried out using Autodock Vina plugin in PyRx software (Trott and Olson, 2010). BIOVIA Discovery studio 2020 and Chimera 1.14 were used to perform post docking analysis and visualization of the molecular interactions between the proteins and ligands respectively.

2.9 Animal material

A total of 72 female albino rats weighing 120–150 g were obtained from the Animal House of the Faculty of Biological Sciences, University of Nigeria, Nsukka. They were acclimatized for 7 days under standard environmental conditions and maintained on a regular feed and water ad libitum. They were grouped into 9 groups of 8 rats each and administered with crude and degummed Citrullus lanatus seed oil for a total of 14 days. Controls were treated with 2% Tween 80 and levamisole as standard drugs. The rats were sacrificed on the 14th day, blood samples used for biochemical analyses were collected through ocular puncture.

2.10 Candida albicans preparation

2.11 Histological examination

2.12 Renal function test

Renal function test was determined by the method of Kozitsina et al. (2014).

2.13 Statistical analysis

The results were analyzed using a statistical software package – SPSS Version 21. Data were expressed as mean ± standard deviation (mean ± SD). Where the variables to be compared are three or more, one-way analysis of variance (ANOVA) was used, the Duncan test was used for post-hoc. p < 0.05 was considered statistically significant

3.1 In vitro antimicrobial test

Table 1 shows that crude and degummed oil (CO and DO respectively) of watermelon seed oil exhibited at different concentrations (63.5, 125.0, 250.0, and 500 mg/ml). There was inhibition against Escherichia coli at all the concentrations except for CO at 500 mg/ml while standard drug (Ciprofloxacin) showed inhibition of 14 mm higher than that of the test groups. However, there was only two inhibitions (6 and 8 mm) exhibited by CO at the concentrations of 250 and 500 mg/ml for Staphylococcus aureus, the inhibition by ciprofloxacin was at 8 mm. Pseudomonas aeruginosa resisted the antimicrobial activity of the oil at all concentrations while Ciprofloxacin inhibited Pseudomonas aeruginosa with a diameter of 10 mm. In the same vein, Candida albicans was sensitive to antifungal activity of CO at all concentration except at the concentration of 63.5 mg/ml; however C. albicans was sensitive to DO at all the concentrations of 63.5–500 mg/ml.

Docking analysis in Table 2 showed that fluconazole formed aromatic interactions with hydrophobic amino acid residues of secreted aspartic proteinases-5 (SAP-5). The residue interacting with linoleic acid formed strong $\pi$ alkyl hydrophobic interactions with oxygen of the carbonyl group of the compound at a docking energy of −5.1 kcal/mol with amino acids of Tyr84, ILE30, ILE123, TRP51 and conventional hydrogen bond with ARG120. Residue ASP86 and linoleic acid formed unfavourable acceptor with the hydrogen of O—H group of linoleic acid. In another development, palmitic acid formed hydrogen bond interaction with hydrophilic ASP86 and Van der waals interaction with SER88 at the docking energy of −7.2 k/cal/mol. Fluconazole interacted with pi anion and aromatic ring that interacted with lone pair of pair of fluorine atom of residue ASP32 and GLY220 as well as pi-alkyl interaction at the binding pocket of ILE 123 at the binding energy of −6.8 kcal/mol (Figs. 1–3).

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Fig. 1

2D and 3D representation of palmitic acid and 2QZX interaction involving Pi-alkyl and conventional hydrogen bond at amino acid residue of TYR84 and ILE123 as well as ASP86 respectively.

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Fig. 2

2D and 3D representation of interaction between fluconazole and 2QZX involving amino acid residue of ILE123, ASP32 and GLY220.

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Fig. 3

2D and 3D representation of interaction between linoleic acid and 2QZX involving ILE30, ILE123, TYR64 and TRP51 as well as ARG120 and ASP86.

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Table 3 show an interaction between ligands (linoleic acid, palmitic acid and fluconazole) and lanosterol 14-demethylase (CYP51) which indicated strong interactions leading to high binding affinities. Docking analysis showed a strong $\pi$ alkyl hydrophobic interactions at the aromatic ring of PHE384, PHE241, LEU95, LEU96, TYR72, MET509 and PRO238 pockets that led to high binding affinity (−8.0 kcal/mol) with palmitic acid and formed another Van der waal interaction with the hydrogen bond of —OH group of palmitic acid. In another development, the aromatic group of PHE384, PHE241 and HIS381 formed pi-pi- stacking interaction with fluconazole. The residues interacting with fluconazole formed pi-sigma interaction with aliphatic amino acid LEU95 and pi-alkyl hydrophobic interaction with amino acid PRO238 of two aromatic rings with a binding affinity of −6.2 kcal/mol. Furthermore, linoleic acid formed hydrophobic alkyl and pi-alkyl interactions only with amino acid PHE384, PHE241, LEU95, HIS381, TYR72, PRO238 of CYP51 at the binding energy of −6.8 kcal/mol (Fig. 6). Palmitic acid exhibited the best binding energy of −8.0 kcal/mol when compared with fluconazole (reference drug) which docked at the energy of −6.2 kcal/mol (Table 4, Figs. 4 and 5).

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Fig. 4

2D and 3D structure of pi-alkyl hydrophobic interaction between palmitic acid and 5EAD involving amino acid residue of PHE384, PHE241, LEU95, LEU96, TYR72, MET509. PRO238.

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Fig. 5

2D and 3D structure of pi-sigma & pi-pi stacked interaction between fluconazole and 5EAD involving PHE384, PHE241, HIS381, LEU96, PRO238.

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Fig. 6

2D and 3D structure alkyl and pi-alkyl interaction between linoleic acid and 5EAD involving amino acid residue of PHE384, PHE241, LEU95, HIS381, TYR72, PRO238.

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3.2 Effect of the crude and degummed oil on Candida albican colonization of kidney

The result of colony forming unit (CFU) {at 1 in 100 dilutions} counted in group 2 {suppressed, inoculated but not treated} was 689.67 ± 4.93 which shows significant increase (p < 0.05) when compared with groups 3 (326.67 ± 5.13), 4 (477.67 ± 3.51), 5 (278.33 ± 6.03), 6 (336.00 ± 4.00), 7 (273.33 ± 3.06) and 8 (186.00 ± 4.00). There was a significant increase (p < 0.05) CFU counted in group 4 when compared to group 3 (treated with 500 mg/kg b. w and 300 mg/kg b. w of degummed oil extract respectively). In the same vein, group 6 recorded a significant increase (p < 0.05) in CFU counted when compared with group 5 (treated with 500 mg/kg b. w and 300 mg/kg b. w of crude oil extract). It could be that the CFU recorded in group 9 (1.33 ± 0.58) was as a result of contaminations, this is because it was only in one of the triplicates that gave four colonies. Groups 5 and 6 (crude) gave a lower (p > 0.05) count than groups 4 and 5 (degummed). When group 8 (treated with 5 mg/kg bw of Fluconazole) was compared with other treatment groups, it gave a marked decrease (p < 0.05) in CFU when compared with other treatment groups. It also revealed that increase (p < 0.05) in CFU recorded in group 2 was as a result of immunosuppression when compared with group 1 that was not suppressed.

At the 14th day, there was a significant increase (p < 0.05){at 1 in100 dilutions} in CFU counted in group 2 (882.33 ± 8.50) when compared with groups 3 (145.33 ± 5.86), 4 (62.00 ± 18.52), 5 (165.67 ± 2.08), 6 (174.00 ± 2.65), 7 (226.33 ± 7.51) and 8 (210.33 ± 8.50). There was no significant difference (p > 0.05) between group 5 and 6 (treated with crude oil extract), same was the case between groups 7 and 8. While there was a reduction (p < 0.05) in CFU count in the groups treated with oil extracts, there was no significant reduction (p > 0.05) in CFU count in group 8 when compared with the 7th day CFU counts. When group 8 of day-14 was compared with group 8 of day-7, there was a slight increase (p > 0.05) in day-14 CFU count (Fig. 7).

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Fig. 7

Fungal Burden of Kidney Colonized with C. albicans.

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Colony forming unit of 10⁻² dilutions of tissue homogenate on SDA

• Group 1 = inoculated without immunosuppression

• Group 2 = inoculated + suppresses without treatment

• Group 3 & 4 = inoculated + suppressed + treated with 300 & 500 mg/kg b.w.of DO respectively

• Group 5 & 6 = inoculated + suppressed + treated with 300 & 500 mg/kg b.w.of CO respectively

• Group 7 = inoculated + suppressed + treated with 25 mg/kg bw levamisol

• Group 8 = inoculated + suppressed + treated with 5 mg/kg bw Fluconazole

• Group 9 = Control

3.3 Result of body and kidney weight

After 7 days of treatment, there was a significant increase (p < 0.05) in the body weight of rats in groups 3, 4, 5 and 7 when compared with group 9 (normal control). This significant increase was sustained up to the 14th days of the study. There were no uniform variations within the group and between the days of the study. The organ weight of all treated groups decreased between 7th and 14th day, except in groups 3 and 5 (which were treated with 300 mg/kg bw of degummed and crude oil extract respectively) which experienced a slight increase (p > 0.05). This could also imply that immunosuppression and Candida albican inoculation affected the kidney more than that of group 9 which was not suppressed at all thus were the only groups that experienced increase in organ weight after 14th day of the study (Table 5).

3.4 Effect of Candida albican on the renal function

There was no significant difference in urea value in all the treated groups when compared with the controls in the first 7 days. But the treated groups recorded a lower (p > 0.05) urea value when compared with group 9 {7.33 ± 2.58} (normal control). However, the trend slightly changed on the 14th day which recorded a significant increase (p < 0.05) in the treated groups as well as group 9 when compared with group 1 (not suppressed but inoculated). There was also an elevation of urea value in group 2 (immunosuppressed, inoculated but not treated) on day 14. But group 1 (24.47 ± 19.38) increased (though not significant) more than group 2 (18.27 ± 2.02) and group 2 increased more than the treated groups. Groups 7 and 8 that were treated with standard drugs Levamisol and Fluconazole respectively were also reduced (not significant) more than the treatment groups.

After 7 days of treatment, the creatinine value gave a significant increase (p < 0.05) in all treated groups except group 4 (64.67 ± 16.33) when compared with groups 1 (42.43 ± 4.92) and 9 (48.91 ± 12.07). While on the 14th day, there was a significant increase (p < 0.05) in all treated groups when compared with group 1 (52.95 ± 12.06). But group 2 (117.73 ± 6.42) showed a significant increase (p < 0.05) when compared with the treated groups (Table 6).

3.5 Histopathologic findings

Histologic results revealed several features of the degenerative characteristics of C. albicans. Results demonstrated that the severity of tissue pathology is related to the integrity of the immune system and the concentration of C. albicans inoculated as observed in our pilot study.

3.5.9

3.5.9 Photomicrograph showing C. Albicans spores on the kidney in group 9 (normal control)

Plate 9: Sections of the kidney collected from this animal showed the normal histomorphology of the kidney. No fungal activity was observed in both the cortex and the medulla as well as the pelvis.**

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Glomerulus (G); Bowman’s capsule (arrow); Renal tubules (RT). (PAS stain: ×400).

3.6 Discussions

The antimicrobial activity of both crude Citrullus lanatus seed oil (CO) and degummed Citrullus lanatus seed oil (DO) (Table 1) showed varied antimicrobial activity of the oils at different concentrations. The antimicrobial activity of both crude and degummed oil could be due to the activity of palmitic acid and linoleic acid in the oil as shown in Figs. 1–6, or maybe due to conventional hydrogen bond interaction of —OH group of the carboxyl group and ASP86 of Secreted Aspartic Protein 5 as shown in Fig. 1. It could also be due to inhibition of the enzyme activities in the microorganisms (Zheng et al., 2005). Free fatty acids are potent inhibitors of enzymes, unsaturated FFAs can also inhibit bacterial activity (Joffre et al., 2019). This could alter cell membrane fluidity and permeability causing destabilization of the membrane and leakage of the internal contents from the cell and death at the end. Zheng et al., (2005) observed the importance of —OH group in the antimicrobial activity of free fatty acid which is in line with docking analysis performed. Zheng et al. (2005) also reported that unsaturated FFAs has demonstrated greater antimicrobial action than saturated FFAs, however our docking analysis showed that palmitic acid exhibited the greatest binding affinity when compared with the reference drug (fluconazole). In another development Wille and Kydonieus (2003) reported that the position of double bonds can affect efficacy and spectrum of antimicrobial activity; the present docking studies also demonstrated that linoleic acid effectively inhibited lanosterol 14-demethylase (CYP51) enzyme which is an important component of Candida albicans membrane. Linoleic acid which has two double bonds were distinctly more active than other fatty acids with three double bonds (Giancarlo et al., 2021).

Body weight of an individual is influenced by the type and quality of food and other metabolic factors. There were no uniform variations in weight gain within the groups and between the days of the study. The organ weight of all treated groups decreased between 7th and 14th day, except in groups 3 and 5 (which were treated with 300 mg/kg b. w of degummed and crude oil extract respectively) which experienced a slight increase (p > 0.05) (Table 5). This could also imply that immunosuppression and Candida albicans inoculation affected the kidney more than groups1 and 9 which was neither suppressed nor treated. The reason for this development could not be understood but the oil might have reduced the rate at which food empties from stomach. Tanaka et al. (2008) indicated that administration (intra-gastric) of long-chain FFAs increased plasma cholecystokinin (CCK) levels, it was observed that intravenous injection of CCK into the vessels of the brain induces satiety (lack of hunger) in laboratory animals. Considering this mode of secretion relative to feeding, it would make a physiologic sense that this hormone plays a significant role in control of food intake in the rats treated with watermelon (Citrullus lanatus) seed oil which is rich in linoleic acid and palmitic acid. Sidhu et al., (2000) observed that fatty acid-induced CCK secretion in humans and from the enteroendocrine cell line which depends essentially on acyl chain length and that the potency of the fatty acid was directly proportional to its chain length; while both C10 and C12 stimulated CCK secretion over this range but C12 was significantly more effective. The chain length of linoleic acid and palmitic acid was above the minimum acyl chain length required to stimulate CCK secretion. CCK might have increased the sensation of short-term fullness, thereby reducing appetite during a meal. Invariably this suggests that the Citrullus lanatus seed oil could have appetite and weight reducing potentials.

Docking is of biological and pharmacological significance given its ability to predict the affinity and activity of the small drug molecules by allowing ligand position itself in a protein binding site. When linoleic acid and palmitic acid were docked against Secreted Aspartic protein-5 (SAP-5), the docking scores of the compounds as well as the standard drug were between −7.2 and −5.1 kcal/mol respectively. Palmitic acid achieved the highest binding affinity score of −7.2 kcal/mol, closely followed by fluconazole (standard drug) with a score of −6.8 kcal/mol. While linoleic acid interacted with SAP-5 at the energy of −5.1 kcal/mol. This suggests that palmitic acid is capable of inhibiting the production Candida albicans SAP-5 enzyme which is more virulent than other SAP strains. The interaction with hydrogen atom of —OH group could also be responsible for the high binding affinity of palmitic acid with SAP-5 enzyme. It has been suggested by Zheng et al (2005) that —OH groups could account for antimicrobial activity of free fatty acids.

While some amino acid residue enhances the activity of the ligands during ligand-receptor interaction, some downregulates the activity of ligands. Molecular docking study showed that the compounds and reference drug interacted with these same amino acid residues such as PHE384, PHE241, LEU96, PRO238 and HIS381. However palmitic acid exceptionally interacted with LEU95, TYR72 and MET509, this suggests that the antifungal action of palmitic acid is drawn from the ligand-receptor interaction at these amino acid binding pockets; giving it the best docking score. Similarly, linoleic acid exceptionally interacted with LEU95 and TYR72 to give it additional antifungal activity compared with the fluconazole. The best binding energies with lanosterol 14-demethylase (CYP51) were shown by palmitic acid and linoleic acid selected in this study (Table 3). The docking scores of palmitic acid and linoleic acid were −8.0 and −6.8 kcal/mol respectively while fluconazole was −5.2 kcal/mol.

Urea and creatinine measurements were used to evaluate renal function in rats challenged with pathogenic SC5314 strain of C. albicans. Increase in urea and creatinine in group 2 (when compared with its treated counterpart) could be due to immunosuppression and C. albicans infection. Marchenko et al. (2015) had reported that serum BUN level after Candida infection was significantly lower than that observed prior to the challenge. This report is in line with our findings. Renal disease is usually connected with reduced urea excretion and eventual increase in blood concentration. This result suggests that CLSO treatments could ameliorate the effect of Candida albicans on renal function when compared with group 3.

Creatinine showed a significant increase (p < 0.05) in all the treated groups when compared with group 9 on both the 7th and 14th day. There was no significant increase (p > 0.05) in creatinine concentration between 7th and 14th day. Both urea and creatinine are delivered from endogenous or exogenous protein but their independent rate of excretion denotes differing metabolic pathway. Our result also showed that colonisation in the tissues and organs was minimised after 14 days of the study as shown in the histopathology studies.

In order to explore the invasive characteristics of C. albicans, we studied tissue microbiology and pathology in female albino wistar rats. This model is similar to human invasive infection occurring at gastrointestinal tract under immunocompromised condition; fungal cells are found in all organs, but the disease progresses mostly in the kidneys and brain (Ghosh et al., 2019). Kidney has been established to be one of the primary target organs in systemic candidiasis; it is also a major organ for the multiplication of Candida species (Ghosh et al., 2019). Therefore, the in vivo activity of Citrullus lanatus seed oil (CLSO) was investigated in experimental systemic candidiasis, by estimating the fungal burden in this organ histologically and microbiologically. The term “multifocal candidiasis” is used to describe the lesions/evasion in histopathologic diagnosis.

In group 1 during first seven (7) days, the number of viable C. albicans cells in the kidneys (homogenised and cultured on Saboraud Dextrose Agar) from animals that were inoculated but not treated nor immmunosuppressed, revealed a significantly low (p < 0.05) number Candida albican cells; after 14 days of treatment the number of viable count increased when compared with the group 2 (immunosuppressed and inoculated but not treated) which had a significantly higher (p < 0.05) number of viable C. albicans cells after 14 days period; suggesting that immunosuppression is a strong tool for multiplication of C. albicans. Similarly, histopathological slides showed a minimal fungal activity with no degeneration and necrosis of the renal tubules when compared to the group 2 which showed multifocal aggregation of fungal blastospores within the renal parenchyma which is also widespread in the glomeruli lumen of renal tubules and blood vessels with degenerate epithelial lining cells of the cytoplasm. It is believed that immunosuppression facilitated fungal colonization and evasion in group 2 rats. The suppression of immune status of the animals probably paved way for severe degeneration of epithelium and clusters of blastospores within the nephron; by enhancing fungal penetration of keratin epithelial linings and enhanced adherence (van de Veerdonk et al., 2010).

Groups 3 and 4 (immunosuppressed and treated with 300 and 500 mg/kg bw of degummed oil respectively) showed a significant decrease (p < 0.05) in colony forming units after 14 days of treatment when compared with group 2. This reduction in the blastospore could be due to antifungal activity of palmitic and linoleic acids as revealed in molecular docking (Tables 2 and 3). Linoleic acid and palmitic acid which are contained in oil has been reported to show antifungal activity (Annamaria et al., 2015). However, Group 5 and 6 (immunosuppressed and treated with 300 and 500 mg/kg b. w of crude oil respectively) showed a minimal fungal blastospore with single presence in the renal tubules and glomeruli; and also, a significantly reduced (p < 0.05) fungal burden in the colony counted when compared to group 2 after 14 days of treatment. Palmitic and linoleic acid restricted the penetration by hyphae and minimize inflammatory changes as well as inhibited colonization as shown in Figs. 1–6. The ameliorative effect witnessed in these treated groups could also be a result of the protective effect of antioxidant minerals (manganese, selenium, and zinc) present in both oils (Apeh et al. 2020). Pyrogallol may have also aided the oxidative stress and inflammatory reactions through its reducing properties leading to immunosuppression. Joharapurkar et al. (2004) had demonstrated that pyrogallol is an immunosuppressive agent through the induction of oxidative stress while Yu (2006) had observed that selenium and zinc inhibited oxidative stress and apoptosis.

In another development, it was observed that the animals showed some clinical presentations (swollen eyelid, persistent stridor, and occasional cough) during the experiment. This is purely a manifestation of respiratory tract infection suspected to have emanated from candidaemia. Stridor could be as a result of lesions coming from the central nervous system (CNS), the gastrointestinal (GI) tract, or the respiratory tract (April, 2016).

Group 8 (immunosuppressed and treated with fluconazole) showed minimal fungal blastospores increased at day-14 of post infection treatment. There could also be possible interaction between fluconazole and pyrogallol (reducing agent) which might mimic fluconazole at its receptor site(s). Findings suggest that fluconazole could act as a blockade to certain cytochrome P-450 isoenzymes involved in drug metabolism in humans (Debruyne, and Ryckelynck, 1993). Redding et al. (2003) reported that Candida glabrata does not respond to doses of fluconazole in cases of oral infection.

Group 9 (normal control: no Candida albicans inoculation and no treatment of any kind) showed the normal histomorphology of the kidney. No fungal activity was observed in both the cortex and the medulla. Likewise, no fungal activity was observed in the renal pelvis. In C. albicans cell counted, there was no count except for one single colony seen which might be suspected to be contamination.

This, therefore, has shown an increased level of colonization in immunosuppressed groups compared to Candida control (group 1); suggesting that increased risk of mucosal infections with Candida albicans was ameliorated by Citrullus lanatus seed oil as treatment progressed. Systemic/physiologic factors antagonistic to Candida organisms could constitute a barrier that inhibited fungal penetration apart from treatment with the oil; such factors might be absent in immunosuppressed rats and this allowed the fungus to superficially colonize the organs.