Hydroethanolic Extracts of Haplopappus baylahuen Remy and Aloysia citriodora Palau Have Bactericide Activity and Inhibit the Ability of Salmonella Enteritidis to Form Biofilm and Adhere to Human Intestinal Cells.

We analysed whether the hydroethanolic extracts from leaves of Haplopappus baylahuen Remy (bailahuen) and Aloysia citriodora Palau (cedron) inhibit the growth and ability of Salmonella Enteritidis to form biofilms and to adhere to human intestinal epithelial cells. Herein, we first determined the total phenolic content and antioxidant and antibacterial activities of the extracts. Then, Salmonella Enteritidis was treated with the extracts to analyse biofilm formation by scanning electronic microscopy and the violet crystal test. We also measured the efflux pump activity of Salmonella Enteritidis since biofilm formation is associated with this phenomenon. Furthermore, the human intestinal cell line Caco-2 was infected with Salmonella Enteritidis pretreated with the extracts, and 30 min later, the number of bacteria that adhered to the cell surface was quantified. Finally, we determined by qPCR the expression of genes associated with biofilm formation, namely, the diguanilate cyclase AdrA protein gene (adrA) and the BapA protein gene (bapA), and genes associated with adhesion, namely, the transcriptional regulator HilA (hilA). The phenolic content and antioxidant and bactericide activities were higher in bailahuen than in the cedron extract. Biofilm formation was inhibited by the extracts in a dose-dependent manner, while the activity of efflux pumps was decreased only with the cedron extract. Adhesion to Caco-2 cells was also inhibited without differences between doses and extracts. The extracts decreased the expression of adrA; with the cedron extract being the most efficient. The expression of hilA is affected only with the cedron extract. We concluded that hydroethanolic extracts of bailahuen and cedron differentially inhibit the growth of Salmonella Enteritidis and affect its the ability to form biofilms and to adhere to human intestinal epithelial cells. These results highlight the presence of molecules in bailahuen and cedron with a high potential for the control of the Salmonella Enteritidis pathogenesis.

Antifungal activity against Botrytis cinerea of labdane-type diterpenoids isolated from the resinous exudate of Haplopappus velutinus Remy (Asteraceae).

Two labdane diterpenoids were isolated, from the resinous exudate of Haplopappus velutinus Remy (Asteraceae); the main compound was identified as 7,13-(E)-labdadien-15,18-dioic-acid-18-methyl ester (1) and the minor compound identified as 7-labden-15,18-dioic-acid-18-methyl ester (2). Their structures were obtained using FTIR, MS, HRMS and NMR data: 1D NMR (1H, 13C and DEPT-135), 2D homonuclear NMR (COSY and NOESY) and heteronuclear NMR (HSQC and HMBC). The trans stereochemistry of the decalin moiety of compounds 1 and 2 was established through NOESY experiments of the reduction product of 1; 7-labden-15,18-diol (1a). Diterpenoids 1 and 1a are described for the first time and showed antifungal activity, inhibiting approximately 40% mycelial growth of Botrytis cinerea.

Phenolic profiling of the South American "Baylahuen" tea (Haplopappus spp., Asteraceae) by HPLC-DAD-ESI-MS.

The aerial parts of several Haplopappus species (Asteraceae), known under the common name "baylahuen", are used as herbal teas in Chile and Argentina. In Chile, "baylahuen" comprises H. multifolius, H. taeda, H. baylahuen and H. rigidus. Little is known about the chemical identity of the infusion constituents in spite of widespread consumption. The aim of the present work was the characterization of phenolics occurring in the infusions and methanol extracts of "baylahuen" by HPLC-DAD-ESI-MS. A simple HPLC-DAD-ESI-MS method was developed for the fast identification and differentiation of Haplopappus spp. used as a tea source, based on the phenolics from the tea and methanol extracts. Some 27 phenolics were tentatively identified in the infusions and methanol extract, including 10 caffeoyl quinic and feruloyl quinic acid derivatives and 17 flavonoids. The HPLC patterns of the Haplopappus tea and methanol extract allow a clear differentiation at the species level. The occurrence of hydroxycinnamic acid derivatives and flavonoids can explain the reputed nutraceutical and health beneficial properties of this herbal tea.

Lipophilicity and antibacterial activity of flavonols: antibacterial activity of resinous exudates of Haplopappus litoralis, H. chrysantemifolius and H. scrobiculatus / Lipofilia y actividad antibacteriana de flavonoles: actividad antibacteriana de los exudados resinosos de Haplopappus litoralis, H. chrysantemifolius y H. scrobiculatus

The antibacterial properties of the resinous exudates from Haplopappus litoralis, H. chrysantemifolius and H. scrobiculatus from Central Chile were assessed against Gram negative and Gram-positive bacteria, and proved active against the latter. The results show that the antibacterial activities of the resinous exudates are independent from the flavonols isolated from each extract that proved to be inactive. The estimated lipophilicity of the flavonols isolated from the Haplopappus resinous exudates were compared with the lipophilicity of known antibacterial flavonols. This analysis showed that lipophilicity is an important variable to predict the antibacterial activity of flavonols.

La actividad antibacteriana de los exudados resinosos de Haplopappus litoralis, H. chrysantemifolius y H. scrobiculatus de la Zona Central de Chile fueron evaluadas frente a bacterias Gram-negativas y Gram-positivas, y resultaron activos frente a estas últimas. Los resultados mostraron que la actividad antibacteriana de los exudados resinosos es independiente de los flavonoles aisladas de cada extracto que no mostraron actividad antibacteriana. La lipofilia estimada de los flavonoles aislados de los exudados resinosos de Haplopappus se comparó con la lipofilia de conocidos flavonoles antibacterianos. Este análisis mostró que la lipofilia es una variable importante para predecir la actividad antibacteriana de los flavonoles.

Biomass, resin and essential oil content and their variability in natural populations of the Chilean crude drug “bailahuén” (Haplopappus spp) / Biomasa, contenido de resina y aceite esencial y su variabilidad en poblaciones naturales de las especies de droga cruda chilena “bailahuén” (Haplopappus spp)

Bailahuén (Haplopappus rigidus, Haplopappus baylahuen, Haplopappus multifolius and Haplopappus taeda; Asteraceae) are medicinal shrubs native to the Andes Mountains of Chile widely used to treat hepatic ailments. At present, exploitation of bailahuén is based on wild collections, affecting the abundance of natural populations. Variability of biomass production and concentration of active compounds in different wild populations of the four Haplopappus species was studied in order to select the best plant material for cultivation. Resins were extracted with dichloromethane and essential oils by distillation. Biomass production of the populations was highest for H. baylahuen and H. rigidus compared with the other species, reaching between 0.56 and 1.61 kg and 1.11-1.48 kg per plant, respectively. No differences were found among populations of the same species. In some H. rigidus populations, the resin content was about a third of the dry weight, whereas plants of H. multifolius had mean values of about 8 percent. In H. baylahuen (11.3-27.7 percent) the resin content of leaves differed significantly among populations, whereas the essential oil ranged from 0.02-0.38 mL*100g DM-1 in H. baylahuen, 0.03-0.5 mL*100 g DM-1 for H. rigidus, and 0.08-0.35 mL*100 g DM-1 for H. taeda. Resin content in stems differed only among H. baylahuen (6.3-15.5 percent) and H. rigidus (10.7-21.2 percent) populations. No significant differences in essential oil content among species could be detected because of the large amount of variation observed among populations. The variation between plants of the same population may indicate favorable selection potential for future breeding programs.

Bailahuén (Haplopappus rigidus, Haplopappus baylahuen, Haplopappus multifolius y Haplopappus taeda; Asteraceae) son arbustos medicinales de la Cordillera de los Andes Chilena ampliamente usados para tratar malestares hepáticos. Actualmente, la explotación del bailahuén se basa en la recolección silvestre, afectando su abundancia. La variabilidad en diferentes poblaciones silvestres de las cuatro especies de Haplopappus fue usada en orden a determinar la potencialidad para seleccionar el mejor material vegetal para cultivo en producción de biomasa y contenido de principios activos. La resina se extrajo por inmersión en diclorometano y el aceite esencial por destilación. Los mayores rendimientos de biomasa se observaron en H. rigidus y H. baylahuen, con valores poblacionales que fluctuaron entre 1,11-1,48 y 0,56-1,61 kg planta-1, respectivamente, sin presentar diferencias entre poblaciones de la misma especie. El contenido de resinas alcanzó en algunas poblaciones de H. rigidus hasta un tercio del peso seco, mientras que en H. multifolius fue de aproximadamente 8 por ciento. En hojas, sólo se observaron diferencias significativas en el contenido de resinas entre poblaciones de H. baylahuen (11,3-27,7 por ciento). El contenido de aceites esenciales en hojas permitió diferenciar poblaciones de H. baylahuen (0,02-0,38 mL*100g MS-1), H. rigidus (0,03-0,50 mL*100g MS-1) y H. taeda (0,08-0,35 mL*100g MS-1). En tallos, el contenido de resina difirió significativamente entre poblaciones de H. baylahuen (6,3-15,6 por ciento) y H. rigidus (10,7-21,2 por ciento). La alta variación del contenido de aceite esencial entre poblaciones no permitió establecer diferencias entre especies. La variación entre poblaciones de la misma especie sugiere un buen pronóstico para futuras selecciones y mejoramiento genético.

Antioxidant activity of coumarins and flavonols from the resinous exudate of Haplopappus multifolius.

The antioxidant activity of eight coumarins and two flavonols isolated from Haplopappus multifolius was studied with the DPPH radical method. Results show that a high concentration of phenolic coumarins and the presence of quercetin and rhamnetin in the exudates could account for the protection of the plant against oxidative stress. Structures for the coumarins 6-hydroxy-7-[(E,E)-3',7'-dimethyl-2',4',7'-octatrienyloxy] coumarin and 7-[(E)-3'-methyl-4'-hydroxy-2'-butenyloxy] coumarin are proposed on the basis of spectroscopic evidence.

Characterization of volatile constituents of Haplopappus greenei and studies on the antifungal activity against phytopathogens.

Essential oil of Haplopappus greenei A. Gray was obtained by hydrodistillation of aerial parts, which were subsequently analyzed by gas chromatography and gas chromatography-mass spectrometry. Major components were identified as carvacrol (8.7%), beta-pinene (7.6%), trans-pinocarveol (6.2%), and caryophyllene oxide (5.8%), respectively. In total, 104 components representing 84.9% of the investigated essential oil were characterized. Furthermore, the essential oil was evaluated for antimalarial, antimicrobial, and antifungal activities. However, only antifungal activity was observed against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides using the direct overlay bioautography assay. Major essential oil components were also evaluated for antifungal activity; the carvacrol standard demonstrated nonselective activity against the three Colletotrichum species and the other compounds were inactive.

Antibacterial new clerodane diterpenes from the surface of Haplopappus foliosus.

A biologically monitored fractionation of the resinous exudate extract of Haplopappus foliosus DC. is reported. Purification of the two active fractions yielded 2-alpha-hydroxy- cis-cleroda-3,13(Z),8(17)-trien-15-oic acid (1) and 2-alpha-acetoxy- cis-cleroda-3,13(Z),8(17)-trien-15-oic acid (2), two new clerodane diterpenes.

Labdane diterpenes from Haplopappus illinitus.

A chemical study of aerial parts of Haplopappus illinitus afforded five labdane diterpenoids whose structures were elucidated by high field NMR spectroscopy and 2D techniques. Four of them are dicarboxylic acids previously reported as the corresponding dimethyl ester derivatives and the fifth is a new compound.