SPP1 and UMOD gene variants are synergistically associated with risk of renal stone disease.

OBJECTIVE: Calcium and oxalate are the most abundant metabolites present in the stone matrix. The SPP1 and UMOD gene has specific expression in kidneys and are involved in various stages of stone formation. Therefore, genetic variants in the SPP1 and UMOD genes may enhance the development of renal stone disease. This study has been designed to understand the association of genetic variants of SPP1 and UMOD genes with renal stone disease. MATERIALS AND METHOD: A prospective study has been carried out, including 150 renal stone disease patients and 150 healthy individuals. Biochemical parameters were performed, including serum calcium levels, creatinine, parathyroid hormone, and 24-Hour urine metabolites. The genotyping of SPP1 (rs1126616) and UMOD (rs4293393) gene variants were performed using a customized TaqMan probe. T-test was used for continuous biochemical data analysis. The Chi-square test has been applied to assess the risk of a particular genotype associated with renal stone disease. In addition, correlation analysis for biochemical parameters and genetic variants with the renal stone disease has been performed using Shapley additive explanations (SHAP) values calculated with the help of the pycaret library. RESULT: Renal stone patients had significantly higher levels of parathyroid hormone (93.37 ± 52.78 pg/ml vs 64.67 ± 31.50 pg/ml, P=<0.0001), serum creatinine (0.94 ± 0.38 mg/dl vs 0.77 ± 0.17 mg/dl, P=<0.0001) and 24hr urine metabolites in comparison to the healthy controls. Heterozygous (CT) variant of SPP1 and homozygous (GG) variant of UMOD genes were significantly associated with an increased risk of developing the renal stone disease (p = 0.0100, OR = 2.06, 95 %CI = 1.13-3.75; p=<0.0001, OR = 5.773, 95 % CI = 2.03-16.38, respectively). Individuals with hyperparathyroidism and CC (SPP1) and GG (UMOD) genotypes have a high risk (P = 0.0055, OR = 2.75, 95 %CI = 1.35-5.67; P = 0.0129, OR = 10.03, 95 %CI = 1.60-110.40, respectively) of developing a renal stone. In addition, individuals with hypercalciuria and TT genotype of SPP1 (P = 0.0112, OR = 2.92, 95 % CI = 1.33-6.35), AG genotype of UMOD (P=<0.0001, OR = 5.45, 95 %CI = 2.24-13.96) and GG genotype of UMOD (P=<0.0001, OR = 10.02, 95 %CI = 3.53-24.63) have high risk of developing renal stones. Moreover, Individuals with hyperoxaluria and AG + GG (UMOD) genotype have a greater risk (P=<0.0001, OR = 7.35, 95 % CI = 3.83-13.68) of developing a renal stone. The renal stone risk was persistent (P=<0.0002, OR = 2.44, 95 % CI = 1.52-3.86) when analyzed for the synergistic effect of risk genotypes of SPP1 (CT) and UMOD (GG) gene. Further, correlation analysis also confirmed the strong association between genetic variants and renal stone development. CONCLUSION: Genetic variants of the SPP1 and UMOD genes were associated with renal stone disease. In the presence of risk genotype and hyperparathyroidism, hypercalciuria, and hyperoxaluria, the susceptibility to develop the renal stone disease risk gets modulated.

Estimation of Scopoletin from Roots of Argyreia nervosa (Burm. fill.) Bojer Using a Validated HPLC-Fluorescence Method Optimized Using the Design of Experiment Approach.

BACKGROUND: Argyreia nervosa (Burm. Fil.) Bojer., a woody climber, is indicated in Ayurveda to treat debilities of the male reproductive system, diseases of the nervous system, and chronic ulcers. OBJECTIVE: A sensitive analytical method was developed to estimate bioactive scopoletin from methanolic extract prepared from the medicinally active dried roots of Argyreia nervosa (Burm. fil.) Bojer using HPLC equipped with a fluorescence detector. METHODS: Chromatographic separation was achieved using a LunaTM (C18, 250 × 4.6 mm, id: 5 µm) column using an isocratic mobile phase comprising phosphate buffer (pH 3.5)-acetonitrile (80 + 20, by volume) at a flow rate of 1.0 mL/min. The excitation wavelength was 345 nm, and the emission wavelength was 444 nm. The chromatographic parameters were optimized using the design of the experiment approach after determining the combined effects of selected independent variables on area, retention time, and tailing factor (TF) for the peak corresponding to scopoletin, and the experimental design was validated by navigating through the design space. RESULTS: The developed method was found linear in the range 10-140 ng/mL. The results of the studies confirmed the accuracy, precision, and robustness of the developed analytical method. The plant material was found to contain 0.0125 ± 0.0001% w/w scopoletin on a dried weight basis when estimated using the developed method. CONCLUSION: The method was developed using the HPLC-fluoresence detection by adopting the design of experiment approach and simple sample preparation for the estimation of scopoletin from roots of A. nervosa. HIGHLIGHT: This extremely sensitive analytical method with one-step sample preparation has the potential to be adapted for routine QC procedures.

Development and Validation of a Green Stability-Indicating HPTLC Method for Estimation of Curcumin, Gallic Acid, and Ursolic Acid From Polyherbal Formulation Jatyadi Taila.

BACKGROUND: Jatyadi taila (JT) is a well-known Ayurvedic wound-healing product, comprising 16 different medicinally important plants, including Curcuma longa, Terminalia chebula, and Jasminum officinale. OBJECTIVE: The proposed work discusses the development and validation of the green and economical stability-indicating HPTLC method for quantification of the key marker phytoconstituents, curcumin (CUR), gallic acid (GA), and ursolic acid (UA), from JT. METHOD: Quality standard parameters for JT were determined following standard procedures. The marker constituents CUR, GA, and UA were resolved from JT using toluene-ethyl acetate-formic acid (6:2:1, v/v/v) as the mobile phase and subsequently derivatized to estimate UA. The developed plates were subjected to HPTLC-MS analysis. All constituents were subjected to forced degradation to determine the proposed technique's stability-indicating property and the accelerated stability studies of marketed formulation and marker constituents. Greenness evaluation of the method was aided by the AGREE methodology. RESULTS: The Rf values of CUR, GA, and UA were found to be 0.60 and 0.60; 0.27 and 0.28; and 0.74 and 0.77 from reference standard and oil samples respectively, when analyzed at 366 nm, 290 nm, and 366 nm, respectively. HPTLC-MS was carried out to verify the active constituents present in JT. The constituents followed first-order degradation kinetics. The quantity of CUR, GA, and UA in JT was reduced at the end of accelerated stability studies. The developed approach was validated in compliance with the International Conference on Harmonization (ICH) Q2 (R2) guideline. CONCLUSIONS: Among the chosen key markers, GA was highly unstable during forced degradation. JT should be stored at a controlled temperature using more protective packaging material to ensure its quality and efficacy. HIGHLIGHTS: The developed method can be used as a quality control tool for JT as it can be used to determine the stability of the key marker compounds the herbal formulation.

Haplotype of CaSR gene is associated with risk of renal stone disease in West Indian population.

Calcium is the most abundant metabolite involved in the stone matrix. The CaSR gene controls calcium homeostasis, and genetic variation in the CaSR gene could lead to the development of renal stone disease. Therefore, the current study has been designed to assess the association of genetic variants of CaSR gene polymorphisms with renal stone disease. A single-centric prospective study has been carried out on a total of 300 participants (150 cases and 150 controls). Serum levels of calcium, creatinine, parathyroid hormone, and 24 h urine metabolites were measured. Two polymorphisms, rs1801725 and rs1042636, of the CaSR gene, have been genotyped for each participant. T test, binary logistic regression, and Chi-square analysis were used for statistical analysis. Renal stone patients had significantly higher levels of serum parathyroid hormone, creatinine, and 24-h urine metabolites in comparison to the controls. CaSR gene variants, rs1801725 (GG) and rs1042636 (AA), both have shown significant association with renal stone disease. In addition, individuals having specific genotypes along with metabolic abnormalities such as hypercalcemia and hyperparathyroidism are found to be at a higher significant risk of developing the renal stone disease. In the present study, the haplotype of the CaSR gene has shown an association with renal stone disease. Individuals with hyperparathyroidism and hypercalcemia and risk genotype have a higher susceptibility to developing renal stone disease.

Development and Validation of Stability Indicating High-Performance Thin-Layer Chromatographic (HPTLC) Method for Quantification of Asiaticoside from Centella asiatica L. and its Marketed Formulation.

Background: Ayurvedic medicines help in healing disease with fewer undesirable effects in comparison with an allopathic system of medicine to treat central nervous system (CNS) disorders, as the latter is more expensive. Centella asiatica L. is often used in Ayurvedic formulations for the treatment of CNS disorders. Objective: A stability test using an HPTLC method for the estimation of an important marker asiaticoside (ASI) from C. asiatica powder and marketed formulation was developed. Methods: The marker compound ASI from plant powders and marketed formulations were resolved using toluene-ethyl acetate-methanol-glacial acetic acid (2+7+3+1, v/v/v/v) as the mobile phase and then was derivatized. The plant powder and marketed formulation were also subjected to stability studies. Results: The Rf value of ASI was found in range of 0.43-0.47 for the standard ASI, plant powder, and marketed formulation. It was found that the plant powder and formulation exhibited first-order degradation kinetics. Conclusions: The contents of ASI in the formulation (Churna) and its flow characters reduced at the end of the 6 months during an accelerated stability study. The developed method can be used to quantify ASI in the presence of its degradation products. Highlights: The developed method helps in determining batch to batch variation in the content of ASI in herbal formulations.

Aphrodisiac and spermatogenic potential of alkaloidal fraction of Hygrophila spinosa T. Ander in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds of Hygrophila spinosa T. Ander (Acanthaceae) are traditionally used as aphrodisiac and spermatogenic in Indian System of medicine. AIM OF THE STUDY: Preliminary phytochemical screening of plant revealed the presence of alkaloids in seeds. As, alkaloidal fractions of several plants showed aphrodisiac and spermatogenic potential, set of experiments were designed to assess alkaloid enriched fraction of seeds of the plant for spermatogenic and aphrodisiac activity using in vitro and in vivo methods. MATERIALS AND METHODS: Alkaloid enriched fraction was prepared and assessed for spermatogenic activity using isolated rat Leydig cells in vitro. The fraction was further evaluated in vivo for spermatogenic and aphrodisiac potential using rat as an experimental animal. Increase in weight of reproductive organs, biochemical evaluation of selected parameters, histological studies of testes and sexual behavioral studies were selected as evaluation parameters for in vivo studies. RESULTS: Isolated rat Leydig cells treated with the fraction showed increased amount of testosterone present in culture media (14.7µg/ml) as compared to that of control (0.8µg/ml). Results of in vivo studies showed increase in serum testosterone level in treated animals (50mg/kg) by (115%), increase in weight of testes (8.0%) as compared to control. Marked improvement in testis histo-architecture of rats evident preliminarily by observing overcrowding of spermatozoa in enlarged lumen of seminiferous tubules in animals treated with testosterone and test fraction. Sertoli cells in treated animals were enlarged with highly granulated cytoplasm. Leydig cells also showed enlarged nucleus and darkly stained cytoplasm as compared to control. Mounting behavior of test animals improved, while latency period was decreased, as observed in behavioral studies. CONCLUSION: The set studies confirmed the ability of the fraction to stimulate Leydig cells and increased serum testosterone level. Increased testosterone level might be responsible for higher number of spermatozoa in testicular lumen as seen in testicular histology as well as increased libido as observed in behavioral studies.