Khalil, H. M. A., I. A. Khalil, A. K. Al-Mokaddem, M. Hassan, R. A. El-Shiekh, H. A. Eliwa, A. M. Tawfek, and W. H. El-Maadawy, "Ashwagandha-loaded nanocapsules improved the behavioral alterations, and blocked MAPK and induced Nrf2 signaling pathways in a hepatic encephalopathy rat model.", Drug delivery and translational research, 2022. Abstract

Ashwagandha (ASH), a vital herb in Ayurvedic medicine, demonstrated potent preclinical hepato- and neuroprotective effects. However, its efficacy is limited due to low oral bioavailability. Accordingly, we encapsulated ASH extract in chitosan-alginate bipolymeric nanocapsules (ASH-BPNCs) to enhance its physical stability and therapeutic effectiveness in the gastrointestinal tract. ASH-BPNC was prepared by emulsification followed by sonication. The NCs showed small particle size (< 220 nm), zeta-potential of 25.2 mV, relatively high entrapment efficiency (79%), physical stability at acidic and neutral pH, and in vitro release profile that extended over 48 h. ASH-BPNC was then investigated in a thioacetamide-induced hepatic encephalopathy (HE) rat model. Compared with free ASH, ASH-BPNC improved survival, neurological score, general motor activity, and cognitive task-performance. ASH-BPNC restored ALT, AST and ammonia serum levels, and maintained hepatic and brain architecture. ASH-BPNC also restored GSH, MDA, and glutathione synthetase levels, and Nrf2 and MAPK signaling pathways in liver and brain tissues. Moreover, ASH-BPNC downregulated hepatic NF-κB immunohistochemical expression. Moreover, the in vivo biodistribution studies demonstrated that most of the administered ASH-BPNC is accumulated in the brain and hepatic tissues. In conclusion, chitosan-alginate BPNCs enhanced the hepatoprotective and neuroprotective effects of ASH, thus providing a promising therapeutic approach for HE.

Ali, M. - A., M. M. Khalil, A. K. Al-Mokaddem, S. H. Aljuaydi, M. M. Ahmed, and H. M. A. Khalil, "Differential effects of cancer modifying agents during radiation therapy on Ehrlich solid tumor-bearing mice: A comparative investigation of metformin and ascorbic acid.", Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine, vol. 187, pp. 110305, 2022. Abstract

BACKGROUND: This work was carried out to compare the modifying roles of ascorbic and metformin during Ehrlich (ESC) tumor-bearing mice irradiation.

METHODS: Fifty Swiss albino male mice were segmented into seven groups, including one control group and six Ehrlich induced tumors treated with ascorbic, ascorbic plus radiation, metformin, metformin plus radiation, and radiation only. Many tests, including behavioral, biochemical, immunohistochemistry, gene expression, DNA fragmentation, oxidative stress markers, and EPR, were performed to interrogate the modifying effects on tumor and liver tissues.

RESULTS: Remarkable apoptosis was found in metformin irradiated animals compared to irradiated ascorbic counterparts. The irradiated metformin mice showed the greatest reduction in PCNA. There was a significant reduction of DNA fragmentation in the liver tissues of the irradiated metformin group. Irradiated metformin and irradiated ascorbic acid animals showed a reduced signal of ERK as well as c-Fos genes. There was a tendency of metformin and metformin irradiated animals to reduce MDA levels in liver tissues. ESC-bearing mice treated with ascorbic or metformin showed an improvement in the spontaneous alternation percentage (SAP%) and improved short-term memory. There was also an improvement in long memory tests.

CONCLUSIONS: The study added more preclinical evidence on the utility of metformin in cancer treatment during radiotherapy. Metformin was shown to reduce lipid peroxidation in irradiated healthy tissues, increase tumor cytotoxicity, downregulate critical pathways involved in tumor progression and proliferation, and enhance tumor apoptosis. Controlled clinical trials using metformin are highly warranted.

Hamdan, D. I., S. S. Hafez, W. H. B. Hassan, M. M. Morsi, H. M. A. Khalil, Y. H. Ahmed, O. A. Ahmed-Farid, and R. A. El-Shiekh, "Chemical profiles with cardioprotective and anti-depressive effects of Miq. leaves and stem branches dichloromethane fractions on isoprenaline induced post-MI depression.", RSC advances, vol. 12, issue 6, pp. 3476-3493, 2022. Abstract

This study was conducted to explore the potential cardioprotective and anti-depressive effects of dichloromethane (DCM) fractions of leaves (L) and stem branches (S) on post-myocardial infarction (MI) depression induced by isoprenaline (ISO) in rats in relation to their metabolites. The study was propped with a UPLC-ESI-MS/MS profiling and chromatographic isolation of the secondary metabolites. Column chromatography revealed the isolation of lupeol palmitate (6) that was isolated for the first time from nature with eight known compounds. In addition, more than forty metabolites belonging, mainly to flavonoids, and anthocyanins groups were identified. The rats were injected with ISO (85 mg kg, s.c) in the first two days, followed by the administration of DCM-L and DCM-S fractions (200 mg kg p.o) for 19 days. Compared with the ISO exposed rats, the treated rats displayed a reduction in cardiac biomarkers (LDH and CKMB), anxiety, and depressive-like behaviour associated with an increase in the brain defense system (SOD and GSH), neuronal cell energy, GABA, serotonin, and dopamine, confirmed by histopathological investigations. In conclusion, DCM-L and DCM-S fractions' cardioprotective and anti-depressive activities are attributed to their metabolite profile. Therefore, they could serve as a potential agent in amending post-MI depression.

Khalil, H. M. A., H. A. Eliwa, R. A. El-Shiekh, A. K. Al-Mokaddem, M. Hassan, A. M. Tawfek, and W. H. El-Maadawy, "Ashwagandha (Withania somnifera) root extract attenuates hepatic and cognitive deficits in thioacetamide-induced rat model of hepatic encephalopathy via induction of Nrf2/HO-1 and mitigation of NF-κB/MAPK signaling pathways.", Journal of ethnopharmacology, vol. 277, pp. 114141, 2021. Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Ashwagandha (ASH) is one of the medicinal plants used in traditional Indian, Ayurvedic, and Unani medicines for their broad range of pharmacological activities including, tonic, aphrodisiac, energy stimulant, and counteracting chronic fatigue. Besides, it is used in the treatment of nervous exhaustion, memory-related conditions, insomnia, as well as improving learning ability and memory capacity. ASH is preclinically proven to be efficient in hepatoprotection and improving cognitive impairment, however, its beneficial effects against hepatic encephalopathy (HE) is still unclear. Therefore, this study aimed at investigating the protective effects of ASH root extract against thioacetamide (TAA)-induced HE and delineate the underlying behavioral and pharmacological mechanisms.

MATERIALS AND METHODS: ASH metabolites were identified using UPLC-HRMS. Rats were pretreated with ASH (200 and 400 mg/kg) for 29 days and administrated TAA (i.p, 350 mg/kg) in a single dose. Then, behavioral (open field test, Y-maze, modified elevated plus maze and novel object recognition test), and biochemical (ammonia and hepatic toxicity indices) assessments, as well as oxidative stress markers (MDA and GSH) were evaluated. The hepatic and brain levels of glutamine synthetase (GS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme-oxygenase (HO)-1, inducible nitric oxide synthase (iNOS) were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of p38/ERK½ were determined using real-time polymerase chain reaction (PCR). Moreover, histopathological investigations and immunohistochemical (NF-κB and TNF-α immunohistochemical expressions) examinations were performed.

RESULTS: Metabolite profiling of ASH revealed more than 45 identified metabolites including phenolic acids, flavonoids and steroidal lactone triterpenoids. Compared to the TAA-intoxicated group, ASH improved the locomotor and cognitive deficits, serum hepatotoxicity indices and ammonia levels, as well as brain and hepatic histopathological alterations. ASH reduced hepatic and brain levels of MDA, GS, and iNOS, and increased their GSH, Nrf2, and HO-1 levels. Also, ASH downregulated p38 and ERK½ mRNA expressions, and NF-κB and TNF-α immunohistochemical expressions in brain and hepatic tissues.

CONCLUSIONS: Our results provided insights into the promising hepato- and neuroprotective effects of ASH, with superiority to 400 mg/kg ASH, to ameliorate HE with its sequential hyperammonemia and liver/brain injuries. This could be attributed to the recorded increase in the spontaneous alternation % and recognition index, antioxidant and anti-inflammatory activities, as well as upregulation of Nrf2 and downregualtion of MAPK signaling pathways.

Hamdan, D. I., S. Salah, W. H. B. Hassan, M. Morsi, H. M. A. Khalil, O. A. - H. Ahmed-Farid, R. A. El-Shiekh, M. A. E. Nael, and A. M. Elissawy, "Anticancer and Neuroprotective Activities of Ethyl Acetate Fractions from Miq. Plant Organs with Ultraperformance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Profiling.", ACS omega, vol. 7, issue 18, pp. 16013-16027, 2022. Abstract

Column chromatography afforded the isolation of seven secondary metabolites (1-(2,4,6-trihydroxy phenyl)-ethanone-4--β-d-glucopyranoside, naringenin-7--β-d-glucopyranoside, kaempferol-3--α-l-rhamnoside, kaempferol-3--β-d-glucopyranoside, quercetin-3--β-d-glucopyranoside, quercetin-3--β-d-galactopyranoside, rutin) from the ethyl acetate (ET) fractions of Miq. stems (S), leaves (L), and fruits (F). Their identification based on ultraviolet (UV), electron ionization (EI), electrospray ionization-mass spectrometry (ESI-MS), and 1D and 2D NMR data. In addition, profiling of ET fractions using ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) resulted in the identification of 82 compounds belonging to different classes, mainly polyphenolic constituents. Chemical profiling as well as molecular docking directed us to biological evaluation. Interestingly, the ET-L fraction exhibited a robust cytotoxic activity against HepG-2, MCF-7, and HELA cell lines. Also, it displayed a neuromodulatory activity against cisplatin neurotoxicity in rats by ameliorating the neurobehavioral dysfunction visualized in the open field and Y-maze test and modulating the neurochemical parameters such as brain amino acid levels (glutamate, aspartate, serine, and histidine), oxidative stress markers (GSH, MDA, and 8-hydroxy-2'-deoxyguanosine), and purinergic cell energy (adenosine triphosphate (ATP) and adenosine monophosphate (AMP)). In conclusion, the isolated compounds (kaempferol-3--β-glucoside and quercetin-3--β-glucoside) from the ET-L fraction could serve as potent anticancer agents due to their strong antioxidant, cytotoxicity, and neuroprotective activity.

Orabi, M. A. A., H. M. A. Khalil, M. E. Abouelela, D. Zaafar, Y. H. Ahmed, R. A. Naggar, H. S. Alyami, E. - S. Abdel-Sattar, K. Matsunami, and D. I. Hamdan, "Carissa macrocarpa Leaves Polar Fraction Ameliorates Doxorubicin-Induced Neurotoxicity in Rats via Downregulating the Oxidative Stress and Inflammatory Markers", Pharmaceuticals, vol. 14, no. 12, 2021. AbstractWebsite

Chemotherapeutic-related toxicity exacerbates the increasing death rate among cancer patients, necessitating greater efforts to find a speedy solution. An in vivo assessment of the protective effect of the C. macrocarpa leaves polar fraction of hydromethanolic extract against doxorubicin (Dox)-induced neurotoxicity was performed. Intriguingly, this fraction ameliorated Dox-induced cognitive dysfunction; reduced serum ROS and brain TNF-α levels, upregulated the brain nerve growth factor (NGF) levels, markedly reduced caspase-3 immunoexpression, and restored the histological architecture of the brain hippocampus. The in vivo study results were corroborated with a UPLC-ESI-MS/MS profiling that revealed the presence of a high percentage of the plant polyphenolics. Molecular modeling of several identified molecules in this fraction demonstrated a strong binding affinity of flavan-3-ol derivatives with TACE enzymes, in agreement with the experimental in vivo neuroprotective activity. In conclusion, the C. macrocarpa leaves polar fraction possesses neuroprotective activity that could have a promising role in ameliorating chemotherapeutic-induced side effects.

Abdelatty, A. M., M. I. Mandouh, A. K. Al-Mokaddem, H. A. Mansour, H. M. A. Khalil, A. A. Elolimy, H. Ford, O. A. A. Farid, A. Prince, O. G. Sakr, et al., "Influence of level of inclusion of Azolla leaf meal on growth performance, meat quality and skeletal muscle p70S6 kinase α abundance in broiler chickens.", Animal : an international journal of animal bioscience, vol. 14, issue 11, pp. 2423-2432, 2020. Abstract

The interest in biodiesel production from oil-bearing seeds rather than soybean necessitates the scientific validation of other good quality protein sources that could substitute soybean meal in animal diets, particularly, broiler chickens where soybean meal constitutes a large portion of their diet. Therefore, the present study was conducted to investigate the effect of sun-dried Azolla leaf meal (ALM) as an unconventional dietary protein source in broiler chicken diet on growth performance, meat quality, skeletal muscle cell growth and protein synthesis through regulation of ribosomal protein S6 kinase (p70S6 kinase α). A total of 120 male Ross 308 broiler chicks were randomly allocated to three dietary treatments. Each treatment had four cages (i.e. replicates) with 10 birds/cage. The control group was fed with a corn-soy-based diet, the AZ5 group was supplemented with 5% ALM and the AZ10 group was supplemented with 10% ALM for 37 days. A 5-day trial was also conducted to measure the apparent nutrient digestibility. Growth performance parameters were measured weekly. At the end of the experiment, 12 birds from each group (3/cage) were euthanized and used for samplings. Inclusion of ALM tended to improve BW gain (P = 0.06) and increased feed intake (P < 0.01). Additionally, ALM decreased the percentage of breast meat cooking loss linearly (P < 0.01). In addition, ALM at a dose of 5% increased the production of propionate in the cecum (P = 0.01). Activation of breast muscle p70S6 kinase was higher when ALM was included in a dose-dependent manner (P < 0.01). The inclusion of ALM increased breast meat redness (P < 0.01); however, the lightness was within the normal range in all groups. Findings from our study suggest that ALM could be included in a broiler chicken diet up to 5% without any major negative effect on meat quality or performance, and it regulates muscle protein synthesis through activation of mammalian target of rapamycin/6S kinase signaling.

Hozyen, H. F., H. M. A. Khalil, R. A. Ghandour, A. K. Al-Mokaddem, M. Samer, and R. A. Azouz, "Nano selenium protects against deltamethrin-induced reproductive toxicity in male rats.", Toxicology and applied pharmacology, vol. 408, pp. 115274, 2020. Abstract

Greater understanding of the efficiency of nanoparticles will assist future research related to male reproductive performance. The current study was performed to assess the potency of selenium nanoparticles (SeNPs) in alleviating deltamethrin (DLM)-induced detrimental effects on sperm characteristics, oxidative status, sexual behavior, and the histological structure of the testes and epididymis in male rats. Thirty-two male Wister rats were divided into four groups according to treatment received orally by gavage 3 times/week for 60 days; control, DLM (0.6 mg/kg bwt), SeNPs (0.5 mg/kg bwt), and DLM-SeNPs groups. DLM caused a significant reduction in sperm count, motility, and viability percent, as well as in body weight and serum testosterone level, blood total antioxidant capacity (TAC), and glutathione peroxidase (GPx) activity. The DLM-treated group showed a significant increase in blood malondialdehyde (MDA) concentration and sperm abnormalities (%), as well as a significant reduction in sexual activity, manifested as an increase in mount, intromission, or ejaculation latency and a reduction in mount or intromission frequency. These toxic effects were confirmed by histological alterations, represented by a significant reduction in the diameter of the seminiferous tubules and spermatogenesis. Conversely, treatment with SeNPs improved DLM-induced negative effects on sperm characteristics, testosterone, and antioxidant biomarkers, as well as behavioral and histopathological alterations. The SeNPs treated group showed improved semen parameters, antioxidant status, and sexual performance. In conclusion, SeNPs may represent an effective treatment for reducing the detrimental effects of DLM on male fertility, and lead to enhanced male reproductive performance.

Hamdan, D. I., R. A. El-Shiekh, M. A. El-Sayed, H. M. A. Khalil, M. R. Mousa, A. A. Al-Gendy, and A. M. El-Shazly, "Phytochemical characterization and anti-inflammatory potential of Egyptian Murcott mandarin cultivar waste (stem, leaves and peel).", Food & function, vol. 11, issue 9, pp. 8214-8236, 2020. Abstract

The stem (S), leaf (L) and fruit peel (P) of Murcott mandarins were separately extracted using 80% ethanol and then fractionated into dichloromethane (DCM) and ethyl acetate (ET). Their metabolic profiles were studied via HPLC-PDA-ESI-MS/MS and afforded a tentative characterization of 98 compounds, including free organic acids, phenolic acid derivatives, flavonoid aglycones, flavonoid glycosides, flavonoids containing 3-hydroxyl-3-methylglutaroyl (HMG) units, coumarin derivatives and limonoids. Column chromatography resulted in isolation of six metabolites for the first time that were identified as nobiletin (C1), isosinensetin (C2), limonin (C3), 4'-demethylnobiletin (C4), stigmasterol-O-glucoside (C5) and hesperidin (C6). In vitro studies of the anti-inflammatory activity of DCM-L against cyclooxygenases (COXs) and 5-lipoxygenase (5-LOX) enzymes revealed that DCM-L showed higher activity than the other tested fractions. The in vivo gastroprotective effects of that fraction were evaluated using alcohol-induced gastric ulcers in rats. The obtained findings validated the gastroprotective and anti-ulcerogenic activities of DCM-L through its anxiolytic, anti-inflammatory, antioxidant and anti-apoptotic effects. Therefore, we recommend the use of Murcott mandarin leaves as a part of a protection strategy for gastric ulcer.

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