UPLC-QToF-MS based fingerprinting of polyphenolic metabolites in the bark extract of Boehmeria rugulosa Wedd.

Boehmeria rugulosa Wedd. is an evergreen tree of Urticaceae family. Its bark has been extensively used in ethno-medicinal system for various ailments such as bone fracture, sprain, snakebite, and wound healing. Phyto-metabolites, which are considered as the principle components for biological activities, have been least explored for this plant. The present work investigated metabolite profiling of the stem bark of B. rugulosa in water extract using Ultra Performance Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry (UPLC-QToF-MS) technique coupled with the UNIFI platform. We identified, for the first time, 20 polyphenolic metabolites belonging to seven groups: caffeoylquinic acids, coumaroylquinic acids, flavan-3-ols, oligomeric flavonoids, caffeic acid derivatives, coumaric acid derivative, and flavone glycoside in the B. rugulosa extract. UNIFI informatics-coupled UPLC-QToF-MS platform aids in the quick identification and fragmentation pattern of metabolites, with higher degree of reproducibility. The present study provides a chemical and therapeutic basis for further exploration of B. rugulosa as a valuable source of phytochemicals that could be instrumental in deciphering its ethno-medicinal utility for various human diseases.

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.

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.

Modulation of psoriatic-like skin inflammation by traditional Indian medicine Divya-Kayakalp-Vati and Oil through attenuation of pro-inflammatory cytokines.

Background and aim: Psoriasis (Ps) is a chronic skin inflammatory disorder, that progresses to scaly-red dermal plaque formations associated with inflammation. Divya-Kayakalp-Vati (DKV) and Divya-Kayakalp-Oil (DKO) are traditional Ayurveda herbo-mineral formulations, that are prescribed for the treatment of inflammatory dermal ailments. In the present study, we evaluated the efficacy of Divya-Kayakalp-Vati and Divya-Kayakalp-Oil (DKV-O) combined treatment in ameliorating Ps-like skin inflammation under in-vitro and in-vivo conditions. Experimental procedure: Efficacy of DKV-O was analyzed in λ-carrageenan-treated Wistar rats paw edema and 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated CD-1 mouse ear edema models through physiological and histopathological analysis. Mode of action for the DKV-O was studied in LPS-stimulated THP-1 cells through pro-inflammatory cytokine analysis. Observed effects were correlated to the phytochemicals constituents of DKV-O analyzed using the HPLC method. Result and conclusion: DKV and DKO formulations were individually found to contain phytochemicals Gallic acid, Catechin, Berberine, Curcumin, Phenol and Benzoic acid. DKV-O treatment significantly reduced the paw volume and edema in Wistar rats stimulated through λ-carrageenan-treatment. Furthermore, DKV-O treatment significantly reduced the ear edema and enhanced biopsy weight, epidermal thickness, inflammatory lesions and influx of neutrophils stimulated by TPA-treatment in CD-1 mice. DKV alone ameliorated the LPS-stimulated release of Interleukin (IL)-6, IL-17A, IL-23, and Tumor Necrosis Factor-alpha cytokines in the THP-1 cells.Taken together, DKV-O showed good efficacy in ameliorating acute systemic inflammation stimulated by effectors such as, λ-carrageenan and TPA in animal models. Hence, Divya-Kayakalp-Vati and Divya-Kayakalp-Oil co-treatment can be further explored as an anti-inflammatory treatment against dermal diseases like psoriasis and atopic dermatitis.

Phytometabolite profiling of Coronil, a herbal medicine for COVID-19, its identification by mass-spectroscopy and quality validation on liquid chromatographic platforms.

Coronil is a tri-herbal medicine consisting of immunomodulatory herbs, Withania somnifera, Tinospora cordifolia, and Ocimum sanctum. The formulation has been developed specifically as the supporting measure for COVID-19. Current investigation is aimed to identify the phytoconstituents in Coronil utilizing ultra-performance liquid chromatography-mass spectrometry coupled with quadrapole time of flight and to establish its quality standardization using high-performance liquid chromatography and high performance thin layer chromatography. Out of 52 identified compounds, cordifolioside A, magnoflorine, rosmarinic acid, palmatine, withanoside IV, withanoside V, withanone, betulinic acid, and ursolic acid were quantified in 15 different batches of Coronil on validated high-performance liquid chromatography method. Similarly, withanoside IV, withaferin A, magnoflorine, palmatine, rosmarinic acid, and ursolic acid were analyzed on high performance thin layer chromatography. Methods were validated as per the International Council for Harmonization guidelines. These methods were specific, reproducible, accurate, precise, linear (r2 > 0.99), and percent recoveries were within the prescribed limits. The content uniformity of Coronil was ascertained using Fourier transform infrared spectroscopy. Results indicated that, validated methods were fit for their intended use and the analytical quality of Coronil was consistent across the batches. Taken together, these developed methods could drive the analytical quality control of herbal medicines such as Coronil, and other formulations containing similar chemical profiles.

Comprehensive and Rapid Quality Evaluation Method for the Ayurvedic Medicine Divya-Swasari-Vati Using Two Analytical Techniques: UPLC/QToF MS and HPLC-DAD.

Divya-Swasari-Vati (DSV) is a calcium-containing herbal medicine formulated for the symptomatic control of respiratory illnesses observed in the current COVID-19 pandemic. DSV is an Ayurvedic medicine used for the treatment of chronic cough and inflammation. The formulation has shown its pharmacological effects against SARS-CoV-2 induced inflammation in the humanized zebrafish model. The present inventive research aimed to establish comprehensive quality parameters of the DSV formulation using validated chromatographic analytical tools. Exhaustive identification of signature marker compounds present in the plant ingredients was carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/QToF MS). This was followed by simultaneous estimation of selected marker components using rapid and reliable high-performance liquid chromatography (HPLC) analysis. Eleven marker components, namely gallic acid, protocatechuic acid, methyl gallate, ellagic acid, coumarin, cinnamic acid, glycyrrhizin, eugenol, 6-gingerol, piperine and glabridin, were selected out of seventy-four identified makers for the quantitative analysis in DSV formulation. Validation of the HPLC method was evaluated by its linearity, precision, and accuracy tests as per the International Council of Harmonization (ICH) guidelines. Calibration curves for the eleven marker compounds showed good linear regression (r2 > 0.999). The relative standard deviation (RSD) value of intraday and interday precision tests were within the prescribed limits. The accuracy test results ranged from 92.75% to 100.13%. Thus, the present inclusive approach is first of its kind employing multi-chromatographic platforms for identification and quantification of the marker components in DSV, which could be applied for routine standardization of DSV and other related formulations.

Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation.

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1ß induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

Herbal decoction Divya-Swasari-Kwath attenuates airway inflammation and remodeling through Nrf-2 mediated antioxidant lung defence in mouse model of allergic asthma.

BACKGROUND AND PURPOSE: Asthma is a chronic respiratory disease orchestrated by immune and structural cells. Identification of novel therapeutic strategies are needed for asthma due to the limitations of existing therapies. We have validated the anti-inflammatory, anti-asthmatic and immunomodulatory therapeutic properties of herbal decoction, Divya-Swasari-Kwath (DSK) using mouse model of ovalbumin (OVA) induced allergic asthma. METHODS AND RESULTS: HPLC analysis identified the presence of Rutin, Glycyrrchzin, Gallic acid, Cinnamic acid, Chlorogenic acid, Caffeic acid and Piperine as bioactive herbal metabolites in DSK. Therapeutic treatment with herbal decoction DSK significantly alleviated the pathological features of allergic asthma including inflammatory cell accumulation in Broncho-Alveolar Lavage (BAL) fluids, specifically lymphocytes and eosinophils, lung inflammation, oxidative stress, airway remodelling, and pro-inflammatory cytokine levels. H&E analysis of lung tissue sections identified attenuated inflammatory cell infiltration and thickening of bronchial epithelium by DSK. PAS staining and MT staining identified decrease in OVA-induced mucus hyper secretion and peri-bronchial collagen deposition respectively, upon DSK treatment. Treatment with DSK increased the mRNA expression of antioxidative defence gene Nrf-2 and its downstream target genes HO-1 and NQO-1. In the same line, biochemical analysis for the markers of oxidative/antioxidant system confirmed the restoration of activity of Catalase, GPx, SOD and EPO and the levels of GSH, GSSG, MDA and Nitrite in whole lungs. In line with PAS staining, DSK treatment decreased the OVA-induced expression of Muc5AC and Muc5B genes. DSK treatment reduced the steady state mRNA expression levels of IL-6, IL-1ß, TNF-α, IL-4, -5, -33, IFN-γ in whole lung; and IL-6, TNF-α and IL-1ß protein levels in BALF. CONCLUSION: Collectively, our results suggest that herbal decoction DSK is effective in protecting against allergic airway inflammation and remodelling by regulating anti-oxidant mechanisms. We postulate that DSK could be the potential therapeutic option for allergic asthma management.

Validation of a Novel Zebrafish Model of Dengue Virus (DENV-3) Pathology Using the Pentaherbal Medicine Denguenil Vati.

Dengue is a devastating viral fever of humans, caused by dengue virus. Using a novel zebrafish model of dengue pathology, we validated the potential anti-dengue therapeutic properties of pentaherbal medicine, Denguenil Vati. At two different time points (at 7 and 14 days post infection with dengue virus), we tested three translational doses (5.8 µg/kg, 28 µg/kg, and 140 µg/kg). Dose- and time-dependent inhibition of the viral copy numbers was identified upon Denguenil Vati treatment. Hepatocyte necrosis, liver inflammation, and red blood cell (RBC) infiltration into the liver were significantly inhibited upon Denguenil treatment. Treatment with Denguenil Vati significantly recovered the virus-induced decreases in total platelet numbers and total RBC count, and concomitantly increasing hematocrit percentage, in a dose- and time-dependent manner. Conversely, virus-induced white blood cell (WBC) counts were significantly normalized. Virus-induced hemorrhage was completely abrogated by Denguenil after 14 days, at all the doses tested. Gene expression analysis identified a significant decrease in disease-induced endothelial apoptotic marker Angiopoetin2 (Ang-2) and pro-inflammatory chemokine marker CCL3 upon Denguenil treatment. Presence of gallic acid, ellagic acid, palmetin, and berberine molecules in the Denguenil formulation was detected by HPLC. Taken together, our results exhibit the potential therapeutic properties of Denguenil Vati in ameliorating pathological features of dengue.

Polyherbal Medicine Divya Sarva-Kalp-Kwath Ameliorates Persistent Carbon Tetrachloride Induced Biochemical and Pathological Liver Impairments in Wistar Rats and in HepG2 Cells.

Divya Sarva-Kalp-Kwath (SKK) is a poly-herbal ayurvedic medicine formulated using plant extracts of Boerhavia diffusa L. (Nyctaginaceae), Phyllanthus niruri L. (Euphorbiaceae), and Solanum nigrum L. (Solanaceae), described to improve liver function and general health. In the present study, we have explored the hepatoprotective effects of SKK in ameliorating carbon tetrachloride (CCl4) induced liver toxicity using in-vitro and in-vivo test systems. Chemical analysis of SKK using Liquid Chromatography-Mass Spectroscopy (LC-MS-QToF) and High-Performance Liquid Chromatography (HPLC) revealed the presence of different bioactive plant metabolites, known to impart hepatoprotective effects. In human hepatocarcinoma (HepG2) cells, co-treatment of SKK with CCl4 effectively reduced the hepatotoxicity induced by the latter. These effects were confirmed by studying parameters such as loss of cell viability; release of hepatic injury enzymatic biomarkers- aspartate aminotransferase (AST), and alkaline phosphatase (ALP); and changes in reactive oxygen species and in mitochondrial membrane potentials. In-vivo safety analysis in Wistar rats showed no loss in animal body weight, or change in feeding habits after repeated oral dosing of SKK up to 1,000 mg/kg/day for 28 days. Also, no injury-related histopathological changes were observed in the animal's blood, liver, kidney, heart, brain, and lung. Pharmacologically, SKK played a significant role in modulating CCl4 induced hepatic injuries in the Wistar rats at a higher dose. In the 9 weeks' study, SKK (200 mg/kg) reduced the CCl4 stimulated increase in the release of enzymes (ALT, AST, and ALP), bilirubin, total cholesterol, and uric acid levels in the Wistar rats. It also reduced the CCl4 stimulated inflammatory lesions such as liver fibrosis, lymphocytic infiltration, and hyper-plasticity. In conclusion, SKK showed pharmacological effects in improving the CCl4 stimulated liver injuries in HepG2 cells and in Wistar rats. Furthermore, no adverse effects were observed up to 10× higher human equivalent dose of SKK during 28-days repeated dose exposure in Wistar rats. Based on the literature search on the identified plant metabolites, SKK was found to act in multiple ways to ameliorate CCl4 induced hepatotoxicity. Therefore, polyherbal SKK medicine has shown remarkable potentials as a possible alternative therapeutics for reducing liver toxicity induced by drugs, and other toxins.

Calcio-Herbal Medicine Divya-Swasari-Vati Ameliorates SARS-CoV-2 Spike Protein-Induced Pathological Features and Inflammation in Humanized Zebrafish Model by Moderating IL-6 and TNF-α Cytokines.

PURPOSE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection has grown into a pandemic and without a specific cure, disease management is the need of the hour through symptomatic interventions. Studies with severe acute respiratory syndrome-coronavirus (SARS-CoV) have highlighted the role of herbal medicines either in combination with antiviral drugs or by themselves in curtailing the severity of infection and associated inflammation. Divya-Swasari-Vati is an Indian ayurvedic formulation used in the treatment of chronic cough and lung inflammation, which is one of the first symptoms of SARS-CoV-2 infections. METHODS: In this study, we used a A549 cell xenotransplant in the swim bladder of zebrafish and modeled the SARS-CoV-2 infection by injecting the fish with a recombinant spike protein. The different groups were given normal feed or feed mixed with either dexamethasone (as the control drug) or Divya-Swasari-Vati. The changes in behavioral fever, infiltration of pro-inflammatory cells in the swim bladder, degeneration or presence of necrotic cells in the kidney, and gene expression of pro-inflammatory cytokines were studied to determine the rescue of the diseased phenotype. RESULTS: Challenge with the spike protein caused changes in the swim bladder cytology with infiltrating pro-inflammatory cells, skin hemorrhage, and increase in behavioral fever. This was also accompanied by increased mortality of the disease control fish. Treatment with Divya-Swasari-Vati reversed most of the disease symptoms including damage to the kidney glomerulocytes, and complete reversal of behavioral fever. Dexamethasone, used as a comparator, was only able to partly rescue the behavioral fever phenotype. Divya-Swasari-Vati also suppressed the pro-inflammatory cytokines, IL-6 and TNF-α, levels in a dose-dependent manner, under in vivo and in vitro conditions. CONCLUSION: The study showed that the A549 xenotransplanted zebrafish injected with the recombinant spike protein of SARS-CoV-2 is an efficient model for the disease; and treatment with Divya-Swasari-Vati medicine rescued most of the inflammatory damage caused by the viral spike protein while increasing survival of the experimental fish.

Anti-Acetylcholinesterase Activities of Mono-Herbal Extracts and Exhibited Synergistic Effects of the Phytoconstituents: A Biochemical and Computational Study.

Alzheimer's disease (AD), a neurodegenerative disease, is the most common form of dementia. Inhibition of acetylcholinesterase (AChE) is a common strategy for the treatment of AD. In this study, aqueous, hydro-methanolic, and methanolic extracts of five potent herbal extracts were tested for their in vitro anti-AChE activity. Among all, the Tinospora cordifolia (Giloy) methanolic fraction performed better with an IC50 of 202.64 µg/mL. Of the HPLC analyzed components of T. cordifolia (methanolic extract), palmatine and berberine performed better (IC50 0.66 and 0.94 µg/mL, respectively) as compared to gallic acid and the tool compound "galantamine hydrobromide" (IC50 7.89 and 1.45 µg/mL, respectively). Mode of inhibition of palmatine and berberine was non-competitive, while the mode was competitive for the tool compound. Combinations of individual alkaloids palmatine and berberine resulted in a synergistic effect for AChE inhibition. Therefore, the AChE inhibition by the methanolic extract of T. cordifolia was probably due to the synergism of the isoquinoline alkaloids. Upon molecular docking, it was observed that palmatine and berberine preferred the peripheral anionic site (PAS) of AChE, with π-interactions to PAS residue Trp286, indicating that it may hinder the substrate binding by partially blocking the entrance of the gorge of the active site or the product release.

Colchicine: Isolation, LC-MS QTof Screening, and Anticancer Activity Study of Gloriosa superba Seeds.

Colchicine was extracted from Gloriosa superba seeds using the Super Critical Fluid (CO2) Extraction (SCFE) technology. The seeds were purified upto 99.82% using column chromatography. Colchicine affinity was further investigated for anticancer activity in six human cancer cell lines, i.e., A549, MCF-7, MDA-MB231, PANC-1, HCT116, and SiHa. Purified colchicine showed the least cell cytotoxicity and antiproliferation and caused no G2/M arrest at clinically acceptable concentrations. Mitotic arrest was observed in only A549 and MDA-MB231 cell lines at 60nM concentration. Our finding indicated the possible use of colchicine at a clinically acceptable dose and provided insight into the science behind microtubule destabilization. However, more studies need to be conducted beforethese findings could be established.