Suchada Sukrong

Antinociceptive and anti-inflammatory effects of rosmarinic acid isolated from Thunbergia laurifolia Lindl.

Rosmarinic acid (RA) was isolated from an ethanolic extract of Thunbergia laurifolia leaves. The antinociceptive activity of RA was assessed in mice using hot-plate, acetic acid-induced writhing, and formalin tests. The anti-inflammatory effects of RA were determined in two mouse models of carrageenan-induced paw edema and cotton pellet-induced granuloma formation. Orally administered RA (50, 100, and 150 mg/kg) showed significant (p<0.001) antinociceptive activity in the hot-plate test and this effect was reversed by naloxone. RA at doses of 50 and 100mg/kg significantly reduced acetic acid-induced writhing by 52% (p<0.01) and 85% (p<0.001), respectively, and RA at 100mg/kg also caused significant inhibition of formalin-induced pain in the early and late phases (p<0.01 and p<0.001, respectively). RA at 100mg/kg significantly suppressed carrageenan-induced paw edema at 3, 4, 5 and 6h after carrageenan injection (p<0.01, p<0.05 p<0.01, and p<0.05, respectively) and showed significant activity against PGE2-induced paw edema. RA at 100mg/kg also inhibited cotton pellet-induced granuloma formation in mice. Taken together, these results demonstrate that RA possesses both central and peripheral antinociceptive activities and has anti-inflammatory effects against acute and chronic inflammation. While further evaluation regarding the safety profile of RA is needed, these data may provide a basis for the rational use of RA and T. laurifolia for treatment of pain and inflammatory disorders.

Sequencing Analysis of the Medicinal Plant Stemona tuberosa and Five Related Species Existing in Thailand Based on trnH-psbA Chloroplast DNA

In Thailand, Stemona tuberosa Lour. , S. phyllantha Gagnep. , S. collinsae Craib , S. burkillii Prain , S. aphylla Craib and S. sp. are found. The identification based on morphological characters alone is difficult and can lead to confusion regarding chemical constituents and biological activities. The tuberous roots of S. tuberosa have long been used for treatment of respiratory diseases and as anthelmintics. However, accurate identification of S. tuberosa is needed to ensure efficacy. Sequence comparison indicated that these Stemona spp. could be identified from the sequence of the trnH- psbA locus. As a result of different sequence lengths, the PCR products generated from newly designed primers could be used to preliminarily group the two species, S. tuberosa and S. phyllantha, apart from others. However, these products could be further sequenced to discriminate among Stemona spp.

Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name “Kwao Khruea” is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating “Kwao Khruea” species.

Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation

The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

Correlation of camptothecin-producing ability and phylogenetic relationship in the genus Ophiorrhiza.

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.

A topoisomerase II poison screen of ethnomedicinal Thai plants using a yeast cell-based assay

ETHNOPHARMACOLOGICAL RELEVANCE The plants used in this study had previously been identified and used by Thai folk practitioners, who had knowledge of Thai traditional medicine, as alternative treatments for cancer. Investigation into the mechanism of the Topoisomerase II (Top2) poison of these plants may give rise to new drug leads for cancer treatment. AIM OF THE STUDY This study aimed to screen ethnomedicinal plants used in Thai traditional medicine for Top2 poison activity using a yeast cell-based assay and also to validate the traditional uses of these plants by examining the Top2 poison activity. MATERIALS AND METHODS Thirty Thai medicinal plants were harvested and identified. Plant methanol extracts were prepared and screened in vitro using a yeast cell-based assay. Mutant yeast strains carrying the top2-1 allele, which encodes a temperature-sensitive topoisomerase, were used to establish the yeast spot test. Strains carrying this mutation grow normally at 25°C and generally have a wild-type drug sensitivity. These yeast strains are able to grow at 30°C, but the Top2 activity is greatly reduced at this temperature, which causes the strains to be highly drug resistant to anti-Top2 agents. Cell growth was measured by colony survival after exposure to various concentrations of extracts at different temperatures. RESULTS The extracts of six out of thirty ethnomedicinal plants, Curcuma longa, C. zedoaria, Derris scandens, Grangea maderaspatana, Stephania pierrei and S. suberosa, were found to have Top2 poison activity against the yeast cells. CONCLUSIONS The yeast screening system confirmed the proposed anti-cancer mechanisms of plants used in Thai traditional medicine by traditional doctors.

Full-length genome analysis of two genetically distinct variants of porcine epidemic diarrhea virus in Thailand

Abstract Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007 and a pandemic variant containing an insertion and deletion in the spike gene was responsible for outbreaks. In 2014, there were further outbreaks of the disease occurring within four months of each other. In this study, the full-length genome sequences of two genetically distinct PEDV isolates from the outbreaks were characterized. The two PEDV isolates, CBR1/2014 and EAS1/2014, were 28,039 and 28,033 nucleotides in length and showed 96.2% and 93.6% similarities at nucleotide and amino acid levels respectively. In total, we have observed 1048 nucleotide substitutions throughout the genome. Compared to EAS1/2014, CBR1/2014 has 2 insertions of 4 (56GENQ59) and 1 (140N) amino acid positions 56–59 and 140, and 2 deletions of 2 (160DG161) and 1 (1199Y) amino acid positions 160–161 and 1199. The phylogenetic analysis based on full-length genome of CBR1/2014 isolate has grouped the virus with the pandemic variants. In contrast, EAS1/2014 isolate was grouped with CV777, LZC and SM98, a classical variant. Our findings demonstrated the emergence of EAS1/2014, a classical variant which is novel to Thailand and genetically distinct from the currently circulating endemic variants. This study warrants further investigations into molecular epidemiology and genetic evolution of the PEDV in Thailand.

New 2-arylbenzofurans from the root bark of Artocarpus gomezianus and their α-glucosidase inhibitory activity

Abstract Two new 2-arylbenzofurans, namely 13-O-methyllakoochin B (1) and artogomezianin (2), were isolated from the root bark of Artocarpus gomezianus, along with six known compounds (3–8). The structures of new compounds were determined by spectroscopic and chemical methods. All of the isolates were evaluated for their α-glucosidase inhibitory activity. Artogomezianin (2) and lakoochin A (3) exhibited strong α-glucosidase inhibitory activity with IC50 values of 18.25 and 26.19 µM, respectively, as compared with the positive control acarbose.

Cytotoxic stilbenes from the roots of Paphiopedilum godefroyae.

Abstract Two new stilbenes, 2-(3′,5′-dimethoxyphenyl)-6-hydroxy-5-methoxybenzofuran (1) and 3′-hydroxy-2,5′-dimethoxystilbene (2), together with seven known stilbenes (3, 5–10) and one flavanone (4), were isolated from the roots of Paphiopedilum godefroyae. Their chemical structures were determined on the basis of their spectroscopic data. These isolated compounds were evaluated for their cytotoxicity against human small cell lung cancer (NCI-H187) cell lines and an arylbenzofuran derivative, 5,6-dimethoxy-2-(3-hydroxy-5-methoxyphenyl)benzofuran (6), was shown to be strongly cytotoxic with an IC50 value of 5.10 μM.

Wound Healing Effect of Flavonoid Glycosides from Afgekia mahidolae B.L.Burtt & Chermsir. Leaves

Five known flavonoid glycosides namely, juglanin (1), astragalin (2), nicotiflorin (3), isoquercetin (4) and apigenin-7-O--glucuronide (5), and a phenolic glucoside, arbutin (6), were isolated for the first time from the leaves of Afgekia mahidolae (Fabaceae). Their structures were elucidated using oneand two-dimensional NMR spectroscopic methods and comparison with the literature. The ability of 1-4 to promote the migration of CCD1064sk fibroblasts into a scratch-wound area was evaluated. The results indicated that juglanin (1) and nicotiflorin (3) significantly increased the migration of these cells and, hence, supporting the wound healing effect of flavonol glycosides.