Phytochemical Screening, Antifungal, and Anticancer Activities of Medicinal Plants Thymelaea Hirsuta, Urginea Maritima, and Plantago Albicans.

Ethyl acetate, ethanol, and acetone extracts of the medicinal plants Thymelaea hirsuta L., Urginea maritima L., and Plantago albicans L. (aerial parts) were evaluated for their phytochemical compositions, antimycotic activity against dermatophytes, and antiproliferative activity against different human cancer cell lines. Among them, the ethanolic extracts showed the highest phytochemical contents along with hyperactivities and were then selected for gas chromatography-mass spectrometry and Fourier-transform infrared spectroscopy analysis. The Fourier-transform infrared spectroscopy analysis confirmed the presence of different characteristic peak values with various functional chemical groups of the active components. However, U. maritima extracts through Fourier-transform infrared spectroscopy analysis showed distinctive peaks related to phenolic, amines, amides, aromatic, alkanes, alkyne, cyclopentanone, conjugated aldehyde, nitro, methoxy, uronic acids, aromatic esters, tertiary alcohol or ester, secondary and primary alcohols, aliphatic ether, sulfoxide, vinylidene, and halo compounds. Many bioactive main compounds with reported biological activities were detected by GC/MS (%) in the ethanolic extract of T. hirsuta, U. maritima, and P. albicans. All studied dermatophytes included a diverse set of virulence factors, including phospholipase, protease, keratinase, hemolysis, and melanoid production, all of which play vital roles in dermatophytic infection. Ethanolic extract of P. albicans inhibited the growth of Trichophyton soudanense totally and Trichophyton erinacei in addition to all Microsporum species. In contrast, the ethanolic extract of Trichophyton hirsuta at concentrations of 25 g/mL totally prevented the growth of all Trichophyton species. EtOH extract of U. maritima completely prevented the growth (100% inhibition) of all dermatophytic strains under study at the lowest concentration of 12.5 µg/mL. Scanning electron microscope analysis revealed considerable morphological modifications and structural alterations in dermatophyte species exposed to ethanolic extract of these plants. The viability of HCT-116, HepG-2, MCF-7, and HeLa cell lines was reduced after treatment with the ethanolic extracts of T. hirsuta, U. maritima, and P. albicans individually with IC50 values (10.0, 9.97, 48.5, and 56.24 µg/mL), (26.98, 25.0, 17.11, and 9.52 µg/mL), and (9.32, 7.46, 12.50, and 16.32 µg/mL), respectively. Our work revealed the significance of these traditional ethnomedical plants as potent sources for biologically active pharmaceuticals with potential applicability for the treatment of fungal and cancer diseases.

Production, purification, characterization, antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4.

L-Asparaginase is an antileukemic agent that depletes L-asparagine "an important nutrient for cancer cells" through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL-1 at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH2PO4. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg-1 of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC50 69.12 µg mL-1) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC50 equal to 2.0, 5.0, 12.40, 8.26 and 22.8 µg mL-1, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.

Purification, characterization, and anticancer and antioxidant activities of L-glutaminase from Aspergillus versicolor Faesay4.

L-Glutaminase is an amidohydrolase which can act as a vital chemotherapeutic agent against various malignancies. In the present work, L-glutaminase productivity from Aspergillus versicolor Faesay4 was significantly increased by 7.72-fold (from 12.33 ± 0.47 to 95.15 ± 0.89 U/mL) by optimizing submerged fermentation parameters in Czapek's Dox (CZD) medium including an incubation period from 3 (12.33 ± 0.47 U/mL) to 6 days (23.36 ± 0.58 U/mL), an incubation temperature from 30 °C (23.36 ± 0.49 U/mL) to 25 °C (31.08 ± 0.60 U/mL), initial pH from pH 5.0 (8.49 ± 0.21 U/mL)  to pH 7.0 (32.18 ± 0.57 U/mL), replacement of glucose (30.19 ± 0.52 U/mL) by sucrose (48.97 ± 0.67 U/mL) as the carbon source at a concentration of 2.0% (w/v), increasing glutamine concentration as the nitrogen source from 1.0% (w/v, 48.54 ± 0.48 U/mL) to 1.5% (w/v, 63.01 ± 0.60 U/mL), and addition of a mixture of KH2PO4 and NaCl (0.5% w/v for both) to SZD as the metal supplementation (95.15 ± 0.89 U/mL). Faesay4 L-glutaminase was purified to yield total activity 13,160 ± 22.76 (U), specific activity 398.79 ± 9.81 (U/mg of protein), and purification fold 2.1 ± 3.18 with final enzyme recovery 57.22 ± 2.17%. The pure enzyme showed a molecular weight of 61.80 kDa, and it was stable and retained 100.0% of its activity at a temperature ranged from 10 to 40 °C and pH 7.0. In our trials, to increase the enzyme activity by optimizing the assay conditions (which were temperature 60 °C, pH 7.0, substrate glutamine, substrate concentration 1.0%, and reaction time 60 min), the enzyme activity increased by 358.8% after changing the assay temperature from 60 to 30 °C and then increased by 138% after decreasing the reaction time from 60 to 40 min. However, both pH 7.0 and glutamine as the substrate remain the best assay parameters for the L-glutaminase activity. When the glutamine in the assay as the reaction substrate was replaced by asparagine, lysine, proline, methionine, cysteine, glycine, valine, phenylalanine, L-alanine, aspartic acid, tyrosine, and serine, the enzyme lost 23.86%, 29.0%, 31.0%, 48.3%, 50.0%, 73.6%, 74.51%, 80.42%, 82.5%, 83.43%, 88.36%, and 89.78% of its activity with glutamine, respectively. Furthermore, Mn2+, K+, Na+, and Fe3+ were enzymatic activators that increased the L-glutaminase activity by 25.0%, 18.05%, 10.97%, and 8.0%, respectively. Faesay4 L-glutaminase was characterized as a serine protease enzyme as a result of complete inhibition by all serine protease inhibitors (PMSF, benzamidine, and TLCK). Purified L-glutaminase isolated from Aspergillus versicolor Faesay4 showed potent DPPH scavenging activities with IC50 = 50 µg/mL and anticancer activities against human liver (HepG-2), colon (HCT-116), breast (MCF-7), lung (A-549), and cervical (Hela) cancer cell lines with IC50 39.61, 12.8, 6.18, 11.48, and 7.25 µg/mL, respectively.