Densitometric Analysis Research Articles

Determination of Dried Root Extract of Rheum emodi and its Commercial Products Using Rhein and Chrysophanol as Standard Markers by HPTLC Densitometric Analysis

Determination of Dried Root Extract of Rheum emodi and its Commercial Products Using Rhein and Chrysophanol as Standard Markers by HPTLC Densitometric Analysis

TLC with densitometric and image analysis in the control of anthocyanin content in fruits of Rubus occidentalis and Rubus idaeus cultivars and hybrids

A TLC method was developed for the determination of anthocyanins in the fruits of some cultivated varieties of Rubus occidentalis (black raspberries) and Rubus idaeus (red raspberries) and their hybrids. Anthocyanin sets in fruits’ extracts were determined on TLC silica gel 60 F254 plates using mobile phase consisted of ethyl acetate-water-formic-acid (100:30:30, V/V), at 4°C (±1°C), at a distance of 70 mm with the use of densitometry and videoscanning. The obtained results were comparable to the results of quantitative analysis performed using by the HPLC-UV/VIS method performed on a Kinetex C-18 column using gradient elution comprising an increase concentration of acetonitrile-water-trifluoroacetic acid (50:50:0.1, V/V) in water-trifluoroacetic acid (99.9:0.1, V/V) from 12 % to 40 % within 40 minutes at 520 nm. The TLC method with videoscanning provides immediate capture of data for unstable compounds like anthocyanins before their partial degradation.

Mycobacterium avium inhibits protein kinase C and MARCKS phosphorylation in human cystic fibrosis and non-cystic fibrosis cells.

In cystic fibrosis (CF), there is abnormal translocation and function of the cystic fibrosis transmembrane conductance regulator (CFTR) and an upregulation of the epithelial sodium channel (ENaC). This leads to hyperabsorption of sodium and fluid from the airway, dehydrated mucus, and an increased risk of respiratory infections. In this study, we performed a proteomic assessment of differentially regulated proteins from CF and non-CF small airway epithelial cells (SAEC) that are sensitive to Mycobacterium avium. CF SAEC and normal non-CF SAEC were infected with M. avium before the cells were harvested for protein. Protein kinase C (PKC) activity was greater in the CF cells compared to the non-CF cells, but the activity was significantly attenuated in both cell types after infection with M. avium compared to vehicle. Western blot and densitometric analysis showed a significant increase in cathepsin B protein expression in M. avium infected CF cells. Myristoylated alanine rich C-kinase substrate (MARCKS) protein was one of several differentially expressed proteins between the groups that was identified by mass spectrometry-based proteomics. Total MARCKS protein expression was greater in CF cells compared to non-CF cells. Phosphorylation of MARCKS at serine 163 was also greater in CF cells compared to non-CF cells after treating both groups of cells with M. avium. Taken together, MARCKS protein is upregulated in CF cells and there is decreased phosphorylation of the protein due to a decrease in PKC activity and presumably increased cathepsin B mediated proteolysis of the protein after M. avium infection.

Characterization of high Fischer ratio oligopeptides produced from casein

High Fischer ratio oligopeptides mixture is a mixed peptide system which possess many beneficial effects to the human body. Casein, a milk protein, was used as feedstock to prepare high Fischer ratio oligopeptides via enzymatic hydrolysis, adsorption and purification. Gel electrophoresis, densitometric analysis, amino acid assay and molecular weight measurement were used to visualize and quantify the prepared peptides. Casein, with Fischer ratio of 2.17, was subjected to hydrolysis by alcalase® (2.4 AU/g, pH 8.5, temperature 55 °C, enzymolysis time 2 h) and flavorzyme® (3.4 AU/g, temperature 50 °C, enzyme amount 120 μL, enzymolysis time 3 h), followed via AK-220 activated carbon adsorption (carbon-hydrolysate ratio: 1:10, adsorption time 3 h, pH 8.0, temperature 55 °C) and 3 KDa membrane ultrafiltration. In conclusion, the processing parameters for alcalase® / flavorzyme® and AK-220 activated carbon helped to obtain oligopeptides with Fischer ratio 9.87 which is significantly higher than feedstock casein F value (p < 0.05) and could potentially be used as food supplement to enhance product nutritional property.

Amine-modified magnetic particles: An efficient tool for enhanced RNA extraction

Magnetic particles are an effective tool for simple, time-saving, and labour-saving nucleic acid extraction. In this study, we investigated the isolation of nucleic acids (NA) using 11 variants of magnetic nanoparticles (MPs, 52 ± 6.8 nm) with a surface concentration of amine groups up to 20.8 nmol · mg−1. All results were compared with morphologically identical magnetic material modified with SiO2 grafted with (3-aminopropyl)triethoxysilane (APTES). The properties of these materials were characterized by transmission electron microscopy, scanning electron microscopy, dynamic and electrophoretic light scattering, and magnetometry. Concentrations of amine groups on MPs-APTES were determined by the chemical bind and release method with photometric quantification.The isolation potential of the proposed materials toward NAs was evaluated using gel electrophoresis with photometric determination of NAs concentrations and RT-qPCR. Our results show that the NAs yields of MPs-APTES are higher than the reference MPs-SiO2, regardless of the amine group concentrations. Although the total yield decreased with the concentration of amine groups, a different affinity towards genomic DNA (gDNA) was observed. A high concentration of grafted amine groups induced a preference for ribosomal RNA (rRNA) over gDNA and mediated effective NA elution. Densitometric image analysis of gDNA bands showed that NAs isolated by MPs-SiO2 contained significantly higher DNA levels than MPs with 1/32 %, 1/2 %, and 16 % APTES modification, which was subsequently confirmed by qPCR. Gene expression analysis performed by RT-qPCR revealed that unwanted gDNA contamination did not significantly affect the threshold cycles (Ct) of target genes when cDNA-specific primers were used, but may lead to overestimation when targeting genes with low expression and no possibility to design cDNA-unique primers. From a practical point of view, MPs-APTES provided better dose-dependent NA isolation performance with stable NA quality.

HPTLC Densitometric Analysis for Quantification of Sennocide-B and Rhein in Leaf Extracts of Senna alexandrina Mill. and Their Commercial Formulations.

The leaf extract of Senna alexandrina Mill., a plant from the Fabaceae family, contains bioactive anthraquinones sennoside-B and rhein, traditionally used in Indian medicine for various health issues. However, standardizing the quality of S. alexandrina and its commercial products is challenging. To address this, we established two high-performance thin-layer chromatography (HPTLC) methods for accurate analysis of these biomarkers in S. alexandrina leaf extract and its commercial formulations. For sennoside-B, a mobile phase combination of solvent for example, butanol, water, and glacial acetic acid (6:3.5:0.5, v/v/v) was used, with detection at 254 nm, producing spots at an Rf value of 0.37 ± 0.006. The method showed linearity between 100 to 2000 ng/spot (R² = 0.9983 ± 0.0017), with a limit of detection (LoD) of 22.84 ± 0.554 ng/band and a limit of quantitation (LoQ) of 69.22 ± 0.859 ng/band. For rhein, a mobile phase combination of solvent toluene, ethyl acetate, and glacial acetic acid in the ratio (7:2.5:0.5, v/v/v) was utilized and was also detected at 254 nm. The obtained spots had Rf value of 0.67 ± 0.004. The linearity was confirmed within the same range (R² = 0.9985 ± 0.0005), with an LoD of 15.49 ± 0.645 ng/spot and an LoQ of 46.94 ± 1.172 ng/spot. Herein, the methods have followed the guidelines of ICH (Q2) R1. These developed HPTLC methods are selective, simple, sensitive, accurate, and economical, making them suitable for everyday assessment of these bioactive markers in S. alexandrina and its commercial products. This study supports the quality control of S. alexandrina, ensuring product consistency, efficacy, and safety.

An eco-friendly smartphone based HPTLC method versus conventional densitometric one for determination of Naltrexone and Bupropion

The rapid uprising technologies of smartphone applications and software introduced a new era for analytical detection techniques. It has transformed bench-top laboratory methods into simpler ones depending on cost-effective, portable, and widely accessible devices. In this work, two high performance thin layer chromatographic (HPTLC) methods were developed based on smartphone’s camera detection and either ImageJ desktop software or Color-Picker smartphone’s application as alternative techniques to conventional densitometric detection. A mixture of Naltrexone hydrochloride (NAL) and Bupropion hydrochloride (BUP) was chromatographed on HPTLC- plates using ethyl acetate, methanol, acetone, and glacial acetic acid (3:6:1:0.5, by volume) as a developing system. The developed plates were scanned at 203 nm for the densitometric analysis, then visualized by modified Dragendorff’s reagent and shot by a smartphone’s camera. The captured images were uploaded to either ImageJ software or Color-Picker application to detect the separated spots. The results derived from the three detection methods were compared over the concentration range of 0.4–24 & 0.6–18 µg/band for the densitometric method, 0.4–24 & 2–24 µg/band for ImageJ built method and 0.8–20 & 5–20 µg/band for Color Picker built method for NAL and BUP, respectively. The methods were found to be appropriate for assaying both active drug substances in pure forms and combined in marketed pharmaceutical formulations. The excellent sustainability of densitometric and ImageJ-based methods enabled also the assessment of their dosage form content uniformity. The greenness and sustainability of the methods were assessed by three metric tools, namely Green Analytical Procedure Index (GAPI), Analytical GREEnness Metric Approach (AGREE), and White Analytical Chemistry (WAC). The assessments results confirmed the sustainability and superiority of the proposed methods in terms of sample treatment, waste mount, energy consumption, cost, and number of analyzed samples per an hour.

Evaluation of a semi-automated test for quantification of von Willebrand multimers

The assessment of von Willebrand factor (VWF) multimer distribution, particularly following the implantation of circulatory support devices, is a crucial parameter in hemostasis. Our study aimed to evaluate the semi-automated quantification of VWF multimers using the Sebia Hydrasys analyzer. Our analysis focused on quantifying high molecular weight, intermediate weight, and low molecular weight VWF multimers. Electrophoretic migration was performed using the Hydrasys 2 scan, and interpretation was carried out using densitometric analysis with the Phoresis software. The Hydrasys scan 2 successfully separated all the expected VWF multimer profiles based on the type of von Willebrand disease. The analysis revealed that in patients with circulatory support devices, elevated levels of plasma VWF rendered multimer migration unanalyzable using the methodology recommended by the manufacturer. Therefore, adjustment to a 100% VWF antigenic level improved gel precision. We also suggest using as a standardized control the Cryocheck™ plasma, and have established reference values. Overall, this semi-automated, standardized, and optimized VWF multimer analysis system allows for an effective assessment of the VWF multimeric profile.

Pulmonary emphysema: the assessment of lung perfusion with Dual-Energy CT and pulmonary scintigraphy.

To assess the correlation of quantitative data of pulmonary Perfused Blood Volume (PBV) on Dual-Energy CT (DECT) datasets in patients with moderate - severe Pulmonary Emphysema (PE) with Lung Perfusion Scintigraphy (LPS) as the reference standard. The secondary endpoints are the correlation between the CT densitometric analysis and the visual assessment of parenchymal destruction with PBV. Patients with moderate - severe PE candidate to Lung Volumetric Reduction (LVR), with available a pre-procedural LS and a contrast-enhanced DECT were retrospectively included. DECT studies were performed with a 3rd generation Dual-Source CT and the PBV was obtained with a 3-material decomposition algorithm. The CT densitometric analysis was performed with a dedicated commercial software (Pulmo3D). The Goddard Score was used for visual assessment. The perfusion LS were performed after the administration of albumin macroaggregates labeled with 99mTechnetium. The image revision was performed by two radiologists or nuclear medicine physicians blinded, respectively, to LS and DECT data. The statistical analysis was performed with nonparametric tests. Thirty-one patients (18 males, median age 69 y.o., interquartile range 62-71 y.o.) with moderate - severe PE (Median Goddard Score 14/20 and 31% of emphysematous parenchyma at quantitative CT) candidate to LVR were retrospectively included. The median enhancement on PBV was 17 HU. Significant correlation coefficients were demonstrated between lung PBV and LS, poor in apical regions (Rho = 0.1-0.2) and fair (Rho = 0.3-0.5) in middle and lower regions. No significant correlations were recorded between the CT densitometric analysis, the visual score, and the PBV. Lung perfusion with PBV on DECT is feasible in patients with moderate - severe PE candidate to LVR, and has a poor to fair agreement with LPS.

Polychlorinated Biphenyls (PCBS)-induced oxidative stress and inflammation in human thyrocytes: involvement of AhR and NRF-2/HO-1 pathway.

In this in vitro study, we investigated the effects of polychlorinated biphenyls (PCBs) on human thyrocytes, with a focus on the involvement of AhR, a key player in xenobiotic response, and the anti-oxidant Nrf-2/HO-1 pathway. Primary cultured thyrocytes were exposed to the dioxin-like congeners PCB118 and PCB126 at 2.5 and 5 µM concentrations. mRNA expression was assessed by real-time PCR, and protein expression by Western Blot and ELISA, while protein quantification was assessed by densitometric analysis. In cultured thyrocytes, PCB118 and PCB126 induced a significant (P < 0.01) increase of mRNA and protein levels of the pro-inflammatory cytokines IL-1beta and IL-6, while reducing those of thyroglobulin (TG) and NIS (p < 0.05), indicating down-regulation of these thyroid-specific genes in PCB-induced inflammation. ROS production also increased (p < 0.001). mRNA levels of AhR and the downstream molecules cytochrome P4501A, Nrf-2/HO-1 increased (p < 0.001), as well as related protein levels (p < 0.01), suggesting the activation of AhR and Nrf-2 pathways in response to PCBs exposure. AhR silencing decreased AhR-related gene expression and restored NIS and TG expression, while reducing inflammatory cytokines and oxidative stress markers (p < 0.05). Dioxin-like PCBs (PCB118 and PCB126) may promote inflammation and oxidative stress in thyrocytes, impairing the expression of genes that are key players of thyroid function. These effects can be partially attributed to the activation of the AhR and Nrf-2 pathways. These data may contribute to explain the mechanisms underlying thyroid toxicity of PCBs, highlighting the potential role of these pollutants as a trigger of autoimmune thyroid inflammation and damage.

Age-related decline in goblet cell numbers and mucin content of the human colon: Implications for lower bowel functions in the elderly

Background & aimsOlder people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content. MethodsChanges in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis. ResultsCompared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age. ConclusionsAgeing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

An enhanced and rapid method for von Willebrand factor multimer analysis for mechanical circulatory device testing.

Von Willebrand factor (VWF) is a critical glycoprotein in hemostasis and is an important factor in diagnosing bleeding disorders. Albeit the analysis of VWF is often compromised by inconsistent methodologies and challenges quantifying multimeric size. Current VWF multimer analysis methods are costly, time-consuming, and often inconsistent; thus, demanding skilled professionals. This study aimed to streamline and optimize the VWF multimer analysis technique, making it more efficient and reproducible, particularly for identifying or predicting mechanical circulatory support (MCS) induced bleeding disorders. Blood samples from healthy volunteers were exposed to high shear forces via a Medtronic HeartWare ventricular assist device. VWF multimers were analyzed using vertical-gel agarose electrophoresis and Western blotting. Differences in VWF distribution were determined using densitometry, and two methods of densitometric analysis were compared: proprietary software against open-source software. Using the developed method: (i) protocol duration was accelerated from three days (in classical methods) to ~ eight hours; (ii) the resolution of the high molecular weight (HMW) VWF multimers were substantially improved; and (iii) densitometric analysis tools were validated. Additionally, the densitometry analysis using two software types showed a strong correlation between results, with the proprietary software reporting slightly higher HMW VWF percentages. This methodology is recommended for affordable, accurate, and reproducible VWF multimer evaluations during MCS use and testing. Further research comparing this method with semi-automated methods would provide additional insight and improve inter-laboratory comparisons.

Estrogen receptor alpha (ER-α) antagonistic activity of phytoconstituents from Potentilla atrosanguinea and Potentilla fulgens in breast cancer

The Potentilla genus has long been used traditionally as food and a folklore medicine. In the present study, aerial parts of two Potentilla species, Potentilla fulgens and Potentilla atrosanguinea, of western Himalayan origin, were studied for their anti-breast cancer activity. Ethyl acetate (PAA-EA, PFA-EA), methanolic (PAA-ME, PFA-ME) and hydro-methanolic extract (PAA-HM, PFA-HM) of the plants were tested for their antiproliferative activities against MCF-7 and T-47D breast cancer cell lines. The extracts showed good antiproliferative activity against ER-α dominant breast cancer cell line T-47D, having IC50 values 6.19 ± 0.01 to 33.23 ± 0.04 μg/ml. Eight compounds were isolated, characterized, and quantified from ethyl acetate and methanolic extracts by column chromatography, 1D, 2D-NMR, HRMS and TLC densitometric analysis. Two compounds (4 and 6) have shown better antiproliferative activity than standard bazedoxifene and were further evaluated for their ER-α binding affinity via—fluorescence polarization-based competitive binding assay. The antiestrogenic properties of both compounds were assessed using western blotting. Compounds 4 and 6 were found to have significant affinity for the ER-α and managed to decrease its expression by 38 and 54% respectively. Compounds 4 and 6 also had good stability and reactivity as measured by minimal fluctuations in molecular dynamic simulation analysis, a good dock score in molecular docking, and a respectable HOMO-LUMO energy gap in DFT calculations. Compounds 4 and 6 have shown reliable results and can be used in the development of natural product-based anti-breast cancer agents.

Snakehead Fish, Channa striata (Bloch, 1973), Protein Concentrate: Excellent Recovery of Fish-Based Albumin Source and Its Possible Application for Sperm Capacitation

Snakehead fish protein concentrate (SFC) shows promising recovery of excellent protein content that has broad potential applications, including the future use of its albumin content as a sperm capacitation promoter. However, the quantity of snakehead fish albumin recovered depends on several variables, including the habitat in which the fish thrives, extraction procedures, and the assay employed. To optimise the albumin content retrieved from snakehead fish flesh with more reliable results, multiple protein extraction methods were undertaken through the protein concentrate preparation and analysed using the Bromocresol purple (BCP) albumin assay. Total soluble protein (TSP), albumin level, and albumin proportion were recovered at a significant reduction in an acid solvent when exposed to up to 50 °C heating compared to extractions undertaken without heating. Moreover, the small number of protein bands identified indicates that the combination of acid solvent and heating treatment was similarly damaging. In this study, we detected the presence of albumin in SFC at 32 %, the highest percentage ever recorded employing the BCP assay, following extraction with water solvent without thermal treatment. Using SDS-PAGE and densitometric analysis, the identified putative albumin-related bands from the two major bands at 40 kDa and 47 kDa, containing respective proportions of 57 % and 16 % of the total protein loaded. Although this exploratory work requires additional analysis and purification steps, further research employing the SFC is worth identifying its ability and how this alternative albumin source can replace serum albumin to modulate the capacitation process in mammalian sperm.

Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells.

The endothelial sodium channel (EnNaC) plays an important role in regulating vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoEC. mAoEC transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, our results suggest constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, while deletion of PIP2 decreases MARCKS expression and overall membrane fluidity.

Development and validation of stability indicating HPTLC method for simultaneous estimation of Dapagliflozin and Linagliptin

For the treatment of Type 2 Diabetes Mellitus, Dapagliflozin selectively inhibits the sodium-glucose co-transporter-2, while Linagliptin competitively and reversibly inhibits dipeptidyl peptidase-4 in a fixed dose combination (1:1). Dapagliflozin and Linagliptin estimation in bulk and tablet formulation was accomplished by the development and validation of a precise and accurate HPTLC method. The procedure used Toluene: Chloroform: Methanol: Triethylamine (7:2:1:0.2 v/v/v) as the mobile phase and HPTLC aluminum plates pre-coated with silica gel 60 F254 as the stationary phase used. Dapagliflozin and Linagliptin were determined to have Rf values of 0.23 and 0.40 correspondingly. At 224 nm, densitometric analysis was performed in the absorbance mode. For Dapagliflozin and Linagliptin 200–1200 ng/band were the linear ranges in which the procedure produced results. Forced degradation studies were performed on both Dapagliflozin and Linagliptin active pharmaceutical ingredients like acid hydrolysis, base hydrolysis, chemical oxidation, dry heat and photodegradation studies. Dapagliflozin and Linagliptin are susceptible to acid hydrolysis, base hydrolysis, chemical oxidation while both the chemical substances are stable to dry heat and photolytic studies.

The effect of selinexor on prostaglandin synthesis in virus-positive Merkel cell carcinoma cell lines.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with high rates of metastasis and mortality. In vitro studies suggest that selinexor (KPT-330), an inhibitor of exportin 1, may be a targeted therapeutic option for MCC. This selective inhibitor prevents the transport of oncogenic mRNA out of the nucleus. Of note, 80% of MCC tumors are integrated with Merkel cell polyomavirus (MCPyV), and virally encoded tumor-antigens, small T (sT) and large T (LT) mRNAs may require an exportin transporter to relocate to the cytoplasm and modulate host tumor-suppressing pathways. To explore selinexor as a targeted therapy for MCC, we examine its ability to inhibit LT and sT antigen expression in vitro and its impact on the prostaglandin synthesis pathway. Protein expression was determined through immunoblotting and quantified by densitometric analysis. Statistical significance was determined with t-test. Treatment of MCPyV-infected cell lines with selinexor resulted in a significant dose-dependent downregulation of key mediators of the prostaglandin synthesis pathway. Given the role of prostaglandin synthesis pathway in MCC, our findings suggest that selinexor, alone or in combination with immunotherapy, could be a promising treatment for MCPyV-infected MCC patients who are resistant to chemotherapy and immunotherapy.

Development of Quantifiable Assays for Detection of Hypersialylation in Oral Cancer Diagnosis

Aim: The aim of this study is to Quantify sialic acid for oral cancer diagnosis using different method using densitometric analysis. Background: Early detection of OSCC is critical for improving survival rates and reducing morbidity, which has prompted researchers to look for potential biomarkers to aid in early diagnosis. Saliva is an excellent diagnostic tool due to its noninvasive nature. Sialic acid levels in saliva have been found to be higher in cancer patients. Hypersialylation is associated with poor prognosis and accelerated cancer progression, as well as immune escape, tumor growth and spread, angiogenesis, and resistance to apoptosis and treatment. This study addresses the imperative need for adaptive, effective, and non-invasive strategies in the diagnosis and treatment of oral cancer, emphasising the importance of early detection for improved prognosis. This study introduces various methods for quantify levels of sialic acid in saliva, including resorcinol and orcinol assays, using colorimetric and densitometric assays. Material and Method: In this study, sialic acid detection methods have been standardized using standards as well as spent media from cancer cell lines to develop noninvasive diagnostic tools for early detection. Two methods used for the detection of sialic acid Resorcinal and Orcinol method by densitometric analysis using Image J software. Results: The study has successfully quantified sialic acid from spent media and saliva using resorcinol and orcinol method by densitometric analysis. Conclusion: In this study we aim to develop a reliable and cost-effective method for quantification of sialic acid using Image j software. The approach presents a non-invasive and efficient alternative for early cancer diagnosis showcasing its potential impact on advancing diagnostic technologies.

Development and Validation of HPTLC and First Derivative Spectrophotometric Methods for Determination of Levetiracetam in Bulk and Tablet Dosage Form

A simple, sensitive high performance thin layer chromatographic (HPTLC) method and first order derivative spectrophotometric methods were developed for estimation of US FDA drug levetiracetam in bulk and its tablet dosage form. Chromatographic estimation was carried out on silica gel 60 F245 TLC plate as stationary phase and toluene: methanol: Ethyl acetate (4:4:2v/v/v) as mobile phase. Densitometric analysis was carried out at wavelength 218nm. The Rf values for Levetiracetam was found to be 0.47±0.03. Linearity was observed over range of 400-3200ng/spot for Levetiracetam (R2 0.998). The %recovery of Levetiracetam was found to be 99.25-100.7%. The LOD and LOQ were found to be 0.0917ng/ml and 0.278ng/ml respectively indicating sensitivity of the method. The first order derivative spectra were obtained at n=4, Δλ=3nm and determinations was made at 215 nm using methanol as solvent. Linearity was observed in the range of 80μg/ml - 280μg/ml with good regression values. Percent mean recovery of Levetiracetam was obtained as 101.5%. Both proposed methods were simple, fast, accurate, precise and reproducible and hence can be applied for routine quality control analysis of levetiracetam in bulk and its tablet dosage form.

Alzheimer's Disease-associated Region-specific Decrease of Vesicular Glutamate Transporter Immunoreactivity in the Medial Temporal Lobe and Superior Temporal Gyrus

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.