Self-medication by orangutans using bioactive properties of a plant

Study site

We carried out field research in the Sabangau Peat-swamp forest (600 km²) as part of the OUTROP – CIMTROP multi-disciplinary research project within the LAHG (Laboratorium Alam Hutan Gambut: Natural Laboratory for the Study of Peat Swamp Forest), a 50 km² area in the Northern Sabangau Catchment. The LAHG was designated for the purpose of scientific research in 1997, and is managed by the Indonesian Research and Conservation Organisation CIMTROP (the Centre for International Co-operation in Management of Tropical Peatland). This forest is a protected tropical peat-swamp forest in the southern area of the Central Kalimantan province in Indonesia. The base camp is located 20 km southwest of Palangkaraya, the provincial capital.

Plant identification and morphology

This species of Dracaena was identified from samples collected after each orang-utan performed the fur-rubbing act. Photographs and dried specimens including both flowering and fruiting parts of the plant were used to identify this species as Dracaena cantleyi by botanists at the herbaria in Palangkaraya, in Bogor, Indonesia and at Kew Gardens in the United Kingdom.

This species forms a woody shrub or small tree. It is part of Asparagaceae subfamily Nolinoideae. It is distributed throughout Borneo to Peninsular Malaysia⁴, where it is sometimes know under the illegitimate name D. aurantiaca (Baker) Wall. ex Hook.f. As is typical for the genus, it produces leaves in rosettes on woody stems. Leaf shape in D. cantleyi is rather variable, and the leaf blades are sometimes variegated with circular to elliptic, discrete to contiguous paler markings. The systematics of this genus have not been studied in tropical South-East Asia since Ridley’s era (1924), but it is suggested that D. sara’wakensis (W.W.Sm.) Jankalski from Borneo is a conspecific of D. cantleyi Baker based on its morphology. D. cantleyi is thought to be relatively infrequent in Central Kalimantan.

Biological activities

Extract preparation

The dried plant material was homogenised to a fine powder in liquid nitrogen. Portions of the ground material (0.35 g) were then extracted separately in 35 ml of water, methanol and/or methanol:tetrahydrofurane (MeOH:THF, 1:1). After 16 hours (overnight) extraction at −20 °C, the resulting homogenates were centrifuged (15,000 rpm, 4 °C, 20 min). The sediments were re-extracted for 1 hour in the same way and then centrifuged. These two supernatants were pooled and dried in vaccuo at 35 °C, then dissolved in 200 μl of methanol and 800 μl of 0.1 M Tris buffer (pH 7.2).

Cell culture

Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in ECGM medium (Endothelial Cell Growth Medium, Provitro, Berlin, Germany) and supplemented with 10% fetal bovine serum (Sigma Aldrich, Munich, Germany). Cells were maintained under standard cell culture conditions at 37 °C and 5% CO₂ in a humid environment. Cells were subcultured two or three times a week using the standard trypsinization procedure. Calcein AM was obtained from Molecular Probes (Life Technologies, Carlsbad, CA, USA). HUVECs were a kind gift from Prof Jitka Ulrichová (Medical Faculty, Palacky University, Olomouc).

ELAM

CD62E (E-selectin, ELAM)-induction assays: Each well of the 96-well plates were coated with gelatine by applying 200 µl of 1.0% gelatine for 10 minutes at room temperature. Outer wells (A1-A12, H1-H12, 1-H1 and A12-H12) contained only 200 µl/well medium and served as an evaporation barrier. 1 × 10⁴ HUVECs were seeded in each of the other wells in a 200 µl medium and grown for 48 hours to optimal confluence. Increasing concentrations of extracts were then added to the HUVEC-containing wells in triplicates, and the cells were incubated for 30 minutes, after which 10 ng/ml TNFα was added per well to stimulate NF-κB, and thus ELAM. After another 4 hours of incubation, the levels of ELAM in each of the HUVEC-containing wells were determined by enzyme-linked activity assays (ELISAs) as described below.

Cell-surface ELISA ELAM: Cells were washed once with phosphate buffered saline (PBS) and fixed with 0.1% glutaraldehyde (Sigma-Aldrich, Munich, Germany) for 15 minutes at room temperature. Then, cells were washed three times with 200 µl per well PBS/0.05% Tween 20, blocked with 200 µl/well 5% bovine serum albumin (BSA)/PBS for 1 hour, and washed again three times with 200 µl per well PBS/0.05% Tween 20. Then anti-ELAM-antibody (clone BBA-1, R&D Systems, Minneapolis, MN, USA) diluted 1:5000 in 0.1% BSA/PBS (100 µl per well) was added for 1 hour at room temperature and washed thereafter five times with 200 µl per well PBS/0.05% Tween 20. Subsequently, goat anti-mouse horseradish-peroxidase (HRP) antibody (Sigma-Aldrich, Munich, Germany) diluted 1:10000 in 0.1% BSA/PBS (100 µl per well) was applied and the cells were incubated for 1 hour in a dark at room temperature and, after decanting, washed five times with 200 µl per well PBS/0.05% Tween 20. The HRP-activity of the cells in each of the wells was estimated using Fast-OPD (o-phenylenediamine dihydrochloride) (Sigma-Aldrich, Munich, Germany) assay and absorbance was measured at OD_492nm in a vertical spectrophotometer.

Cytotoxicity testing: For the ELAM expression assay, the toxicity of tested extracts was assessed in HUVECs by Calcein AM or sulforhodamine B (Sigma Aldrich, Munich, Germany) cytotoxicity assays in 96-well microtitre plates³³. 20 µL portions of each of the extract concentrations were added in triplicate to the cells, which were then incubated at 37 °C in an atmosphere containing 5% CO₂ for 4 hours. After this, Calcein AM solution (Molecular Probes, Invitrogen, Karlsruhe, Germany) was added for 1 hour, or sulforhodamine B after fixation with 50% trichloracetic acid, according to the manufacturer’s instructions. The fluorescence of viable cells stained with Calcein was quantified using a Fluoroskan Ascent instrument (Labsystems, Vantaa, Finland) reader and on the basis of triplicate experiments the cytotoxic concentrations were calculated. The absorbance of total protein mass related to the cell number stained with sulforhodamine B was measured using Tecan Infinite reader (Tecan Group, Männedorf, Switzerland).

ICAM-1, IL-6

Sandwich CD54 (ICAM-1) and IL-6 ELISAs: HUVEC cells were seeded into 24 well-plates and treated immediately with tested extracts for 30 minutes. MeOH/Tris was used as a vehicle for controls and TNFα (10 ng/ml) as inflammation stimuli. After 24 hours of treatment, culture medium was removed and collected into microfuge tube. Concentrations of cytokines (IL-6, ICAM-1) produced by cells into the medium was assessed using sandwich ELISA kits (PeproTech, Rocky Hill, NJ, USA). 96 well-plates for ELISA (Nunc, Thermo Fisher Scientific, Rockford, USA) were coated with capture antibody. After overnight incubation, plates were washed with 0.05% Tween in PBS and blocked with 1% BSA in PBS (Sigma-Aldrich, Munich, Germany) for 1 hour. Subsequently, plates were washed with PBS with Tween (0.05%) and standard or sample was added for 2 h. After washing, detection antibody was applied for another 2 hours. Avidin-HRP conjugate was incubated for 30 minutes and thereafter HRP activity was estimated by ABTS substrate (Sigma-Aldrich, Munich, Germany) and absorbance was measured at OD_405nm in a multimode plate reader (Infinite 200 PRO, Tecan Group, Männedorf, Switzerland).

ICAM-1, VCAM-1

Flow cytometric analysis of cell surface adhesion proteins (ICAM-1, VCAM-1): Treated HUVECs were trypsinized, seeded in 100-mm culture dishes, and immediately incubated with the test extracts. After 30 minutes, TNFα (10 ng/ml) was used as an inflammation stimulus. After 24 hours of incubation, the cells were again detached with trypsin, washed, fixed (4% formaldehyde), and stained with anti-ICAM-1 or anti-VCAM-1 antibody labelled with FITC (Becton Dickinson, NJ, USA) in PBS. Signal were then assessed with a flow cytometer (FACS Verse^TM, Becton Dickinson, NJ, USA). In each experiment, the fluorescence of cells exposed to all treatments was expressed relative to the mean fluorescence of cells treated with TNFα alone (set as 100%), and changes in the expression of ICAM-1 or VCAM-1 on the cells’ surfaces were expressed in terms of relative changes in the mean (logarithmic) index of fluorescence intensity using BD FACSuite software (Becton Dickinson, NJ, USA). At least three different sets of experiments were performed in triplicates.

Data Availability

Data for these analyses are available from the corresponding author.

from Hacker News https://ift.tt/2j38iaR