Kittisak Buddhachat

Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product

The Phyllanthus genus, a plant used in traditional Thai medicine, has according to several pharmacopeias hepatoprotective properties. Not only is the anatomical morphology of these species relatively similar but they also share the Thai common names Look-Tai-Bai (ลูกใต้ใบ) and Yah-Tai-Bai (หญ้าใต้ใบ), which might cause confusion for laypersons. This study attempted to develop a method for accurate identification of Phyllanthus species, especially Phyllanthus amarus, and to detect contaminants in P. amarus products by using DNA barcoding coupled with high resolution melting (HRM) analysis (bar-HRM). Two plastid loci (rbcL and trnL) were chosen for DNA barcoding to generate a suitable primer for distinguishing Phyllanthus species by HRM analysis. The five species of Phyllanthus were subjected to amplification for testing the specificity and discrimination power of the designed primers derived from rbcL and trnL regions. Sensitivity of the method (DNA barcoding conjugated with HRM) to detect adulterant in P. amarus samples was evaluated. The commercial P. amarus products obtained from a local market were authenticated. The primer pair derived from trnL DNA barcoding (PhylltrnL) had more specificity and power of discrimination for Phyllanthus species than that derived from rbcL DNA barcoding (PhyllrbcL). The result showed that Tm of P. amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus airy-shawii, and Phyllanthus virgatus was 74.3±0.08, 73.04±0.07, 73.36±0.05, 72.21±0.06, 72.77±0.15°C, respectively. This method proved to be a very sensitive tool that can be used for rapid detection of contamination as low as 1% of other Phyllanthus species in P. amarus admixtures. All commercial products of P. amarus obtained from a local market in Thailand were found to contain pure raw materials of P. amarus without any substitution or contamination. Our results indicated that the use of DNA barcoding coupled with HRM was an efficient molecular tool for correct species identification. This molecular tool provides a noteworthy benefit for quality control of medicinal plants and industry plants for pharmacological prospects.

Elemental Analysis of Bone, Teeth, Horn and Antler in Different Animal Species Using Non-Invasive Handheld X-Ray Fluorescence.

Mineralized tissues accumulate elements that play crucial roles in animal health. Although elemental content of bone, blood and teeth of human and some animal species have been characterized, data for many others are lacking, as well as species comparisons. Here we describe the distribution of elements in horn (Bovidae), antler (Cervidae), teeth and bone (humerus) across a number of species determined by handheld X-ray fluorescence (XRF) to better understand differences and potential biological relevance. A difference in elemental profiles between horns and antlers was observed, possibly due to the outer layer of horns being comprised of keratin, whereas antlers are true bone. Species differences in tissue elemental content may be intrinsic, but also related to feeding habits that contribute to mineral accumulation, particularly for toxic heavy metals. One significant finding was a higher level of iron (Fe) in the humerus bone of elephants compared to other species. This may be an adaptation of the hematopoietic system by distributing Fe throughout the bone rather than the marrow, as elephant humerus lacks a marrow cavity. We also conducted discriminant analysis and found XRF was capable of distinguishing samples from different species, with humerus bone being the best source for species discrimination. For example, we found a 79.2% correct prediction and success rate of 80% for classification between human and non-human humerus bone. These findings show that handheld XRF can serve as an effective tool for the biological study of elemental composition in mineralized tissue samples and may have a forensic application.

A modified colorimetric method of gelatinolytic assay using bacterial collagenase type II as a model

Coomassie brilliant blue (CBB) heated by microwave was used as a staining dye for measuring gelatinolytic activity. The quantity of gelatin remaining after incubation with bacterial collagenase was determined using the heated CBB, resulting in visible blue pellets. Dimethyl sulfoxide was added to dissolve the dye and measurement of the absorbance at 600 nm was done to detect the level of gelatin (up to 10 μg), with the limit of detection for the amount of collagenase at 50 ng. This approach is rapid, simple, and economic for the purpose of screening for pharmaceutical agents that possess inhibitory activity on collagenase.

Use of handheld X-ray fluorescence as a non-invasive method to distinguish between Asian and African elephant tusks.

We describe the use of handheld X-ray fluorescence, for elephant tusk species identification. Asian (n = 72) and African (n = 85) elephant tusks were scanned and we utilized the species differences in elemental composition to develop a functional model differentiating between species with high precision. Spatially, the majority of measured elements (n = 26) exhibited a homogeneous distribution in cross-section, but a more heterologous pattern in the longitudinal direction. Twenty-one of twenty four elements differed between Asian and African samples. Data were subjected to hierarchical cluster analysis followed by a stepwise discriminant analysis, which identified elements for the functional equation. The best equation consisted of ratios of Si, S, Cl, Ti, Mn, Ag, Sb and W, with Zr as the denominator. Next, Bayesian binary regression model analysis was conducted to predict the probability that a tusk would be of African origin. A cut-off value was established to improve discrimination. This Bayesian hybrid classification model was then validated by scanning an additional 30 Asian and 41 African tusks, which showed high accuracy (94%) and precision (95%) rates. We conclude that handheld XRF is an accurate, non-invasive method to discriminate origin of elephant tusks provides rapid results applicable to use in the field.

The Distribution of Elements in 48 Canine Compact Bone Types Using Handheld X-Ray Fluorescence

A major question when we talk about the elements in the bone is whether all bones contain the same elements. To answer this question, this study was designed for determination of the elemental levels in 48 various canine compact bones using handheld X-ray fluorescence technique. From a total of 26 elements that could be detected, only 13 elements were found in all 48 bones. The sternum and os penis were significantly different from the other bones in that they contained the highest number of elements. The ratio of Ca and P was significantly different when comparing certain bones: there was a higher Ca/P ratio in the patella (right), calcaneus (right and left), and sternum compared with a lower ratio in the radius (left), rib (left), phalanx (left forelimb), and carpus (left). These results are the first to demonstrate that different types of bones have different elemental profiles, even for major elements such as Ca and P. Moreover, the Ca/P ratio was also different between bone types. This data is important for the selection of bones appropriate to the element studied. In addition, the results proved that the elements were not equally distributed in every bone in the body.

Two fluoroquinolones and their combinations with hyaluronan: comparison of effects on canine chondrocyte culture

Fluoroquinolones (FQs) are frequently used for septic arthritis. Increased antibacterial activity has been associated with mammalian cell cytotoxicity that may increase the risk of developing osteoarthritis. This study compared the direct effects of two different FQs, enrofloxacin (Enro) and marbofloxacin (Mar), on normal primary canine chondrocytes and inflammatory-stimulated chondrocytes, in addition to their administration in combination with hyaluronan (HA). Cell viability, cell apoptosis, s-GAG production, and expression patterns of inflammatory, extracellular matrix (ECM) component and protease genes were measured. Enro co-culturing with HA could modify s-GAG synthesis compared with the negative control group. Co-treatment with both FQs and HA significantly decreased cell viability and induced more total apoptotic cell death compared with the negative control and pre-IL-1β-stimulated group. Enro regulated IL-1β-stimulated cells to overexpress IL-1β, TNF, and MMP3, whereas Mar induced upregulation of PTGS2 and NFKB1 and enhanced the expression of ECM component genes HAS1, COL2A1, and ACAN as well as TIMP1 and MMP9. Simultaneous use of HA with Enro can effectively reduce the expression of IL-1β, TNF, and MMP3 in pre-IL-1β-stimulated chondrocytes. These results suggest the beneficial effects of HA in reducing the adverse effects of Enro treatment at the transcriptional level.

Gender-Related Differences in Pelvic Morphometrics of the Retriever Dog Breed.

This study presents the results from a morphometric analysis of 52 dry Retriever dog pelvic bones (30 male, 22 female). A total of 20 parameters were measured using an osteometric board and digital vernier caliper. Six parameters were found to be significantly higher (P < 0.05) in males than in females, while one parameter was significantly higher (P < 0.05) in females than in males. However, none of the measured parameters demonstrated clear cut‐off values with no intersect between males and females. Therefore, we generated a stepwise discriminant analysis from all 20 parameters in order to develop a possible working equation to discriminate gender from a dog pelvic bone. Stepwise discriminant analysis was used to create a discrimination function: Y = [82.1*PS/AII] − [50.72*LIS/LI] − [23.09*OTD/SP] + [7.69*SP/IE] + [6.52*IC/OW] + [7.67*ISA/OW] + [20.77*AII/PS] + [504.71*OW/ISA] − [90.84*PS/ISA] − [148.95], which showed an accuracy rate of 86.27%. This is the first study presenting an equation/function for use in discriminating gender from a dogs pelvic measurements. The results can be used in veterinary forensic anthropology and also show that a dogs pelvis presents sexual dimorphisms, as in humans.

Determining comparative elemental profile using handheld X-ray fluorescence in humans, elephants, dogs, and dolphins: Preliminary study for species identification.

Species identification is a crucial step in forensic anthropological studies. The aim of this study was to determine elemental profiles in bones from four mammal species, to be used for species discrimination. Human, elephant, dog, and dolphin bones were scanned by X-ray fluorescence (XRF); the differences in elemental profiles between species were determined using discriminant analysis. Dogs had the greatest number of elements (23), followed by humans (22) and elephants (20). Dolphins had the lowest number of elements (16). The accuracy rate of species identification in humans, elephants, dogs, and dolphins was 98.7%, 100%, 94.9%, and 92.3%, respectively. We conclude that element profiles of bones based on XRF analyses can serve as a tool for determining species.

Variation in elemental composition of human teeth and its application for feasible species identification

Identifying human remains is a primary task in forensic science. In this study, we propose a possible new technique, handheld X-ray fluorescence (HHXRF), for determining whether a suspected tooth is an authentic human tooth. A total of 444 teeth obtained from 111 human skulls (male=62, female=49) aged between 30-67 years (51.81±8.37 years) were used as subjects. The teeth were scanned by HHXRF to acquire their elemental profile. Differences in elemental composition were analyzed for different tooth positions (numbers 1-32), between crown and root, and between sexes (male and female); also, the proportion of elements in relation to different human ages was examined. Teeth from 20 different animal species, serving as non-human teeth samples, were used to distinguish between human and non-human teeth through a stepwise discriminant analysis. Our results revealed that different tooth positions, different regions (crown and root) of a tooth, and different sexes demonstrated disparities in the proportion of several elements. The accuracy rate of predicting sex based on the elemental profile of human teeth was 65.5%. Likewise, a dissimilar distribution of elements between human and non-human teeth was observed, leading to a high degree of correctness of 83.2% for distinguishing them. In conclusion, elemental analysis by HHXRF could serve as a promising candidate tool for identifying human teeth in forensic science, but is ineffective for sex determination.

Feline (Felis catus) Skull and Pelvic Morphology and Morphometry: Gender-Related Difference?

This study demonstrates sexual dimorphism of feline bones, based on a morphometric analysis of 38 dried feline skulls and pelvic bones (20 males, 18 females). A total of 44 parameters (skull = 12, mandible = 10, pelvis = 22) were measured using a digital vernier calliper. In morphological observation of these bones, there were three hallmarks indicating a remarkable difference between sexes: the coronoid process of the mandible (accuracy rate = 88.2%); and the os coxae – caudal ventral iliac spine (accuracy rate = 94.4%), and the angle of the ischiatic arch (accuracy rate = 74.3%). In addition, based on morphometric characteristics, six parameters were found to be significantly different (P < 0.05) between males and females, consisting of one in the mandible and five in the pelvis, but no parameters in the skull. Effective equations to discriminate gender were generated through a stepwise discriminant analysis from feline mandible and pelvic bones. Our findings showed that an equation from the pelvic bones, Y = [−16.066*T/O] + [2.559*IC/PS] + [13.357*TTL/ISA] − [4.478], appeared to be more applicable with a 97.3% accuracy rate, while a function from the mandible gave a 64.9% accuracy rate. In conclusion, we suggest that an equation from feline pelvic measurements and three hallmarks, one on the mandible and two on the os coxae, can be used for sex estimation.