Isolation and enumeration of dermatophytic fungi
Among 300 specimens scanned; 222 (74.00%), 234 (78.00%) and 279 (93.00%) of total samples showed positive results for the evidence of fungal elements by direct microscopic (KOH+), SDA and RSM culture methods, respectively while 78 (26.00%), 66 (22.00%), and 21 (7.00%) of total samples show no evidence of the fungi with these methods, respectively (Table 1). The culture method was better than microscopic method because of KOH preparation not only produce carbonate crystals by adsorbing CO2 but also form fat pellets in the slide after heating slightly, hence decreasing efficacious visualization of fungi (Rebell and Taplin 1970; El-Gendy 2010; El-Bondkly et al. 2012; El- Gendy et al. 2014).
Concerning to the correlation between direct microscopic examination and culture method on SDA or RSM medium, 183 (61.00%) isolates were positive for presence of fungi in both KOH and SDA medium tests. Moreover, 51 (17.00%), 21 (7.00%) and 45 (15.00%) of samples gave negative results with microscopic, culture method on SDA medium and both methods, respectively while by applying both direct microscopic test and culture method on RSM together, 210 (70.00%) and 9 (3.00%) of samples gave positive and negative results, respectively for fungal elements. Thus, these results support RSM adequacy as a direct isolation method for clinical dermatophytic fungi than SDA specifically, it is appropriate for phenotypic characterization. Likewise, the isolation frequency of fungi on SDA (n=234, 78.00%), was less than RSM (n=279, 93.00%) because of RSM medium containing different antibiotics that provide a differentiating quality medium for isolating dermatophytes and inhibited non-dermatophytes (El-Gendy 2010; El-Bondkly et al. 2012; El-Gendy et al. 2014). As stated by Madhavi et al. (2011) 33% of total samples showed no fungal element on both direct microscopic and culture methods but 43 and 58% of the samples were distinguished using direct microscopic and culture method, respectively.
Specification and validation of dermatophytic differentiation media
Based on phenotypic and chemotypic characteristics, 253 isolates (90.68%) of 279 isolates obtained after culturing on RSM were actual dermatophytic fungi. While 45 dermatophytic fungi were unable to alter the RSM medium coloration, 19 non dermatophytic fungi; 11 Candida sp.; 1 spore-forming bacterium of Bacillus sp.; 1 Gram negative strain of Escherichia coli and 2 Gram positive isolates of Staphylococcus aureus were able to change RSM medium color (Table 2).
Moreover, our data proved that DTM medium is not completely specified for dermatophytes because of 11, 9, 4, 2, and 2 non-dermatophytic fungi, Candida sp., spore-forming bacilli, S. aureus and Escherichia coli respectively were able to grow and change DTM medium color but 33 dermatophytic isolates were unable to change the color of DTM. Many non-dermatophytic fungi have culture morphology like that of dermatophytes routinely isolated from clinical samplings and they are able to change DTM color which can be attributed to these fungi have resistance to the concentration of antifungal antibiotic applied in DTM and thus leading to false positive dermatophytes results (El-Bondkly et al. 2012; El-Gendy et al. 2014). These finding suggested that RSM and DTM media failed to be the preferred medium for fast differentiation of true dermatophytic fungi among clinical isolates and reflect the argent demand another confirmatory and selective medium. In this regard the ability of DIM medium to eliminate untruthful positive results obtained with commercial market media like RSM and DTM was evaluated. All dermatophytic isolates (n=253) recovered from RSM were capable of growing on DIM as well as turning its color to dark purple after 96 h of incubation (Table 2). Moreover, 6 isolates of non-dermatophytic fungi and 3 Candida species grown in DIM without altering its color while one isolate belong to Candida changed the color of DIM medium. The positive color change result of Candida strain on DIM medium can be rule out depending on it was much less intense those produced by different dermatophytic species the growth of Candida as creamy bacteria like colonies easy distinguishing from dermatophytes. Hence, when we take these two criteria into consideration, DIM medium specificity and sensitivity for dermatophytic fungi can be rose from 99.61% to 100%. Effectiveness and validation of DIM toward eliminating problems of untruthful positive results occurred with RSM and DTM media owing to its higher content of cycloheximide, 4 mg/mL, and incubation at 37 °C in comparison to 0.5 mg/mL and 30 °C in DTM as previously reported by Gromadzki et al. (2003), El-Gendy (2010), El-Bondkly et al. (2012) and El-Gendy et al. (2014).
Evaluation of different virulent factors in dermatophytes
Dermatophytes secrete many enzymes to hydrolyze the host tissue components as a source of nutrients. Moreover, released enzymes act as antigens and stimulate different degrees of inflammation. Interestingly all endophytic genera and species in this evaluation were potent enzymatic and non enzymatic virulent factors producers, which reflect the high pathogenicity of these isolates (Table 3). High keratinase activity was determined in M. cookie, E. floccosum, T. rubrum and Trichophyton vaoundei (23.56, 22.08 20.35 and 20.14 U/mL, respectively) on stratum corneum followed by (21.62, 19.07, 18.3 and 17.41 U/mL, respectively) on human hair and (22.10, 17.90, 16.00 and 15.88 U/mL, respectively) on human nail but the maximum yield of protease activity estimated in T. rubrum, M. ferrogenium, T. tonsurance, M. canis (29.91, 28.63, 27.59 and 26.91 U/mL, respectively) on stratum corneum followed by (28.00, 25.31, 27.45 and 25.09 U/mL, respectively) on human hair or (29.54, 28.11, 27.00 and 25.41 U/mL, respectively) on human nail. Moreover, stratum corneum was the best substrate for the induction of phospholipase in fungal strains M. persicolor, M. racemosum, M. ferrogenium, T. erinacei and T. mentagrophytes (62.25, 60.34, 60.11, 57.37 and 54.55, U/mL, respectively). Hewitt and Vincent (1989) and El-Gendy et al. (2010, 2014) in terms of the capability of dermatophytes to parasitize the host relies on the function of keratinase, protease, phospholipase, lipase, etc… not only for elastin, keratin, collagen and phospholipids degradation to provide substrates to fungi but also to stimulate delayed hypersensitivity.
On the other hand, all human substrates supported the melanoid pigment formation as one of the most active non enzymatic virulence factor by these dermatophytic fungi under study but the highest amounts of melanin were formed by E. floccosum, T. rubrum, M. gallinae, T. tonsurance, T. erinacei and M. gypseum (0.772, 0.771, 0.762, 0.704, 0.697 and 0.683, mg/mL, respectively) on human nail as the best inducer for melanin formation. Consequently, understanding the specific virulence factors responsible for pathogenicity of dermatophytes will help develop new therapeutic strategies. Our data are in line with El- Gendy et al. (2014) who stated that dermatophytic fungi synthesize a wide range of enzymatic virulence factors for instance keratinases, proteases and phospholipases upon different substrate specificities as well as non enzymatic factors like melanoid pigments, which are involved in the adherence process and pathogenicity of dermatophytes.
Evaluation the antifungal activity of different plant extracts against dermatophytes
In Table (4) we reported that hexane extract of all plants under study exhibited the lowest inhibitory activity percentage against all dermatophytic fungi obtained in this work but the maximum activity was noticed with methanol and acetone extracts, thus there is compatibility between the polarity of both solvent and antifungal metabolites and their solubility is effective in methanol or acetone but not in hexane (El-Gendy 2010).
Growth of M. audouinii, M. canis, T. soudanense,T. erinacei, M. persicolor, M. gypseum, M. ferrogenium, M. cookie, M. gallinae and M. racemosum isolates totally repressed by the methanolic extract of P. albicans at concentrations of 100 and 200 µg/mL but T. vaoundei isolate at 200 µg/mL. Moreover, the methanolic extract of P. albicans at concentrations of 50, 100 and 200 µg/mL gave the lowest growth inhibition activity toward the growth of E. floccosum (9.3 ± 0.1%, 30.0 ± 0.3% and 33.0 ± 0.7%) followed by T. rubrum (33.3 ± 0.0%, 56.7 ± 0.0% and 80.0 ± 0.0%), T. tonsurance (11.8 ± 0.7%, 66.0 ± 0.9% and 88.0 ± 0.0%) and T. mentagrophytes (56.7 ± 0.4%, 80.0 ± 0.0% and 93.0 ± 0.0%), respectively. Furthermore, the acetone extract of P. albicans gave similar results to the methanol extract except for T. soudanense and T. rubrum strains due to both of them were totally inhibited at 200 µg/mL of acetone extract. From data in Table (4) the P. albicans have potent anti-dermatophytic activity against all Microsporum species (100% of isolates were inhibited) followed by Trichophyton species (50% and 66.67% of isolates were inhibited by methanol and acetone extracts, respectively) with poor activity against E. floccosum. Sepahvand et al. (2018) and El-Demerdash (2018) reported these medicinal plants extracts as the most effective medicinal plants for dermatophytosis in development of plant-based medicines against fungi due to their potent fungitoxic properties.
Furthermore, whereas the highest reducing power against the growth of Trichophyton andEpidermophyton species was achieved after treatment with the methanolic extract of the ancient Egyptian medical plant T. hirsuta. The growth of all Trichophyton species (T. soudanense,T. erinacei, T. rubrum, T. tonsurance, T. vaoundei and T. mentagrophytes) and E. floccosum was 100% inhibited by the methanolic extract of T. hirsiuta at concentrations of 100 and 200 µg/mL as well as acetone extract at 100 µg/mL. growth of M. audouinii, M. gypseum, M. ferrogenium, M. cookie, M. canis, M. persicolor, M. gallinae, M. racemosum isolates was inhibited by (28.3 ± 0.5% and 40.7 ± 0.0%), (80.0 ± 2.0% and 90.0 ± 0.0%), (29.3 ± 0.4% and 50.0 ± 0.0%), (50.0 ± 1.2% and 67.0 ± 0.0%), (70.0 ± 1.3% and 83.0 ± 0.0%), (80.0 ± 0.4% and 88.0 ± 2.4%), (42.0 ± 2.1% and 45.0± 1.9%) and (28.3 ± 0.1% and 46.0 ± 2.5%) after treatment with methanol and acetone extracts individually at a concentration of 200 µg/mL , respectively (Table 4). Egyptian medical plant T. hirsuta extracts were reported to have antimicrobial, antitumor, antihypoglycemic and antioxidant activities due to their high phenolic compound contents (Yahyaoui et al. 2017). Study of Felhi et al. (2017) proved that extracted aerial parts of T. hirsuta exhibited a moderate to strong antimicrobial action against yeasts and bacteria. Abed et al. (2014) reported that among hexane, acetone and methanolic extracts of T. hirsuta, methanolic extract was the most active toward bacteria with inhibition zones equal to 11 to 25 mm in diameter which refer to the presence of biologically active metabolites of different chemical types in its MeOH extract for instance phenolic compounds.
Data in Table (4) showed that no growth was detected for T. rubrum, T. tonsurance, T. erinacei, M. audouinii, M. gypseum, M. cookie, M. persicolor, M. gallinae and E. floccosum isolates with 50, 100 and 200 µg/mL of U. maritime extracted with methanol or acetone which is much lesser than that of the P. albicans and T. hirsiuta extracts that might be indicates the existence of broad spectrum metabolic inhibitors in U. maritime extracts against pathogenic fungi. Furthermore, with increasing concentrations to 100 or 200 µg/mL of both extracts individually each of T. mentagrophytes, T. soudanense, T. vaoundei, M. ferrogenium, M. canis and M. racemosum strains failed to grow. Aboelsoud (2010) and El-Demerdash (2018) reported that U. maritima is one of the best leading therapeutic plants in ancient Egypt. Antibacterial activity of U. maritima methanolic extract against various pathogens showed different antimicrobial properties of plant extracts. B. cereus ATCC10876 was the most inhibited pathogen with adiameter of inhibition zone 11 mm, followed by Acinetobacter baumanii (10 mm) and Salmonella typhimurium (9 mm) and this activity attributed to the presence of secoiridoid, glucosides, phenylethanoids and flavonoids contained in the extract (Belhaddad et al. 2017). The potent inhibitory activity of these plants against dermatophytic fungi is important because these fungi are the main reason of death and infectious diseases globally each year.
Determination of the antitumor activity of different plant extracts against human cancer cell lines
A dose-dependent inhibtion in cells viability after treating with each extract for each plant as illustrated in Figures 1, 2, and 3. The methanolic extract of P. albicans reduce the viability of HCT-116, HepG-2, MCF-7 and HeLa cell lines to (41%, 40%, 80% and 81%); (30%, 20%, 66% and 70%); (19%, 0%, 49% and 57%); (5%, 0%, 35% and 45%) and (0, 0, 21, and 40%) compared to (47, 50, 88, and 89%); (33, 30, 79 and 80%); (22, 0, 60, and 70%); (16%, 0%, 40% and 59%) and (0%, 0%, 36% and 50%) with acetone extract of P. albicans at dosage of 25, 50, 100, 200 and 300 µg/mL, respectively. Similar to our results Nazarizadeh et al. (2013) reported that P. major methanol extract (1 µg/mL) was cytotoxic against cervix and ovary carcinoma with stimulating the proliferation of nasopharynx carcinoma along with other activities like wound healing, anti-inflammatory, anti-fatigue and hematopoietic. Interestingly, Gálvez et al. (2003) evaluated the cytotoxic activity of Plantago species against MCF-7 and UACC-62 cells and the strong cytotoxicities were detected in methanolic extracts of Plantago coronopus and Plantago bellardii with IC50 equal to 32 and 34 µg/mL against MCF-7 and UACC-62, respectively.
However, death in HCT-116, HepG-2, MCF-7, and HeLa carcinoma cell lines equal to (10%, 20%, 23% and 60%); (16%, 40%, 58% and 80%); (40%, 50%, 80% and 100%); (51%, 65%, 100% and 100%) and (49%, 82%, 100% and 100%) was reported after treatment with methanol extract of T. hirsuta but with acetone extract it has been estimated to be (20%, 26%, 15% and 53%); (25%, 50%, 31% and 70%); (33%, 60%, 60% and 89%); (60%, 71%, 81% and 100%) and (67%, 89%, 97% and 100%) at a concentrations of 25, 50, 100, 200, and 300 µg/mL, respectively (Fig. 2). HCT-116 showed the highest resistant to T. hirsuta but while HeLa followed by MCF-7 cell line showed the highest sensitivity for both methanolic and acetone extracts of T. hirsiuta. Previously, polar extracts of T. hirsuta exhibited dose dependent cytotoxic effect on human colon cancer cell growth (Akrout et al. 2011) and HeLa cell viability with IC50 value175 µg/mL (Felhi et al. 2017).
Overall, U. maritima methanolic extract exhibited the highest cytotoxicity against HCT-116 followed by MCF-7 and HepG-2 cell lines but the lowest activity was recorded with cervical cancer. The viability of HCT-116, MCF-7 and HepG-2 cell lines was totally repressed with 100 µg/mL (TGI~100 µg/mL ) whereas the growth of HeLa cell line was reduced to 10% at 100 µg/mL of U. maritima methanolic extract and totally killed at 200 µg/mL . On the other hand, HCT-116 and MCF-7 cancer cell lines failed to survive at 100 µg/mL of U. maritima acetone extract but HeLa and HepG-2 cell lines killed by 200 and 300 µg/mL of acetone extract, respectively. Urginea maritima is endemic to Egypt, well known for its traditional medicine applications and it is typical for treating malignancies by reason of their selective killing of cancer cell by apoptosis instead of necrosis. Urginea maritima selectively repressed the proliferation of SH-SY5Y malignant cells in dose-dependent mode (Elghuol et al. 2016). Al‑Dabbagh et al. (2019) noticed the same observations in HepG-2 cells treated with the ethanolic extract of Matricaria recutita L. with IC50 ~300 µg/mL. Mohamed et al. (2014) isolated urgineaglyceride A and quercetin-3'-O-β-d-glucopyranoside from U. maritima extract with repression activity toward lung, glioblastoma and prostate cancer cell lines. Then these plants, P. albicans; T. hirsuta and U. maritima appear to be among the preferred drugs in the future to treat these deadly diseases.
Phytochemical analysis of selected plant extracts
All the investigated Egyptian medical plants exhibited an amazing presence of significant phytochemicals that involved in several biological activities and the quantities of secondary metabolites were increased through increasing the polarity of solvents used, so the maximum yields were detected in methanol extract followed by acetone while the lowest amount was detected in hexane extract (Table 5). These results highlighting the chemical differences between various extracts of each plant obtained in accordance with solvent. Yahyaoui et al. (2017, 2018a, b) reported that the content of polyphenols, flavonoids tanins, alkaloids and other phytochemicals are varying considerably according to the solvents used, polarity of solvent, extraction methods, particle size and geographical origin. The phytochemical investigations of P. albicans revealed the presence of various chemical constituents of great clinical importance with a history of pharmacological effects, i.e. polyphenols (31.25 ± 0.011, 100.61 ± 0.010 and 100.58 ± 0.018 mg/g); flavonoids (25.00 ± 0.18, 53.19 ± 0.40 and 50.92 ± 0.39 mg/g); condensed tannins (1.22 ± 0.00, 7.80 ± 0.30 and 6.71 ± 0.18 mg/mL); carotenoids (0.43 ± 0.20, 2.99 ± 0.15 and 2.46 ± 0.12 mg/mL); lutein (0.25 ± 0.01, 0.85 ± 0.05 and 0.76 ± 0.04 mg/mL), crude saponins (1.0 ± 0.05%, 1.90 ± 0.09% and 1.80± 0.09%) and alkaloids (0.00 ± 0.00%, 0.50 ± 0.00% and 0.44 ± 0.00%) in hexane, methanol and acetone extracts, respectively. These data are in line with Francoise et al. (2008) investigation of Plantago species phytochemicals which revealed the occurrence of various chemical ingredients, i.e. steroids, tannins, coumarins, lipids, flavonoids, iridoids, polysaccharides, sterols and volatile substances of medication importance.
Phytochemical composition of T. hirsuta was total polyphenols (48.15 ± 0.01, 150.09 ± 0.05 and 142.16 ± 0.01 mg/g); total flavonoids (15.76 ± 1.65, 41.83 ± 0.31 and 40.39 ± 0.26 mg/g); condensed tannins (2.31 ± 0.05, 9.00± 0.22 and 7.63 ± 0.19 mg/mL); carotenoids (0.32 ± 0.05, 3.53 ± 0.10 and 3.04 ± 0.14 mg/mL); lutein (0.03 ± 0.01, 0.17 ± 0.01 and 0.17 ± 0.02 mg/mL); crude saponins (1.2 ± 0.04%, 2.4 ± 0.05% and 2.4 ± 0.05%) and alkaloids (0.32 ± 0.00%, 1.70 ± 0.04% and 1.58 ± 0.04%) in hexane, methanol and acetone extracts, respectively. Our data are in harmony with Yahyaoui et al. (2017) with a view to the methanolic extracts of T. hirsuta aerial parts in their study gave the high amount of flavonoids ranged from 88.76 ± 7.24 to 163.64 ± 3.32 mg QE/gdw, followed by hexane extract (30.45 ± 2.15 to 37.26 ± 0.51 mg QE/gdw). Phytochemical composition of the aerial part of T. hirsuta methanolic extract performed by Yahyaoui et al. (2018a, b) possessed the highest total phenol contents (191.59 ± 2.09 to 362.74 ± 3.49 mg EAG/gdw) followed by the hexane (53.81 ± 2.19 to 165.47 ± 4.95 mg EAG/gdw) and Abed et al. (2014) who reported that the amount of condensed tannins in the methanolic extract of T. hirsuta ranged between 9.45± 1.22 and 0.09 ± 0.02 mg EAG/gdw.
The secondary metabolites of U. maritima were composed of total polyphenols (23.92 ± 0.021, 362.74 ± 3.49 and 329.58 ± 0.05 mg/g); total flavonoids (17.63 ± 0.41, 65.30 ± 0.51 and 45.24 ± 0.50 mg/g); condensed tannins (5.13 ± 0.11, 13.96 ± 0.20 and 9.96 ± 0.22 mg/mL); total carotenoids (2.93 ± 0.15, 5.84 ± 0.13 and 3.21 ± 0.18 mg/mL); lutein (0.18 ± 0.01, 0.61 ± 0.05 and 0.43 ± 0.04 mg/mL); crude saponins (0.8 ± 0.05%, 2.35 ± 0.10% and 1.22 ± 0.07%) and alkaloids (0.1 ± 0.01%, 1.87 ± 0.09% and 1.39 ± 0.06%) in hexane; methanol and acetone extracts, respectively. Our data are consistent with Belhadda et al. (2017) who recommended that the bulb of U. maritima methanolic extract contained the highest level of flavonoid, glycoside, tannin, anthraquinone, anthocyanin, triterpenoide, steroid and reducing compounds. Phenolic compounds are associated with a broad spectrum of pharmacological and health promoting effects and they are important essential constituent in a diverse nutraceutical, therapeutical and cosmetic applications (Elghuol et al. 2016). Tannins have been found to have antiviral, antibacterial, anti-parasitic, anti-inflammatory, antiulcer and antioxidant effects for possible therapeutic applications (Elghuol et al. 2016). Therefore, the concentration of these compounds could contribute synergistically to the significant pharmacological actions (Al‑Dabbagh et al. 2019).