Identification, Validation and Standardization of Bioactive Molecules Using UPLC/MS-QToF, UHPLC and HPTLC in Divya-Denguenil-Vati: A Penta-Herbal Formulation for Dengue Fever.

For the last fifty years, Dengue has been one of the most common mosquito-borne arboviral infections which has spread over the tropical and subtropical world. Divya-Denguenil-Vati (DNV) has been formulated by blending five specific herbs for effective resolution of Dengue fever. In the present study, we aimed to identify, develop, validate, and standardize methods for Divya-Denguenil-Vati (DNV), on UHPLC and HPTLC analytical platforms, with rapid, sensitive, accurate and rugged attributes. At first, 97 phyto-constituents were identified by UPLC/MS-QToF in Divya-Denguenil-Vati. UHPLC method was then developed and validated for simultaneous determination of gallic acid, 5-HMF, protocatechuic acid, magnoflorine, methyl gallate, berberine, rutin, ellagic acid, ß-ecdysone and rosmarinic acid in DNV. Four selected markers, gallic acid, rosmarinic acid, magnoflorine and rutin were further developed and validated on HPTLC. Analytical processes were validated as per ICH Q2 (R1) guidelines; and were found linear (r 2 > 0.99), sensitive, precise (%RSD < 5%), and accurate, as indicated by high recovery values (88-105%). The limit of detection and quantification were also established for these phyto-metabolites, with their respective RSDs within 5% limits. Finally, these validated methods were employed to test twenty six different commercial batches of DNV. The quality, reproducibility and consistency of DNV have been well established using these developed and reliable analytical tools. These analytical strategies successfully set a path forward for robust quality evaluation and standardization of Divya-Denguenil-Vati, and other related herbal formulations. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04183-7.

Penta-O-Galloyl-ß-D-Glucose in Pistacia integerrima Targets AMPK-ULK1 and ERK/STAT3 Signaling Axes to Induce ROS-Independent Autophagic Cell Death in Human Lung Cancer Cells.

Natural molecules have promising perspectives as adjuvants to chemotherapies against cancer. Pistacia chinensis subsp. Integerrima (hereafter, Pistacia integerrima) traditionally known for medicinal values in respiratory disorders was tested for anti-lung cancer properties. The extract prepared from Pistacia integerrima (PI) selectively impaired the viability of lung cancer cells, A549 and NCI-H460, compared to non-cancer cells. At non-lethal concentrations, PI mitigated colony-forming, spheroid formations and metastatic properties of lung cancer cells. As a step toward identifying the phytomolecule that is imparting the anti-lung cancer properties in PI, we subjected the extract to extensive characterization through UPLC/QToF-MS and further validated the findings with UHPLC. The gallotannin, penta-O-galloyl-ß-D-glucose (PGG), among others, was identified through UPLC/QToF-MS. PGG exhibits potential chemopreventive effects against various cancer types. However, a defined mechanism of action of PGG in restricting lung cancer progression is still unexplored. Bioactivity-guided column fractionations enabled the determination of PGG as the major phytochemical that governed PI-mediated AMPK-ULK1-dependent autophagy and apoptosis, albeit independent of intracellular ROS activation. Interestingly, the autophagy flux when inhibited restored the cell viability even in the presence of PI. The study further delineated that PI and PGG activated ERK and inhibited STAT3 to trigger apoptosis through caspase-3 and PARP 1 pathways. Collectively, the finding demonstrates that plant extract, PGG, in the PI extract effectively combats lung cancer progression through autophagic cell death by altering ERK/AMPK-ULK1/STAT3 signaling axes. The study proposes PGG as a potential AMPK activator and STAT3 inhibitor that can be exploited further in developing adjuvant chemotherapeutics against lung cancer.

Withania somnifera (L.) Dunal whole-plant extracts exhibited anti-sporotrichotic effects by destabilizing peripheral integrity of Sporothrix globosa yeast cells.

Chronic topical cases of Sporotrichosis, a chronic fungal infection caused by the ubiquitously present cryptic members of the Sporothrix species complex, are treated with oral administrations of itraconazole. However, severe pulmonary or disseminated cases require repeated intra-venous doses of amphotericin B or even surgical debridement of the infected tissue. The unavoidable adverse side-effects of the current treatments, besides the growing drug resistance among Sporothrix genus, demands exploration of alternative therapeutic options. Medicinal herbs, due to their multi-targeting capacity, are gaining popularity amidst the rising antimicrobial recalcitrance. Withania somnifera is a well-known medicinal herb with reported antifungal activities against several pathogenic fungal genera. In this study, the antifungal effect of the whole plant extract of W. somnifera (WSWE) has been explored for the first time, against an itraconazole resistant strain of S. globosa. WSWE treatment inhibited S. globosa yeast form growth in a dose-dependent manner, with IC50 of 1.40 mg/ml. Minimum fungicidal concentration (MFC) was found to be 50 mg/ml. Sorbitol protection and ergosterol binding assays, revealed that anti-sporotrichotic effects of WSWE correlated well with the destabilization of the fungal cell wall and cell membrane. This observation was validated through dose-dependent decrease in overall ergosterol contents in WSWE-treated S. globosa cells. Compositional analysis of WSWE through high performance liquid chromatography (HPLC) exhibited the presence of several anti-microbial phytochemicals like withanone, withaferin A, withanolides A and B, and withanoside IV and V. Withanone and withaferin A, purified from WSWE, were 10-20 folds more potent against S. globosa than WSWE, thus, suggesting to be the major phytocompounds responsible for the observed anti-sporotrichotic activity. In conclusion, this study has demonstrated the anti-sporotrichotic property of the whole plant extract of W. somnifera against S. globosa that could be further explored for the development of a natural antifungal agent against chronic Sporotrichosis.

Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model.

PURPOSE: SARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals. AIM: This study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone. METHODS: Through molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction. RESULTS: Withanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein. CONCLUSION: In conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.