Atul S. Rathore

Alleviating exercise-induced muscular stress using neat and processed bee pollen: oxidative markers, mitochondrial enzymes, and myostatin expression in rats

Background The current study was designed to investigate the influence of monofloral Indian mustard bee pollen (MIMBP) and processed monofloral Indian mustard bee pollen (PMIMBP) supplementation on chronic swimming exercise-induced oxidative stress implications in the gastrocnemius muscle of Wistar rats. Methods MIMBP was processed with an edible lipid-surfactant mixture (Captex 355:Tween 80) to increase the extraction of polyphenols and flavonoid aglycones as analyzed by UV spectroscopy and high performance liquid chromatography-photo diode array. Wistar rats in different groups were fed with MIMBP or PMIMBP supplements at a dose of 100 mg/kg, 200 mg/kg and 300 mg/kg individually, while being subjected to chronic swimming exercise for 4 weeks (5 d/wk). Various biochemical [superoxide dismutase (SOD), glutathione (GSH), malonaldehyde (MDA), nitric oxide (NO), and total protein content], mitochondrial (Complex I, II, III, and IV enzyme activity), and molecular (myostatin mRNA expression) parameters were monitored in the gastrocnemius muscle of each group. Results Administration of both MIMBP (300 mg/kg) and PMIMBP (100 mg/kg, 200 mg/kg, and 300 mg/kg) wielded an antioxidant effect by significantly improving SOD, GSH, MDA, NO, and total protein levels. Further MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) significantly improved impaired mitochondrial Complex I, II, III, and IV enzyme activity. Significant down-regulation of myostatin mRNA expression by MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) indicates a muscle protectant role in oxidative stress conditions. Conclusion The study establishes the antioxidant, mitochondrial upregulatory, and myostatin inhibitory effects of both MIMBP and PMIMBP in exercise-induced oxidative stress conditions, suggesting their usefulness in effective management of exercise-induced muscular stress. Further, processing of MIMBP with an edible lipid-surfactant mixture was found to improve the therapeutic efficiency of pollen.

Application of HPLC for the simultaneous determination of aceclofenac, paracetamol and tramadol hydrochloride in pharmaceutical dosage form.

A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of aceclofenac (ACF), paracetamol (PCM) and tramadol hydrochloride (TRM) in pharmaceutical dosage form. The chromatographic separation was achieved on a HiQ-Sil™ HS C18 column (250×4.6 mm i.d., 5 μm particle size), kromatek analytical column at ambient temperature. The mobile phase consisted of 40: 60 (v/v); phosphate buffer (pH 6.0): methanol. The flow rate was set to 1.0 mL min−1 and UV detection was carried out at 270 nm. The retention time (tR) for ACF, PCM and TRM were found to be 14.567 ± 0.02, 3.133 ± 0.01 and 7.858 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, speci city, accuracy and system suitability. The linear dynamic ranges were from 40–160 μg mL−1 for ACF, 130–520 μg mL−1 for PCM and 15–60 μg mL−1 for TRM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form.

Investigation of the nutraceutical potential of monofloral Indian mustard bee pollen.

OBJECTIVE This study was designed to investigate the nutraceutical potential of monofloral Indian mustard bee pollen (MIMBP). METHODS The nutritional value of MIMBP was examined in terms of proteins, fats, carbohydrates, and energy value. Its chemical composition in terms of total polyphenol and flavonoid content was determined. MIMBP was screened for free flavonoid aglycones by developing and validating a high-performance liquid chromatography-photo diode array (HPLC-PDA) method. MIMBP was analyzed for in vitro antioxidant effect in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity. RESULTS MIMBP was found to be comprised of proteins ((182.2±5.9) g/kg), fats ((137.7±6.8) g/kg) and carbohydrates ((560.6±17.4) g/kg), which result in its high energy value ((17 616.7±78.6) kJ/kg). MIMBP was found to contain polyphenols ((18 286.1±374.0) mg gallic acid equivalent/kg) and flavonoids ((1 223.5±53.1) mg quercetin equivalent/kg). The HPLC-PDA analysis revealed the presence of kaempferol ((65.4±0.5) mg/kg) and quercetin ((51.4±0.4) mg/kg) in MIMBP, which can be used as markers for determining the quality of bee pollen. The MIMBP extract showed DPPH free radical-scavenging activity with a half maximal inhibitory concentration of 54.79 μg/mL. CONCLUSION The MIMBP was found to be a rich source of nutrients providing high caloric value, which makes it a candidate for a potential nutraceutical agent. The study also illustrated the high antioxidant content of MIMBP, especially in the principle polyphenols and flavonoids, which suggests its potential role in the prevention of free radical-implicated diseases. The DPPH-scavenging effect of MIMBP further confirmed its antioxidant potential. Additionally, we developed a simple, specific and accurate HPLC-PDA method for the identification and quantification of free flavonoid aglycones. This can be applied in future screenings of the quality of pollen collected by honeybees.

Method Development and Validation for the Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium in Drug Formulation Using Normal Phase High-Performance Thin-Layer Chromatography

A simple, precise, specific, and accurate high-performance thin-layer chromatographic method has been developed for the simultaneous determination of fexofenadine hydrochloride (FEX) and montelukast sodium (MTKT) in pharmaceutical dosage form. The separation was carried out on Merck HPTLC aluminum plates of silica gel G60 F254, (20×10 cm) with 250 μm thickness using toluene: ethyl acetate: methanol: ammonia (30%) (0.5: 7: 2: 0.5, v/v/v/v) as mobile phase. HPTLC separation of the two drugs followed by densitometric measurement was carried out in the absorbance mode at 220 nm. The drugs were resolved satisfactorily with 𝑅𝑓 values of 0.21±0.01 and 0.59±0.01 for FEX and MTKT, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with 𝑟2=0.9996 and 0.9998 for FEX and MTKT, respectively, in the concentration range of 2400–10800 ng spot−1 for FEX and 200–900 ng spot−1 for MTKT. The method was validated for precision, robustness, specificity, and accuracy. The limits of detection and quantitation were 100 and 300 ng spot−1, respectively, for FEX and 50 and 100 ng spot−1, respectively, for MTKT. The proposed developed HPTLC method can be applied for identification and quantitative determination of FEX and MTKT in bulk drug and drug formulation.

Stability-Indicating High-Performance Thin-Layer Chromatographic Method for Quantitative Estimation of Emtricitabine in Bulk Drug and Pharmaceutical Dosage Form

A simple, sensitive, precise, specific and stability indicating high-performance thin-layer chromatographic (HPTLC) method for the determination of emtricitabine both in bulk drug and pharmaceutical dosage form was developed and validated. The method employed aluminium plates precoated with silica gel G60 F254 as the stationary phase. The solvent system consisted of toluene : ethyl acetate : methanol (2 : 8 : 1, v/v/v). This solvent system was found to give compact spots for emtricitabine with 𝑅𝑓 value 0.26±0.01. Densitometric analysis of emtricitabine was carried out in the absorbance mode at 284 nm. Linear regression analysis showed good linearity (𝑟2=0.9997) with respect to peak area in the concentration range of 30–110 ng spot−1. The method was validated for precision, limit of detection (LOD), limit of quantitation (LOQ), robustness, accuracy and specificity. Emtricitabine was subjected to acid and alkali hydrolysis, oxidation, neutral hydrolysis, photodegradation and dry heat treatment. Also the degraded products peaks were well resolved from the pure drug with significantly different 𝑅𝑓 values. Statistical analysis proved that the method is repeatable and specific for the estimation of the said drug. As the method could effectively separate the drugs from their degradation products, it can be employed as a stability indicating method.

Application of HPLC for the Simultaneous Determination of Paracetamol, Chlorzoxazone, and Nimesulide in Pharmaceutical Dosage Form

A simple, precise, and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CHZ), and nimesulide (NIM) in pharmaceutical dosage form. The chromatographic separation was achieved on a Thermo Hypersil GOLD C18 column (250 × 4.6 mm i.d., 5 μm particle size). The mobile phase consisted of water : acetonitrile (55 : 45 v/v). The flow rate was set to 1.2 mL min−1 and UV detection was carried out at 275 nm. The retention time () for PCM, CHZ, and NIM was found to be 2.69 ± 0.02, 4.61 ± 0.01, and 9.55 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, specificity, and accuracy. The linear dynamic ranges were 32.5–65.0 μg mL−1 for PCM, 37.5–75.0 μg mL−1 for CHZ, and 10.0–20.0 μg mL−1 for NIM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form.

Rapid and sensitive determination of major polyphenolic components in Euphoria longana Lam. seeds using matrix solid-phase dispersion extraction and UHPLC with hybrid linear ion trap triple quadrupole mass spectrometry.

A rapid and sensitive method for the extraction and determination of four major polyphenolic components in Euphoria longana Lam. seeds is presented for the first time based on matrix solid-phase dispersion extraction followed by ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry. Matrix solid-phase dispersion method was designed for the extraction of Euphoria longana seed constituents and compared with microwave-assisted extraction and ultrasonic-assisted extraction methods. An Ultra high performance liquid chromatography with hybrid triple quadrupole linear ion-trap mass spectrometry method was developed for quantitative analysis in multiple-reaction monitoring mode in negative electrospray ionization. The chromatographic separation was accomplished using an ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with gradient elution of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. The developed method was validated with acceptable linearity (r2 > 0.999), precision (RSD ≤ 2.22%) and recovery (RSD ≤ 2.35%). The results indicated that matrix solid-phase dispersion produced comparable extraction efficiency compared with other methods nevertheless was more convenient and time-saving with reduced requirements on sample and solvent volumes. The proposed method is rapid and sensitive in providing a promising alternative for extraction and comprehensive determination of active components for quality control of Euphoria longana products.

Determination of Major Polyphenolic Components in Euphoria longanaLam. by Validated High Performance Thin-Layer ChromatographyMethod and Direct Analysis in Real Time Mass Spectrometry

In the current study, a simple, sensitive, precise, and specific method has been developed and validated for the determination of four major polyphenolic compounds in Euphoria longana Lam. seeds based on high performance thin-layer chromatography (HPTLC) and confirmed by direct analysis in real time mass spectrometry (DART-MS). The chromatographic separation was accomplished on Merck HPTLC aluminum plates precoated with silica gel G60 F254 (20 cm × 10 cm) with 250 μm thickness as stationary phase using a mobile phase composed of n-butanol: water: methanol: formic acid (7.59: 1.27: 0.13: 1.01, v/v/v/v). Densitometric measurement was performed in the absorbance mode at 280 nm. The compounds were resolved satisfactorily with Rf values of 0.40 ± 0.01, 0.57 ± 0.02, 0.69 ± 0.01, and 0.79 ± 0.01 for corilagin, ellagic acid, epicatechin, and gallic acid, respectively. The method developed was validated with acceptable linearity (r2>0.995), sensitivity, precision (RSD ≤ 1.60%), robustness, and recovery (RSD ≤ 1.77%). The proposed method was successfully applied for the determination of active components for comprehensive quality control of E. longana products. Furthermore, peak identities of compounds from each band were confirmed by direct analysis in real time mass spectrometry.

Characteristic Fingerprint Analysis of Mallotus philippinensis by UltraPerformance Liquid Chromatography Electrospray Ionization MassSpectrometry

The developing countries mostly trust on traditional remedies, which include the use of different plant extracts or the bioactive phytoconstituents. For this purpose, analysis such as chemical fingerprinting strongly represents one of the best possibilities in searching of new economic and therapeutically effective plants for medicine. Mallotus philippinensis Muell. Arg (Euphorbiaceae) is a large genus of the trees and shrubs mainly distributed in the tropical and subtropical regions and are reported to have widespread range of pharmacological activities. A new, simple and rapid ultraperformance liquid chromatography (UPLC) method with photodiode array (PDA) detector has been developed. Further confirmation was performed by electrospray ionization mass spectrometry (ESI-MS) for the chemical fingerprint analysis in extracts of Mallotus philippinensis. The chromatographic separations were obtained on a Waters ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column using a gradient elution with 0.1% (v/v) formic acid/water and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The UPLC-PDA-ESI-MS characteristic fingerprints were established and 7 characteristic peaks were identified along with 5 unknown peaks within 4.5 min by comparing the retention times, λ max (nm), and MS spectra with the literature data. Therefore, this fingerprint analysis method can be applied for the identification and quality control of Mallotus philippinensis.

Separation and Determination of Lamivudine, Tenofovir Disoproxil Fumarate and Efavirenz in Tablet Dosage Form by Thin-Layer Chromatographic-Densitometric Method

This paper describes the development and validation of high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of lamivudine (LAM), tenofovir disoproxil fumarate (TDF) and efavirenz (EFV). The separation was carried out on Merck HPTLC aluminum plates precoated with silica gel G60 F254 (20 × 10 cm) with 250-µm thickness using chloroform-methanol-toluene (9:1.2:0.3, v/v) as mobile phase. HPTLC separation of the three drugs followed by densitometric measurement was carried out in the absorbance mode at 260 nm. The drugs were resolved satisfactorily with RF values of 0.20 ± 0.02, 0.61 ± 0.01 and 0.73 ± 0.02 for LAM, TDF and EFV, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9997, 0.9991 and 0.9990 for LAM, TDF and EFV, respectively, in the concentration range of 400–800 ng spot−1 for LAM and TDF and 800–1600 ng spot−1 for EFV. The limit of detection and quantitation were 180 and 300 ng spot−1, respectively for LAM, 150 and 210 ng spot−1, respectively for TDF and 300 and 400 ng spot−1, respectively, for EFV. The method was validated for precision, robustness, limit of detection (LOD), limit of quantitation (LOQ), specificity and accuracy. The method can be used for routine analysis of these drugs in various formulations in quality control laboratories.