Network pharmacology-based strategic prediction and target identification of apocarotenoids and carotenoids from standardized Kashmir saffron (Crocus sativus L.) extract against polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects women of reproductive age, characterized by a range of symptoms, including irregular menstrual cycles, excess male hormones (androgens), metabolic abnormalities such as hyperinsulinemia, hyperlipidemia, and metabolic disturbances like glucose imbalance. Botanical supplements are perceived first and safe choice over available regimens to regulate PCOS. There are several reports available stating that apocarotenoids, carotenoids, and whole extracts of Crocus sativus were identified to have a potential role in the management of women health. This study aimed to propose a network pharmacology-based method to determine the potential therapeutic pathways of phytoconstituents (apocarotenoids and carotenoids) of UHPLC-PDA standardized stigma-based Crocus sativus extract (CSE) for the management of PCOS. Furthermore, to validate the potential targets and signaling pathways, these apocarotenoids, and carotenoids were screened for molecular docking and in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions. The information regarding PCOS-related genes was retrieved from the PCOS knowledge database (PCOSKB), resulting in an established network between putative targets of PCOS and Crocus sativus extract phytochemicals to prevail the mechanism of action. Based on the screening conditions, 4 prominent targets namely, serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription (STAT3), mitogen-activated protein kinase 3 (MAPK3), and mitogen-activated protein kinase 1 (MAPK1), were identified through network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that MAP kinase and serine-threonine pathways were found prominent targets in PCOS. Further, a molecular docking study shows that crocetin, picrocrocin, and safranal had the best binding affinity for the identified targets. In silico ADMET results revealed that carotenoids and apocarotenoids were found to have the maximum bioavailability and were able to cross the blood-brain barrier without any toxic effects. The combined results revealed that the apocarotenoids and carotenoids of Crocus sativus extract could act on various targets to regulate multiple pathways related to PCOS.

Pharmacokinetic Study of Withanosides and Withanolides from Withania somnifera Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS).

Withania somnifera is a traditional Indian herb described under the 'Rasayana' class in Ayurveda, which gained immense popularity as a dietary supplement in the USA, Europe, Asia, and the Indian domestic market. Despite enormous research on the pharmacological effect of withanosides and withanolides, bioanalytical method development and pharmacokinetics remained challenging and unexplored for these constituents due to isomeric and isobaric characteristics. In current research work, molecular descriptors, pharmacokinetic, and toxicity prediction (ADMET) of these constituents were performed using Molinspiration and admetSAR tools. A rapid, selective, and reproducible bioanalytical method was developed and validated for seven withanosides and withanolides as per USFDA/EMA guidelines, further applied to determine pharmacokinetic parameters of Withania somnifera root extract (WSE) constituents in male Sprague Dawley rats at a dose of 500 mg/kg. Additionally, an ex vivo permeability study was carried out to explore the absorption pattern of withanosides and withanolides from the intestinal lumen. In silico, ADMET revealed oral bioavailability of withanosides and withanolides following Lipinski's rules of five with significant absorption from the gastrointestinal tract and the ability to cross the blood-brain barrier. Upon oral administration of WSE, Cmax was found to be 13.833 ± 3.727, 124.415 ± 64.932, 57.536 ± 7.523, and 7.283 ± 3.341 ng/mL for withanoside IV, withaferin A, 12-Deoxy-withastramonolide, and withanolide A, respectively, with Tmax of 0.750 ± 0.000, 0.250 ± 0.000, 0.291 ± 0.102, and 0.333 ± 0.129 h. Moreover, at a given dose, withanoside V, withanolide B, and withanone were detected in plasma; however, the concentration of these constituents was found below LLOQ. Thus, these four major withanoside and withanolides were quantified in plasma supported by ex vivo permeation data exhibiting a time-dependent absorption of withanosides and withanolides across the intestinal barrier. These composite findings provide insights to design a clinical trial of WSE as a potent nutraceutical.

Reversal of TGF-ß-induced epithelial-mesenchymal transition in hepatocellular carcinoma by sorafenib, a VEGFR-2 and Raf kinase inhibitor.

The epithelial-mesenchymal transition (EMT) is considered an essential process for cancer development and metastasis. Sorafenib, a RAF kinase and VEGFR-2 inhibitor, exhibits efficacy against advanced hepatocellular carcinoma (HCC), renal carcinoma, and thyroid cancer. It is well established that transforming growth factor-ß (TGF-ß) activated EMT is involved in the invasion and metastasis of Hep G2 cells in HCC. In this study, we investigated the effects of sorafenib on various biomarkers associated with EMT using flow cytometry. We found that sorafenib upregulated the epithelial marker E-cadherin and downregulated the mesenchymal marker vimentin. Furthermore, sorafenib downregulated the level of the EMT-inducing transcription factor SNAIL. Our findings provide insights into the mechanisms associated with the anti-EMT effects of VEGFR-2/RAF kinase inhibitors.

Quantitative Determination and Characterization of a Kashmir Saffron (Crocus sativus L.)-Based Botanical Supplement Using Single-Laboratory Validation Study by HPLC-PDA with LC-MS/MS and HPTLC Investigations.

Food ingredients hold a higher nutritional value as a botanical supplement playing a vital role in modifying and maintaining the physiological conditions that improve human health benefits. The Kashmir saffron (Crocus sativus L; KCS) obtained from dried stigmas is known for its aroma precursors and apocarotenoid derivatives, imparting a wide range of medicinal values and therapeutic benefits. In the present study, a simultaneous determination of apocarotenoids and flavonoids in stigma-based botanical supplements was carried out using analytical investigations. The high-performance thin-layer chromatography-based qualitative analysis of the raw material (stigmas, stamens, and tepals) and stigma extract has been carried out to identify apocarotenoids and flavonoids. The rapid HPLC-PDA method for the simultaneous quantification of KCS apocarotenoids was robust, precise (<5.0%), linear (R 2 > 0.99), and accurate (80-110%) as per the single-laboratory validation data. Furthermore, the combined-expanded uncertainty (95%; K = 2) was calculated and found as 0.0035-0.007% (<5.0%) as per the EURACHEM guide for this HPLC analysis. Additionally, an untargeted identification of 36 compounds in the botanical supplement was based on the elution order, UV-vis spectra, mass fragmentation pattern, and standards by ESI-MS/MS analysis with comprehensive chromatographic fingerprinting. Thus, these analytical approaches enable a composite profile of the stigma-based extract as a potential supplement for human health benefits.

Evaluation of piperine against cancer stem cells (CSCs) of hepatocellular carcinoma: Insights into epithelial-mesenchymal transition (EMT).

Cancer stem cells (CSCs) are involved in recurrent hepatocellular carcinoma (HCC), yet there is a lack of effective treatment that targets these CSCs. CD44+ and CD133+ CSCs are markedly expressed in HepG2 cells and were isolated and characterized using fluorescence-activated cell sorting (FACS) analysis. Since piperine is known as an effective molecule against metastasis, we thought to investigate the effect of piperine against CD44+/CD133+ CSCs. Herein, piperine was found to be active against these CSCs. Also, it was found appropriate to respite at the 'subG0/G1 and G0/G1' phase of the cell cycle analysis, respectively. TGF-ß activated epithelial-mesenchymal transition (EMT) has been involved in the invasion and metastasis of HepG2 cells in hepatocellular carcinoma. Therefore, we next investigated the effect of piperine on different biomarkers that remarkably takes part in the process of EMT using flow cytometric analysis. Piperine was found able to repress the epithelial marker (E-cadherin) but was unable to restore the level of Vimentin (mesenchymal marker) and SNAIL (EMT-inducing transcription factor). Therefore, the findings of this study revealed that piperine could be an effective treatment strategy for recurrent hepatocarcinogenesis.