Removal of Heavy Metals from Mine Tailings in Central Chile Using Solidago chilensis Meyen, Haplopappus foliosus DC, and Lycium chilense Miers ex Bertero.

Mining activities have been a part of the history of Chile since time immemorial, generating pollution and environmental liabilities. Due to the lack of regulation, many tailings are deposited close to rivers or/and on unstable ground, near which towns have been built, generally in locations with no budget for their treatment. This study tested three plant species from Northern and Central Chile to remove total chromium, nickel, and zinc from tailings: Solidago chilensis, Haplopappus foliosus, and Lycium chilense, which complements the few existing studies on heavy metals removal with native or endemic Chilean shrubs. The experiments were conducted ex situ, and the initial and final concentrations of metals were determined in tailings and plants to obtain the removal efficiency, translocation and bioconcentration factors. Among these species, the best performance was obtained using Solidago chilensis, achieving removal efficiencies of 24% for Cr, 19% for Ni, and 17% for Zn, showing the ability to phytostabilize chromium and the higher resistance concerning the toxicity threshold. Haplopappus foliosus and Lycium chilense presented a slight tendency to stabilize chromium. Only Solidago chilensis showed little ability to extract Zn.

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.

Lioptilodes friasi (Lepidoptera: Pterophoridae) Niche Breadth in the Chilean Mediterranean Matorral Biome: Trophic and Altitudinal Dimensions.

Understanding the factors driving the diet breadth of phytophagous insects remains one of the main questions in ecological research. In this study we explored the diet breadth and plant-insect associations in the plume moth Lioptilodes friasi Vargas & Parra (Lepidoptera: Pterophoridae). This phytophagous insect was originally described in association with a single host species, Haplopappus foliosus (Asteraceae), a native shrub of the Chilean Mediterranean matorral. In order to address the breadth of host plant choice, we surveyed other Haplopappus species growing along the elevation gradient of central Chile from sea level to 2600 m. We were able to obtain L. friasi adults from five additional Haplopappus species: Haplopappus chrysantemifolius and Haplopappus decurrens from the coastal zone, Haplopappus multifolius and Haplopappus schumanii from the mid-elevation zone, and Haplopappus scrobiculatus at high elevation. Our results demonstrate that the genus-specialized endophagous herbivore L. friasi has a wider distribution and climatic tolerance than previously described. Its biogeographical range extends from the lowland coastal habitats up to the Andean subnival level. We propose that shared flower phenotypic traits such as morphology and chemical composition may have allowed the colonization of closely related Haplopappus species in central Chile, the expansion of which is limited by the harsh high elevation conditions.

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.

The intergenic spacer region of the rDNA in Haplopappus gracilis (Nutt.) Gray.

In this paper, we provide further information on the genome organisation of Haplopappus gracilis, one of the six angiosperms showing the lowest chromosome number, i.e. 2n = 4, by determining the nucleotide sequence of the intergenic spacer region of the ribosomal RNA genes and its cytological localization on metaphase chromosomes. DNA sequence analysis reveals the occurring of a product of 4,382 bp in length, characterised by the presence of four blocks of different repeated sequences. Our analysis also evidenced putative promoter regions with three transcription initiation sites for polymerase I, as previously reported in Artemisia absinthium, belonging to the same Asteraceae family. A fluorescent in situ hybridization with the intergenic spacer probe indicates the presence of rDNA genes only in the satellited chromosomes of H. gracilis; besides, differences in the signal intensity between homologous chromosomes were frequently observed, thus suggesting for these chromosome sites the presence of a variable number of rDNA gene copies, even if a divergent chromatin organisation in corresponding regions cannot be ruled out.

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.

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion.

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.

New phenolic esters from the resinous exudate of Haplopappus taeda.

Two new phenolic esters 9-trans-p-coumaroyloxy-alpha-terpineol (1) and 7-trans-p-coumaroyloxy-taedol (2), both endowed with free radical scavenger activity and cleroda-3,13 (E)-dien-15,18-diol (3) for which a cis stereochemistry at the decalin junction was found, were isolated from the resinous exudate from Haplopappus taeda upper parts.

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.

A new antibacterial clerodane diterpenoid from the resinous exudate of Haplopappus uncinatus.

The antibacterial activity of Haplopappus uncinatus is attributed to a new clerodane diterpenoid, 18-acetoxy-cis-cleroda-3-en-15-oic acid (10 betaH, 16 xi 19 beta, 17beta, 20 alpha form) (1), isolated as a major component from the resinous exudates of its twigs and leaves, together with the inactive 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone, n-alkanes and a few sesquiterpenoids.

Antioxidant properties and TLC characterization of four Chilean Haplopappus-species known as bailahuén.

"Bailahuén" is the common name of a medicinal shrub native to Chile where this resinous herb is widely used for its liver stimulating properties. Although the official species is Haplopappus baylahuen Remy (Asteraceae), other species of the same genus are also used in different regions as "bailahuén". A thin layer chromatography (TLC) method for rapid identification of different species and detection of adulterations is described for four of the species: Haplopappus baylahuen, Haplopappus taeda Reiche, Haplopappus multifolius Phil., and Haplopappus remyanus Wedd. To confirm efficiency in all species antioxidant properties were screened in resins, infusions, and methanolic extracts by tests of lipid peroxidation in erythrocytes and free radical scavenging activity by DPPH. In both studies Haplopappus baylahuen showed the lowest antioxidant capacity. In DPPH analyses, infusion and resins of Haplopappus baylahuen also showed the lowest and Haplopappus remyanus the major inhibiting activity of free radicals, while Haplopappus multifolius proved to have the highest result when the methanolic extracts were used. The chemical characterization of the studied species showed important levels of flavonoids and coumarins, with flavonoids predominating in Haplopappus taeda, coumarins in Haplopappus multifolius and both of them in Haplopappus baylahuen and Haplopappus remyanus.

Two new O-geranyl coumarins from the resinous exudate of Haplopappus multifolius.

From the resinous exudate of leaves of Haplopappus multifolius two new coumarins were isolated and assigned the structures 6-hydroxy-7-(5'-hydroxy-3',7'-dimethylocta-2',6'-dien)-oxycoumarin (1) and 6-hydroxy-7-(7'-hydroxy-3',7'-dimethylocta-2',5'-dien)-oxy coumarin (2).

Argentinian plant extracts with relaxant effect on the smooth muscle of the corpus cavernosum of guinea pig.

Extracts of different polarity from Baccharis trimera, Haplopappus rigidus Huperzia saururus, Maytenus ilicifolia, Satureja parvifolia and Senecio eriophyton were tested for their relaxant activity on smooth muscle using L-phenylephrine precontracted strips of corpus cavernosum obtained from Guinea pigs. Highly significant and dose dependent results were obtained with the dichloromethane extracts of H. saururus (87% of relaxation at the dose of 10 mg/ml), S. parvifolia (95% of relaxation at 2.5 mg/ml) and S. eriophyton (94% of relaxation at 5 mg/ml). Similar effects were observed with the methanol extracts of H. saururus (88% of relaxation at 10 mg/ml) and S. parvifolia (84% of relaxation at 10 mg/ml). These results were comparable to those obtained with the dichloromethane and methanol extracts of the well known Mexican species Turnera diffusa. Moreover, the aqueous extract of H. rigidus and the aqueous and methanol extracts of S. eriophyton were highly effective in a dose dependent manner (more than 90% of relaxation at the dose of 10 mg/ml). Significant results, but with a lower overall relaxant activity (about 70% of relaxation at 10 mg/ml), could also be obtained with the aqueous extract of S. parvifolia and with the dichlormethane and methanol extracts of B. trimera and M. ilicifolia. The positive controls with Sildenafil citrate at doses ranging from 0.35 to 35 microg/ml yielded moderate effects (up to 46% of relaxation at 35 microg/ml). The effects observed in the present study seem to validate the folk medicinal use of the tested plants and open new ways in the search for natural products with vasodilatory effects.

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.

Antimycobacterial flavones from Haplopappus sonorensis.

Crude extracts of Haplopappus sonorensis (A. Gray) S.F. Blake (Asteraceae), showed activity against Mycobacterium tuberculosis H(37)Rv. By assay-guided fractionation, 5-hydroxy-3,7,4'-trimethoxyflavone (1). 5,7-dihydroxy-3,4'-dimethoxyflavone (2). and 5,4'-dihydroxy-3,7-dimethoxyflavone (3). were identified as the antimycobacterial principles. Compound 2 was the most active compound.

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.