In Vitro and In Silico Evaluation of Betulin on Calcium Oxalate Crystal Formation.

Objective: The medicinal plant Betula alba has been used for prevention and treatment of kidney stones. Betulin is one of the main phytochemicals of Betula alba. The aim of this study is to investigate the antioxidant and antiurolithiatic activity of betulin in vitro and in silico. For antioxidant activity, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), total reducing capacity, nitric oxide (NO) radical scavenging assay, and superoxide radical scavenging assay were studied. Method: In order to study antiurolithiatic activity, three assays such as crystallization, nucleation, and aggregation of oxalate crystal in urine were performed. In silico experiments were performed by using AutoDock 4.2 tools in order to establish affinity of phytochemicals toward antioxidant enzyme and matrix metalloproteinase (MMP-2 and 9). Results: The results obtained clearly demonstrate the significant scavenging activity of betulin and cystone against DPPH, NO, and superoxide radicals in comparison to standard antioxidant L-ascorbate (L-AA). It has also been observed that betulin has the capacity to inhibit the crystallization, nucleation, and aggregation in comparison to cystone. On the other hand, betulin and L-AA showed strong affinity toward antioxidant enzymes and matrix metalloproteinase as determined by in silico experiments. Conclusions: From this, it may be concluded that the antiurolithiatic activity of betulin is, at least in part, mediated by its antioxidant property.

Homologous ELISA for measurement of medroxyprogesterone acetate in serum.

In order to develop ELISA for medroxyprogesterone acetate, medroxyprogesterone acetate-3-carboxymethyloxime (MPA-3-CMO) was coupled to bovine serum albumin (BSA) for immunogen preparation and to horseradish peroxidase (HRP) for enzyme conjugate preparation by N-hydroxysuccinimide mediated carbodiimide reaction. The immunogen was used to raise the antiserum in New Zealand white rabbit. The immunoreactivity of MPA-3-CMO-BSA-antibody and MPA-3-CMO-HRP enzyme conjugate was checked by checkerboard assay. The MPA-3-CMO-HRP enzyme conjugate and MPA-3-CMO-BSA-antibody were used for further development, standardization and validation of the assay. Sensitivity, ED50 and affinity of the assay were found to be 0.114 ng/mL, 2.75 ng/mL and 9.9 × 10⁻8 L/mol respectively. The % cross-reaction of analogous steroids with MPA-3-CMO-BSA-antibody was less than 0.025%. The recovery of the exogenously spiked MPA serum pools were in the range of 96.83-105.47%. The intra- and inter-assay coefficients of variation was less than 7.02%. The correlation coefficient of the serum level of MPA measured by the developed assay with the commercially available kit was found to be 0.95 (n = 37). This developed ELISA was further validated by measuring serum level of MPA in rat after administering them different doses of MPA intramuscularly.