Metabolic profiling of milk thistle different organs using UPLC-TQD-MS/MS coupled to multivariate analysis in relation to their selective antiviral potential.

INTRODUCTION: Silybum marianum commonly known as milk thistle is one of the most imperative medicinal plants due to its remarkable pharmacological activities. Lately, the antiviral activities of S. marianum extract have been studied and it showed effectiveness against many viruses. OBJECTIVE: Although most previous studies were concerned mainly with silymarin content of the fruit, the present study provides comprehensive comparative evaluation of S. marianum different organs' chemical profiles using UPLC-MS/MS coupled to chemometrics to unravel potentially selective antiviral compounds against human coronavirus (HCoV-229E). METHODOLOGY: UPLC-ESI-TQD-MS/MS analysis was utilized to establish metabolic fingerprints for S. marianum organs namely fruits, roots, stems and seeds. Multivariate analysis, using OPLS-DA and HCA-heat map was applied to explore the main discriminatory phytoconstituents between organs. Selective virucidal activity of organs extracts against coronavirus (HCoV-229E) was evaluated for the first time using cytopathic effect (CPE) inhibition assay. Correlation coefficient analysis was implemented for detection of potential constituents having virucidal activity. RESULTS: UPLC-MS/MS analysis resulted in 87 identified metabolites belonging to different classes. OPLS-DA revealed in-between class discrimination between milk thistle organs proving their significantly different metabolic profiles. The results of CPE assay showed that all tested organ samples exhibited dose dependent inhibitory activity in nanomolar range. Correlation analysis disclosed that caffeic acid-O-hexoside, gadoleic and linolenic acids were the most potentially selective antiviral phytoconstituents. CONCLUSION: This study valorizes the importance of different S. marianum organs as wealthy sources of selective and effective antiviral candidates. This approach can be extended to unravel potentially active constituents from complex plant matrices.

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.

Computational biology and in vitro studies for anticipating cancer-related molecular targets of sweet wormwood (Artemisia annua).

BACKGROUND: Cancer is one of the leading causes of death worldwide. Recently, it was shown that many natural extracts have positive effects against cancer, compared with chemotherapy or recent hormonal treatments. A. annua is an annual medicinal herb used in the traditional Chinese medicine. It has also been shown to inhibit the proliferation of various cancer cell lines. METHODS: Multi-level modes of action of A. annua constituents in cancer therapy were investigated using an integrated approach of network pharmacology, molecular docking, dynamic simulations and in-vitro cytotoxicity testing on both healthy and cancer cells. RESULTS: Network pharmacology-based analysis showed that the hit Artemisia annua constituents related to cancer targets were 3-(2-methylpropanoyl)-4-cadinene-3,11-diol, artemisinin G, O-(2-propenal) coniferaldehyde, (2-glyceryl)-O-coniferaldehyde and arteamisinin III, whereas the main cancer allied targets were NFKB1, MAP2K1 and AR. Sixty-eight significant signaling KEGG pathways with p < 0.01 were recognized, the most enriched of which were prostate cancer, breast cancer, melanoma and pancreatic cancer. Thirty-five biological processes were mainly regulated by cancer, involving cellular response to mechanical stimulus, positive regulation of gene expression and transcription. Molecular docking analysis of the top hit compounds against the most enriched target proteins showed that 3-(2-methylpropanoyl)-4-cadinene-3,11-diol and O-(2-propenal) coniferaldehyde exhibited the most stabilized interactions. Molecular dynamics simulations were performed to explain the stability of these two compounds in their protein-ligand complexes. Finally, confirmation of the potential anticancer activity was attained by in-vitro cytotoxicity testing of the extract on human prostate (PC-3), breast (MDA-MB-231), pancreatic (PANC-1) and melanoma (A375) cancerous cell lines. CONCLUSION: This study presents deeper insights into A. annua molecular mechanisms of action in cancer for the first time using an integrated approaches verifying the herb's value.

Alleviation of liver cirrhosis and associated portal-hypertension by Astragalus species in relation to their UPLC-MS/MS metabolic profiles: a mechanistic study.

Liver cirrhosis is a late-stage liver disease characterized by excessive fibrous deposition triggering portal-hypertension (PH); the prime restrainer for cirrhosis-related complications. Remedies that can dually oppose hepatic fibrosis and lower PH, may prevent progression into decompensated-cirrhosis. Different Astragalus-species members have shown antifibrotic and diuretic actions with possible subsequent PH reduction. However, A.spinosus and A.trigonus were poorly tested for eliciting these actions. Herein, A.spinosus and A.trigonus roots and aerial parts extracts were subjected to comprehensive metabolic-fingerprinting using UHPLC-MS/MS resulting in 56 identified phytoconstituents, followed by chemometric untargeted analysis that revealed variable metabolic profiles exemplified by different species and organ types. Consequently, tested extracts were in-vivo evaluated for potential antifibrotic/anticirrhotic activity by assessing specific markers. The mechanistic prospective to induce diuresis was investigated by analyzing plasma aldosterone and renal-transporters gene-expression. Serum apelin and dimethylarginine-dimethylaminohydrolase-1 were measured to indicate the overall effect on PH. All extracts amended cirrhosis and PH to varying extents and induced diuresis via different mechanisms. Further, An OPLS model was built to generate a comprehensive metabolic-profiling of A.spinosus and A.trigonus secondary-metabolites providing a chemical-based evidence for their efficacious consistency. In conclusion, A.spinosus and A.trigonus organs comprised myriad pharmacologically-active constituents that act synergistically to ameliorate cirrhosis and associated PH.

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.

The effect of topical chamomile in the prevention of chemotherapy-induced oral mucositis: A randomized clinical trial.

OBJECTIVE: To clinically assess the effectiveness of topical chamomile oral gel in the prevention of chemotherapy-induced oral mucositis. MATERIAL AND METHODS: A parallel single-blind randomized clinical trial conducted on 45 patients who were undergoing chemotherapy. Patients were assigned to three equal groups. Group I received conventional symptomatic treatment that included antifungal agents (Miconaz oral gel, Medical Union Pharmaceuticals), topical anesthetics, and anti-inflammatory agent (BBC oral spray, Amoun Pharmaceutical Company) three times per day for three weeks, group II received 3% chamomile topical oral gel, whereas group III patients were given both conventional symptomatic treatment and chamomile topical oral gel. All patients were clinically assessed for pain and oral mucositis severity at three separate time intervals: 1 week, 2 weeks, and 3 weeks. RESULTS: Most patients experienced oral mucositis with more severity reported in the conventional group (grade III = 6.7%) compared to the other two groups, neither of which developed more than grade II. Mean pain scores showed no significant difference between the groups, but intragroup analysis showed that pain score increased in the conventional treatment group more than the other two groups. CONCLUSION: Topical chamomile 3% gel has demonstrated in this study to lower the severity of the mucositis with lower pain scores compared to the other two groups.

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.

Chemometric evaluation of alfalfa sprouting impact on its metabolic profile using HPTLC fingerprint-efficacy relationship analysis modelled with partial least squares regression.

Sprouting is a commonly applied food processing practice specially in Western countries. Tracking the impact of sprouting of Medicago sativa L. (alfalfa) seeds on their phytochemical composition and curative efficacy was implemented in the current study. Sprouting of alfalfa seeds under controlled conditions for eleven days was performed in a biochemical incubator and three samples were randomly taken each day. A total of thirty-six samples (three ungerminated seeds and thirty-three sprouts samples) were collected, extracted and their cytotoxic, antioxidant and antimicrobial activities against five pathogenic microbial strains were measured. Samples were subjected to High performance thin layer chromatography (HPTLC) as a pattern-oriented strategy for metabolite fingerprinting to discover the fluctuations occurring during the sprouting process accompanied by multivariate chemometric analysis. Unsupervised pattern recognition was carried out using Principal Component Analysis (PCA) after extracting the chromatographic fingerprints from HPTLC chromatograms using ImageJ® software. PCA- loading plots demonstrated that luteolin-7-O-glucoside, ferulic acid and P-coumaric acid were the metabolically significant markers. Thus, simultaneous quantification of these crucial three markers in different aged alfalfa seeds/ sprouts extracts was performed using a newly developed and validated HPTLC-image analysis method. The results of the biological activities together with the quantitative data were further subjected to a Partial Least Squares Regression (PLSR) model for implementing HPTLC fingerprint-efficacy relationship analysis. The results obtained from metabolic pool profiling revealed that sprouting can cause remarkable changes in the phytochemical, nutritional and efficacy characteristics of alfalfa seeds.

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.

Targeted and untargeted-metabolite profiling to track the compositional integrity of ginger during processing using digitally-enhanced HPTLC pattern recognition analysis.

Tracking the impact of commonly applied post-harvesting and industrial processing practices on the compositional integrity of ginger rhizome was implemented in this work. Untargeted metabolite profiling was performed using digitally-enhanced HPTLC method where the chromatographic fingerprints were extracted using ImageJ software then analysed with multivariate Principal Component Analysis (PCA) for pattern recognition. A targeted approach was applied using a new, validated, simple and fast HPTLC image analysis method for simultaneous quantification of the officially recognized markers 6-, 8-, 10-gingerol and 6-shogaol in conjunction with chemometric Hierarchical Clustering Analysis (HCA). The results of both targeted and untargeted metabolite profiling revealed that peeling, drying in addition to storage employed during processing have a great influence on ginger chemo-profile, the different forms of processed ginger shouldn't be used interchangeably. Moreover, it deemed necessary to consider the holistic metabolic profile for comprehensive evaluation of ginger during processing.