Effects of Preharvest Methyl Jasmonate Elicitation and Electrical Stimulation on Camptothecin Production by In Vitro Plants of Ophiorrhiza ridleyana Craib

To enhance plant camptothecin (CPT) production in vitro, 5-month-old Ophiorrhiza ridleyana Craib plant cultures were treated with solutions of methyl jasmonate (MeJA) dissolved in ethanol, which were applied to the surface of the solid culture medium. It was demonstrated that the maximum CPT content in the tissue-cultured plants was achieved after 12 h elicitation with 50 µM MeJA. The mean CPT contents in roots and stems were 50.8 and 67.0 µg/g DW, respectively, which were approximately 1.8- and 2.6-fold higher, respectively, than those of the control. However, MeJA elicitation showed no significant effect on CPT accumulation in O. ridleyana leaves. Moreover, it was found that direct electric current (DC) stimulation also significantly increased CPT accumulation in O. ridleyana. The treatment with DC at 20 mA for 3 min of stimulation enhanced 3-fold the CPT content in roots, stems, and leaves to 41.9, 36.0 and 19.6 µg/g DW, respectively, which were approximately 1.5-, 1.7- and 1.4-fold higher, respectively, as compared to those of the control. The results demonstrate that preharvest treatment by MeJA elicitation and electrical stimulation can be beneficial for secondary metabolite production of CPT in tissue-culture plants of O. ridleyana

DNA barcoding of Aristolochia plants and development of species-specific multiplex PCR to aid HPTLC in ascertainment of Aristolochia herbal materials.

The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai "Krai-Krue". "Krai-Krue" herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination

Antimicrobial Activity against Foodborne Pathogens and Antioxidant Activity of Plant Leaves Traditionally Used as Food Packaging

In accordance with Thai wisdom, indigenous plant leaves have been used as food packaging to preserve freshness. Many studies have demonstrated that both antioxidant and antimicrobial activities contribute to protecting food from spoilage. Hence, the ethanolic extracts of leaves from selected plants traditionally used as food packaging, including Nelumbo nucifera (1), Cocos nucifera (2), Nypa fruticans (3), Nepenthes mirabilis (4), Dendrocalamus asper (5), Cephalostachyum pergracile (6), Musa balbisiana (7), and Piper sarmentosum (8), were investigated to determine whether they have antioxidant and antimicrobial activities against spoilage microorganisms and foodborne pathogens that might be beneficial for food quality. Extracts 1–4 exhibited high phenolic content at 82.18–115.15 mg GAE/g and high antioxidant capacity on DPPH, FRAP and SRSA assay at 14.71–34.28 μg/mL, 342.92–551.38 μmol Fe2+/g, and 11.19–38.97 μg/mL, respectively, while leaf extracts 5–8 showed lower phenolic content at 34.43–50.08 mg GAE/g and lower antioxidant capacity on DPPH, FRAP, and SRSA at 46.70–142.16 μg/mL, 54.57–191.78 μmol Fe2+/g, and 69.05–>120 μg/mL, respectively. Extracts 1–4 possessed antimicrobial activities against food-relevant bacteria, including Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, and Escherichia coli. Only N. mirabilis extract (4) showed antimicrobial activities against Salmonella enterica subsp. enterica serovar Abony and Candida albicans. Extracts 5–8 showed slight antimicrobial activities against B. cereus and E. coli. As the growth and activity of microorganisms are the main cause of food spoilage, N. fruticans (3) was selected for bioassay-guided isolation to obtain 3-O-caffeoyl shikimic acid (I), isoorientin (II) and isovitexin (III), which are responsible for its antimicrobial activity against foodborne pathogens. N. fruticans was identified as a new source of natural antimicrobial compounds I–III, among which 3-O-caffeoyl shikimic acid was proven to show antimicrobial activity for the first time. These findings support the use of leaves for wrapping food and protecting food against oxidation and foodborne pathogens through their antioxidant and antimicrobial activities, respectively. Thus, leaves could be used as a natural packaging material and natural preservative

A validated TLC-image analysis method for detecting and quantifying bioactive phyllanthin in Phyllanthus amarus and commercial herbal drugs

Phyllanthus amarus Schum. and Thonn. has long been used for the treatment of liver diseases. The hepatoprotective compound presented in P. amarus was phyllanthin. In this study, the fast determination and quantitation of bioactive phyllanthin in P. amarus and its commercial herbal drugs were developed using a simple thin-layer chromatographic (TLC)- image analysis method. Chromatographic separation of phyllanthin was carried out and the intensity of phyllanthin was examined by TLC-image analysis. The linear equation line showed good relationship for the calibration curve in the various concentrations of phyllanthin. The limits of detection and limits of quantitation were 0.16 and 0.49 µg/spot, respectively. The contents of phyllanthin from fifteen plant materials collected from different locations and twelve commercial herbal drugs determined using the TLC-image analysis method were not significantly different from those determined using the TLCdensitometric method. These results suggest that the proposed TLC-image analysis method can be used as an effective method for quantitating phyllanthin in P. amarus plants and commercial products

DNA barcoding of Bacopa species coupled with high-resolution melting analysis

In Thailand, there are three species of Bacopa, namely, B. monnieri, B. caroliniana and B. floribunda. Among these Bacopa species, B. monnieri is the only medicinal species used for the treatment of cognitive impairment and improvement of cognitive abilities because of its bioactive constituents, bacoside A and B. However, due to the similar characteristics of plants, it is difficult to differentiate among related species, resulting in confusion during identification. For this reason, to ensure therapeutic quality for consumers, authentication is important. In this study, the three abovementioned Bacopa species were evaluated using barcoding coupled with high-resolution melting (Bar-HRM) analysis based on primers designed for the trnL-F sequences of the three species. The melting profiles of the trnL-F amplicons of B. caroliniana and B. floribunda were clearly different from the melting profile of the trnL-F amplicon from B. monnieri; thus, the species could be discriminated by Bar-HRM analysis. Bar-HRM was then used to authenticate commercial products in various forms. The melting curves of the six commercial samples indicated that all the tested products contained genuine B. monnieri species. This method provides an efficient and reliable authentication system for future commercial herbal products and offers a reference system for quality control.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and β-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7–9 expression and downregulation of Bcl-2 and full-length caspase-7–9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis