Ashour, R. M. S., M. O. N. A. M. OKBA, E. T. Menze, and R. A. El Gedaily, "Eucalyptus Sideroxylon Bark Anti-inflammatory Potential, Its UPLC-PDA-ESI-qTOF-MS Profiling, and Isolation of a New Phloroglucinol.", Journal of chromatographic science, vol. 57, issue 6, pp. 565-574, 2019. Abstract

Eucalyptus barks contain complex biomass of constituents with considerable chemical and structural diversity. Reports about Eucalyptus sideroxylon Cunn. ex Woolls bark composition and biological activities are limited. Non-targeted metabolomic analysis via ultra-performance liquid chromatography-quadrupole-time-of-flight-photodiode array-mass spectrometry (UPLC-qTOF-PDA-MS) enabled first-time detection of 41 secondary metabolites of which 31 were identified including; 6 flavonoids, 4 ellagic acid derivatives, 8 triterpenes, 10 fatty acids and 3 miscellaneous. The isolation and structure elucidation of methyl morolate, β-sitosterol, syringaldeyhde and 7'-deoxyguajavadial A were reported. The bark methylene chloride: methanol (8:2) extract demonstrated significant (P < 0.01) in vitro anti-inflammatory activity through membrane stabilization, protein denaturation inhibition, anti-lipoxygenase, and proteinase inhibition assays. The strongest anti-inflammatory activity was via membrane stabilization (34.4%) as compared to diclofenac sodium (26%) at the same concentration (125 μg/mL). Our study represents the sole complete map for E. sideroxylon bark components and represents it as new anti-inflammatory drug.

Shehabeldine, A. M., R. M. Ashour, M. O. N. A. M. OKBA, and F. R. Saber, "Callistemon citrinus bioactive metabolites as new inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation.", Journal of ethnopharmacology, vol. 254, pp. 112669, 2020. Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: The development of new inhibitors of bacterial virulence factors from natural origin has recently received significant attention. Callistemon citrinus Skeels is an important plant of great medicinal value. Its antimicrobial activity is well documented. Although several compounds were isolated from this plant, the actual bioactive compounds responsible for its antimicrobial activity are still unrevealed.

AIM OF THE STUDY: To evaluate the effect of C. citrinus crude extract and isolated compounds on methicillin-resistant and sensitive Staphylococcus aureus.

MATERIALS AND METHODS: The methylene chloride-methanol extract (MME) of C. citrinus leaves was prepared by Soxhlet apparatus. Biologically guided fractionation of MME was accomplished using several normal and reversed phase silica gel columns. The potency of MME and its isolated compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) was evaluated. In addition, the mechanism of resistance was studied using three virulence factors; antibiofilm activity, inhibition of staphyloxanthin biosynthesis and effect on acid tolerance. Ultrastructural changes in MRSA and MSSA were observed by TEM to understand mode of action of these compounds.

RESULTS: Pulverulentone A (C1), 8- desmethyl eucalyptin (C2) and eucalyptin (C3) were isolated from the most bioactive fraction of MME. Confocal scanning laser microscopy images revealed that C. citrinus isolated compounds destroyed the intact architecture of biofilm, thickness and reduced its biomass. Pulverulentone A (C1) showed the most potent anti-biofilm activity up to 71% and 62.3% against MRSA and MSSA, respectively. It also exhibited the highest inhibition of staphyloxanthin biosynthesis of MRSA and MSSA by 55.6% and 54.5%, respectively. The bacterial cell membrane was compromised, losing its integrity and releasing important cellular constituents when exposed to C1-C3 CONCLUSIONS: C. citrinus phenolics and acylphloroglucinols may serve as potential source of plant-based antibacterials and thus could be implicated to control MRSA biofilm formation.

OKBA, M. O. N. A. M., R. A. El-Shiekh, M. Abu-Elghait, M. Sobeh, and R. M. S. Ashour, "HPLC-PDA-ESI-MS/MS Profiling and Anti-Biofilm Potential of Eucalyptus sideroxylon Flowers.", Antibiotics (Basel, Switzerland), vol. 10, issue 7, 2021. Abstract

The development of multidrug-resistant bacterial strains is a worldwide emerging problem that needs a global solution. Exploring new natural antibiofilm agents is one of the most important alternative therapies in combating bacterial infections. This study aimed at testing the antimicrobial potential of flowers extract (ESFE) against , , , and prior to testing the antibiofilm activity against , and . ESFE demonstrated antimicrobial activity and promising inhibition activity against methicillin-resistant (MRSA) biofilm formation up to 95.9% ( < 0.05) at a concentration of 0.05 mg/mL and eradicated biofilm formation up to 71.2% ( < 0.05) at a concentration of 0.7 mg/mL. LC-MS analysis allowed the tentative identification of eighty-three secondary metabolites: 21 phloroglucinol, 18 terpenes, 16 flavonoids, 7 oleuropeic acid derivatives, 7 ellagic acid derivatives, 6 gallic acid derivatives, 3 phenolic acids, 3 fatty acids and 2 miscellaneous. In conclusion, is a rich source of effective constituents that promote its valorization as a promising candidate in the management of multidrug-resistant bacterial infections.

El-Shiekh, R. A., R. M. Ashour, E. A. Abd El-Haleim, K. A. Ahmed, and E. Abdel-Sattar, "Hibiscus sabdariffa L.: A potent natural neuroprotective agent for the prevention of streptozotocin-induced Alzheimer's disease in mice.", Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, vol. 128, pp. 110303, 2020. Abstract

Hibiscus sabdariffa L. (Malvaceae) is one of the well-known traditionally used remedy worldwide. It exhibited numerous pharmacological properties including antioxidant, antidepressant, sedative, anti-inflammatory, antiproliferative, antimicrobial and neuroprotective activities. The aim of this study is to highlight the mechanisms underlying the neuroprotective effects of anthocyanin-enriched extracts of two Hibiscus varieties (white and red calyces) in the management of Alzheimer's disease (AD) in addition to their metabolic profiling. The anthocyanin contents were determined quantitatively using the pH-differential technique and qualitatively by LC/MS/MS. The extracts were tested in vitro for their antioxidant potential as well as acetylcholinesterase inhibition activity and both showed promising activities. The LC/MS/MS analysis allowed the tentative identification of 26 and 24 metabolites in red and white calyces, respectively, represented by anthocyanins, flavonoids, aliphatic and phenolic acids. In vivo, streptozotocin induced AD in mice model was established and Hibiscus extracts were tested at a dose of 200 mg kg compared to celecoxib (30 mg/kg). Histopathology of cerebral cortex and hippocampus, immunohistochemistry for tau- protein and caspase-3 with behavioral tests and measurement of several biochemical parameters were done. Hibiscus prevented memory impairment, and this could be attributed to the amelioration of STZ-induced neuroinflammation and amyloidogenesis. Consequently, Hibiscus represents a promising safe agent that can be repurposed for AD through exerting anti-inflammatory, anti-acetylcholinesterase, antioxidant, and anti-amyloidogenic activities.

Ashour, R. M. S., R. A. El-Shiekh, M. Sobeh, M. A. O. Abdelfattah, M. M. Abdel-Aziz, and M. O. N. A. M. OKBA, "Eucalyptus torquata L. flowers: a comprehensive study reporting their metabolites profiling and anti-gouty arthritis potential.", Scientific reports, vol. 13, issue 1, pp. 18682, 2023. Abstract

Gouty arthritis is one of the most common metabolic disorders affecting people. Plant based drugs can lower the risk of this health disorder. The anti-gouty potential of Eucalyptus torquata flowers methanol extract (ETME) was evaluated in vitro via measuring the inhibitory effects of five pro-inflammatory enzymes; xanthine oxidase (XO), hyaluronidase, lipoxygenase (5-LOX), cyclooxygenases COX-1, and COX-2, in addition to evaluating the inhibition of histamine release, albumin denaturation, membrane stabilization, tyrosinase, and protease inhibitory activities. Also, its antioxidant potential was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays and ferric reducing power assay (FRAP). HPLC-PDA-MS/MS was used to identify the metabolites in the tested extract. The latter exhibited substantial anti-arthritic properties in all assays with comparable potential to the corresponding reference drugs. HPLC-MS/MS analysis of this bioactive extract tentatively annotated 46 metabolites including phloroglucinols, gallic and ellagic acids derivatives, terpenes, flavonoids, fatty acids, and miscellaneous metabolites. Our study highlights the medicinal importance of E. torquata as an anti-gouty candidate and opens new avenues of gouty management.

El-Shiekh, R. A., R. M. S. Ashour, M. O. N. A. M. OKBA, A. A. Mandour, O. Kutkat, Y. Moatasim, and R. Elshimy, "Natural compounds as possible anti-SARS-CoV-2 therapeutic agents: an and study.", Natural product research, pp. 1-6, 2023. Abstract

WHO declared severe acute respiratory syndrome coronavirus-2' (SARS-CoV-2) was global health emergency since 2020. In our study eighteen natural compounds were investigated for possible anti-SARS-CoV-2 potential, where the most potent natural compounds were ursolic acid and dioscin with IC value of 4.49 µg/mL and 7.11 µg/mL, respectively. Hesperidin, catechin, diosmin, isorhamnetin-3--glucoside and hyperoside showed medium antiviral activity with IC value of 20.87, 22.57, 38.92, 39.62 and 47.10 µg/mL, respectively. Molecular modelling studies including docking study and predictive ADME study were performed on all tested molecules. Their binding energies after docking were calculated and their orientations at the active sites of both SARS-CoV-2 main protease (Mpro) and spike (S) receptors were visualised and compared to the downloaded ligands. Also, the predictive ADME studies showed good pharmacokinetic properties of most of the tested compounds. The obtained results obtained confirmed that many of the tested compounds are promising SARS-CoV-2 inhibitors.

Ali, N. B., S. S. Abdelhamid Ibrahim, M. A. Alsherbiny, E. Sheta, R. A. El-Shiekh, R. M. Ashour, A. A. El-Gazar, G. M. Ragab, S. H. El-Gayed, C. G. Li, et al., "Gastroprotective potential of red onion (Allium cepa L.) peel in ethanol-induced gastric injury in rats: Involvement of Nrf2/HO-1 and HMGB-1/NF-κB trajectories.", Journal of ethnopharmacology, vol. 319, issue Pt 1, pp. 117115, 2024. Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: The utilization of plants with therapeutic properties in traditional medicine has a longstanding practice. Among them, the well-known Allium cepa L. commonly known as onion has been valued for its anti-inflammatory and antioxidant potential in the treatment of various ailments, including gastric ulcers.

AIM OF THE STUDY: This study investigated the gastroprotective potential of red onion peel extract and its fractions in a rat model of ethanol-induced gastric ulcer. Moreover, their phytochemical profiles were compared to identify the active metabolites.

MATERIALS AND METHODS: Mass spectrometry-based metabolomics and chemometrics were performed for phytochemical analysis. Ethanol-induced gastric ulcer model was used to assess the gastroprotective activity. Nine groups of rats were allocated as follows: Group 1 was the normal control; Group 2 rats were used as a positive control/model and received 1 mL of absolute ethanol; and Group 3 rats were treated with famotidine at a dose of 20 mg/kg orally. Group 4 and 5 rats were treated with total acidified ethanolic extract (T1, T2). Group 6 and 7 rats were treated with anthocyanins-rich fractions (P1, P2). Groups 8 and 9 were the flavonoids-rich fraction (S1, S2) treatment. Prior to scarification, the ulcer index in mm was obtained from gastric tissues photographed beside a ruler with further analysis using ImageJ software.

RESULTS: Seventy key major and discriminatory metabolites were identified including flavonoids, anthocyanins, phenolic acids, and miscellaneous compounds. The examined extract and its fractions significantly reduced the ulcer index and inflammatory cytokines via downregulating HMGB-1/NF-κB. Also, they augmented the expression of Nrf2/HO-1 and reduced NOX1/4 mRNA expression. Moreover, there was a significant reduction in the oxidative stress and apoptotic biomarkers as well as a noticeable enhancement in histopathological changes of the stomach tissues.

CONCLUSION: Red onion peels have a promising dose dependent gastroprotective potential in alcohol-induced ulcers via modulating Nrf2/HO-1 and HMGB-1/NF-κB trajectories. This highlights the potential of red onion peels in treating gastric ulcers.

Ali, N. B., R. A. El-Shiekh, R. M. Ashour, S. H. El-Gayed, E. Abdel-Sattar, and M. Hassan, "In Vitro and In Vivo Antibiofilm Activity of Red Onion Scales: An Agro-Food Waste.", Molecules (Basel, Switzerland), vol. 28, issue 1, 2023. Abstract

Red onion wastes (ROW) are valuable sources of bioactive metabolites with promising antimicrobial effects. Methicillin-resistant (MRSA) infections are a growing risk in hospitals and communities. This study aims to investigate the in vitro and in vivo antibiofilm activities of the acidified ethanolic extract of red onion scales (RO-T) and its fractions against an MRSA vaginal colonization model. The RO-T extract, as well as its anthocyanin-rich fraction (RO-P) and flavonoid-rich fraction (RO-S), recorded a promising antibacterial activity against highly virulent strains of bacteria (MRSA, , and ). RO-S showed the highest antibacterial activity (MBC of 0.33 ± 0.11 mg/mL) against MRSA USA300 and significantly eradicated its biofilm formation with an IC of 0.003. Using a rat model, in vivo assessment on all samples, which were formulated as a hydrogel, revealed a significant reduction of MRSA bacterial load recovered from an infected vagina compared to that of the negative control group (NCG). RO-T extract and vancomycin groups recorded the highest antibacterial activity with a bacterial load 2.998 and 3.358 logs lower than the NCG, respectively. The histopathological investigation confirmed our findings. RO-T and RO-S were standardized for their quercetin content. Finally, ROW offers a new potent antibiofilm agent mostly due to its high quercetin content.

El Gaafary, M., F. R. Saber, E. A. Mahrous, R. M. Ashour, M. O. N. A. M. OKBA, L. Jin, S. J. Lang, M. Schmiech, T. Simmet, and T. Syrovets, "The phloroglucinol calcitrinone A, a novel mitochondria-targeting agent, induces cell death in breast cancer cells.", Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, vol. 162, pp. 112896, 2022. Abstract

Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. From the leaves of Callistemon citrinus, we have isolated a novel phloroglucinol dimer, calcitrinone A, and analyzed its potential anticancer activity using the triple-negative breast cancer cell line MDA-MB-231. Calcitrinone A decreased the total intracellular ATP levels, inhibited proliferation, and induced apoptosis in MDA-MB-231 cells, but was less toxic to peripheral blood mononuclear cells. The antiproliferative and apoptosis-inducing effects of calcitrinone A were confirmed in vivo using breast cancer xenografts grown on chick chorioallantoic membranes. Mechanistic analysis showed mitochondrial membrane-potential dissipation and interference with energy-yielding processes resulting in cell accumulation in the S phase of the cell cycle. Seahorse assay analysis revealed an early inhibition of mitochondrial oxidative phosphorylation (OXPHOS). At the molecular level, calcitrinone A inhibited activity of the succinate-coenzyme Q reductase (SQR) (mitochondrial complex II). In silico docking identified the coenzyme Q binding pocket as a possible high affinity binding site for calcitrinone A in SQR. Inhibition of complex II was accompanied by strong elevation of mitochondrial superoxide and cytoplasmic ROS. Calcitrinone A might be a promising anticancer lead compound acting through the interference with the mitochondrial complex II activity.

Ismail, M. M., M. Hassan, S. S. Moawad, M. O. N. A. M. OKBA, R. M. Ashour, N. m Fayek, and F. R. Saber, "Exploring the Antivirulence Activity of Pulverulentone A, a Phloroglucinol-Derivative from Leaf Extract, against Multi-Drug Resistant .", Antibiotics (Basel, Switzerland), vol. 10, issue 8, 2021. Abstract

(1) Background: Bacterial resistance to antibiotics is a global life-threatening issue. Antivirulence therapy is a promising approach to combat bacterial infections as it disarms the bacteria from their virulence factors with reduced selective pressure and a lower chance of resistance. (2) Methods: leaf extract and its major constituent, Pulverulentone A, were tested for their ability to inhibit biofilm, exopolysaccharides, pyocyanin and proteases produced by MDR . In addition, a larvae model was employed to evaluate the in vivo cytotoxicity of Pulverulentone A and its ability to combat infection. Docking study was further performed to investigate Pulverulentone A druggability against main quorum sensing (QS) targets expressed by ; (3) Results: Both extract and the isolated compound could inhibit biofilm formation, extracellular polymeric substances (EPS) and pigment production by the tested isolates. Unexpectedly, no significant inhibition was observed on proteases production. The in silico docking analysis revealed good interactions of Pulverulentone A with all QS targets examined (LasR, MyfR/PqsR, QscR). Pulverulentone A was safe up to 400 µg·mL in caterpillars. Moreover, -treatment of with Pulverulentone A slightly enhanced the survival of the infected larvae. (4) Conclusions: The present study proves Pulverulentone A safety with significant in vitro and in silico antivirulence potential against .