Integrated Multiplatform Analysis and Separation of Thirteen Flavonoids and Anthraquinones in Seven Medicinal Cassia Species.

BACKGROUND: Cassia (Family: Fabaceae) species are a large group of flowering plants rich in bioactive anthraquinone and flavonoids used in botanical supplements and nutraceuticals. OBJECTIVE: A simple and reliable high-performance liquid chromatography-photodiode array (HPLC-PDA) method was developed and validated for separating and quantifying thirteen anthraquinone and flavonoids. These compounds were further confirmed using an LC-based electrospray ionization mass spectrometry (ESI-MS/MS) method in the leaves and flowers of selected Cassia species. A simple and rapid HPTLC method was developed for chemical fingerprint analysis of all Cassia species. METHOD: All thirteen compounds were chromatographically separated on a Zorbax TC18 (4.6 x 250, 5 µm particle size) analytical column, and 0.1% formic acid and acetonitrile as elution solvents at a flow rate of 0.8 mL/min with detection at 259 nm. For HPTLC fingerprinting, the mobile phase compositions of toluene, ethyl acetate, and formic acid (5.5:4.2:0.6, v/v/v) were optimized to separate and identify all compounds using silica gel 60F254 aluminum plates. RESULTS: The validation data for the developed HPLC-PDA method for thirteen compounds showed good linearity (r2 > 0.99) with a sensitive LOD (0.082-1.969 mg/mL), LOQ (0.250-5.967 mg/mL), and excellent recoveries (85.22-100.32%). The quantification results were found to be precise and accurate (<5.0% and relative error), -0.77-0.44 with ESI-MS/MS confirmation in the Cassia samples. The novel HPTLC method was excellent separation for thirteen compounds, with Rf values ranging between 0.12- 0.61. CONCLUSIONS: The developed HPLC-PDA method was simple, and precise and could separate and quantify anthraquinones and flavonoids along with confirmation, using a novel LC-based ESI-MS/MS. The HPTLC method was found to be simple and precise, with excellent separation capabilities for these compounds. HIGHLIGHTS: This novel multiplatform approach successfully identified and quantified thirteen compounds simultaneously using an integration of data strategy in seven medicinally important Cassia species' leaves and flowers.

Saffron (Crocus sativus L.) extract attenuates chronic scopolamine-induced cognitive impairment, amyloid beta, and neurofibrillary tangles accumulation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Crocus sativus L. known as saffron, is a popular food condiment with a high aroma, deep colour, and long and thick threads (stigmas) cultivated in Iran, Morocco, Spain, Italy, China, Japan, France, Turkey, and India. In 'Ayurveda', saffron is acknowledged for its immunostimulant, aphrodisiac, cardiotonic, liver tonic, nervine tonic, carminative, diaphoretic, diuretic, emmenagogue, galactagogue, febrifuge, sedative, relaxant, and anxiolytic activities. The renowned Persian physician and philosopher, Avicenna, delineated saffron as an antidepressant, hypnotic, anti-inflammatory, hepatoprotective, bronchodilator, and aphrodisiac in his book, the Canon of Medicine. Within traditional Iranian Medicine (TIM), saffron is characterized as a mood elevator and a rejuvenator for the body and senses. Further, the ethnopharmacological evidence indicates that saffron has shown an effect against neurodegenerative disorders namely, dementia, Alzheimer's, and Parkinson's with its bioactive constituents i.e., carotenoids and apocarotenoids. AIM: The present study aimed to investigate the potential of standardized (Kashmir Saffron, India) Crocus sativus extract (CSE) in chronic scopolamine-induced cognitive impairment, amyloid beta (Aß) plaque, and neurofibrillary tangles (NFT) accumulation in rat brains by targeting AChE inhibition and scopolamine mechanistic effect. METHODS: The experimental animals were divided into six groups: group 1: normal control, group 2: scopolamine, group 3,4 and 5 rivastigmine tartrate, CSE (p.o. 10 mg/kg, 15 mg/kg, and 20 mg/kg) respectively. Each treatment group received scopolamine after 20 min of dosing, till 4 weeks. The effects of different treatments on learning, acquisition, and reversal memory were performed using a Morris water maze test. In addition to behavioral assessments, biochemical parameters such as AChE, IL-6, and antioxidants were measured in isolated brains. Histological observations were also conducted to assess the presence of Aß plaques and NFT. Furthermore, molecular docking was performed to explore the potential AChE inhibitory activity of the bioactive constituents of standardized CSE. RESULTS: Scopolamine produces memory impairment, and its chronic administration forms Aß plaque and NFT in rat brains. Supplementation with CSE in presence of scopolamine has shown remarkable effects on behavioural activity, special acquisition, and reversal memory. The CSE has also shown promising effects on AChE inhibition and antioxidant activity. The results of the docking study also indicate that trans-crocetin, i.e., a biologically active metabolite of Crocins, has strong AChE inhibitory activity, supported by an in vivo animal experiment. CONCLUSION: Supplementation with CSE significantly attenuates the formation of Aß plaque and NFT in the hippocampus at a dose of 20 mg/kg per day. In addition, CSE also counters scopolamine-induced neuroinflammation.

Withania Somnifera Extract Mitigates Experimental Acute Graft versus Host Disease Without Abrogating Graft Versus Leukemia Effect.

Acute graft versus host disease (aGvHD) is the major contributor of nonrelapse mortality in alloHSCT. It is associated with an inflammatory immune response manifesting as cytokine storm with ensuing damage to target organs such as liver, gut, and skin. Prevention of aGvHD while retaining the beneficial graft versus leukemia (GvL) effect remains a major challenge. Withania somnifera extract (WSE) is known for its anti-inflammatory, immune-modulatory, and anticancer properties, which are appealing in the context of aGvHD. Herein, we demonstrated that prophylactic and therapeutic use of WSE in experimental model of alloHSCT mitigates aGvHD-associated morbidity and mortality. In the prophylaxis study, a dose of 75 mg/kg of WSE offered greatest protection against death due to aGvHD (hazard ratio [HR] = 0.15 [0.03-0.68], P ≤ .01), whereas 250 mg/kg was most effective for the treatment of aGvHD (HR = 0.16 [0.05-0.5], P ≤ .01). WSE treatment protected liver, gut, and skin from damage by inhibiting cytokine storm and lymphocytic infiltration to aGvHD target organs. In addition, WSE did not compromise the GvL effect, as alloHSCT with or without WSE did not allow the leukemic A20 cells to grow. In fact, WSE showed marginal antileukemic effect in vivo. WSE is currently under clinical investigation for the prevention and treatment of aGvHD.

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.

Clinical safety and tolerability evaluation of Withania somnifera (L.) Dunal (Ashwagandha) root extract in healthy human volunteers.

BACKGROUND: Withania somnifera (L.) Dunal, known as Ashwagandha, is an adaptogen with significant importance in Ayurveda for its potential health benefits in strength ('balavardhan') and muscle growth ('mamsavardhan'). Despite numerous studies on its efficacy, limited research is reported on its clinical safety and tolerability in healthy individuals. OBJECTIVE: This research evaluated the tolerability and safety of standardized Withania somnifera root extract (WSE) capsules (AgeVel®/Witholytin®) at 1000 mg/day dose upon oral administration in healthy male participants. METHOD: A non-randomized, open-label, single-treatment clinical study included eighteen healthy male participants aged 18 to 60. The participants were administered a dose of 500 mg of the WSE capsules twice daily for four weeks. Each capsule contained not less than 7.50 mg of total withanolides. The study evaluated various indicators in a cohort of healthy participants throughout the trial, including vital signs, organ function tests, urine analysis, X-ray and ECG, cardiorespiratory endurance, body fat percentage, lean body weight, adverse events profile, and tolerability of the WSE capsules. RESULTS: The participant's physical, hematological, and biochemical characteristics were normal, and no significant alterations or irregularities were observed in safety metrics like liver, kidney, and thyroid functions after administering AgeVel®/Witholytin®. CONCLUSION: This study found that healthy male participants could consume a standardized WSE at a daily dosage of 1000 mg for four weeks without any adverse effects. Future research should focus on long-term safety assessments in male and female participants.

Clinical pharmacokinetic evaluation of Withania somnifera (L.) Dunal root extract in healthy human volunteers: A non-randomized, single dose study utilizing UHPLC-MS/MS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal; (Solanaceae), commonly known as Ashwagandha, is one of the most significant medicinal herbs in 'Ayurveda', a traditional Indian medicine used for centuries with evidence in scriptures. Ashwagandha was mentioned in old Ayurvedic medical literature such as Charaka Samhita and Sushruta Samhita for improving weight and strength, with multiple citations for internal and exterior usage in emaciation and nourishing the body. Ethnopharmacological evidence revealed that it was used to relieve inflammation, reduce abdominal swelling, as a mild purgative, and treat swollen glands. The root was regarded as a tonic, aphrodisiac, and emmenagogue in the Unani tradition of the Indian medicinal system. Further, Ashwagandha has been also described as an Ayurvedic medicinal plant in the Ayurvedic Pharmacopoeia of India extending informed therapeutic usage and formulations. Despite the widespread ethnopharmacological usage of Ashwagandha, clinical pharmacokinetic parameters are lacking in the literature; hence, the findings of this study will be relevant for calculating doses for future clinical evaluations of Ashwagandha root extract. AIM: This study aimed to develop a validated and highly sensitive bioanalytical method for quantifying withanosides and withanolides of the Ashwagandha root extract in human plasma to explore its bioaccessibility. Further to apply a developed method to perform pharmacokinetics of standardized Withania somnifera (L.) Dunal root extract (WSE; AgeVel®/Witholytin®) capsules in healthy human volunteers. METHODS: A sensitive, reliable, and specific ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of five major withanosides and withanolides (withanoside IV, withanoside V, withanolide A, withaferin A, and 12-deoxy-withastramonolide) in human plasma. Further for the study, eighteen healthy male volunteers (18-45 years) were enrolled in a non-randomized, open-label, single period, single treatment, clinical pharmacokinetic study and given a single dose (500 mg) of WSE (AgeVel®/Witholytin®) capsules containing not less than 7.5 mg of total withanolides under fasting condition. Later, pharmacokinetic profiles were assessed using the plasma concentration of each bioactive constituent Vs. time data. RESULTS: For all five constituents, the bioanalytical method demonstrated high selectivity, specificity, and linearity. There was no carryover, and no matrix effect was observed. Furthermore, the inter-day and intra-day precision and accuracy results fulfilled the acceptance criteria. Upon oral administration of WSE capsules, Cmax was found to be 0.639 ± 0.211, 2.926 ± 1.317, 2.833 ± 0.981, and 5.498 ± 1.986 ng/mL for withanoside IV, withanolide A, withaferin A, and 12-deoxy-withastramonolide with Tmax of 1.639 ± 0.993, 1.361 ± 0.850, 0.903 ± 0.273, and 1.375 ± 0.510 h respectively. Further, withanoside V was also detected in plasma; but its concentration was found below LLOQ. CONCLUSION: The novel and first-time developed bioanalytical method was successfully applied for the quantification of five bio-active constituents in human volunteers following administration of WSE capsules, indicating that withanosides and withanolides were rapidly absorbed from the stomach, have high oral bioavailability, and an optimum half-life to produce significant pharmacological activity. Further, AgeVel®/Witholytin® was found safe and well tolerated after oral administration, with no adverse reaction observed at a 500 mg dose.

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.

A randomized, controlled, comparative, proof-of-concept study to evaluate the efficacy and safety of Nisha-Amalaki capsules in prediabetic patients for preventing progression to diabetes.

BACKGROUND: Prediabetes is an intermediate state of hyperglycemia, which acts as a precursor to Diabetes mellitus if left untreated. Nisha (Curcuma longa) and Amalaki (Emblica officinalis) combination has been advocated as drugs of choice to treat the early manifestations of Diabetes mellitus. OBJECTIVE: This prospective, randomized, single-blind, placebo-controlled, comparative study was planned to assess the efficacy and safety of Nisha-Amalaki capsules in preventing progression to Diabetes mellitus in prediabetic patients when administered for 6 months. METHODS: The study was conducted on prediabetic participants randomized to receive either Nisha-Amalaki (500 mg) or placebo one capsule twice a day for six months. The effect of study medications on IDRS (Indian Diabetes Risk Score), BMI (Body Mass Index), blood sugar, serum insulin, HOMA-IR (Homeostasis Model Assessment-Estimated Insulin Resistance), HbA1c (glycated hemoglobin), oxidative markers, Ayurvedic symptoms and Quality of Life (QoL) scores was assessed at regular intervals. RESULTS: 58 of the 62 participants enrolled completed the study. Significant fall in IDRS score [p < 0.001], BMI [p < 0.001], fasting, and 2 h post-OGTT sugar, insulin, HbA1c, HOMA-IR, and oxidative stress markers [p < 0.001] was observed in patients receiving Nisha-Amalaki at 6 months. Ayurvedic symptoms and QoL scores also improved at 6 months in the treatment group. CONCLUSION: Treatment with Nisha-Amalaki capsules improved all study parameters including insulin sensitivity at 6 months as compared to placebo in prediabetic patients. Thus Nisha-Amalaki should be considered as prophylactic therapy in prediabetics to delay progression to diabetes.

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.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

A glucuronated flavone TMMG spatially targets chondrocytes to alleviate cartilage degeneration through negative regulation of IL-1ß.

Chondrocytes are the only resident cell types that form the extracellular matrix of cartilage. Inflammation alters the anabolic and catabolic regulation of chondrocytes, resulting in the progression of osteoarthritis (OA). The potential of TMMG, a glucuronated flavone, was explored against the pathophysiology of OA in both in vitro and in vivo models. The effects of TMMG were evaluated on chondrocytes and the ATDC5 cell line treated with IL-1ß in an established in vitro inflammatory OA model. An anterior cruciate ligament transection (ACLT) model was used to simulate post-traumatic injury in vivo. Micro-CT and histological examination were employed to examine the micro-architectural status and cartilage alteration. Further, serum biomarkers were measured using ELISA to assess OA progression. In-vitro, TMMG reduced excessive ROS generation and inhibited pro-inflammatory IL-1ß secretion by mouse chondrocytes and macrophages, which contributes to OA progression. This expression pattern closely mirrored osteoclastogenesis prevention. In-vivo results show that TMMG prevented chondrocyte apoptosis and degradation of articular cartilage thickness, subchondral parameters, and elevated serum COMP, CTX-II, and IL-1ß which were significantly restored in 5 and 10 mg.kg-1day-1 treated animals and comparable to the positive control Indomethacin. In addition, TMMG also improved cartilage integrity and decreased the OARSI score by maintaining chondrocyte numbers and delaying ECM degradation. These findings suggest that TMMG may be a prospective disease-modifying agent that can mitigate OA progression.

A standardized extract of Coleus forskohlii root protects rats from ovariectomy-induced loss of bone mass and strength, and impaired bone material by osteogenic and anti-resorptive mechanisms.

Introduction: In obese humans, Coleus forskohlii root extract (CF) protects against weight gain owing to the presence of forskolin, an adenylate cyclase (AC) activator. As AC increases intracellular cyclic adenosine monophosphate (cAMP) levels in osteoblasts that has an osteogenic effect, we thus tested the skeletal effects of a standardized CF (CFE) in rats. Methods: Concentrations of forskolin and isoforskolin were measured in CFE by HPLC. CFE and forskolin (the most abundant compound present in CFE) were studied for their osteogenic efficacy in vitro by alkaline phosphatase (ALP), cAMP and cyclic guanosine monophosphate (cGMP) assays. Femur osteotomy model was used to determine the osteogenic dose of CFE. In growing rats, CFE was tested for its osteogenic effect in intact bone. In adult ovariectomized (OVX) rats, we assessed the effect of CFE on bone mass, strength and material. The effect of forskolin was assessed in vivo by measuring the expression of osteogenic genes in the calvarium of rat pups. Results: Forskolin content in CFE was 20.969%. CFE increased osteoblast differentiation and intracellular cAMP and cGMP levels in rat calvarial osteoblasts. At 25 mg/kg (half of human equivalent dose), CFE significantly enhanced calcein deposition at the osteotomy site. In growing rats, CFE promoted modeling-directed bone formation. In OVX rats, CFE maintained bone mass and microarchitecture to the level of sham-operated rats. Moreover, surface-referent bone formation in CFE treated rats was significantly increased over the OVX group and was comparable with the sham group. CFE also increased the pro-collagen type-I N-terminal propeptide: cross-linked C-telopeptide of type-I collagen (PINP : CTX-1) ratio over the OVX rats, and maintained it to the sham level. CFE treatment decreased the OVX-induced increases in the carbonate-to-phosphate, and carbonate-to-amide-I ratios. CFE also prevented the OVX-mediated decrease in mineral crystallinity. Nanoindentation parameters, including modulus and hardness, were decreased by OVX but CFE maintained these to the sham levels. Forskolin stimulated ALP, cAMP and cGMP in vitro and upregulated osteogenic genes in vivo. Conclusion: CFE, likely due to the presence of forskolin displayed a bone-conserving effect via osteogenic and anti-resorptive mechanisms resulting in the maintenance of bone mass, microarchitecture, material, and strength.

A Simple HPTLC Approach of Quantification of Serratol and Tirucallic Acid with Boswellic Acids in Boswellia serrata by Validated Densitometric Method with MS/MS Characterization.

This study was planned to develop a simple high-performance thin-layer chromatography method for qualitative and quantitative estimation of 3-acetyl-11-keto-ß-boswellic acid (AKBBA), ß-boswellic acid (BBA), 3-oxo-tirucallic acid (TCA) and serratol (SRT) with HPTLC-ESI-MS/MS for characterization in Boswellia serrata Roxb. oleo gum resin extract. The method was developed with hexane-ethyl acetate-toluene-chloroform-formic acid as mobile phase. RF values observed for AKBBA, BBA, TCA and SRT were 0.42, 0.39, 0.53 and 0.72, respectively. The method was validated according to International Council for Harmonisation guidelines. The concentration range for linearity was 100-500 ng/band for AKBBA and 200-700 ng/band for the other three markers with r2 > 0.99. The method resulted in good recoveries as 101.56, 100.68, 98.64 and 103.26%. The limit of detection was noticed as 25 , 37, 54 and 38 ng/band, with a limit of quantification as 76, 114, 116 and 115 ng/band, for AKBBA, BBA, TCA and SRT, respectively. The four markers were identified and confirmed in B. serrata extract using TLC-MS by indirect profiling by LC-ESI-MS/MS and were identified as terpenoids, TCA and cembranoids: AKBBA (mass/charge (m/z) = 513.00), BBA (m/z = 455.40), 3-oxo-tirucallic acid (m/z = 455.70) and SRT (m/z = 291.25), respectively.

Comparative Morpho-micrometric Investigations in Six Indigenous Ocimum Species of India with DOE Based HPTLC Method for Multi-class Component Analysis.

The Ocimum genus is one of India's prominent botanical classes of traditional medicinal culture comprising medicinally and agronomically important plants. Morphological resemblances, overlapping geographical distribution, and history of traditional nomenclature have necessitated a comprehensive qualitative report for effective quality control and removing the species ambiguity pertaining to this genus. This paper provides detailed morpho-micrometric characteristics used to differentiate between six indigenous Ocimum species of India. Among them, O. gratissimum was distinguished as the only shrub with a fleshy petiole. In green and purple forms, O. tenuiflorum leaves had serrate margins and showed no particular anatomical differences except for the anthocyanins containing epidermal cells of the latter. O. basilicum had glabrous leaves except for the veins, which were puberulous. O. filamentosum had tenuous anther filaments and was the least aromatic while O. africanum had a citrusy odour, which along with the number of xylary rows, size of mesophyll cells, and epidermal cell wall architecture, distinguished it from O. americanum. An HPTLC method was developed using experimental design and validated for quantification of multi-class compounds from terpenoic, phenolic acids, and flavonoids in Ocimum leaves. It was found linear (r 2 > 0.99) with recoveries between 95 - 100% for all compounds. The eluted bands of marker compounds were subjected to HPTLC-MS analysis as a confirmative tool. This is the first anatomical and analytical report of O. filamentosum Forssk. The obtained results could be effectively used for species identification using vegetative characters alone with the anatomical-HPTLC data backing up the former as a rapid and economical tool.

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.

Caviunin glycoside (CAFG) from Dalbergia sissoo attenuates osteoarthritis by modulating chondrogenic and matrix regulating proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: Dalbergia sissoo DC. (Indian rosewood or Sheesham) is a traditional medicinal plant, reported since time immemorial for its analgesic, anti-nociceptive, anti-inflammatory, and immuno-modulatory properties. D. sissoo DC (DS). is being used traditionally to cure joint inflammation and joint pain. AIM: To study the potential of DS leaves and its derived novel compound CAFG to treat the clinical symptoms of osteoarthritis (OA) and its underlying mechanism. METHODS: The chemical profile of DS extract (DSE) with isoflavonoids and isoflvaonoid glycosides from the DS was established by UHPLC-PDA and UHPLC-MS/MS. Monosodium iodoacetate (MIA) was injected into the knee joint to develop the OA model in rats. DSE was given orally for 28 days daily at 250 and 500 mg.kg-1day-1. For in-vitro experiments, chondrocytes isolated from joint articular cartilage were negatively induced with interleukin-1ß (IL-1ß) and CAFG was given to the cells as a co-treatment. RESULTS: Chondrocytes undergo apoptosis following inflammation and proteoglycan synthesis affected in MIA injected knees. DSE administration prevented these effects as assessed by H&E and Toluidine blue staining. Micro-CT analysis showed that subchondral bone loss was restored. DSE decreased elevated serum levels of cartilage-bone degradation (CTX-I, CTX-II, and COMP), inflammation markers IL-1ß, and matrix-degrading MMP-3 and 13. The effects of IL-1ß on gene expression of chondrocytes were reversed by CAFG treatment at 1 µM. CONCLUSION: Data showed that DSE protected joint cartilage and deterioration in subchondral bone in vivo while in in-vitro, its active ingredient CAFG prevented interleukin-1ß induced effects and inhibited OA. This finding suggest that DSE and CAFG could be used as a possible therapeutic to treat osteoarthritis.

Effect of Withania somnifera hydroalcoholic extract and other dietary interventions in improving muscle strength in aging rats.

BACKGROUND: Aging leads to loss of skeletal muscle, diminished muscle strength, and decline in physical functions. OBJECTIVE: This study evaluates Withania somnifera and some dietary interventions to combat muscle weakness in aging rats. MATERIALS AND METHODS: Rats (12-13 months old) corresponding to a human age of 60-65 years were assigned to various groups and given orally a standardized W. somnifera extract (WSE, 500 mg/kg) or a protein cocktail comprising soybean (1.5 g/kg) and quinoa (1 g/kg) or a combination of WSE and the protein cocktail or whey protein (1 g/kg) as a reference standard or only resistance exercise for 60 days. Grip strength and blood glucose levels were monitored weekly. At the end of the treatment, total protein, inflammatory markers (CRP, IL-6 and TNF-α), AMPK, malondialdehyde, glutathione, antioxidant enzymes and apoptotic regulator genes (Bax and Bcl-2) were assayed. The biceps brachii muscle of all animals was subjected to histomorphological study. RESULTS: All treatments successfully attenuated aging-elevated glucose, CRP, IL-6, TNF-α, AMPK, malondialdehyde, and Bax levels. A significant restoration of the aging-depleted total protein levels, glutathione, superoxide dismutase, catalase, and Bcl-2 was noted in the treatment groups. An increase in grip strength and greater biceps mass with all treatments indicated regaining of the frail aging muscle's strength and functionality. The WSE + protein treatment elicited the best results among all treatment groups to optimize muscle strength. CONCLUSION: All the interventions curbed muscle loss and strengthened the skeletal muscle by reducing inflammation, oxidative stress and apoptosis, and increasing ATP availability to the muscle.

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.

An Ayurvedic formulation of Emblica officinalis and Curcuma longa alleviates insulin resistance in diabetic rats: Involvement of curcuminoids and polyphenolics.

BACKGROUND: Nishamalaki is an Ayurvedic herbal formulation used to treat type 2 diabetes mellitus (T2DM), comprises Emblica officinalis and Curcuma longa. OBJECTIVE(S): One of the main cause of T2DM is Insulin Resistance (IR) hence, this study was planned to evaluate IR lowering effect of a standardized Nishamalaki extract "EmbliQur" in high-fat diet (HFD) and streptozotocin (STZ) induced T2DM rats. MATERIALS AND METHODS: Curcuminoids (23.89% w/w), gallic acid (5.27% w/w) and tannins (25.44% w/w) were quantified from EmbliQur. Rats were fed HFD throughout the study of 45 days and received STZ (40 mg/kg, i.p) on the 15th day of the study. Rats with more than 250 mg/dl of fasting blood glucose level (FBGL) were considered diabetic and selected for administration of EmbliQur (500 mg and 1000 mg/kg) or the standard drug metformin (120 mg/kg, p.o) from the 18th day of the study for the next 27 days. FBGL and insulin levels of all rats were measured weekly and an oral glucose tolerance test (OGTT) was done at the end of the study. The values of FBGL and insulin were used to calculate IR by the HOMA-IR, QUICKI and Matsuda methods. RESULTS: Rats treated with STZ/HFD had significantly higher than normal FBGL and insulin levels throughout the study and exhibited skewed IR indices in the above three methods of IR assessment. EmbliQur treatment successfully lowered the HFD/STZ-elevated BGL and insulin levels, and ameliorated IR in all models of IR evaluation. CONCLUSION: EmbliQur 1000 mg/kg was noted to be more effective than EmbliQur 500 mg/kg in alleviating IR.

Pharmacokinetics of solid lipid Boswellia serrata particles in healthy subjects.

OBJECTIVES: The anti-inflammatory activity of Boswellia serrata extracts (BSE) is well known. BSE comprises boswellic acids (BA) such as 3-O-acetyl-11-keto-beta-boswellic acid (AKBA) and 11-keto-boswellic acid (KBA) as major constituents. One of the limitations of BAs is their poor oral bioavailability. The aim of the study was to prepare solid lipid particles of Boswellia serrata extract (SLBSP) to enhance the bioavailability of BAs. METHODS: The pharmacokinetic profile of BAs was studied in 10 healthy human volunteers following a single oral dose of 333 mg of SLBSP. Pharmacokinetic blood samples were collected at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 12 h post drug administration. Plasma KBA and AKBA levels were measured using a validated LC-MS/MS method. Pharmacokinetics parameters were estimated using Pheonix WinNonlin (Build 6.4.0.768) software. RESULTS: Ten healthy human volunteers were included and peak plasma concentration was achieved in 1.5 and 2.3 h for AKBA and KBA respectively. Maximum plasma concentration (Cmax) was 8.04 ± 1.67 ng/mL for AKBA and 23.83 ± 4.41 ng/mL for KBA whereas the corresponding area under the concentration-time curve (AUC) was 136.7 ± 56.77 ng/mL*h and 165.7 ± 24.5 ng/mL*h respectively. The elimination half-life (t1/2) of AKBA and KBA was 6.8 ± 3.0 h and 2.45 ± 0.3 h respectively. CONCLUSIONS: The SLBSP formulation of BSE showed enhanced oral bioavailability of BAs compared with historically reported data of unformulated BSE.