Siriwadee Chomdej

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.

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI).

OBJECTIVE To analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand. METHODS Based on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program. RESULTS The phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera. CONCLUSIONS The sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution.

Red River barrier and Pleistocene climatic fluctuations shaped the genetic structure of Microhyla fissipes complex (Anura: Microhylidae) in southern China and Indochina

South China and Indochina host striking species diversity and endemism. Complex tectonic and climatic evolutions appear to be the main drivers of the biogeographic patterns. In this study, based on the geologic history of this region, we test 2 hypotheses using the evolutionary history of Microhyla fissipes species complex. Using DNA sequence data from both mitochondrial and nuclear genes, we first test the hypothesis that the Red River is a barrier to gene flow and dispersal. Second, we test the hypothesis that Pleistocene climatic cycling affected the genetic structure and population history of these frogs. We detect 2 major genetic splits that associate with the Red River. Time estimation suggests that late Miocene tectonic movement associated with the Red River drove their diversification. Species distribution modeling (SDM) resolves significant ecological differences between sides of the Red River. Thus, ecological divergence also probably promoted and maintained the diversification. Genogeography, historical demography, and SDM associate patterns in southern China with climate changes of the last glacial maximum (LGM), but not Indochina. Differences in geography and climate between the 2 areas best explain the discovery. Responses to the Pleistocene glacial–interglacial cycling vary among species and regions.

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.

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

BackgroundMatrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the MMP-3 gene in in vitro human chondrosarcoma using siRNA.MethodsCells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and MMP-3 siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).ResultsIn IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (p < 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (p < 0.05). MMP-3 gene expression was downregulated significantly (p < 0.05).ConclusionMMP-3 specific siRNA can inhibit the expression of MMP-3 in chondrosarcoma. This suggests that MMP-3 siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.

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.

Effects of corticosteroids and their combinations with hyaluronanon on the biochemical properties of porcine cartilage explants

BackgroundIntra-articular injection of corticosteroids is used to treat the inflammatory pain of arthritis and osteoarthritis (OA), but our previous study found a deleterious effect of these steroids on chondrocyte cells. Hyaluronic acid (HA) injection has been suggested as a means to counteract negative side effects through replenishment of synovial fluid that can decrease pain in affected joints. To better understand the effects of corticosteroids on these processes, dexamethasone (Dex) and prednisolone (Pred) were administered to porcine cartilage explants at several concentrations with and without HA. We examined corticoid effects by determining sulfate-glycosaminoglycan (s-GAG) and uronic acid (UA) content of the explant media, and safranin-O staining of the cells. Analysis of lactate dehydrogenase (LDH) activity was conducted to assess cell cytotoxicity.ResultsDex treatment significantly reduced cellular cytotoxicity compared to the other treatment groups, especially with regards to the release of s-GAG, and protects against superficial proteoglycan damage. However, there was no difference between Pred and Dex, with and without HA, in the UA content remaining in porcine cartilage explants.ConclusionsThe data suggest that combinations of Dex and Pred with HA did not have a significant effect on protection or enhancement of the articular cartilage matrix under the current conditions.

Differences in osteon structure histomorphometry between puppyhood and adult stages in the Golden Retriever

Osteon structure has been widely studied in mammals, but osteon structure in dogs has received relatively little attention, especially in terms of whether aging has any effect on osteon structure. The aim of this study was to compare the osteon structure of both flat (scapula and os coxae) and long bones (humerus, radius, ulna, metacarpus, femur and tibia) of male puppy and adult Golden Retrievers. We examined five parameters: Haversian canal diameter, Haversian canal area, osteon diameter, osteon area, and number of lacunae per osteon. Our results show that the values for Haversian canal diameter were significantly higher in the os coxae and tibia, but significantly lower in the femur of adult dogs as compared to those of puppies. The Haversian canal diameter of the other bones investigated did not show any significant differences between puppies and adult dogs. The Haversian canal area was significantly greater in the os coxae, radius and femur of adult dogs than in those of puppies. The osteon diameter and area of every bone examined were significantly smaller in puppies than in adult dogs. Lastly, the number of lacunae per osteon showed the same trend as osteon diameter and area. Plexiform bone could be found in three bones in puppies, i.e. the femur, humerus and tibia. Overall, the results of this study should provide basic knowledge on the microanatomy of cortical bone in dogs and on the possible influence age.

Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1β-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1β, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1β-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.

Articular Cartilage Gene Expression after Coxofemoral Joint Luxation in the Dog

This study examined the relationship between days of hip luxation and the expression of various mRNA. Twenty-six articular cartilages were used in the experiment: 3 samples were from normal dogs and 23 samples were collected from the femoral heads of hips that had been luxated for different lengths of time. Ten mRNA, including nonapoptotic genes (AGG, COL2A1, MMP-3, HAS-1, HAS-2, and TIMP-1) and apoptotic genes (BAX, BCL-2, CAS-3, and CAS-9), were studied for their expression using real-time PCR. We found very high correlation between expression level and luxation days (r 2 > 0.9) in COL2A1, MMP-3, HAS-1, HAS-2, TIMP-1, BAX, and CAS-9, while the others (AGG, BCL-2, and CAS-3) also showed high correlation (r 2 = 7–9). And we found a significant difference (P < 0.05) in the expression of transcripts depending on the number of luxation days. In conclusion, a delay in joint reduction may increase the chances of development of osteoarthritis.