Development and Validation of Novel Quality Evaluation Methods to Differentiate Two Closely Related Species of Tinospora: A Rapid HPTLC- and HPLC-Based Assessment with MS/MS Characterization.

BACKGROUND: The sympatric occurrence of the species that often resulted in different gatherings of plant material, ambiguous history on traditional use, and taxonomic flux due to similarities within the Tinospora (Menispermaceae) taxa are some of the reasons that triggered the necessity to develop robust analytical methods for efficient QC, especially to recognize dry and powder forms. OBJECTIVE: To develop novel HPTLC-based fingerprinting of two closely resembling Tinospora species followed by HPTLC-MS analysis and identification of compounds differentiating Tinospora crispa (TCP) and Tinospora cordifolia (TCR) and a rapid and quantitative assessment by HPLC with a photodiode array detector (HPLC-PDA) with MS/MS characterization of specific TCP and TCR analytical markers. METHODS: An HPTLC-based method was developed using chloroform-toluene-methanol-formic acid (7 + 4 + 2 + 0.2, by volume). The TCP compounds could be distinguished and isolated using successive column chromatography with complete characterization. Further these used in the reverse phase (RP)-HPLC-PDA coupled with LC-ESI (electrospray ionization)-MS/MS to quantify and confirmation in TCP and TCR. RESULTS: The fingerprinting showed distinct bands in TCP stems, confirmed as clerodane- furanoditerpenoids with indirect profiling by the HPTLC-MS technique. Systematic isolation confirmed these compounds as borapetosides B and E. Thus, the RP-HPLC-PDA method was developed for these borapetosides B and E, with tinosporide to differentiate these two species. The quantitation method was well validated with good linearity (r2 >0.99) with sensitive LOD (0.49-3.71 mcg/mL) and LOQ (1.48-11.23 mcg/mL) with recoveries of 92.34-96.19%. CONCLUSION: A novel, validated HPLC-PDA method showed good resolution and reliability (up to 1% adulteration) in quantification for targeted major analytical markers from TCP to differentiate TCR. Thus, HPTLC and HPLC-PDA-based techniques are helpful with MS/MS-based characterization to identify and quantify these analytical markers from TCP (borapetoside B and E) and TCR (tinosporide) in dry and powder form. HIGHLIGHTS: This article reports on the systemic use of HPTLC-MS for separating and identifying analytical markers in Tinospora species, distinguishing TCR and TCP with quantitative HPLC-PDA and MS/MS assessment.

Recent Advancements in Extraction Techniques of Ashwagandha (Withania somnifera) with Insights on Phytochemicals, Structural Significance, Pharmacology, and Current Trends in Food Applications.

Ashwagandha, also known as Withania somnifera (WS), is an ayurvedic botanical plant with numerous applications in dietary supplements and traditional medicines worldwide. Due to the restorative qualities of its roots, WS has potent therapeutic value in traditional Indian (Ayurvedic, Unani, Siddha) and modern medicine recognized as the "Indian ginseng". The presence of phytochemical bioactive compounds such as withanolides, withanosides, alkaloids, flavonoids, and phenolic compounds has an important role in the therapeutic and nutritional properties of WS. Thus, the choice of WS plant part and extraction solvents, with conventional and modern techniques, plays a role in establishing WS as a potential nutraceutical product. WS has recently made its way into food supplements and products, such as baked goods, juices, beverages, sweets, and dairy items. The review aims to cover the key perspectives about WS in terms of plant description, phytochemistry, structural significance, and earlier reported extraction methodologies along with the analytical and pharmacological landscape in the area. It also attempts to iterate the key limitations and further insights into extraction techniques and bioactive standardization with the regulatory framework. It presents a key to the future development of prospective applications in foods such as food supplements or functional foods.

Recent insights into the hepatoprotective potential of medicinal plants and plant-derived compounds.

Liver problems are a worldwide concern, and conventional medicinal therapies are ineffective. Hence, safeguarding the healthy liver is vital for good health and well-being. Infections due to virus, immune problems, cancer, alcohol abuse, and an overdose of drugs are some of the causes of liver diseases. Antioxidants derived from medicinal plants and conventional dietary sources can protect the liver from damages caused by oxidative stress system and various chemicals. Plants and plant-derived phytochemicals are appealing hepatoprotective agents since they have less side effects and still there is a lot of interest shown in using herbal tonics for treating liver disorders. This review therefore primarily focuses on newly discovered medicinal plants and compounds produced from plants that fall under the classifications of flavonoids, alkaloids, terpenoids, polyphenolics, sterols, anthocyanins, and saponin glycosides, all of which have the potential to be hepatoprotective. Hosta plantaginea, Ligusticum chuanxiong, Daniella oliveri, Garcinia mangostana, Solanum melongena, Vaccinium myrtillus, Picrorhiza kurroa, and Citrus medica are some potential plants having hepatoprotective effects. We conclude that these phytochemicals and the plant extracts listed above are used in the future to treat a variety of liver diseases, additional research is still needed to develop safer and more potent phytochemical drugs.

Bioanalytical Method Development and Validation Study of Neuroprotective Extract of Kashmiri Saffron Using Ultra-Fast Liquid Chromatography-Tandem Mass Spectrometry (UFLC-MS/MS): In Vivo Pharmacokinetics of Apocarotenoids and Carotenoids.

Kashmir saffron (Crocus sativus L.), also known as Indian saffron, is an important Asian medicinal plant with protective therapeutic applications in brain health. The main bioactive in Kashmir or Indian Saffron (KCS) and its extract (CSE) are apocarotenoids picrocrocin (PIC) and safranal (SAF) with carotenoids, crocetin esters (crocins), and crocetins. The ultra-fast liquid chromatography(UFLC)- photodiode array standardization confirmed the presence of biomarkers PIC, trans-4-GG-crocin (T4C), trans-3-Gg-crocin (T3C), cis-4-GG-crocin (C4C), trans-2-gg-crocin (T2C), trans-crocetin (TCT), and SAF in CSE. This study's objectives were to develop and validate a sensitive and rapid UFLC-tandem mass spectrometry method for PIC and SAF along T4C and TCT in rat plasma with internal standards (IS). The calibration curves were linear (R2 > 0.990), with the lower limit of quantification (LLOQ) as 10 ng/mL. The UFLC-MS/MS assay-based precision (RSD, <15%) and accuracy (RE, -11.03-9.96) on analytical quality control (QC) levels were well within the acceptance criteria with excellent recoveries (91.18-106.86%) in plasma samples. The method was applied to investigate the in vivo pharmacokinetic parameters after oral administration of 40 mg/kg CSE in the rats (n = 6). The active metabolite TCT and T4C, PIC, SAF were quantified for the first time with T3C, C4C, T2C by this validated bioanalytical method, which will be useful for preclinical/clinical trials of CSE as a potential neuroprotective dietary supplement.

Applications of natural product-derived carbon dots in cancer biology.

Natural products have contributed conspicuously to the development of innovative nanomedicines. Hence, the interface between nanomaterial science and plant natural products may bestow comprehensive diagnostic and therapeutic strategies for tackling diseases such as cancer and neurological disorders. Natural product-derived carbon dots (NPdCDs) have revealed noteworthy attributes in the fields of cancer theranostics, microbial imaging, drug sensing and drug delivery. As plants consist of a cocktail of bioactive phytomolecules, the NPdCDs can be anticipated to have medicinal properties, biocompatibility, photo-stability and easy functionalization. NPdCDs have wide-ranging applications. The primary objective of this review is to comment on recent developments in the use of NPdCDs, with special reference to their application in cancer biology. The future of the use of NPdCDs has also been considered.

Investigating 11 Withanosides and Withanolides by UHPLC-PDA and Mass Fragmentation Studies from Ashwagandha (Withania somnifera).

Withania somnifera (WS), also known as ashwagandha or Indian ginseng, is known for its pharmacological significance in neurodegenerative diseases, stress, cancer, immunomodulatory, and antiviral activity. In this study, the WS extract (WSE) from the root was subjected to ultrahigh-performance liquid chromatography with photodiode array detection (UHPLC-PDA) analysis to separate 11 withanoside and withanolide compounds. The quantification validation was carried out as per ICHQ2R1 guidelines in a single methodology. The calibration curves were linear (r 2 > 0.99) for all 11 compounds within the tested concentration ranges. The limits of detection and quantification were in the range of 0.213-0.362 and 0.646-1.098 µg/mL, respectively. The results were precise (relative standard deviation, <5.0%) and accurate (relative error, 0.01-0.76). All compounds showed good recoveries of 84.77-100.11%. For the first time, withanoside VII, 27-hydroxywithanone, dihydrowithaferin A, and viscosalactone B were quantified and validated along with bioactive compounds withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, and withanolide B simultaneously in WS. This UHPLC-PDA method has practical adaptability for ashwagandha raw material, extract, and product manufacturers, along with basic and applied science researchers. The method has been developed on UHPLC for routine analysis. The 11 withanosides and withanolides were confirmed using the fragmentation pattern obtained by the combined use of electrospray ionization and collision-induced dissociation in triple-quadrupole tandem mass spectrometry (TQ-MS/MS) in the WSE.

Assessment of Curcuma longa extract for adulteration with synthetic curcumin by analytical investigations.

"Curcumin (CUR)" is the principal active phytoconstituent present in Curcuma longa (CL), also known as Turmeric, is a popular natural product used in food and dietary supplements industries. For economic advantage, CUR is manufactured synthetically. The synthetic curcumin (SC) could be mislabeled, mistaken, or mixed with natural origin CL or CL extract (CLE) or CL products for replenishing CUR. The study aimed to differentiate CLE and SC by targeting CIMP-1,i.e. (1E,4Z)-5-hydroxy-1-(3-hydroxy-4-methoxyphenyl) hexa-1,4-dien-3-one by HPLC-PDA (photodiode array) and HPTLC-DS (densitometry) based on unique patterns. The validated HPLC-PDA method for CIMP-1 and CUR in SC showed robustness and sensitivity up to 1% adulteration with recovery, precision, and linearity of compounds as per guidelines. All four compounds were identified and confirmed by ESI-MS/MS. In this research, the presence of Boron (B) found as a qualitative indicator of SC (> 250.0 mg/kg) and CLE (< 2.0 mg/kg) by ICP-MS. Further, this HPLC-PDA method was successfully applied for sixteen samples of CLE procured across India, out of which four samples showed the presence of synthetically origin curcumin. This research is the first report of simple, lab-based methods for profiling of CUR based on natural or synthetic origin and identification of SC.