Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the ß-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Design, Synthesis, and Pharmacological Evaluation of Embelin-Aryl/alkyl Amine Hybrids as Orally Bioavailable Blood-Brain Barrier Permeable Multitargeted Agents with Therapeutic Potential in Alzheimer's Disease: Discovery of SB-1448.

The complex and multifaceted nature of Alzheimer's disease has brought about a pressing demand to develop ligands targeting multiple pathways to combat its outrageous prevalence. Embelin is a major secondary metabolite of Embelia ribes Burm f., one of the oldest herbs in Indian traditional medicine. It is a micromolar inhibitor of cholinesterases (ChEs) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) with poor absorption, distribution, metabolism, and excretion (ADME) properties. Herein, we synthesize a series of embelin-aryl/alkyl amine hybrids to improve its physicochemical properties and therapeutic potency against targeted enzymes. The most active derivative, 9j (SB-1448), inhibits human acetylcholinesterase (hAChE), human butyrylcholinesterase (hBChE), and human BACE-1 (hBACE-1) with IC50 values of 0.15, 1.6, and 0.6 µM, respectively. It inhibits both ChEs noncompetitively with ki values of 0.21 and 1.3 µM, respectively. It is orally bioavailable, crosses blood-brain barrier (BBB), inhibits Aß self-aggregation, possesses good ADME properties, and protects neuronal cells from scopolamine-induced cell death. The oral administration of 9j at 30 mg/kg attenuates the scopolamine-induced cognitive impairments in C57BL/6J mice.

Techniques for analytical estimation of COVID-19 clinical candidate, niclosamide in pharmaceutical and biomedical samples.

Niclosamide is a well-known broad-spectrum antiparasitic drug used for human as well as veterinary tapeworm infections. Recently, it attracted attention as an antiviral agent for treating coronavirus disease 2019. It is administered orally in humans to treat tapeworm infections. Furthermore, it is a registered pesticide and molluscicide to control infections in the aquaculture industry. Its chronic environmental exposure has potential toxicities when such contaminated seafood is consumed. Therefore, monitoring its residual concentration in food products (seafood, water, water waste, etc.) and pharmaceuticals (active pharmaceutical ingredients, bulk drugs, and formulations) is imperative. The present review critically investigates the sophisticated techniques employed for analyzing niclosamide, its degradation products, and metabolites in various samples and matrices. The future scope for green analytical methods, green sample extraction and preparation is also deliberated.

Estimating thermodynamic equilibrium solubility and solute-solvent interactions of niclosamide in eight mono-solvents at different temperatures.

Niclosamide is an FDA-approved oral anthelmintic drug currently being repurposed for COVID-19 infection. Its interesting applicability in multiple therapeutic indications has sparked interest in this drug/ scaffold. Despite its therapeutic use for several years, its detailed solubility information from Chemistry Manufacturing & Controls perspective is unavailable. Thus, the present study is intended to determine the mole fraction solubility of niclosamide in commonly used solvents and cosolvents at a temperature range of 298.15-323.15 K. The polymorphic changes from crystalline to monohydrate forms post-equilibration in various solvents were observed. The maximum mole fraction solubility of niclosamide at 323.15 K is 1.103 × 10-3 in PEG400, followed by PEG200 (5.272 × 10-4), 1-butanol (3.047 × 10-4), 2-propanol (2.42 × 10-4), ethanol (1.66 × 10-4), DMSO (1.52 × 10-4), methanol (7.78 × 10-5) and water (3.27 × 10-7). The molecular electrostatic potential showed a linear correlation with the solubility. PEG400 has higher electrostatic potential, and H-bond acceptor count, which forms a hydrogen bond with phenolic -OH of niclosamide and thus enhances its solubility. This data is valuable for the drug discovery and development teams working on the medicinal chemistry and process chemistry of this scaffold.

Molecular interactions of niclosamide with hydroxyethyl cellulose in binary and ternary amorphous solid dispersions for synergistic enhancement of water solubility and oral pharmacokinetics in rats.

The cellulose-based polymers are extensively employed in oral formulations for addressing ADMET issues of API. Herein, we report the synergistic effect of hydroxyethyl cellulose in solubility/dissolution enhancement of BCS class II, anthelmintic drug niclosamide. The low solubility and poor oral bioavailability are the primary reasons for its high daily dose. The amorphous solid dispersions (ASDs) developed herein demonstrated reproducible solubility and dissolution enhancement in smaller-to-pilot batches. The significant boost in niclosamide solubility in HEC-based binary SD was rationalized as a result of intermolecular H-bonding as indicated by in-silico studies and further supported by characterization data. HEC is plausibly inhibiting the precipitation of drug and thereby enabling high dissolution and permeation across the membrane. The comparative oral pharmacokinetics in Wistar rats at 25 mg/kg provided 4.4-fold higher plasma exposure of niclosamide in SD formulation SB-ASD-N2 over the plain drug. The results presented herein warrant validation of this ASD under clinical settings.

Analytical Methods for Furanochromone Natural Product, Khellin and Its Inspired Drug Candidates, Amiodarone and Sodium Cromoglycate.

Khellin is the key furanocoumarin of Ammi visnaga L. (Lam.) that exhibits various biological properties. This unique natural product has inspired the discovery of two first-in-class drugs, amiodarone and sodium cromoglycate. A wide range of analytical methods were generated while translating khellin scaffold into clinically used drugs; however, they have never been reviewed and critically assessed. The present review aims to review and evaluate the analytical techniques for the natural products, khellin, visnagin, and their inspired drugs, amiodarone and sodium cromoglycate. High-performance liquid chromatography (HPLC) is the extensively used technique in most analytical methods reported for these compounds; however, other techniques including the fluorimetry, luminescence spectrophotometry, potentiometry, voltammetry, FT-Raman spectroscopy, and ELISA were also employed. The review will be helpful for further basic and translational research on furanochromone and related scaffolds.

Phosphate moiety in FDA-approved pharmaceutical salts and prodrugs.

The salification and prodrug approaches modulate the physicochemical properties and absorption, distribution, metabolism, excretion, and toxicity parameters of drugs and lead candidates. The "phosphate" is one of the key counterions/promoiety used in the salt formation and prodrug synthesis. Salification with phosphoric acid enhances the aqueous solubility and thereby facilitates the administration of a drug by the parenteral route. Phosphate moiety in prodrug synthesis mainly improves permeability by lipophilic substitution. Histamine phosphate is the first phosphate salt, and hydrocortisone phosphate was the first prodrug approved by FDA in 1939 and 1952, respectively. The orange book enlists 12 phosphate salts and 17 phosphate prodrugs. Phosphate prodrugs, namely combretastatin A-4 diphosphate, combretastatin A-4 phosphate, lufotrelvir, TP-1287, pyridoxal phosphate, riboflavin phosphate, and psilocybin are clinical candidates. This review focuses on the FDA-approved phosphate salts and prodrugs from 1939 to 2021. The biopharmaceutical advantage of phosphate salts and prodrugs over the parent molecule is also deliberated.

Recent developments in the management of Huntington's disease.

Huntington's disease (HD) is a rare, incurable, inheritedneurodegenerative disorder manifested by chorea, hyperkinetic, and hypokinetic movements. The FDA has approved only two drugs, viz. tetrabenazine, and deutetrabenazine, to manage the chorea associated with HD. However, several other drugs are used as an off-label to manage chorea and other symptoms such as depression, anxiety, muscle tremors, and cognitive dysfunction associated with HD. So far, there is no disease-modifying treatment available. Drug repurposing has been a primary drive to search for new anti-HD drugs. Numerous molecular targets along with a wide range of small molecules and gene therapies are currently under clinical investigation. More than 200 clinical studies are underway for HD, 75% are interventional, and 25% are observational studies. The present review discusses the small molecule clinical pipeline and molecular targets for HD. Furthermore, the biomarkers, diagnostic tests, gene therapies, behavioral and observational studies for HD were also deliberated.

Analysis of clinical trials on biomaterial and therapeutic applications of chitosan: A review.

Chitosan is a modified natural carbohydrate polymer derived from chitin that occurs in many natural sources. It has a diverse range of applications in medical and pharmaceutical sciences. Its primary and permitted use is biomaterial in medical devices. Chitosan and its derivatives also find utility in pharmaceuticals as an excipient, drug carrier, or therapeutic agent. The USFDA has approved chitosan usage as a biomaterial but not for pharmaceutical use, primarily because of the concerns over its source, purity, and immunogenicity. A large number of clinical studies are underway on chitosan-based materials/ products because of their diverse applications. Herein, we analyze clinical studies to understand their clinical usage portfolio. Our analysis shows that >100 clinical studies are underway to investigate the safety/efficacy of chitosan or its biomaterials/ nanoparticles, comprising ~95% interventional and ~ 5% observational studies. The regulatory considerations that limit the use of chitosan in pharmaceuticals are also deliberated. TEASER: Clinical Trials of Chitosan.

Enhancing Biopharmaceutical Attributes of Khellin by Amorphous Binary Solid Dispersions.

Khellin, a furanochromone isolated from fruits and seeds of Ammi visnaga, is traditionally used in many eastern Mediterranean countries. The plant decoction and the crystalline substance khellin have many pharmacological activities. For instance, it acts as a bronchodilator and also relieves renal colic and urethral stones, etc. However, the low water solubility (~ 120 µg/mL) and low bioavailability limit its therapeutic application. Thus, the present research explores the development of its binary and ternary solid dispersion formulations to improve its solubility and dissolution behavior. A 24-well plate miniaturized protocol was established to identify the optimal hydrophilic polymer to prepare its solid dispersions. PEG-4000 was recognized as the favorable hydrophilic carrier in preparation of solid dispersion, SSB17. The formulation displayed ~ five-fold enhancement in the aqueous solubility of khellin. The binary solid dispersion SSB17 was manufactured at a gram scale and evaluated using 1H-NMR, 13C-NMR, FT-IR, p-XRD, SEM, DSC, in vitro dissolution, and predicted pharmacokinetics. The quantitative dissolution data of SSB17 demonstrated ~ 2-3-fold improvement in AUC at physiological pH conditions. These conclusions highlight the basis for further preclinical studies on solid dispersions of khellin with improved biopharmaceutical properties.

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.

Modulation of biopharmaceutical properties of acidic drugs using cationic counterions: A critical analysis of FDA-approved pharmaceutical salts.

Salification has a successful track record in modulating the biopharmaceutical properties of drugs. This is evident from the significant share (40%) of pharmaceutical salts in FDA-approved drugs in the past 80-years. Based on the ionic nature of drugs, the corresponding cationic or anionic counterions are employed for salification. This review aims to provide the contribution of cationic counterions in FDA-approved drugs from 1939 to 2020. The analysis of 80-years data has shown that the 7.1% of the FDA-approved drugs comprise cationic counterions (98 pharmaceutical salts). Heparin sodium is the pioneering drug in the history of pharmaceutical salts that was approved in 1939 as an anticoagulant medication. Inorganic (sodium, calcium, potassium, magnesium, silver), as well as organic (tromethamine, meglumine, erbumine) cationic counterions, were used in FDA-approved drugs with a major share by sodium (76 drugs). The technical superiority of cationic salts over other salt forms and the parent drug is also exemplified using case studies.

Identification of plant-based multitargeted leads for Alzheimer's disease: In-vitro and in-vivo validation of Woodfordia fruticosa (L.) Kurz.

BACKGROUND: Alzheimer's disease (AD) is a complex neurodegenerative disease with no availability of disease-modifying therapeutics. The complex etiology and recent failures in clinical trials indicate the need for multitargeted agents. PURPOSE: The present study aims to discover new plant-based multitargeted anti-AD leads. METHODS: A library of plant extracts was screened for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1). The secondary metabolites of active extracts were also tested, followed by enzyme-kinetics and molecular modeling to understand the mechanism of inhibition. The most active extract was investigated for in-vivo anti-dementia activity in behavioral mice models. RESULTS: Among the library of 105 extracts, Woodfordia fruticosa (SBE-80) and Bergenia ciliata (SBE-65) extracts displayed significant inhibition of all three enzymes. Gallic acid, one of the constituents of both plants, shows moderate inhibition of AChE and BACE-1. Catechin-3-O-gallate (CG), another constituent of SBE-65, inhibits EeAChE, rHuAChE, and eqBChE with IC50's of 29.9, 1.77, and 8.4 µM, respectively; along with a mild-inhibition of BACE-1. Ellagic acid, the constituent of SBE-80, inhibits BACE-1 with an IC50 value of 16 µM. The W. fruticosa extract SBE-80 at the dose of 25 mg/kg QD × 9 (PO) displayed memory-enhancing activity in Morris Water Maze and Passive Avoidance Test in Swiss albino mice. Treatment with SBE-80 also inhibits AChE in-vivo; whereas, a non-significant decrease in the serum TBARS was observed. CONCLUSION: W. fruticosa is identified for the first time as an anti-AD lead candidate. The in-vitro and in-vivo data presented herein and the documented safety profile of W. fruticosa indicate its strong potential for preclinical development as a botanical drug for dementia/AD.

Carboxylic Acid Counterions in FDA-Approved Pharmaceutical Salts.

Salification is one of the powerful and widely employed approaches to improve the biopharmaceutical properties of drugs. The FDA's eighty-year trajectory of new drug approvals depicts around one-third of the drugs clinically used as their pharmaceutical salts. Among various cationic and anionic counterions used in FDA-approved pharmaceutical salts, the carboxylic acids have significantly contributed. A total of 94 pharmaceutical salts discovered during 1943-2020 comprises carboxylic acids as counterions with a major contribution of acetate, maleate, tartrate, fumarate, and succinate. Hydrocodone tartrate is the first FDA-approved carboxylate salt approved in 1943. Overall, the analysis shows that fifteen carboxylic acid counterions are present in FDA-approved pharmaceutical salts with a major share of acetate (18 drugs). This review provides an account of FDA-approved carboxylate salts from 1939 to 2020. The decade-wise analysis indicates that 1991-2000 contributed a maximum number of carboxylate salts (24) and least (3) in 1939-1950. The technical advantage of carboxylate salts over free-base or other counterions is also discussed. Graphical Abstract.

Critical Analysis of Drug Product Recalls due to Nitrosamine Impurities.

A product recall is the outcome of a careful pharmacovigilance; and it is an integral part of drug regulation. Among various reasons for product recall, the detection of unacceptable levels of carcinogenic impurities is one of the most serious concerns. The genotoxic and carcinogenic potential of N-nitrosamines raises a serious safety concern, and in September 2020, the FDA issued guidance for the pharmaceutical industry regarding the control of nitrosamines in drug products. The FDA database shows that >1400 product lots have been recalled from the market due to the presence of carcinogenic N-nitrosamine impurities at levels beyond the acceptable intake limit of 26.5 ng/day. The drugs that were present in recalled products include valsartan, irbesartan, losartan, metformin, ranitidine, and nizatidine. This perspective provides a critical account of these product recalls with an emphasis on the source and mechanism for the formation of N-nitrosamines in these products.

Crocetin promotes clearance of amyloid-ß by inducing autophagy via the STK11/LKB1-mediated AMPK pathway.

Alzheimer disease (AD) is usually accompanied by two prominent pathological features, cerebral accumulation of amyloid-ß (Aß) plaques and presence of MAPT/tau neurofibrillary tangles. Dysregulated clearance of Aß largely contributes to its accumulation and plaque formation in the brain. Macroautophagy/autophagy is a lysosomal degradative process, which plays an important role in the clearance of Aß. Failure of autophagic clearance of Aß is currently acknowledged as a contributing factor to increased accumulation of Aß in AD brains. In this study, we have identified crocetin, a pharmacologically active constituent from the flower stigmas of Crocus sativus, as a potential inducer of autophagy in AD. In the cellular model, crocetin induced autophagy in N9 microglial and primary neuron cells through STK11/LKB1 (serine/threonine kinase 11)-mediated AMP-activated protein kinase (AMPK) pathway activation. Autophagy induction by crocetin significantly increased Aß clearance in N9 cells. Moreover, crocetin crossed the blood-brain barrier and induced autophagy in the brains' hippocampi of wild-type male C57BL/6 mice. Further studies in transgenic male 5XFAD mice, as a model of AD, revealed that one-month treatment with crocetin significantly reduced Aß levels and neuroinflammation in the mice brains and improved memory function by inducing autophagy that was mediated by AMPK pathway activation. Our findings support further development of crocetin as a pharmacological inducer of autophagy to prevent, slow down progression, and/or treat AD.Abbreviations: Aß: amyloid-ß; ABCB1/P-gp/P-glycoprotein: ATP-binding cassette, subfamily B (MDR/TAP), member 1; AD: Alzheimer disease; AMPK/PRKAA: AMP-activated protein kinase; APP: amyloid beta (A4) precursor protein; ATG: autophagy related; BBB: blood-brain barrier; BECN1: beclin 1, autophagy related; CAMKK2/CaMKKß: calcium/calmodulin-dependent protein kinase kinase 2, beta; CSE: Crocus sativus extract; CTSB: cathepsin B; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; Kp: brain partition coefficient; LRP1: low density lipoprotein receptor-related protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK/ERK: mitogen-activated protein kinase; MAPT/tau: microtubule-associated protein tau; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTOR: mechanistic target of rapamycin kinase; MWM: Morris water maze; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NMDA: N-methyl-d-aspartic acid; RPTOR: regulatory associated protein of MTOR; RPS6KB1/p70S6K: ribosomal protein S6 kinase 1; SQSTM1: sequestosome 1; SRB: sulforhodamine B; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; TSC2: TSC complex subunit 2; ULK1: unc-51 like kinase 1.

Recent developments in pharmaceutical salts: FDA approvals from 2015 to 2019.

Around half of the new molecular entities approved by the US Food and Drug Administration (FDA) are pharmaceutical salts. The pharmaceutical salts have been on a continuous growth trajectory since the approval of the first salt form in 1939. This review aims to provide updates on pharmaceutical salts approved by the FDA between 2015 and 2019. The five-year drug-approval database contains 61 pharmaceutical salts, featuring a diverse range of counterions; however, hydrochlorides are the most abundant. The chemical structures of all pharmaceutical salts in each class are presented here, along with their therapeutic indications and date of approval. The reason behind the selection of a particular counterion and the technical superiority achieved by the salt form over the free active pharmaceutical ingredient base are also discussed.

Evaluation of rohitukine-enriched fraction of Dysoxylum binectariferum Hook.f. (leaves) as anti-arthritic phytopharmaceutical candidate: Chemical standardization, in-vivo validation, formulation development and oral pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis is a chronic inflammatory disease of joints. Dysoxylum binectariferum Hook.f (Family: Meliaceae) is a Indian medicinal plant which is traditionally being used to heal inflammation of joints. AIM OF THE STUDY: This work was aimed to carry out chemical standardization, in-vitro/in-vivo validation, oral pharmacokinetics and formulation development of anti-arthritic botanical lead, the rohitukine-enriched fraction of D. binectariferum. MATERIALS AND METHODS: The rohitukine-enriched fraction of D. binectariferum was standardized using four chemical markers and was checked for microbial load, heavy metal content, aflatoxins and pesticides. Its in-vitro inhibitory effect on the lipopolysaccharide (LPS) induced production of pro-inflammatory cytokines TNF-α and IL-6 was studied in THP-1 cells. The in-vivo anti-arthritic activity was investigated in collagen-induced arthritis model in DBA/1J mice. The sustained release capsule formulation was developed and characterized for physicochemical and pharmacokinetic properties. RESULTS: Rohitukine and schumaniofioside A were found to be major chemical constituents of the botanical lead. The rohitukine-enriched fraction of D. binectariferum significantly reduced the production of both pro-inflammatory cytokines TNF-α and IL-6 (>50% inhibition at 3.12 µg/mL) in THP-1 cells. In LPS-treated wild-type mice model, the rohitukine-enriched fraction at 200 mg/kg (PO, QD) completely reduced serum TNF-α levels. In transgenic mice model (collagen-induced arthritis in DBA/1J mice), rohitukine-enriched fraction at 100 mg/kg (PO, QD) dose has resulted in >75% reduction of TNF-α/IL-6 serum levels, 68% reduction in anti-mouse type II collagen IgG1 antibody levels, decreased joint proteoglycan loss and reduced paw edema in DBA/1J mice. The sustained release capsule formulation of rohitukine-enriched fraction showed sustained-release of rohitukine over the period of 24 h, and resulted in an improved plasma-exposure of rohitukine in SD rats. CONCLUSIONS: The data presented herein demonstrated anti-arthritic potential of rohitukine-enriched fraction of D. binectariferum and this study will serve as the benchmark for further research on this botanical lead and developed sustained release capsule formulation.

Chemical analysis of saffron by HPLC based crocetin estimation.

Saffron is one of the most expensive and valuable spice having tremendous commercial value in food industry and thus its quality check is of utmost importance. Crocins are unique group of extremely hydrophilic apocarotenoids which are main components of saffron. Crocetin is an aglycone of crocins which do occur naturally in saffron, and is produced in biological system as a bioactive metabolite via hydrolytic cleavage of crocins. Crocins are unstable and tend to undergo isomerisation/ inter-conversions, and therefore their quantitative estimation is difficult. Herein, we have established for the first time, a crocetin-based HPLC method to evaluate the total crocin content of saffron, and thereby analyze the quality of saffron. The present approach comprises alkali-mediated conversion of crocins to crocetin in raw material followed by quantitative estimation of in-situ formed crocetin by HPLC analysis. The unique and efficient protocol for preparation of high purity analytical grade 'crocetin' directly from saffron has also been established. It is simple and efficient way to check the quality of saffron/ saffron-containing products.