The green coffee extract (CE) was produced by spray dryer technique with dry unroasted seeds of coffee (Coffea arabica L) by Centroflora (Botucatu, Brazil). The extract was evaluated for its physical and chemical characteristics by IT2 100 method (standardized in 21.04% of chlorogenic acid, 27-33% of caffeoylquinic acid and 7.5% to 8% of caffeine), according to the report provided by the manufacturer.
Apomorphine, caffeine and 6-hydroxydopamine (6-OHDA) were purchased from Sigma (St. Louis, USA) and haloperidol (Haldol®) from Janssen Pharmaceutica N.V. (Beerse, Belgium). Methamphetamine was donated by the Brazilian Federal Police. For HPLC analysis, caffeine standard was purchased from Fluka (Munich, Germany) and 5-O-caffeoylquinic acid standard from Sigma-Aldrich (Milano, Italy). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany) and HPLC-grade water was prepared from distilled water using a Milli-Q system (Millipore, Waters, Milford, USA).
Caffeine quantification in CE was carried out through phase reversed high-performance liquid chromatography with Diodo Array detector (HPLC/DAD). The HPLC-DAD analyses were conducted on a Hewlett Packard 1090 II equipped with a degasser, an autosampler, and diode array detector. The identification of caffeine was based on retention time, UV spectra comparison with a standard and spiking. Quantitation was obtained through a calibration curve made with known concentrations of caffeine. The calibration curve for caffeine (Y= 2935.6 X) was found to be linear with R2 = 0.99.
The coffee extract was dissolved in water: methanol (80:20) v/v (5 mg / 2mL) and filtered with a 0.45 μm polytetrafluoroethylene (PTFE) filter, prior to injection of 31.2 μL into the HPLC system, using a reverse phase, C18, Spherisorb ODS II (Hewlett Packard) column (4.6 × 250 mm, 5 μm), connected to a guard column. Spectral UV data from all peaks were collected in the spectral range 240 – 400 nm, and chromatograms were recorded at 330 (caffeoylquinic acid derivatives and feruloylquinic acids derivatives) and 270 nm (caffeine)21. The mobile phases consisted of eluent A (0.1% aq. formic acid) and eluent B (methanol), with the following gradient profile: 0 min –20% B in A; 10 min – 30% B in A, 20 min – 50% B in A; 30 min – 70% B in A; 40 min– 90% B in A; 45 min – 40% B in A and finally returned to the initial conditions (20% B in A) to re-equilibrate the column prior to another run. The flow rate was kept constant at 0.8 mL/min, and the temperature of the column was maintained at 28°C.
Male Swiss mice (40 to 50 g) and male Wistar rats (300 to 400 g) 2-5 months old were provided by the bioterium of Psychobiology from Universidade Federal de São Paulo (UNIFESP). Animals were kept in rooms with controlled temperature (23 ± 2ºC) and light/dark cycle of 12 hours with water and food ad libitum. The project was approved by the Ethics Committee of UNIFESP (CEP #1946/06) and we followed the ARRIVE guidelines principles of the use of laboratory animals.
CE (doses of 100 and 400 mg/kg) and caffeine (31.2 mg/kg – the equivalent caffeine in 400 mg/kg of CE) were solubilized in water and orally administered by gavage in the volume of 1 mL/kg (rats) and 10 mL/kg (mice). The control animals received the vehicle water at the same volume by gavage.
Evaluation of antioxidant capacity
Antioxidant activity was evaluated by the lipid peroxidation inhibition measured by malondialdehyde concentration in rat brain homogenate. Tissue homogenate was prepared with phosphate buffer and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and then diluted again in phosphate buffer (1:3). Five concentrations of CE were added to each homogenate tube as follows: 4.17 µg/mL; 8.33 µg/mL; 12.50 µg/mL; 16.67 µg/mL; 33.33 µg/mL (final concentrations), and then incubated in water bath with thiobarbituric acid for 60 minutes at 37 ºC. The experiment was carried out according to the methodology described by Stocks et al.22. The antioxidant activity was determined for each concentration of the extract and the concentration inhibiting 50% of lipid peroxidation (Q1/2) was calculated by linear regression using the log of the concentration and the percentage of inhibition (mean of 4 assays).
Catalepsy induced by haloperidol
Groups of 10-13 mice were acutely or for 15 consecutive days treated by gavage with vehicle (controls), CE at doses of 100 or 400 mg/kg (experimental groups) or caffeine at dose of 31.2 mg/kg (positive control). Thirty minutes after the acute or the last administration of CE, caffeine or vehicle, the animals received haloperidol (5 mg/kg, ip), except the negative control group, which received saline (ip).
Forty-five minutes after the administration of haloperidol (or saline) the animals were placed with their forepaws positioned on a bar suspended 5 cm above the bench and the catalepsy time (immobility) was recorded23. The number of times each animal got off the bar over 10 minutes was recorded with a maximum of 10 times. The catalepsy index was calculated by the rate between the total catalepsy time and the number of times the mice left the bar.
Unilateral lesion induced by 6-OHDA
Male Wistar rats were anesthetized with ketamine (90 mg/kg, ip) and xylazine (5 mg/kg, ip) and placed on a stereotaxic apparatus. After the skin incision and the skull exposition, a hole was performed to the bone using a dentist drill and the neurotoxin 6-OHDA (12 μg/2 μl buffered saline in 0.2% ascorbic acid) was infused with an injection pump24 in the medial forebrain bundle (MFB) of the right hemisphere (experiment 1) or in the right striatum (experiment 2) in the lesioned group, while sham-operated animals were infused with 2 μl of buffered saline. The coordinates for MFB lesion were: AP, -1.9 mm; ML, -1.9 mm; DV, -7.2 mm of bregma; and for striatum: AP, +0.5 mm; ML, -2.5 mm; DV -4.5 mm; according to the brain atlas Paxinos and Watson25. After surgery, dental wax was applied on the skull to close the access hole, the head skin was sutured, and the animals were allowed to recover in individual boxes with food, water and controlled temperature. The experimental design of experiments 1 and 2 is shown on Fig. 1. A pilot study showed that the lesion was effective in inducing neuronal loss in the substantia nigra (Fig. 1 – qualitative analysis).
Experiment 1A: Effect of acute treatment with CE on the rotational behavior of rats with lesion on MFB
Groups of 10-15 rats lesioned on MFB with 6-OHDA were challenged acutely on the 14th day with CE (100 or 400 mg/kg), caffeine (31.2 mg/kg) or vehicle by gavage. An extra group of rats that received buffered saline instead of 6-OHDA (sham-operated group) was challenged with CE (400 mg/kg) on the 14th day. Immediately after the respective treatments, the animals were evaluated for 60 minutes and the number of contralateral and ipsilateral complete rotations (360o) in a cylinder was recorded26. On the 15th day, all animals received apomorphine (0.1 mg/kg, sc), and on the 16th day, methamphetamine (2.5 mg/kg, ip), and the number of ipsilateral and contralateral rotations was recorded again.
Experiment 1B: Effect of repeated treatment with CE on the rotational behavior of rats with lesion on MFB
For this experiment, groups of 12-15 rats lesioned on MFB were orally treated for 30 days with CE (100 or 400 mg/kg), caffeine (31.2 mg/kg) or vehicle, by gavage. An extra group of sham-operated rats received vehicle by gavage for the same period. On the 15th and 29th days after surgery the animals were evaluated for 60 minutes regarding the number of ipsilateral or contralateral rotations, after administration of apomorphine (0.1 mg/kg, sc). On 16th and 30th days, methamphetamine (2.5 mg/kg, ip) was administered and the number of rotations was once again recorded.
Experiment 2: Effect of pre- and post-treatment with CE on the rotational behavior of rats with striatal lesion
Groups of 11-15 rats were pre-treated for 15 days with CE (100 or 400 mg/kg), caffeine (31.2 mg/kg) or vehicle by gavage. One hour after treatment on day 15 the rats were submitted to the stereotaxic surgery and 6-OHDA (lesioned group) or buffered saline (sham-operated group) was administered into the right striatum. The treatment continued for another 15 days and the animals were challenged with apomorphine (15th day after surgery) and methamphetamine (16th day) as previously described. The treatment with CE or caffeine on the days of rotational tests was made after the behavioral evaluation to avoid acute interference with apomorphine or methamphetamine.
Central monoamines dosage by HPLC
After 24 hours of the last behavioral evaluation, the animals from experiments 1B and 2 were euthanized for brain collection, and the brains were frozen at -80oC. Later, the right (lesioned) and left (control) striatum were dissected, weighed, homogenized, centrifuged and then filtered in nitrocellulose membrane as described by Machado et al.27. Precipitates were diluted in a solution of 0.1 N of sodium hydroxide and evaluated for protein concentration using a colorimetric quantification kit (Pierce Chemical, Rockford, USA) and supernatants (20 µL) were used for monoamines quantification through high-performance liquid chromatography (HPLC) according to method described in details by Machado et al.27. Each sample was analyzed in duplicate for concentrations of dopamine (DA), noradrenaline (NE), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA). The recovery rate of the analytes was determined by adding a predetermined amount of internal standard dihydroxybenzylamine for the homogenization of tissue. The peak areas of samples were compared with peak areas of standards of each neurotransmitter or metabolite, which were injected at known concentrations, at intervals between runs.
Statistical comparisons were performed using GraphPad Prism 8.3 software. After checking for normality distribution using Shapiro-Wilks test, Kruskal-Wallis test followed by Mann-Whitney was applied to analyze the results of rotational behavior test along with Wilcoxon to compare ipsilateral and contralateral rotations among each group. Monoamines levels were analyzed using Kruskal-Wallis test and One-way ANOVA followed by Tukey to analyze the catalepsy. The results are expressed as mean ± standard error (SEM) and the raw data is available in the Supplementary Data S1. Statistical significance was considered at p<0.05.