Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines.

Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3-120.2% target recovery at 0.1-10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.

α-glucosidase inhibitors isolated from Mimosa pudica L.

The aim of the study was to isolate digestive enzymes inhibitors from Mimosa pudica through a bioassay-guided fractionation approach. Repeated silica gel and sephadex LH 20 column chromatographies of bioactive fractions afforded stigmasterol, quercetin and avicularin as digestive enzymes inhibitors whose IC50 values as compared to acarbose (351.02 ± 1.46 µg mL-1) were found to be as 91.08 ± 1.54, 75.16 ± 0.92 and 481.7 ± 0.703 µg mL-1, respectively. In conclusion, M. pudica could be a good and safe source of digestive enzymes inhibitors for the management of diabetes in future.

Rapid investigation of α-glucosidase inhibitory activity of Phaleria macrocarpa extracts using FTIR-ATR based fingerprinting.

Phaleria macrocarpa, known as "Mahkota Dewa", is a widely used medicinal plant in Malaysia. This study focused on the characterization of α-glucosidase inhibitory activity of P. macrocarpa extracts using Fourier transform infrared spectroscopy (FTIR)-based metabolomics. P. macrocarpa and its extracts contain thousands of compounds having synergistic effect. Generally, their variability exists, and there are many active components in meager amounts. Thus, the conventional measurement methods of a single component for the quality control are time consuming, laborious, expensive, and unreliable. It is of great interest to develop a rapid prediction method for herbal quality control to investigate the α-glucosidase inhibitory activity of P. macrocarpa by multicomponent analyses. In this study, a rapid and simple analytical method was developed using FTIR spectroscopy-based fingerprinting. A total of 36 extracts of different ethanol concentrations were prepared and tested on inhibitory potential and fingerprinted using FTIR spectroscopy, coupled with chemometrics of orthogonal partial least square (OPLS) at the 4000-400 cm-1 frequency region and resolution of 4 cm-1. The OPLS model generated the highest regression coefficient with R2Y = 0.98 and Q2Y = 0.70, lowest root mean square error estimation = 17.17, and root mean square error of cross validation = 57.29. A five-component (1+4+0) predictive model was build up to correlate FTIR spectra with activity, and the responsible functional groups, such as -CH, -NH, -COOH, and -OH, were identified for the bioactivity. A successful multivariate model was constructed using FTIR-attenuated total reflection as a simple and rapid technique to predict the inhibitory activity.

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain.

Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.

Evaluation of antioxidant properties and mineral composition of Purslane (Portulaca oleracea L.) at different growth stages.

The main objective of this research was to appraise the changes in mineral content and antioxidant attributes of Portulaca oleracea over different growth stages. The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). DPPH scavenging (IC(50)) capacity ranged from 1.30 ± 0.04 to 1.71 ± 0.04 mg/mL, while the ascorbic acid equivalent antioxidant activity (AEAC) values were 229.5 ± 7.9 to 319.3 ± 8.7 mg AA/100 g, total phenol content (TPC) varied from 174.5 ± 8.5 to 348.5 ± 7.9 mg GAE/100 g. AAC 60.5 ± 2.1 to 86.5 ± 3.9 mg/100 g and FRAP 1.8 ± 0.1 to 4.3 ± 0.1 mg GAE/g. There was good correlation between the results of TPC and AEAC, and between IC(50) and FRAP assays (r(2) > 0.9). The concentrations of Ca, Mg, K, Fe and Zn increased with plant maturity. Calcium (Ca) was negatively correlated with sodium (Na) and chloride (Cl), but positively correlated with magnesium (Mg), potassium (K), iron (Fe) and zinc (Zn). Portulaca olerecea cultivars could be used as a source of minerals and antioxidants, especially for functional food and nutraceutical applications.