Semi-Synthesis of N-Aryl Amide Analogs of Piperine from Piper nigrum and Evaluation of Their Antitrypanosomal, Antimalarial, and Anti-SARS-CoV-2 Main Protease Activities.

Piper nigrum, or black pepper, produces piperine, an alkaloid that has diverse pharmacological activities. In this study, N-aryl amide piperine analogs were prepared by semi-synthesis involving the saponification of piperine (1) to yield piperic acid (2) followed by esterification to obtain compounds 3, 4, and 5. The compounds were examined for their antitrypanosomal, antimalarial, and anti-SARS-CoV-2 main protease activities. The new 2,5-dimethoxy-substituted phenyl piperamide 5 exhibited the most robust biological activities with no cytotoxicity against mammalian cell lines, Vero and Vero E6, as compared to the other compounds in this series. Its half-maximal inhibitory concentration (IC50) for antitrypanosomal activity against Trypanosoma brucei rhodesiense was 15.46 ± 3.09 µM, and its antimalarial activity against the 3D7 strain of Plasmodium falciparum was 24.55 ± 1.91 µM, which were fourfold and fivefold more potent, respectively, than the activities of piperine. Interestingly, compound 5 inhibited the activity of 3C-like main protease (3CLPro) toward anti-SARS-CoV-2 activity at the IC50 of 106.9 ± 1.2 µM, which was threefold more potent than the activity of rutin. Docking and molecular dynamic simulation indicated that the potential binding of 5 in the 3CLpro active site had the improved binding interaction and stability. Therefore, new aryl amide analogs of piperine 5 should be investigated further as a promising anti-infective agent against human African trypanosomiasis, malaria, and COVID-19.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Oxyresveratrol: Structural Modification and Evaluation of Biological Activities.

Oxyresveratrol (2,4,3',5'-tetrahydroxystilbene, 1), a phytoalexin present in large amounts in the heartwood of Artocarpus lacucha Buch.-Ham., has been reported to possess a wide variety of biological activities. As part of our continuing studies on the structural modification of oxyresveratrol, a library of twenty-six compounds was prepared via O-alkylation, aromatic halogenation, and electrophilic aromatic substitution. The two aromatic rings of the stilbene system of 1 can be chemically modulated by exploiting different protecting groups. Such a strategy allows for selective and exclusive modifications on either ring A or ring B. All compounds were evaluated in vitro for a panel of biological activities, including free radical scavenging activity, DNA protective properties, antiherpetic activity, inhibition of α-glucosidase and neuraminidase, and cytotoxicity against some cancer cell lines. Several derivatives were comparably active or even more potent than the parent oxyresveratrol and/or the appropriate positive controls. The partially etherified analogs 5'-hydroxy-2,3',4-trimethoxystilbene and 3',5'-dihydroxy-2,4-dimethoxystilbene demonstrated promising anti-herpetic and DNA protective activities, offering new leads for neuropreventive agent research, whereas 5'-hydroxy-2,3',4,-triisopropoxystilbene displayed anti-α-glucosidase effects, providing a new lead molecule for anti-diabetic drug development. 3',5'-Diacetoxy-2,4-diisopropoxystilbene showed potent and selective cytotoxicity against HeLa cancer cells, but the compound still needs further in vivo investigation to verify its anticancer potential.

Complete genome characterization of porcine epidemic diarrhea virus in Vietnam.

Porcine epidemic diarrhea virus (PEDV) first emerged in Vietnam in 2009. In this study, the complete genomes of three Vietnamese PEDV isolates were characterized. These three isolates were isolated from 3-day-old pigs experiencing diarrhea. Two isolates were from swine farms in the south, and the other was from northern Vietnam. The whole genome sequences of these isolates are 28,035 nucleotides in length and have characteristics similar to those of other PEDV isolates. All three Vietnamese PEDV isolates share 99.8 % and 99.6 % sequence identity at the nucleotide and amino acid level, respectively, and have insertions of four amino acids (GENQ) and one amino acid (N) at positions 56-59 and 140, respectively, and one deletion of two amino acids (DG) at positions 160-161. Phylogenetic analysis based on the whole genome revealed that the three Vietnamese PEDV isolates are grouped together with new variants from China from 2011 to 2012 and are genetically distinct from US isolates and the classical PEDV variant. The results suggest that Vietnamese PEDV isolates are new variants, as evidenced by their genetic composition of insertions and a deletion in the spike gene, and they might have originated from the same ancestor as the Chinese PEDV strain. This study provides a better understanding of the molecular characteristics of PEDV in Vietnam.

Replacement of a quinone by a 5-O-acetylhydroquinone abolishes the accidental necrosis inducing effect while preserving the apoptosis-inducing effect of renieramycin M on lung cancer cells.

Renieramycin M (1), a bistetrahydroisoquinolinequinone alkaloid isolated from the marine sponge Xestospongia sp., has been reported to possess promising anticancer effects. However, its accidental necrosis inducing effect has limited further development due to concerns of unwanted toxicity. The presence of two quinone moieties in its structure was demonstrated to induce accidental necrosis and increase reactive oxygen species (ROS) levels. Therefore, one quinone of 1 was modified to produce the 5-O-acetylated hydroquinone derivative (2), and 2 dramatically reduced the accidental necrosis inducing effect while preserving the apoptosis-inducing effect of parent 1 on lung cancer H23 cells. Addition of the antioxidant N-acetylcysteine suppressed the accidental necrosis mediated by 1, suggesting that its accidental necrosis inducing effect was ROS-dependent. The fluorescent probe dihydroethidium revealed that the accidental necrosis mediated by 1 was due to its ability to generate intracellular superoxide anions. Interestingly, the remaining quinone in 2 was required for its cytotoxicity, as the 5,8,15,18-O-tetraacetylated bishydroquinone derivative (3) exhibited weak cytotoxicity compared to 1 and 2. The present study demonstrates a simple way to eliminate the undesired accidental necrosis inducing effect of substances that may be developed as improved anticancer drug candidates.

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.

An Efficient DNA Extraction for a Blue Xestospongia sp. Sponge and Its Associated Microorganisms Containing Cytotoxic Substances.

Extraction of high quantity and quality DNAs from marine sponges, which contain diverse and abundant microbial communities, is important to molecular biology techniques for the analysis of nucleic acids. Several marine sponges and their associated microorganisms have been known to produce cytotoxic natural products on several cancer cell lines via DNA damage mechanisms. These marine cytotoxic substances might be one of the factors that cause the low quantity and quality of DNAs during the DNA extraction from its living origin. Therefore, the extraction of DNA of a Thai blue marine sponge Xestospongia sp. with sufficient purity and quantity for molecular study can be challenging. In this study, we developed an efficient extraction method to prepare DNAs from a Thai blue marine sponge Xestospongia sp. which accumulated a highly potent cytotoxic alkaloid with DNA-damaging activity, named Renieramycin M (RM), as a major constituent in high quantity. We demonstrated that removal of RM from the sponge samples by a simple methanolic extraction before DNA extraction dramatically increased the yield and purity of DNAs compared to the RM-unremoved sponge samples. High molecular weight (HMW) genomic DNA was obtained from sponge samples with 8 times of RM elimination by using modified NaOAc salting-out extraction method. The quantity and quality of the prepared DNAs were comparatively determined via spectrophotometry, electrophoresis, and 16S rRNA gene amplification. Our result suggests that the removal of DNA-damaging constituents from the samples is a crucial step and must be seriously taken as the necessary consideration for the practical protocol of DNA extraction.

Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity.

Human vascular endothelial growth factor (VEGF) is a potent pro-angiogenic growth factor essential for wound healing. Due to its potential applications, many expression strategies have been developed to produce high levels of VEGF. Here, we have optimized the expression conditions for the production of recombinant VEGF in Nicotiana benthamiana by using a geminiviral vector. Four different expression constructs that differ by the location of a C- or N-terminal histidine tag and SEKDEL sequence were developed and utilized for plant transient expression. The recombinant VEGF was further purified by using affinity chromatography and confirmed by SDS-PAGE and Western blotting probed with anti-VEGF antibody. Furthermore, our results showed that the recombinant VEGF in all tested concentrations did not exhibit any cytotoxic effect on HaCaT cells and induced cell migration in vitro. These findings show that the plant-produced VEGF has the potential to be used in regenerative medicine and cosmetic industry.

Anti-herpes simplex virus (HSV-1) activity of oxyresveratrol derived from Thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous HSV-1 infection in mice.

Oxyresveratrol, a major compound purified from Artocarpus lakoocha, a Thai traditional medicinal plant, was evaluated for its mechanism of action and therapeutic efficacy on cutaneous herpes simplex virus (HSV) infection in mice. The inhibitory concentrations for 50% HSV-1 plaque formation of oxyresveratrol, three clinical isolates, thymidine kinase (TK)-deficient and phosphonoacetic acid (PAA)-resistant HSV-1 were 19.8, 23.3, 23.5, 24.8, 25.5 and 21.7microg/ml, respectively. Oxyresveratrol exhibited the inhibitory activity at the early and late phase of viral replication and inhibited the viral replication with pretreatment in one-step growth assay of HSV-1 and HSV-2. Oxyresveratrol inhibited late protein synthesis at 30microg/ml. The combination of oxyresveratrol and acyclovir (ACV) produced synergistic anti-HSV-1 effect, as characterized by the isobologram of plaque inhibition. Mice orally treated with oxyresveratrol (500mg/kg/dose) dose at 8 h before and three times daily had significant delay in herpetic skin lesion development (P<0.05). Topical application of 30% oxyresveratrol ointment five times daily significantly delayed the development of skin lesions and protected mice from death (P<0.0001).

Complete Genome Sequences of Two Genetically Distinct Variants of Porcine Epidemic Diarrhea Virus in the Eastern Region of Thailand.

Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007. Previously, PEDV in Thailand was a new variant containing an insertion and deletion in the spike gene. Herein, full-length genome sequences are reported for two variants of PEDV isolates from pigs displaying diarrhea in Thailand.

Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand.

Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008-2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea.

Oxyresveratrol protects against DNA damage induced by photosensitized riboflavin.

Riboflavin can be photosensitized to produce reactive oxygen species. In the present study, a DNA damage assay was developed based on the photo reaction of riboflavin. In this test system, oxyresveratrol showed higher DNA protective effect than the well-known antioxidants Trolox and ascorbic acid. The results suggest potential applications for oxyresveratrol as an anti-aging agent and a riboflavin stabilizer.