Exploring the dynamics of bioactive metabolites changes in barley grains (Hordeum vulgare L.) during roasting: Insights from UPLC-QqQ-MS/MS analysis combined to chemometrics.

This investigation delves into the dynamic metabolic shifts within barley grains during the roasting process, employing UPLC-QqQ-MS/MS analysis. The complex spectrum of metabolites before and after roasting is revealed. The resulting data, unveils substantial transformations in chemical composition during roasting. A total of 62 chromatographic peaks spanning phenolic compounds, flavones, Millard Reaction Products, amino acids, lignans, vitamins, folates, and anthocyanins were annotated. Leveraging UPLC-QqQ-MS/MS analysis, we scrutinized the intricate metabolite profile before and after roasting where the roasting process was found to trigger dynamic changes across diverse metabolite classes particularly Millard Reaction Products, produced through the Maillard reaction, with dihydro-5-methyl-5H-cyclopentapyrazine, maltol and hydroxy maltol emerging as discerning markers of roasting progression. Amino acids and sugars showed degradation, while beta-glucan, a signature barley sugar, experienced notable decline. Folate derivatives witnessed pronounced reduction, aligning with the heat sensitivity of folates. Harnessing the power of multivariate data analysis, the consequences of roasting materialize through distinct clusters in PCA and OPLS-DA plots. Noteworthy, roasting duration governs the trajectory of metabolic divergence, culminating in the identification of roasting-specific markers. Epigallocatechin, procyanidin B, 10-HCO-H4 folate, and hordatine A emerge as pivotal discriminators. Orthogonal Projection to Latent Structure (OPLS) analysis linked anti-inflammatory activity with 30-min, 1-hour, and 1.5-hour roasted samples, with hordatine B in addition to some Millard Reaction Products being correlated with pro-inflammatory marker downregulation.. This study encapsulates the intricate metabolic metamorphosis ignited by roasting in barley grains, offering a holistic comprehension of their potential health-enhancing attributes. Key metabolites act as poignant indicators of these transformations, substantiating the complex interplay between roasting and the barley grain metabolome.

Metabolomic insights into the therapeutic mechanisms of costus (Saussurea costus (Falc.) Lipsch.) root extract in propylthiouracil-induced hypothyroidism rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea costus (Falc.) Lipschitz. is one of the most reputed medicinal plants as a traditional medicine in the Arab and Middle East regions in the treatment of thyroid disorders, however, more investigations are needed to fully understand its effectiveness and mechanism of action. AIM OF THE STUDY: The primary objective of the study was to assess the impact of Saussurea costus (COST) on the metabolic profiles of propylthiouracil (PTU)-induced hypothyroidism in rats. This involves a comprehensive examination of serum metabolites using UPLC/QqQ-MS analysis aiming to identify differential metabolites, elucidate underlying mechanisms, and evaluate the potential pharmacological effect of COST in restoring metabolic homeostasis. MATERIALS AND METHODS: Hypothyroidism was induced in female Sprague-Dawley rats by oral administration of propylthiouracil (PTU). UPLC/QqQ MS analysis of serum samples from normal, PTU, and PTU + COST rats was utilized for annotation of intrinsic metabolites with the aid of online Human metabolome database (HMDB) and extensive literature surfing. Multivariate statistical analyses, including orthogonal partial least squares discriminant analysis (OPLS-DA), discerned variations between the different groups. Serum levels of T3, T4 and TSH in addition to arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) levels in thyroid gland tissues; Phospholipase A2 group IIA (PLA2G2A), and lipoprotein lipase (LPL) in liver tissues were assessed by specific ELISA kits. Gene expression for key proteins of the primary evolved pathwayswere quantified by one-step qRT-PCR technique. Histopathological evaluation of thyroid gland tissue was performed by an investigator blinded to the experimental group using light microscope. RESULTS: Distinct clustering in multivariate statistical analysis models indicated significant variations in serum chemical profiles among normal, disease, and treated groups. VIP values guided the selection of differential metabolites, revealing significant changes in metabolite concentrations. Subsequent to COST treatment, 43 differential intrinsic metabolites exhibited a notable tendency to revert towards normal levels. Annotated metabolites, such as lysophosphatidylcholine (LPC), L-acetylcarnitine, gamma-glutamylserine, and others, showed differential regulation in response to PTU and subsequent S. costus treatment. Notably, 21 metabolites were associated with polyunsaturated fatty acids (PUFAs) biosynthesis, arachidonic acid (ARA) metabolism, and glycerophospholipid metabolism exhibited significant changes on conducting metabolic pathway analysis. CONCLUSIONS: COST improves PTU-induced hypothyroidism by regulating biosynthesis of PUFAs signified by n-3/n-6, ARA and glycerophospholipid metabolism. The study provides us a novel mechanism to explain the improvement of hypothyroidism and associated dyslipidemia by COST, depicts a metabolic profile of hypothyroidism, and gives us another point cut for further exploring the biomarkers and pathogenesis of hypothyroidism.

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.

Antiangiogenic Pterocarpan and Flavonoid Constituents of Erythrina lysistemon.

The roots of Erythrina lysistemon, growing in Egypt, yielded 24 flavonoid compounds, including 17 pterocarpans, two isoflavanones, one flavanone, two isoflavans, one 2-arylbenzofuran, and an isoflava-3-ene. Nine pterocarpans have not been reported previously (7-9, 11-14, 19, and 20), and 11 are reported here for the first time from this species. Structures were established using HRESIMS, NMR, and circular dichroism techniques. Selected compounds were tested for their ability to block the growth of human retinal endothelial cells and antiangiogenic activity in vitro. The isoflavonoids 5 and 6, and the pterocarpans 1, 2, 4, 20, and 22 demonstrated selective antiproliferative activities on endothelial cells compared to a nonendothelial cell type, with concentration-dependent antiangiogenic effects in vitro against HRECs, a cell type relevant to neovascular eye diseases.

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.

Metabolomics combined to chemometrics reveals the putative α-glucosidase and α-amylase inhibitory metabolites of ground cherry (Physalis pruinosa L.).

In this work, metabolic profiling of the different parts of ground cherry (P. pruinosa) including fruits, calyces, leaves, stems and roots using UPLC-MS/MS analysis combined to chemometric analysis was attempted. A total of 82 chromatographic peaks belonging to different metabolite classes were identified including terpenes, flavonoids genin and glycosides, withanolides, physalins, sucrose esters, fatty acids and other miscellaneous compounds with withanolides being the most predominant class. Roots extracts possessed the highest relative content of the identified 5ß,6ß-epoxy withanolides and intermediate withanolides, while sucrose esters and flavonoidal glycosides were found in a great abundance in calyces extracts. Moreover, physalins were found in all extracts except for roots extracts. Studying the coefficients plots revealed that terpenes and physalins (physanicantriol, loliolide, physalisitin C) were responsible for discrimination of fruits extracts. Calyces, leaves and stems extracts were found to possess antioxidant activity and higher inhibition of α-glucosidase activity. In an attempt to identify the compounds responsible for the hypoglycemic activity using both α-amylase and α-glucosidase inhibition assays, OPLS models coefficient plots were constructed which indicated that physangulide B, physaperuvin G, neophysalin A, and acylsucroses were positively correlated to α-glucosidase inhibition, while guaiacyl-primeveroside, phyperunolide C, physalactone, physalolactone C and perulactone, were positively correlated to α-amylase inhibitory activity.

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.

Tandem mass tag-based proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under ultraviolet-B and dark stress.

Mulberry leaves have been used in traditional Chinese medicine due to their antioxidant, antidiabetic, and antihyperlipidemic properties. A previous study showed that ultraviolet-B radiation followed by dark incubation could improve the contents of active ingredients in mulberry leaves, such as moracin N and chalcomoracin. The endoplasmic reticulum (ER) serves as a protein quality control center and the location for protein synthesis, which is involved in the response to the environmental stress in plants. To investigate the mechanisms in response to ultraviolet-B radiation followed by dark incubation (UV + D), ER proteomics was performed on mulberry leaves. The ER protein markers, glucose-regulated protein (GRP78), and calnexin (CNX), were significantly higher in the ER fraction than in the total protein fraction, indicating that the ER was purified. Compared to the control, the abundance of protein disulfide isomerase, UDP-glucose glycoprotein glucosyltransferase, CNX, and calreticulin proteins decreased, while of the abundance of heat shock-related proteins increased under stress. P450 enzyme system-related proteins and ribosomal proteins showed significant increases. These results suggest that under UV + D stress, mulberry leaves activated the cell redox and ER quality control systems, enhancing protein synthesis and weakening N-glycan biosynthesis in the ER to resist the damage.

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.

Chemical profiling and identification of anti-inflammatory biomarkers of oriental Thuja (Platycladus orientalis) using UPLC/MS/MS and network pharmacology-based analyses.

Platycladus orientalis L. Franco has many folk uses as it is mainly used to treat inflammatory ailments. UPLC-MS/MS was used for the chemical profiling of P. orientalis leaves. Identified metabolites were forwarded to network pharmacology analysis. Networks were constructed based on STITCH, SEA, DAVID, KEGG and STRING databases and using Cytoscape. The identified hit compounds were afzelin, myricetin, apigenin-7-O-hexoside, quercetrin and hyperoside. IL2, VEGFA, AKT1, AKT2, CREB1, IL5, RPS6KB1 and TNF were the main inflammation-related targets identified. Quercetrin and hyperoside were tested for their anti-inflammatory activity. it can be concluded that, the identified hit compounds exhibited strong synergistic interactions with the inflammation and immunity-related targets and pathways.

Comparative metabolomics reveals the cytotoxic and anti-inflammatory discriminatory chemical markers of raw and roasted colocynth fruit (Citrullus colocynthis L.).

Colocynth has a long history of use in traditional medicine for treatment of various inflammatory diseases where it is commonly roasted before being applied for medical purposes to reduce its toxicity. This study aims at tracking the effect of heat processing on the metabolic profile of the peels, pulps and seeds of colocynth fruit using UPLC-QqQ-MS-based metabolomics. The analysis resulted in tentative identification of 72 compounds belonging to different chemical classes. With roasting, a decline was observed in the relative amounts of chemical constituents where 42, 25 and 29 compounds were down-regulated in the peels, pulps and seeds, respectively. EC100 values resulting in 100% cell viability were all higher in roasted samples compared to their relevant raw ones. Correlation analysis indicated that the main cytotoxic chemical markers were cucurbitacin glycosides and their genins. Further, ex vivo anti-inflammatory activity testing multivariate models revealed that unprocessed samples correlated with inhibition of TNF-α, IL-1ß and IFN-γ where quercetrin, calodendroside A, and hexanoic acid methyl ester were the most significant chemical markers, while processed samples showed correlation with IL-6 pro-inflammatory marker inhibition with protocatechuic and protocatechuic acid glycoside being the main correlated chemical markers.

Profiling of acetylcholinesterase inhibitory alkaloids from some Crinum, Habranthus and Zephyranthes species by GC-MS combined with multivariate analyses and in silico studies.

Acetylcholinesterase (AChE) inhibitors remain the class of drugs used for the treatment of Alzheimer disease (AD). For the aim of discovering new sources of potent AChE inhibitors, a combined AChE-inhibitory activity together with alkaloid profiles by GC-MS, combined with multivariate statistical analysis for biomarkers determination and in silico studies were attempted. Strategy was applied on leaves, roots and bulbs of six aquatic and terrestrial Amaryllidaceae species. Thirty alkaloids were identified and the AChE inhibitory activities of the extracts were tested by in-vitro Ellman method. Principal bioactive markers were discovered by correlating AChE inhibitory activity with chemical fingerprints via PLS and OPLS modeling which revealed that galanthamine, lycoramine, caranine, tazettine and N-demethylgalanthamine were the most bio-significant markers. Furthermore, the molecular docking was performed to illustrate binding orientations of the top scoring alkaloids in the active site of human acetylcholinesterase. Suggested strategy revealed that, beside galanthamine, caranine, N-demethylgalanthamine, and lycoramine are promising AChE inhibitors.

Peroxidase inhibitory and antioxidant constituents from Juniperus L. species guided by HPTLC-bioautography and molecular docking studies.

The application of a newly developed HPTLC-bioautography assay for detecting peroxidase enzyme inhibitors in plant extracts in addition to bioautography methods for detecting antioxidant compounds resulted in the isolation of a new biflavonoid 3'-methoxy sahranflavone along with two known biflavonoids and three flavonoids from the leaves and cones of Juniperus communis, J. horizontalis and J. chinensis. The structures of all compounds were elucidated by means of 1 D and 2 D NMR and MALDI-TOF MS technique in addition to comparison to literature data. Quantitative estimation of antiperoxidase and antioxidative capacity based on DPPH free radical scavenging activity and ß-carotene bleaching of extracts, active fraction and constituents was achieved by applying validated high resolution image analyses techniques. 3'-methoxy sahranflavone and quercetrin possessed high mutual antiperoxidase and antioxidant activities. Molecular docking simulations were performed to reveal the interaction of isolated compounds with human myeloperoxidase enzyme on the molecular level indicating the potential anti-inflammatory activity of 3'-methoxy sahranflavone and quercetrin.

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.

Potential role of medicinal plants and their constituents in the mitigation of SARS-CoV-2: identifying related therapeutic targets using network pharmacology and molecular docking analyses.

Since the outbreak of Coronavirus disease (COVID-19) caused by SARS-CoV-2 in December 2019, there has been no vaccine or specific antiviral medication for treatment of the infection where supportive care and prevention of complications is the current management strategy. In this work, the potential use of medicinal plants and more than 16 500 of their constituents was investigated within two suggested therapeutic strategies in the fight against SARS-CoV-2 including prevention of SARS-CoV-2 RNA synthesis and replication, through targeting vital proteins and enzymes as well as modulation of the host's immunity through production of virulence factors. Molecular docking studies on the viral enzymes 3Clpro, PLpro and RdRp suggested rocymosin B, verbascoside, rutin, caftaric acid, luteolin 7-rutinoside, fenugreekine and cyanidin 3-(6''-malonylglucoside) as promising molecules for further drug development. Meanwhile, the medicinal plants Glycyrrhiza glabra, Hibiscus sabdariffa, Cichorium intybus, Chrysanthemum coronarium, Nigella sativa, Anastatica hierochuntica, Euphorbia species, Psidium guajava and Epilobium hirsutum were enriched in compounds with the multi-targets PTGS2, IL2, IL1b, VCAM1 and TNF such as quercetin, ursolic acid, kaempferol, isorhamnetin, luteolin, glycerrhizin and apigenin. Enriched pathways of the molecular targets included cytokine-cytokine receptor interaction, TNF signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and JAK-STAT3 signaling pathway which are all closely related to inflammatory, innate and adaptive immune responses. The present study identified natural compounds targeting SARS-CoV-2 for further in vitro and in vivo studies and emphasizes the potential role of medicinal plants in the mitigation of SARS-CoV-2.

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.

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.