Cytotoxic Activity of Wild Plant and Callus Extracts of Ageratina pichinchensis and 2,3-Dihydrobenzofuran Isolated from a Callus Culture.

Ageratina pichinchensis (Kunth) R.M. King & H. Rob. belongs to the Asteraceae family and is a plant native to Mexico to which several biological properties are attributed. In this study, the cytotoxic effect of four extracts from the wild plants and two extracts from A. pichinchensis callus culture were evaluated against carcinogenic cell lines including prostate carcinoma, cervical cancer, hepatocellular carcinoma, hepatoma human, lung cancer, and cellular keratinocytes. The extracts were obtained with ethyl acetate and methanol using both leaves and stems or the callus. Only the ethyl acetate extract of the callus culture influenced the cervical cancer cell line (HeLa) with an IC50 of 94.79 ± 2.0 µg/mL. From the ethyl acetate callus extract, 2,3-dihydrobenzofuran was isolated and purified and also evaluated against cancer cells. The cytotoxic evaluation of this compound showed a significant effect against the HeLa cell line with an IC50 of 23.86 ± 2.5 µg/mL. Our results contribute to the development of biotechnological alternatives and extraction processes to produce compounds with possible potential against certain types of human cancer.

Jasmonic and Salicylic Acids Enhance Biomass, Total Phenolic Content, and Antioxidant Activity of Adventitious Roots of Acmella radicans (Jacq.) R.K. Jansen Cultured in Shake Flasks.

Acmella radicans (Asteraceae) is a plant native to America. Despite it having medicinal attributes, studies on its phytochemical properties are scarce, and biotechnological studies do not exist for this species. In this study, we established an adventitious root culture from A. radicans internodal segments in shake flasks with indole-3-butyric acid (IBA), and then elicited it with jasmonic acid (JA) and salicylic acid (SA). The total phenolic content and antioxidant activity were evaluated, and a comparison was made using in vitro plantlets and wild plants. Internodal segments with 0.1 mg/L IBA showed 100% root induction and exhibited better growth after transfer to shake flasks with MS liquid culture medium. JA had a significant effect on biomass increase compared to unelicited roots, mainly with 50 µM JA (28%), while SA did not show significant results. Root elicited with 100 µM (SA and JA) showed a 0.34- and 3.9-fold increase, respectively, in total phenolic content (TPC) compared to the control. The antioxidant activity was also significant, and a lower half-maximal inhibitory concentration (IC50) was observed as the AJ concentration increased. Roots elicited with AJ (100 µM) exhibited high antioxidant activity with DPPH (IC50 = 9.4 µg/mL) and ABTS (IC50 = 3.3 µg/mL) assays; these values were close to those for vitamin C (IC50 = 2.0 µg/mL). The TPC and antioxidant activity of in vitro plants and root cultured in shake flasks showed the lowest values in most cases; even the root cultures without elicitation were better than those of a wild plant. In this study, we demonstrated that A. radicans root culture is capable of producing secondary metabolites, while its production and antioxidant activity can be enhanced using jasmonic acid.

Obtaining 2,3-Dihydrobenzofuran and 3-Epilupeol from Ageratina pichinchensis (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.

Phytochemical, Pharmacological, and Biotechnological Study of Ageratina pichinchensis: A Native Species of Mexico.

Ageratina pichinchensis (Asteraceae) has been used for a long time in traditional Mexican medicine for treating different skin conditions and injuries. This review aimed to provide an up-to-date view regarding the traditional uses, chemical composition, and pharmacological properties (in vitro, in vivo, and clinical trials) that have been achieved using crude extracts, fractions, or pure compounds. Moreover, for a critical evaluation of the published literature, key databases (Pubmed, Science Direct, and SciFinder, among others) were systematically searched using keywords to retrieve relevant publications on this plant. Studies that reported on crude extracts, fractions, or isolated pure compounds of A. pichinchensis have found a varied range of biological effects, including antibacterial, curative, antiulcer, antifungal, and anti-inflammatory activities. Phytochemical analyses of different parts of A. pichinchensis revealed 47 compounds belonging to chromenes, furans, glycosylated flavonoids, terpenoids, and essential oils. Furthermore, biotechnological studies of A. pichinchensis such as callus and cell suspension cultures have provided information for future research perspectives to improve the production of valuable bioactive compounds.

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.

Achillin Increases Chemosensitivity to Paclitaxel, Overcoming Resistance and Enhancing Apoptosis in Human Hepatocellular Carcinoma Cell Line Resistant to Paclitaxel (Hep3B/PTX).

Multidrug resistance (MDR) has become a major obstacle in the treatment of cancer, and is associated with mechanisms such as increased drug outflow, reduction of apoptosis, and/or altered drug metabolism. These problems can be mitigated by the coadministration of agents known as chemosensitizers, as they can reverse resistance to anticancer drugs and eventually resensitize cancer cells. We explore the chemosensitizing effect of Achillin, a guaianolide-type sesquiterpene lactone isolated from the Mexican medicinal plant Artemisia ludovisiana, to reverse MDR in Hep3B/PTX cells of hepatocellular carcinoma, which present resistance to paclitaxel (PTX). Achillin showed an important effect as chemosensitizer; indeed, the cytotoxic effect of PTX (25 nM) was enhanced, and the induction of G2/M phase cell cycle arrest and apoptosis were potentiated when combining with Achillin (100 µM). In addition, we observed that Achillin decreases P-gp levels and increases the intracellular retention of doxorubicin in Hep3B/PTX cells; in addition, homology structural modeling and molecular docking calculations predicted that Achillin interacts in two regions (M-site and R-site) of transporter drug efflux P-glycoprotein (P-gp). Our results suggest that the chemosensitizer effect demonstrated for Achillin could be associated with P-gp modulation. This work also provides useful information for the development of new therapeutic agents from guaianolide-type sesquiterpene lactones like Achillin.

Establishment and Phytochemical Analysis of a Callus Culture from Ageratina pichinchensis (Asteraceae) and Its Anti-Inflammatory Activity.

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L-1 NAA with 0.1 mg L-1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 µM), IL-6 (IC50 = 73.71 ± 3.21 µM), and TNF-α (IC50 = 73.20 ± 5.99 µM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 µM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.