ABSTRACT
This study aims to build a standardization method for preparation of effective powder from FSA and to quantify diosgenin in FSA. Methodology: One kg of FS were used in this study. Setting: BMS, KOM and KOP, IIUM Kuantan campus. FS were washed with distilled water to exclude any foreign matter, and were then air dried. FS-powder were put in distilled water in a ratio of 1 g of powder in 20 ml of distilled water and were shaken at room temperature for 24 hours. Ten mg of hydrolyzed extract sample was diluted in 10 ml volumetric flask with methanol for 15 minutes. Chromatographic estimation was performed using an equilibrated reverse phase Eclipse XDB-C18 column (particle size 5 µg, 4.6 mm x 150 mm). Results: One gram of FSA extract was hydrolyzed to produce sapogenins and 46.6% was recovered. A calibration curve that was constructed based on five dilutions of diosgenin standard at concentrations of 2, 5, 10, 20, 30 and 50 ppm produced a linear graft (r = 0.999). The concentration of diosgenin in FSA extract as calculated using the regression analysis was found to be 29.66 µg/ml, 13.81 % w/w on dried weight basis. Conclusion: Preparation and standardization of effective powder from FSA are the corner stone of many scientific researches in IIUM and Malaysia. Diosgenin is available in the FSA in adequate concentration. The adequate amount of diosgenin in the FSA will guide us to do further study in the way of preparation of a natural product that can be used in the field of reversible anti-fertility therapy.
ABSTRACT
Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.