Reagent grade ethanol (ETOH), methanol (MeOH), chloroform, ethyl acetate (EtOAc) and HPLC grade water were obtained from Merck KGaA, (Darmstadt, Germany). Flumazenil (Lanexat®), diazepam (Valium®) clonazepam (Klonopin™) were from Hoffmann-La Roche Inc., [³H]-methyl-flunitrazepam 60-85Ci/mmol were purchased from ARC American Radiolabeled Chemicals Inc. Saint Louis, MO, Trizma® Base 99.9% (Tris [Hydroxymethyl] aminomethane) Sigma-Aldrich Saint Louis, MO. HPLC standards, (rutin, quercetin, isorhamnetin, scopoletin, citric acid, kaempferol-3-O-rutinose, isorhamnetin-3-O-glucoside, atropine and scopolamine all standards with purity higher than 95% by HPLC) were purchased either from Sigma Aldrich (Saint Louis, Mo, USA), ChromaDex (Santa Ana, CA, USA), or Extrasynthèse (Genay, France).

The plant material used in the present study was collected during two summers (December 2010 and 2011) in a location 5 km from Parque Nacional Alerces Costero (40⁰ 10′, 22.5″ S, 73⁰ 26′ 30.5″ W), Valdivia, Chile. Aerial parts and flowers were collected and identified by the botanist Carlos Lehnebach. Leaves and twigs were air dried at room temperature and crushed to powder prior to extraction procedures. A voucher specimen was deposited at the Institute of Pharmacy (voucher number lp1052011), Universidad Austral de Chile.

Rockefeller male mice, 2 months old from the Animal House at the Instituto de Immunología, Universidad Austral de Chile (UACh) were used. They were maintained on a 12:12 light-dark schedule, in a controlled temperature (21°C), were housed in groups of 8 per polycarbonate cages and received free access to food and water. Animals were transported to the laboratory from the animal house 24 h prior to the experimental session in order to their adaptation. The experiments were achieved between 12 and 17 h. Animals were sacrificed by cervical dislocation under pentobarbital anesthesia or CO₂ gas. All behavioral procedures were approved by ethical commission of the University Austral de Chile, UACh- DID S2001-67.

In order to extract all compounds present in the plant, the plant material was extracted with acidified solvent. Thus, the alkaloid-rich extract (ALK) was obtained from 500 g of L. pubiflora chopped leaves using EtOH/HCl, 1% v:v (3 × 1 L) for 24 h. The extract was filtered yielding after evaporation under reduced pressure a syrupy green residue, the residue was later re-suspended in water (0.5 L). Precipitated lipids and chlorophyll were filtered off. The pH of the solution was increased to pH 10-11 using NH₄OH and then CHCl₃ was added (4 times × 300 mL) in order to obtain free alkaloids. The chloroform extracts were combined and evaporated under vacuum and freeze dried to yield 3.0 g of dry extract (0.6% yield w/w) which was suspended in Tween- 80 −10% for further experiments.

Once the alkaloids were extracted, the remaining aqueous solution was neutralized to pH 7 with NaHCO₃ and repeatedly extracted with EtOAc (4 times × 300 mL). The extracts showed no major differences on thin-layer chromatography (TLC) and were combined and evaporated under vacuum to a small volume (50 mL). The absence of alkaloids was confirmed using the Draggendorf reaction in TLC assay and HPLC study. A portion (15 mL) was dried and suspended (0.5 g) in Tween- 80 −10% for the biological assays. The remaining concentrated solution (35 mL) was precipitated, and then filtered to yield a white amorphous powder which was crystallized on MeOH in order to obtain a pure product (65 mg). Direct comparison with a standard sample and spectroscopic analysis (IR, UV, ¹H-NMR and mass spectra, please see Supplementary Material, Table S1) allowed us to identify the isolated compound as the coumarin scopoletin (Simirgiotis et al., 2013).

The alkaloid and non alkaloidal partition extracts from leaves of L. pubiflora were analyzed for organic compounds using UHPLC coupled with high resolution mass spectrometry. Approximately 5 mg of each of the dried material dissolved in 2 mL of ultrapure water, with sonication for 2 min and filtered (PTFE filter, Merck) and 10 microliters were injected in the UHPLC instrument for UHPLC-MS analysis. A Thermo Scientific Ultimate 3000 RS UHPLC system controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, Germany) hyphenated with a Thermo high resolution Q-Exactive focus mass spectrometer (Thermo, Bremen, Germany) were used for analysis. All other conditions and parameters were set as previously reported (Simirgiotis et al., 2016b). Liquid chromatography was performed using an UHPLC C18 column (Acclaim, 150 mm × 4.6 mm ID, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) operated at 25°C. The detection wavelengths were 254, 280, and 320 nm and PDA recorded from 200 to 800 nm for peak characterization. Mobile phases were 1% formic aqueous solution (A) and acetonitrile (B). The gradient program (time (min), % B) was: (0.00, 5); (5.00, 5); (10.00, 30); (15.00, 30); (20.00, 70); (25.00, 70); and 12 min for column equilibration before each injection. The flow rate was 1.00 mL min⁻¹, and the injection volume was 10 μL. Standards and extracts dissolved in methanol were kept at 10°C during storage in the auto-sampler (Simirgiotis et al., 2016c). The Spectrometer and HESI II parameters were optimized as previously reported (Simirgiotis et al., 2016c). For the quantification of atropine and scopolamine all measurements were done in triplicate (n = 3). For the recovery experiments of these compounds, known amounts of standards (stock solutions of 0.5 mg/mL) were added to 50 g of the plant matrix and the spiked sample processed using the same methods described above, in triplicate. The amount of the tested compounds were then measured and the percent ratio between the spiked and non-spiked (set as zero level) sample was calculated. The measured amounts in relation to the theoretically present ones were expressed as percent of recovery. The obtained recovery was 115.12 ± 1.68 and 98.3 ± 0.98% (mean ± RSD) for atropine and scopolamine, respectively.

Rockefeller mice maintained on a 12:12 light-dark schedule, in a controlled temperature (21°C). In the behavioral assessment, all testing performed at 150 mg/kg. For the pentobarbital sleeping test, mice were divided into six groups (n = 10), which included three test groups treated with the different extracts with its vehicle as control. All drugs were injected intraperitoneally (i.p.). Extracts were administered 30 min prior to pentobarbital exposure 40 mg/kg. The three test groups received an injection of 150 mg/kg of the ALK extract, 150 mg/kg of the NON-ALK extract and the pure isolated compound scopoletin (7.5 mg/kg). The elevated plus-maze test (EPM) was performed using a maze of two open arms with transparent floor, two black arms enclosed by walls (6 cm × 30 cm × 10 cm) opposite to each other, joined by a central black platform (8 cm × 8 cm). Three different groups of mice were considered (10 mice each group): group one treated with diazepam, group two treated with the ALK extract and group three treated with the NON-ALK extract. In all groups the anxiolytic and locomotor activities were evaluated on the maze, after receiving flumazenil at 0.3 mg/kg s.c., 10 min prior to evaluation.

[³H]Flunitrazepam binding assays were performed with cortical membranes from female rats of approximately 2 months old (125–150 g) obtained from Analytical Biological Services, Inc. (Wilmington, DE). The reaction was initiated by the addition of tissue (20–24 μg protein) to tubes containing 2 nM [³H]flunitrazepam in a final volume of 400 μL of 50 mM Tris-HCl buffer, pH 7.4. Non-specific binding was determined in the presence of 10⁻⁴ M clonazepam (Ortiz et al., 1999). Different concentrations of extracts were added to tubes for competition studies. Saturation binding was performed using different concentrations of [³H]Flunitrazepam in the presence of 1.25 × 10⁻⁵ g/L of L. pubiflora alkaloid extract. All samples were incubated at 25°C for 40 min. The assay was stopped by rapid filtration of 100 μL of each sample in 24 mm in diameter Millipore AP 40 Glass Fiber prefilters (Millipore Corporation BedFord, MA) in a Millipore manifold (Millipore Corporation BedFord, MA) followed by two - 2.5 mL ice cold buffer washes. Radioactivity of the dried filters was quantified in a Beckman LS 6000 counter with 5 mL of EcoLume scintillation cocktail. Results are shown as percentage of total binding.

In order to determine the experimental dose of the extract, we conducted a dose-response curve in which animals received 800 mg/kg of the ALK extract, and death was induced approximate 5 min after the injections (data not shown). Convulsions were induced at 400 mg/kg i.p. (data not shown). In contrast, 200 mg/kg of the extract induced excitatory effects. Hyperactivity was evident, which caused difficulty in the handling of mice. Therefore, we determined that 150 mg/kg i.p. produced a stable response during a 1 h observation period. Therefore, all behavioral testing was done at 150 mg/kg (i.p.).

Mice were divided into six groups (n = 10), which included three test groups treated with the different extracts with its vehicle as control. All drugs were injected intraperitoneally. Extracts were administered 30 min prior to pentobarbital exposure 40 mg/kg (i.p.). The onset of sleep (or latency time, the time elapsed between injection of pentobarbital and loss of the righting reflex) and sleeping time (lapse between loss and the recovery of righting reflex) was registered according to Matsumoto et al. (1997). The three test groups received an i.p. injection of 150 mg/kg of the ALK extract, 150 mg/kg of the NON-ALK extract and 7.5 mg/kg i.p. of the isolated compound scopoletin (in the same concentration found in the ALK extract). The control groups were injected with vehicle (10% Tween 80).

The elevated plus-maze test was performed according to Maruyama et al. (1998). It consisted of two open arms with transparent floor (6 cm × 30 cm), two black arms enclosed by walls (6 cm × 30 cm × 10 cm) arranged in a way that one pair of identical arms were opposite to each other, joined by a central black platform (8 cm × 8 cm). The apparatus was raised to a height of 40 cm above the floor and located in a room with black walls and floor, illuminated by fluorescent light (40 W). The experimental session was registered by a camera linked to a monitor and a video-recorder in an adjacent room, for further analysis by testers unaware of the treatment conditions.

Three groups of 10 mice were used, a control group (NaCl 0.9%), an extract group (150 mg/Kg of extract i.p.) and a reference group (diazepam 1 mg/Kg i,p). EPM behavior was registered 30 min after dosing. At the beginning of the test, each mouse was individually placed onto the central platform facing a closed arm, and the behavior was recorded during a 5 min session. The criterion for an arm entry was defined as four paws into a given arm. In another set of experiments: diazepam, ALK and NON-ALK treated mice received flumazenil at 0.3 mg/kg s.c., 10 min prior to evaluation of anxiolytic and locomotor activity on the maze following (Kuribara et al., 1998).

Significance was measured using one-way ANOVA. Whenever appropriate, Student's t-test or χ² were also employed. Data are expressed as the mean ± SEM. Statistical significance was attained at p ≤ 0.05. Saturation and inhibition curves were analyzed using GraphPad Prism 4 software (version 4.03, GraphPad Prism, San Diego, CA).

Several compounds including tropane alkaloids, phenolic acids and flavonoids were identified (Table 1) by means of high resolution mass spectrometry (UHPLC-Q-OT-MS), and the UHPLC-TIC chromatogram is depicted in Figure 2. For the tropane alkaloids the retention times of the major components were 11.21 min. for atropine, and 10.34 min. for scopolamine. The contents per g dry weight of the plant L. pubiflora were determined by HPLC to be 344 ± 6.32 μg for scopolamine, 1260 ± 15.24 μg for atropine and 646.8 ± 18.85 μg of scopoletin. The major tropane alkaloids -scopolamine and atropine- were present in 5.7 and 21%, respectively, in the ALK extract used for pharmacological experiments (see below). The coumarin scopoletin was isolated (see Experimental) and its structure obtained by spectroscopic methods (see Supplementary Material) and resulted the major component of the NON-ALK extract (3.90 g). Below is the detailed explanation of the accurate mass identification (Table 1). Several examples of structures and full MS spectra are shown in Supplementary Material (Figures S2).

The administration of the alkaloid (ALK) extract of L pubiflora prior to pentobarbital injection, led to a statistically significant increase of the pentobarbital-induced sleeping time, with no modification of sleep latency (Figure 4). The non-alkaloid (NON-ALK) extract of L pubiflora also showed a sedative effect, increasing the sleeping time by approximately 42% compared to the control group. No changes in latency time or sleeping time were found after administration of the major coumarin isolated, scopoletin (Figure 4).

We studied the effect of the ALK and the NON-ALK extracts of L. pubiflora on anxiety-like behavior using the elevated plus-maze test (EPM). The animals that received the ALK extract spent significantly longer time in the open arms of the maze in comparison to that of the control group, as it was also induced by diazepam (DZ) exposure, a well-known GABA_A receptor agonist. This effect was not retained by the NON-ALK extract (Figure 5, top panel). By looking at the number of closed arm entries as an indicator of activity levels in this task, we found that the ALK, NON-ALK, and DZ injections at the selected dosages induced a decrease in this parameter (Figure 5, bottom panel). In order to confirm whether the GABA_A receptor is involved in open arm behavior, we studied the effects of flumazenil (FLU) (0.3 mg/kg s.c), a high affinity benzodiazepine receptor antagonist. Figure 6 shows that, indeed, FLU counteracted the anxiolytic effect of the alkaloid extract.

Finally, we performed receptor studies in order to determine the interactions of the alkaloid extract with GABA_A receptors. The alkaloid extract of L pubiflora was added from 10⁻⁷ to 10⁻⁴ mg/mL in presence of 2 nM [³H]flunitrazepam. The results of at least two independent experiments performed in triplicate showed that L. pubiflora ALK extract decreases flunitrazepam binding. The estimated IC₅₀ was 2.365 × 10⁻⁵g/L (95% CI 1.6563e-005 to 3.3536e-005). It was not possible to test higher concentrations of the extract due to solubility. The saturation curve of the extract is shown in the Figure 7, which was achieved incubating several concentrations of [³H]Flunitrazepam in presence of 1.25 × 10⁻⁵ g/L of L. pubiflora alkaloid extract. As can be seen, the [³H]Flunitrazepam binding was markedly diminished in the range of concentrations that were tested. The apparent Kd could not be estimated as saturation with the extract was not observed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.