Bio-guided isolation of potential anti-inflammatory constituents of some endophytes isolated from the leaves of ground cherry (Physalis pruinosa L.) via ex-vivo and in-silico studies.

BACKGROUND: Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa. METHODS: The endophytic fungi were isolated from the fresh leaves of P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators (TNF-α, IL-1ß and INF-γ) was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in the docking study. RESULTS: A total of 50 endophytic fungal isolates were separated from P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3'-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate (A. alternata MN615420). Among the tested isolated compounds, 3'-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1ß. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1ß, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis. CONCLUSIONS: The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-γ, IL-1ß and INF-γ.

Impact of Oral Probiotics in Amelioration of Immunological and Inflammatory Responses on Experimentally Induced Acute Diverticulitis.

Acute diverticulitis is inflammation of a colon diverticulum; it represents a major cause of morbidity and mortality. The alteration of gut microbiota contributes to the promotion of inflammation and the development of acute diverticulitis disease. Probiotics can modify the gut microbiota, so they are considered a promising option for managing diverticulitis disease. This study aimed to investigate the potential protective effect of probiotics, alone or in combination with amoxicillin, on the experimentally induced model of acute diverticulitis disease. Forty-two rats were divided into seven groups as follows: control group: received water and food only; DSS group: received 3% dextran sulfate sodium (DSS) daily for 7 days; LPS group: injected with lipopolysaccharide (LPS) enema at the dose of (4 mg/kg); probiotics group: treated with probiotics (Lactobacillus acidophilus and Bifidobacterium lactis) each of which (4 × 108 CFU suspended in 2 ml distilled water) orally for 7 days; DSS/LPS group: received DSS and LPS; DSS/LPS treated with probiotics group; DSS/LPS treated with probiotics and amoxicillin group. The results revealed that both treatments (probiotics and probiotics-amoxicillin) attenuated DSS/LPS-induced diverticulitis, by restoring the colonic antioxidant status, ameliorating inflammation (significantly reduced TNF-α, interleukins, interferon-γ, myeloperoxidase activity, and C-reactive protein), decreasing apoptosis (through downregulating caspase-3), and reduction of the colon aerobic bacterial count. These probiotic strains were effective in preventing the development of the experimentally induced acute diverticulitis through the anti-inflammatory and immunomodulatory effects and have affected gut microbiota, so they can be considered a potential option in treating acute diverticulitis disease.

Integrated serum pharmacochemistry and network pharmacology analyses reveal the bioactive metabolites and potential functional mechanism of ground cherry (Physalis pruinosa L.) in treatment of type 2 diabetes mellitus in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Different Physalis plants have been widely employed in traditional medicine for management of diabetes mellitus. Previous studies with respect to the in vivo antidiabetic activity of Physalis plants illustrated that they improved glucose and lipid metabolism in streptozotocin (STZ) -induced diabetic rats yet the mechanism of action of bioactive constituents of the different organs of Physalis plants on diabetes remains obscure. AIM OF STUDY: Our objective is to study the effects of the different organs of ground cherry (P. pruinosa) on diabetes in rat models and elucidate their mechanism of actions through serum pharmacochemistry combined to network pharmacology analyses and in-vivo testing. MATERIALS AND METHODS: Characterization of the constituents in the drug-dosed serum samples relative to the blank serum after treatment with different extracts was performed by UPLC -MS/MS technique. The absorbed metabolites where then subjected to network pharmacology analysis to construct an interaction network linking "compound-target-pathway". In vivo verification was implemented to determine a hypothesized mechanism of action on a STZ and high fat diet induced type II diabetes mellitus (T2DM) model based on functional and enrichment analyses of the Kyoto Encyclopedia of Genes and Genome and Gene Ontology. RESULTS: Identification of a total of 73 compounds (22 prototypes and 51 metabolites) derived from P. pruinosa extracts was achieved through comparison of the serum samples collected from diabetic control group and extracts treated groups. The identified compounds were found to belong to different classes according to their structural type including withanolides, physalins and flavonoids. The absorbed compounds in the analyzed serum samples were considered as the potential bioactive components. The component-target network was found to have 23 nodes with 17 target genes including MAPK8, CYP1A1 and CYP1B1. Quercetin and withaferin A were found to possess the highest combined score in the C-T network. Integrated serum pharmacochemistry and network pharmacology analyses revealed the enrichment of leaves extract with the active constituents, which can be utilized in T2DM treatment. In the top KEGG pathways, lipid and atherosclerosis metabolic pathways in addition to T2DM pathways were found to be highly prioritized. The diabetic rats, which received leaves extract exhibited a substantial increment in GLUT2, INSR, IRS-1, PI3K-p85 and AKT-ser473 proteins by 105%, 142%, 109%, 81% and 73%, respectively relative to the untreated diabetic group. The immunoblotting performed for MAPK and ERK1/2 part of the inflammatory pathway studied in STZ induced diabetic rats revealed that leaves, calyces and stems extracts resulted in a substantial diminish in p38-MAPK, ERK 1/2, NF-κB, and TNF-α. Histopathological examination revealed that the hepatic histoarchitecture was substantially improved in the leaves, stems, and clayces-treated rats in comparison with untreated diabetic rats. Further, pancreatic injuries, which induced by STZ were dramatically altered by the treatment with P. pruinosa leaves, calyces and stems extracts. ß-cells in diabetic rats received leaves extract disclosed moderate insulin immunostaining with a notable increase in the mean insulin area%. CONCLUSIONS: The study in hand offers a comprehensive study to clarify the bioactive metabolites of the different organs of P. pruinosa. The basic pharmacological effects and underlying mechanism of actions in the management of STZ and high fat diet induced T2DM were specifically covered in this paper.

Protective effects of amoxicillin and probiotics on colon disorders in an experimental model of acute diverticulitis disease.

Acute diverticulitis disease is associated with inflammation and infection in the colon diverticula and may lead to severe morbidity. This study aimed to evaluate and compare the protective effects of amoxicillin antibiotic, either alone or in combination with probiotics (Lactobacillus acidophilus and Bifidobacterium lactis), in a rat model of acute diverticulitis disease. Acute diverticulitis was induced, in albino rats, by adding 3% weight/volume of dextran sulfate sodium (DSS) to the rats' drinking water; daily for 7 days, in addition to injecting lipopolysaccharide (LPS) enema (4 mg/kg). The impact of treatments was assessed by measuring the physiological and immunological parameters and evaluating colon macroscopic and microscopic lesions. The results showed that both treatments (especially probiotics with amoxicillin) alleviated the adverse effects of DSS and LPS. This was obvious through the modulation of the rats' body weight and the colon weight-to-length ratio. Also, there was a significant (p < 0.001) decrease in the colon macroscopic lesion score. The pro-inflammatory cytokines [(TNF)-α, (IL)-1ß, (IFN)-γ, and (IL)-18]; in the colon tissue; were significantly (p < 0.001) decreased. Also, both treatments significantly ameliorated the elevation of myeloperoxidase activity and C-reactive protein levels, in addition to improving the histopathological alterations in the colon tissue. In conclusion, amoxicillin and probiotics-amoxicillin were effective in preventing the development of experimentally induced acute diverticulitis, through their anti-inflammatory and immunomodulatory effects. Furthermore, this study has explored the role of probiotics in preventing DSS/LPS-induced acute diverticulitis, so it can be applied as a promising treatment option for acute diverticulitis disease.

Comprehensive metabolomics unveil the discriminatory metabolites of some Mediterranean Sea marine algae in relation to their cytotoxic activities.

Marine algae have served as a treasure trove of structurally variable and biologically active metabolites. The present study emphasizes on UPLC-MS metabolites fingerprinting for the first systematic broad scale metabolites characterization of three different phyla of marine seaweeds; Ulva fasciata, Pterocladia capillacea and Sargassum hornschuchii along with Spirulina platensis harvested from the Mediterranean Sea. A total of 85 metabolites belonging to various classes including mostly fatty acids and their derivatives, terpenoids, amino acids and dipeptides with considerable amounts of polyphenolic compounds. OPLS-DA model offered a better overview of phylum-based discrimination rapidly uncovering the compositional heterogeneity in metabolite profiles of algae extracts. An OPLS model was constructed using the cytotoxic activities against PC3 and MDA-MB-231 tumor cells to succinctly screen cytotoxic discriminatory metabolites among the tested algae species. The coefficient plot revealed that unsaturated fatty acids as stearidonic acid and linolenic acid, terpenoids namely as rosmanol, campestanol, dipeptides primarily glutamylglycine, glycyltyrosine along with polyphenolic compounds being abundantly present in S. platensis and U. fasciata samples with relatively marked cytotoxic potential might be the significant contributors synergistically meditating their anti-proliferative activity against PC3 and MDA-MB-231 tumor cells. Such results serve as baseline for understanding the chemistry of these species and performing strict correlation between metabolite and activity where a lack of information in this regard is observed.

Comparative metabolomics reveal intraspecies variability in bioactive compounds of different cultivars of pomegranate fruit (Punica granatum L.) and their waste by-products.

BACKGROUND: The different parts of pomegranate fruit are considered a powerful mixture of bioactive compounds yet the peels and pulps of the fruits are usually discarded and considered as industrial waste. In this work, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) was utilized for metabolomics analysis of different parts (peel, pulp, seed and juice) of pomegranate fruit cultivars to verify possible variations among the fruits and their waste products as potential sources of functional constituents. RESULTS: Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) coefficient-plot showed enrichment of phenolic compounds such as punicalagin and ellagic acid derivatives in pulp samples while seeds class was enriched in phlorizin, catechin and quercetin, juice class showed abundance of naringenin and pelargonidin-3-pentoside while peels were enriched in anthocyanins and flavonoids including cyanidin diglycoside, quercetin and luteolin glycosides. Although the juice samples of almost all tested cultivars showed remarkable cytotoxic activity, the pulp samples, particularly the Manfalouti cultivar, exhibited the most potent [half maximal inhibitory concentration (IC50 ) = 2.367 ± 0.14 µg/mL in MCF-7, IC50  = 3.854 ± 0.23 µg/mL in Hep-G2 cell lines]. OPLS models were constructed for determination of cytotoxicity-associated metabolites among where the coefficients plots revealed tannins; granatin A, ellagic acid derivatives, punicalagin α and ß, in addition to anthocyanins and phenolic compounds; cyanidin diglycoside, quercetin, phlorizin, 3-O-caffeoylquinic acid, naringenin and liquiritin were more pertinent with cytotoxicity of the different parts of pomegranate fruit. CONCLUSION: The results obtained allow for the full utilization of the resources of pomegranate fruit and its industrial waste as sources of bioactive compounds. © 2022 Society of Chemical Industry.

De novo Transcriptome Analysis Revealed the Putative Pathway Genes Involved in Biosynthesis of Moracins in Morus alba L.

Moracins, a kind of 2-phenyl-benzofuran compound from Moraceae, serve as phytoalexins with antimicrobial, anti-inflammatory, antitumor, and antidiabetes activities and respond to biotic and abiotic stresses, while their biosynthetic pathway and regulatory mechanism remain unclear. Here, we report a de novo transcriptome sequencing for different tissues of seedlings, as well as leaves under different stresses, in M. alba L. A total of 88 282 unigenes were assembled with an average length of 937 bp, and 82.2% of them were annotated. On the basis of the differential expression analysis and enzymatic activity assays in vitro, moracins were traced to the phenylpropanoid pathway, and a putative biosynthetic pathway of moracins was proposed. Unigenes coding key enzymes in the pathway were identified and their expression levels were verified by real-time quantitative reverse transcription PCR (qRT-PCR). Particularly, a p-coumaroyl CoA 2'-hydroxylase was presumed to be involved in the biosynthesis of stilbenes and deoxychalcones in mulberry. Additionally, the transcription factors that might participate in the regulation of moracin biosynthesis were obtained by coexpression analysis. These results shed light on the putative biosynthetic pathway of moracins, providing a basis for further investigation in functional characterization and transcriptional regulation of moracin biosynthesis in mulberry.

SWATH-based quantitative proteomic analysis of Morus alba L. leaves after exposure to ultraviolet-B radiation and incubation in the dark.

Morus alba is a woody shrub of the family Moraceae and used as traditional Chinese medicine for a long history. Ultraviolet-B (UV-B) radiation, as a kind of abiotic stress factor, affected the growth and secondary metabolism in M. alba. Previous studies indicated that the contents of several secondary metabolites such as moracin N, chalcomaricin were significantly increased under high level UV-B radiation and dark incubation in M. alba leaves. To reveal the response mechanism under UV-B radiation and dark incubation in M. alba leaves, SWATH-based quantitative proteomic analysis was performed. Totally, 716 proteins were identified and quantified in the control, UVB, and UVD groups. Among them, 123 proteins and 96 proteins were identified as differentially abundant proteins in UVB group and UVD groups, respectively. Proteins related to photosynthesis, amino acid biosynthesis, and tocopherol biosynthesis were significantly altered in UVB group, while proteins related to the biosynthesis of phenolic compounds were significantly altered in UVD group. In addition, the abundances of proteins involved in the ubiquitin-proteasome system (UPS) were significantly increased in both UVB and UVD groups, indicating that UPS combined with secondary mechanism participated in the resistance to UV-B radiation and dark incubation. The obtained results provide novel insight into the effects of high level UV-B radiation on M. alba leaves and on the strategies used for maximizing the chemical constituents and the medicinal value of the M. alba leaves.

Development of a validated HPTLC-bioautographic method for evaluation of aromatase inhibitory activity of plant extracts and their constituents.

INTRODUCTION: Aromatase is a CYP450 enzyme that catalyses the conversion of androgens into oestrogens, where the decrease in the production of oestrogens aided by aromatase inhibitors is considered a target in post-menopausal breast cancer therapy. TLC-bioautography is a technique employed for combining chromatographic separations on TLC plates with bioassays. This is the first report to evaluate aromatase inhibitory activity using this technique. OBJECTIVES: The aim of this study is to develop and validate a new TLC-bioautographic method for determination of aromatase inhibitory activity in 14 plant extracts. Two quantitation methods, the peak area method and reciprocal iso-inhibition volume (RIV) method, were compared and investigated to attain reliable results. Factors affecting the enzymatic reaction (temperature, pH, enzyme and substrate concentrations … etc.) were also investigated to attain the optimum parameters. METHODOLOGY: TLC assisted by digital image processing was implemented for quantitative estimation of the aromatase inhibition of 14 plant extracts using chrysin as positive control. The fluorometric substrate dibenzyl fluorescein (DBF) was utilised for the assay, where inhibitory compounds were visualised as dark spots against a blue fluorescent background. Two software programs, Sorbfil® videodensitometer (in the peak area method) and ImageJ® (in the RIV method), were thoroughly validated using the International Council on Harmonisation (ICH) guideline and used for quantitation. RESULTS: The RIV method showed superiority over the peak area method in the quantitation results of the tracks with non-homogenous background with %RSD values of 0.98 and 1.49 compared with 2.86 and 3.58, respectively. Further, the methods allow the comparison of the activity of different unknown inhibitory compounds without the need for a reference or a positive control. CONCLUSION: Using the TLC-bioautographic method by image processing combined with the RIV quantitation method, simultaneous separation and quantitation of aromatase inhibitory components could be applied to estimate the relative activity of various plant extracts.

Oral lichen planus after COVID-19, a case report.

INTRODUCTION: The COVID-19 is a global pandemic that is now responsible for more than 3 million deaths around the world. The oral and dermatological manifestations of COVID-19 are being remarkably reported. This report aims to describe a unique case of oral presentation of erosive lichen planus after COVID-19 infection. CASE PRESENTATION: We present a case of oral lichen planus manifested in the buccal mucosa and the tongue. The lesions were detected one month after COVID-19 infection. Clinical, as well as the histopathological diagnosis, was performed on the oral lesion to confirm oral lichen planus. CLINICAL DISCUSSION: Despite viruses being reported as a triggering factor in oral lichen planus, very few cases of oral lichen planus after COVID-19 infection were noticed. The immunological and psychological changes induced by COVID-19 infection may explain the tendency to find lichen planus lesions in COVID-19 patients. CONCLUSION: Our case suggests the possible association between COVID-19 and oral lichen planus. This report highlights the role of health practitioners to be concerned about the probably rising incidence of lichen planus during the COVID-19 pandemic and consider appropriate therapeutic and protective measures against infection.

Efficacy-directed discrimination of the essential oils of three Juniperus species based on their in-vitro antimicrobial and anti-inflammatory activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Juniperus plants are considered important sources of cedar-wood oil which is used widely in folk medicine as antiseptic and in treatment of inflammatory disorders such as, rheumatoid arthritis but there is not enough scientific evidence to support the claimed uses and there is no specification of a certain Juniperus species as the most active. AIM OF THE STUDY: The aim of this study is volatiles profiling of three Juniperus species; J. communis, J. horizontalis and J. chinensis in addition to efficacy-directed discrimination of the three studied essential oils based on their antimicrobial, and anti-inflammatory activities in LPS (lipopolysaccharide)-stimulated WBCs (White blood cells) to investigate the inter-specific variability effect on the biological activities of each oil. MATERIALS AND METHODS: Volatile components profiling of the three studied plants volatile oils was achieved using GC-FID (Gas chromatography - flame ionization detector) and GC-MS (Gas chromatography - mass spectrometry). The antimicrobial activity of the studied essential oils was investigated and the minimum inhibitory concentration (MIC) was determined for oils. The production of the pro-inflammatory cytokines was evaluated by ELISA (Enzyme linked immunosorbent assay). Identification of the biomarkers responsible for each activity was attempted through construction of orthogonal projection to latent structures model using multivariate statistical analysis. RESULTS: Forty five components were identified in the volatile oils of the three studied plants. J. horizontalis oil displayed the highest activity against E. coli while J. communis showed the highest activity against S. aureus. OPLS model biplot showed the in-between class discrimination of J. chinensis oil sample from J. communis and J. horizontalis. The three oils were found to significantly decrease the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, and gamma interferon (INF- γ) in lipopolysaccharide-activated white blood cells. All studied oils were similar in reduction of TNF-α, and INF-γ, while J. chinensis oil possessed the highest potency against IL-1ß. The coefficient plots of TNF-α and INF-γ pro-inflammatory mediators showed that 1-terpineol, 4-terpineol, bornyl acetate, dl-limonene and α-pinene positive contributors to both activities while ß-thujone, 3-carene and γ-muurolene were the positive contributors to IL-1ß inhibitory activity. CONCLUSION: The differences observed in the volatile profiles among the three studied oils demonstrate the effect of inter-specific variability on the biological activities of the tested oils. It was shown that the tested oils possessed good antibacterial activities against E.coli and S. aureus justifying its folk use as an a topical antiseptic while the observed anti-inflammatory effects in human WBCs is due at least in part to their inhibitory effect on the production of pro-inflammatory cytokines.

Identifying cancer-related molecular targets of Nandina domestica Thunb. by network pharmacology-based analysis in combination with chemical profiling and molecular docking studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Nandina domestica Thunb. have served as folk medicines in Chinese and Japanese tradition for treatment of several tumors including pharynx tumor and tooth abscess for many years, yet its exact mechanism of action is not yet known. AIM OF THE STUDY: The study targets the identification of the main constituents of the fruits extracts and investigation of their mode of action in cancer therapy via pharmacology-based analysis and molecular docking. MATERIALS AND METHODS: The different extracts of N. domestica Thunb. were analyzed via UPLC-MS/MS for identification of their active constituents. STITCH, DAVID, KEGG and STRING database were utilized for construction of compound-target and compound-target-pathway networks using Cytoscape 3.2.1. Molecular docking analysis of the top hit compounds was performed against the identified top hit molecular targets in the constructed networks. In vitro-testing of Nandina domestica Thunb. against colorectal cancer cell lines was carried out and correlated to the chemical profile of the extract to identify important biomarkers. The ADME properties of the active compounds were also evaluated. RESULTS: 22 compounds were identified by UPLC-MS/MS analysis and were forwarded to network pharmacology-based analysis. Results showed the enrichment of 5 compounds and 4 molecular targets in the network namely; AKT1, CASP3, MAPK1 and TP53. The pathway analysis of the identified targets revealed that 15 cancer-related pathways were enriched including colorectal cancer, endometrial cancer and small-cell lung cancer. In-vitro testing of the extracts against colo-rectal cancer cell lines revealed the fractions enriched in the identified hit compounds were indeed the most active as revealed from the HCA-heat-map. ADME results showed that all compounds were drug-like candidates showing acceptable values according to Lipinski's rule. CONCLUSIONS: Network pharmacology analysis revealed that the compounds isoquercitrin, quercitrin, berberine, chlorogenic acid and caffeic acid showed strong synergistic interactions with the cancer-related targets and pathways. It could be concluded that N. domestica Thunb. constituents affect both apoptosis and Akt-signaling pathways during the stages of early and intermediate adenoma through interaction with the targets CASP3 and MAPK1 (ErC2) while during the stages of late adenoma and carcinoma, the compounds acts through the p53 and ErbB signaling pathways.

Synthesis, biological evaluation, and docking study of indole aryl sulfonamides as aromatase inhibitors.

In order to identify new aromatase enzyme inhibitors, thirty aryl sulfonamide derivatives containing an indole nucleus have been synthesized. The enzyme inhibition assay showed that four compounds inhibit aromatase in the sub-micromolar range. Loading concentrations of these four compounds were afterwards tested for cell viability and cytotoxicity on MCF7 human breast cancer cells, revealing a time- and dose-dependent decrease of active metabolizing cells over the time of the culture (0-72 h), starting from a concentration of 100 µM. Likewise LDH released raised up to 40% at early time of exposures (24 h). Finally, the docking study showed that the best active compounds efficiently bound in the active site of the aromatase; high values of HBD and low levels of HBA are the principal requirement evidenced by the QSAR model.

Prediction of potential cancer-related molecular targets of North African plants constituents using network pharmacology-based analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Nowadays, cancer is considered one of the leading causes of death in developing countries. Due to mediocre socioeconomic status of many of the North African countries, people resort to traditional medicine from natural products for cancer therapy which are of great chemical complexity, interacting with several protein targets leading to synergistic effects. A holistic network pharmacology approach is needed for understanding the molecular mechanism of North African plants constituents in the different cancer-related pathways. AIM OF THE STUDY: The aim of this study is the implementation of network pharmacology for identification of the main active constituents of North African plants against cancer molecular targets and to explore their therapeutic mechanism. MATERIALS AND METHODS: Constituents of North African plants were retrieved from public database and ADME screening was implemented for filtration of constituents using Qikprop software. STITCH database was used for predicting the plant constituents target proteins/genes, TDD DB and Uniprot databases were used for identifying genes related to cancer. Constituent-target gene (C-T), constituent-pathway (C-P) and plant-constituent-target gene (P-C-T) networks were constructed using Cytoscape to decipher the anti-cancer mechanism of action of the plants. KEGG pathway and GO enrichment analysis were performed to investigate the molecular mechanisms and pathways related to cancer. RESULTS: 6844 constituent were subjected to ADME filtration resulting in 3194 constituent which were forwarded to target prediction. 53 constituents and 36 targets were linked through 329 edges which constituted the main pathways related to cancer. Luteolin, alternariol, apigenin, aloe-emodin and myricetin had the highest combined score in the C-T network, while the genes CASP3, CYP1A1, CYP1B1, PTGS2, MAPK8, AKT1 and EGFR were the most enriched by the constituents in this network. Euphorbia spp., Hyphaene thebaica, Artemisia herba-alba, bee propolis and Marrubium vulgare possessed the largest number of P-C-T interactions. The identified targets were mainly associated with cell cycle arrest and apoptosis in addition to inhibition of cellular proliferation by revealing a striking functional association with various signal and cancer related pathways CONCLUSIONS: Analysis of the constructed pharmacological networks results allowed for the prediction and interpretation of the multi-constituent, multi-target, and multi-pathway mechanisms of North African plants as potential source for supportive treatment of cancer where their potential molecular mechanism towards cancer-associated targets, biological processes and pathways were revealed.

Evaluation of the influence of green extraction solvents on the cytotoxic activities of Crinum (Amaryllidaeae) alkaloid extracts using in-vitro-in-silico approach.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional use of Amaryllidaceae plants to treat many disease have been known for a very long period of time. The chemical analysis of these plants has yielded a diversity of alkaloids with analgesic, anticholinergic, antitumor and antiviral activities. Crinum bulbispermum (Burm.f.) Milne-Redh. & Schweick in particular has been used by Zulu, Sotho and Tswana people to treat tumors as a form of chemotherapy, while in Madagascar, Crinum powellii Baker Handb. was used in the treatment of abscesses and tumors. Many of the alkaloids spawned by genus Crinum will surely take part in the production of anticancer drugs but their further clinical development is restricted by their limited commercial availability. An emerging area of research is the establishment of green extraction techniques of different targeted compounds. AIM OF THE STUDY: Our comparative study has investigated the possibility of getting improved biological responses by changing extraction solvent to a better and greener one. This study aimed to assess the cytotoxic activity of Crinum powellii and Crinum bulbispermum bulbs, when extracted by different green solvents. MATERIALS AND METHODS: The green solvents Genapol X-80 (a surfactant-aided extraction), DES-3 (Choline chloride: fructose 5:2) mixture (a natural deep eutectic solvent) and purified distilled water were used for extraction of the bulbs. Extracts were tested against two cell lines HEPG-2 and HCT 116, with doxorubicin as a positive reference. Molecular docking studies were carried out to illustrate binding orientations of the alkaloids in the active site of several molecular targets for treatment of hepatic and colorectal cancer. RESULTS: DES aided extraction showed highest cytotoxicity against the two cell lines, followed by surfactant aided extracts and finally aqueous extracts. There is an obvious relationship between alkaloidal content and antiproliferative potency of extracts. Multivariate statistical analyses were performed to aid the prediction of the alkaloids responsible for the activity. The alkaloid crinine showed high correlation coefficient value against HCT colon cancer cell line in the orthogonal projection to latent structures (OPLS) model, suggesting that it could operate with a selective mode of action on this cell line. In addition, the alkaloid lycorine had almost no correlation to anti-proliferative activity against HCT colon cancer cells. Molecular docking studies confirmed the same conclusions. CONCLUSION: Herein, it was demonstrated that natural deep eutectic solvents (NADES) components and surfactant solutions could be chosen to enhance biological activity of extracts prepared.

Integrated in silico-in vitro strategy for screening of some traditional Egyptian plants for human aromatase inhibitors.

ETHNOPHARMACOLOGICAL RELEVANCE: Aromatase enzyme (CYP19) is widely known as a critical target protein for treating hormone-dependent breast cancer. Natural products from traditional medicinal plants continue to be an active source of aromatase inhibitors. Meanwhile, high cost of experimental work and low hit rate associated with HTS have stimulated the implementation of in-silico virtual screening to resolve these pitfalls, where coupling of both classical wet lab procedure and VS may offer a more deepened access to bioactive compounds with less work and time waste. AIM OF THE STUDY: In this work, a sequential structure-based and ligand-based virtual screening strategy was utilized for investigating an in-house database of 1720 phytochemical constituents of 29 medicinal plants and natural products used in traditional Egyptian medicine to search for compounds with the potential to be used as inhibitors of the human aromatase enzyme. MATERIALS AND METHODS: The suggested strategy included using Glide docking with its feature 'extra precision (XP)' for carrying out structure-based virtual screening (SBVS) where the resulting hits were further promoted to ligand-based virtual screening (LBVS) through the development of two pharmacophore and QSAR models; one for steroidal and the other for non-steroidal aromatase inhibitors. RESULTS: The combined results revealed that Artemisia annua, Zingiber officinale, Cicer arietinum, Annona muricata and Vitex agnus castus were the top scoring plants in terms of in-silico activity scores, respectively. The hydro-alcoholic extracts and different solvent fractions of the top scoring plants were subsequently tested experimentally for their aromatase inhibitory activity, by the aid of in-vitro fluorometric assay. The rank ordering of the activities for the plants agreed with the ordering predicted on the basis of SBVS and LBVS workflow implemented. CONCLUSION: The suggested strategy provides a reliable means of prospecting in-silico screening of natural products databases in the search for new dug leads as aromatase inhibitors. The hits so obtained can then be subjected to further phytochemical studies, to isolate and identify suitable compounds for further in-vitro testing.

Fingerprint profile and efficacy-associated markers of Nigella sativa oil for geographical origin determination using targeted and untargeted HPTLC-multivariate analysis.

Efficacy directed-fingerprint analysis of high-performance thin layer chromatography is proposed to set up fingerprint activity relationship modeling for precise discrimination of chemical and effective consistency of Nigella sativa oils from different geographical origins. A whole of 27 samples of N. sativa oils from three geographical area (Egypt, Ethiopia and Syria) were collected and their antimicrobial, cytotoxic, anti-inflammatory and analgesic activities were measured. The results revealed that there was significant difference in the biological activities of the oils collected. The fingerprints of the samples had been established by high performance thin layer chromatography, subsequently the data had been utilized for the discrimination of the samples geographical origin. The loading plots of HPTLC-Principal Component Analysis (PCA) had been used to discover the crucial marker ingredients for classification. Furthermore, targeted chemical fingerprints had been established by HPTLC, and discriminant analyses were calculated depending on five common characteristic peaks. The chosen markers were quantified by validated HPTLC methods, and then the quantitative data as well as the oils bioactive properties were subjected to partial least squares regression (PLSR) analyses. Thymoquinone and free fatty acids (FFA) were revealed as potential markers to distinguish the chemical consistency and efficacy of the oils from the three different geographical origins. The suggested technique provides an applicable integrated strategy to screen for efficacy-associated markers for discrimination of N. sativa oils from distinctive geographical origins exploiting HPTLC fingerprint activity relationship modeling.

Validated HPTLC Method for Dihydrokaempferol-4'-O-glucopyranoside Quantitative Determination in Alcea Species.

Dihydrokaempferol-4'-O-glucopyranoside, a flavanonol glucoside, is the major compound in the flower of Alcea rosea L. which possesses significant antioxidant and anticancer activity against HepG-2 cell line and thus can be considered a marker compound for A. rosea L. We attempted to establish a new simple, validated high-performance thin-layer chromatographic (HPTLC) method for the quantitation of dihydrokaempferol-4'-O-glucopyranoside to help in the standardization of the hydroalcoholic extracts of A. rosea L. flowers and to evaluate the best method for its extraction from the plant material. The separation was carried out on an HPTLC aluminum plate pre-coated with silica gel 60F-254, eluted with ethyl acetate-methanol-water-acetic acid (30:5:4:0.15 v/v). Densitometric scanning was performed using a Camag TLC scanner III, at 295 nm. A linear relationship was obtained between the concentrations (0.9-3.6 mg) and peak areas with the correlation coefficient (r) of 0.9971 ± 0.0002. The percentage relative standard deviations of intra-day and inter-day precisions were 0.22-1.45 and 0.49-1.66, respectively. The percentage w/w of dihydrokaempferol-4'-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively.

In-silico profiling of the biological activities of Amaryllidaceae alkaloids.

OBJECTIVES: The large number of publications about Amaryllidaceae alkaloids reflects the abundance and variety in biological activity of these alkaloids. An in-silico approach was implemented in this work to rationalize the individual alkaloids to molecular biological activity. METHODS: A database was generated containing 313 Amaryllidaceae alkaloids which were then subjected to in-silico-validated structure-based virtual screening using extra precision (XP) approach of Glide docking program. Further pharmacophore detection of the high scorers resulted in a hybrid model considering the structural and spatial characteristics of the molecules. The focus was laid on representative targets against viral infections, acetylcholinesterase and cancer. BEDROC studies were used for validation of the accuracy of docking methods. KEY FINDINGS: As expected, galanthamine-type alkaloids were the most active against hACHE; yet, lycorenine- and tazettine-type alkaloids contributed significantly, while lycorine-type alkaloids dominated the hit list against HIV-1 PR target protein and were significantly active against HIV-1 RT and influenza NA. Surprisingly, belladine-type alkaloids showed the highest number of hits against HDAC2, while lycorine- and narciclasine-type alkaloids dominated the hit lists against Aurora kinase A and VEGFR2. CONCLUSIONS: This report provides useful information on Amaryllidaceae alkaloids and serves as a starting point to access their undiscovered biological activity.

In vitro cytotoxicity of some Narcissus plants extracts.

This study compares the chloroform extracts of bulbs and roots of Narcissus papyraceus Ker Gawl. and Narcissus tazetta L. The cytotoxicity of the plant extracts was evaluated against human hepatocellular carcinoma cell line (HEPG2) and colon carcinoma cell line (HCT116) in comparison to doxorubicin. The extracts from the after-flowering (AF) bulbs of N. tazetta L. and N. papyraceus exhibited strong cytotoxic activity against HEPG2 (IC50: 2.2, 3.5 µg mL(-1)) and HCT116 (IC50: 4.2, 3.9 µg mL(-1)) cell lines, respectively. N. tazetta L. bulbs exhibited the least cell viability percentage in HepG-2 cell line (5.32%), while the AF root extracts of N. papyraceus exhibited the least cell viability percentage in HCT116 cell line (4.93%), when applied at a concentration of 50 µg mL(-1), thereby being more active than doxorubicin at the same concentration.