Cytotoxic activity and chemical composition of the root extract from the mexican species Linum scabrellum: mechanism of action of the active compound 6-Methoxypodophyllotoxin

12 p.-6 fig.-1 tab.The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC50 = 0.57 μg/mL), MCF7 breast (IC50 = 0.56 μg/mL), PC3 prostate (IC50 = 1.60 μg/mL), and SiHa cervical (IC50 = 1.54 μg/mL), as well as toward the normal fibroblasts line HFS-30 IC50 = 1.02 μg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC50 value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant K b = 7.6 × 10(6) M(-1)). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.Ivonne Alejandre-Garc´ıa acknowledges fellowship 226354 from CONACYT. The authors thank Dr. Alfonso Lejia from Centro de Ciencias Genomicas, UNAM, for technical ´assistance. The authors are indebted to Dr. T Fitzgerald from Florida A&M University for his kind gift of the colchicine analog 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). Partial support from CONACYT (Grants CB 156276 and 222714) is acknowledged.The authors thank Laboratorio Nacional de Estructura de Macromoleculas (Conacyt 251613) for the spectroscopic and ´mass analyses and support given to J. F. D. from BIPPED2 and BIO2013-42984R from the Ministry of Economy of Spain.Peer reviewe

High variability of perezone content in rhizomes of Acourtia cordata wild plants, environmental factors related, and proteomic analysis

With the aim of exploring the source of the high variability observed in the production of perezone, in Acourtia cordata wild plants, we analyze the influence of soil parameters and phenotypic characteristics on its perezone content. Perezone is a sesquiterpene quinone responsible for several pharmacological effects and the A. cordata plants are the natural source of this metabolite. The chemistry of perezone has been widely studied, however, no studies exist related to its production under natural conditions, nor to its biosynthesis and the environmental factors that affect the yield of this compound in wild plants. We also used a proteomic approach to detect differentially expressed proteins in wild plant rhizomes and compare the profiles of high vs. low perezone-producing plants. Our results show that in perezone-producing rhizomes, the presence of high concentrations of this compound could result from a positive response to the effects of some edaphic factors, such as total phosphorus (Pt), total nitrogen (Nt), ammonium (NH4), and organic matter (O. M.), but could also be due to a negative response to the soil pH value. Additionally, we identified 616 differentially expressed proteins between high and low perezone producers. According to the functional annotation of this comparison, the upregulated proteins were grouped in valine biosynthesis, breakdown of leucine and isoleucine, and secondary metabolism such as terpenoid biosynthesis. Downregulated proteins were grouped in basal metabolism processes, such as pyruvate and purine metabolism and glycolysis/gluconeogenesis. Our results suggest that soil parameters can impact the content of perezone in wild plants. Furthermore, we used proteomic resources to obtain data on the pathways expressed when A. cordata plants produce high and low concentrations of perezone. These data may be useful to further explore the possible relationship between perezone production and abiotic or biotic factors and the molecular mechanisms related to high and low perezone production.This work was supported by the Programa de Mejoramiento del Profesorado PROMEP/103.5/13/6626 and Consejo Nacional de Ciencia y Tecnología CONACyT-Mexico for Ph.D. scholarship 392123/254165. The University of Alicante lab is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+I 2013-2016, funded by ISCIII and ERDF. Roque Bru-Martínez received financial support from the University of Alicante (VIGROB-105)

Effect of Plant Growth Regulators on Different Explants of Artemisia ludoviciana under Photoperiod and Darkness Conditions and Their Influence on Achillin Production

Species of the genus Artemisia mainly biosynthesize sesquiterpene lactones. Achillin is a guaianolide-type sesquiterpene lactone isolated from Artemisia ludoviciana; it has shown antibacterial and anti-inflammatory activities. In addition, achillin exhibits a significant chemosensitizing effect on hepatocellular carcinoma cells resistant to paclitaxel (PTX). The objective of this study was to establish a callus culture from different explants under conditions of light and total darkness to produce achillin. To obtain in vitro cultures, explants of leaves, nodes, internodes, and roots were used, and they were cultured in MS medium with 0.1 mg/L of kinetin (KIN) or benzyl amino purine (BAP) and/or naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid (PIC) at 0.1 and 1.0 mg/L. Of all treatments, internodes with BAP (0.1 mg/L) and PIC (1.0 mg/L) grown under photoperiod showed the best friable callus induction, however, GC-MS analysis showed higher achillin content (1703.05 µg/mL) in leaf calluses with PIC (1.0) and KIN (0.1) under photoperiod, and in node plantlets (1880.01 µg/mL) with PIC (0.1) and BAP (0.1). From 12.34 g of dry leaves of Artemisia ludoviciana, 257 mg of achillin were isolated and purified, which was used as a reference in the quantification of achillin in the in vitro culture

Establishment of a Cell Suspension Culture of Ageratina pichinchensis (Kunth) for the Improved Production of Anti-Inflammatory Compounds

Ageratina pichinchensis (Kunth) is a plant used in traditional Mexican medicine to treat multiple ailments. However, there have not been biotechnological studies on producing compounds in in vitro cultures. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran (2) and 3-epilupeol (3), evaluate the anti-inflammatory potential of its extracts, and perform a phytochemical analysis. Cell suspension cultures were established in a MS culture medium of 30-g L−1 sucrose, 1.0-mg L−1 α-naphthaleneacetic acid, and 0.1-mg L−1 6-furfurylaminopurine. The ethyl acetate extract of the cell culture analyzed by gas chromatography (GC) revealed that the maximum production of anti-inflammatory compounds 2 and 3 occurs on days eight and 16, respectively, improving the time and previously reported yields in callus cultures. The anti-inflammatory activity of these extracts exhibited a significant inhibition of nitric oxide (NO) production. Furthermore, a phytochemical study of the ethyl acetate (EtOAc) and methanol (MeOH) extracts from day 20 led to the identification of 17 known compounds. The structures of the compounds were assigned by an analysis of 1D and 2D NMR data and the remainder by GC–MS. This is the first report of the production of (-)-Artemesinol, (-)-Artemesinol glucoside, encecalin, and 3,5-diprenyl-acetophenone by a cell suspension culture of A. pichinchensis

Obtaining 2,3-Dihydrobenzofuran and 3-Epilupeol from <i>Ageratina pichinchensis</i> (Kunth) R.King & Ho.Rob. Cell Cultures Grown in Shake Flasks under Photoperiod and Darkness, and Its Scale-Up to an Airlift Bioreactor for Enhanced Production

Ageratina pichinchensis (Kunth) R.King & Ho.Rob. is a plant used in traditional Mexican medicine, and some biotechnological studies have shown that its calluses and cell suspension cultures can produce important anti-inflammatory compounds. In this study, we established a cell culture of A. pichinchensis in a 2 L airlift bioreactor and evaluated the production of the anti-inflammatory compounds 2,3-dihydrobenzofuran (1) and 3-epilupeol (2). The maximum biomass production (11.90 ± 2.48 g/L) was reached at 11 days of culture and cell viability was between 80% and 90%. Among kinetic parameters, the specific growth rate (µ) was 0.2216 days−1 and doubling time (td) was 3.13 days. Gas chromatography coupled with mass spectrometry (GC-MS) analysis of extracts showed the maximum production of compound 1 (903.02 ± 41.06 µg/g extract) and compound 2 (561.63 ± 10.63 µg/g extract) at 7 and 14 days, respectively. This study stands out for the significant production of 2,3-dihydrobenzofuran and 3-epilupeol and by the significant reduction in production time compared to callus and cell suspension cultures, previously reported. To date, these compounds have not been found in the wild plant, i.e., its production has only been reported in cell cultures of A. pichinchensis. Therefore, plant cell cultured in an airlift reactor can be an alternative for the improved production of these anti-inflammatory compounds