Plant extraction and spilanthol isolation
A. ciliata aerial parts were collected at the garden of the Associação dos Funcionários Fiscais do Estado de Santa Catarina (AFFESC, Florianópolis, Santa Catarina, Brazil) in October, 2012. The ethanol extract, hexane fraction and spilanthol were obtained as previously described (Silveira et al. 2016). HR-MS spectrum and chromatogram can be found as supplementary material.
Drugs, Reagents and doses
The following substances were used: STZ, capsazepine (TRPA1 channel antagonist) and galantamine (commonly used drug in the treatment of AD) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents (ethanol and hexane) used to obtain the spilanthol were commercial grade, purchased from VETEC (RJ, BR). Ketamine (Vetbrands, FL, USA), xylazine (Agener União, SP, BR), lidocaine with epinephrine 2% (Cristália, SP, BR). All drugs were dissolved in saline [except STZ which was dissolved in artificial cerebrospinal fluid prepared as described by Jayant et al. (2016) and were infused at room temperature]. The dose of galantamine used in this study as positive control in memory experiments was determined from a preliminary study based on the doses used by Berté et al (2018). The other doses used were determined based on pilot experiments. Due to the low amount of the compound, capsazepine was used only acutely administered 15 minutes before the administration of spilanthol in the inhibitory avoidance experiment.
Animals and Ethical statement
Male swiss albino mice (25 – 30 g), approximately 90 days old, were used for the study. The animals were obtained from the Universidade do Vale do Itajaí (UNIVALI). They were kept at 22 ± 2 °C with free access to food and water, under a 12:12 h natural light (sunlight) and dark cycle, except during pharmacological assays. This study followed the guidelines established by the Research Ethics Committee of UNIVALI, and the Brazilian Law on Animal Experimentation and was approved by the Research Ethics Committee of UNIVALI (CEUA/UNIVALI), protocol 11/18.
Mice were randomly divided into nine groups, each containing 8 – 10 mice:
Group 1 (n = 10) regards the sham-operated animals that did not receive STZ nor oral treatment; however, received the same amount of the vehicle used to solubilize STZ (artificial liquor), 2 μl i.c.v. This group is important to show that i.c.v. does not cause hippocampal lesion interfering with behavioral and biochemical assays.
Group 2 (n = 9) is the negative control, injected with STZ (2 µL of 2.5 mg/ml; i.c.v.) and treated with a vehicle per os (p.o.) route in which extract and spilanthol were solubilized (DMSO 2% and distillated water).
Group 3 (n = 10) represents the positive control, was injected with STZ (2 µL of 2.5 mg/ml; i.c.v.), and followed by injection of galantamine (30mg/kg, p.o.) treatment for 15 days.
Groups 4 to 6 (n = 10) are the animals that were treated for 15 days with ethanolic extract (50 mg/kg, 100 mg/kg e 150 mg/kg, p.o., respectively), dissolved in DMSO 2% and distillated water.
Group 7 (n = 10) received the hexane fraction ofA. ciliata(100 mg/mL; p.o).
Group 8 (n = 10) was treated with spilanthol (10 mg/mL; p.o).
Group 9 (n = 10) was treated with spilanthol (10 mg/mL; p.o). and capsazepine (40mg/kg).
Neurobehavioral tests were carried out within 24 h after the 15th day of treatment (Fig. 1).
Experimental model of SAD induced by STZ
SAD was induced by the freehand i.c.v. procedure previously reported (Pinton et al. 2010) with slightly modifications. Briefly, mice were anesthetized with xylazine/ketamine, than they were submitted to a minor surgery to remove the cutaneous tissue aiming at the exposure of the skull. After cleaning and asepsis of the cranial region, the animals still under anesthesia received STZ (2 µL of 2.5 mg/mL solution; i.c.v.) through a hypodermic needle attached to a cannula, which was linked to a 5 μL Hamilton syringe. After 48 h, each mouse received a second injection of STZ (same way as the first one). Drug treatment started after the second dose of STZ and lasted for more than 15 days, including the days of the behavioral tests. Treatment was done orally (p.o.) and experiments were carried out 1 hour after them.
Novel object recognition (NOR) test
The NOR test is used to evaluate long-term memory and also cognition. It is based on the principle that, in a familiar environment, laboratory animals show an instinctive affinity for novelty, or preference for exploring a new object rather than a familiar (Ennaceur 2010). To this end, animals from Group 1 to 6 were evaluated.
The test consisted of three different phases (habituation phase, training phase, and test phase) conducted on three successive days and followed the protocol described by Myskiw et al. (2008) with slightly modifications. Briefly, in the first phase, mice were exposed to an open field apparatus consisted of a wooden box of 40 x 60 x 50 cm dimensions and were left for 15 min in the absence of stimulus objects to adapt to the apparatus. On the next day animals were placed on the apparatus for 10 min in the presence of two identical in shape, color, and size objects put in opposite corners of the wooden box for familiarization. On day three, mice were tested by placing them again in the open field apparatus but now in the presence of a familiar and a novel object (different in shape, color and size from the other) and were left to explore the objects for 10 min. The time taken by each mouse to explore the two objects during the acquisition and retention phases of the test were recorded manually and separately with two stop watches (by a trained observer). Its Discrimination Index (DI) wasthen calculated for the retention trial as DI=B-A1/B+A1 (B=novelobject, A1 = familiar object).
Effect of treatments on the locomotor activity of animals (Exploratory behavior) –Open Field Test (OFT)
Locomotor activity was assessed using the same open field apparatus used in the NOR test. In this test, during animals' habituation phase, the exploratory behavior of them was evaluated during the first six minutes. Mice were placed individually in the center of the open field and crossing movements were recorded during this time.
Inhibitory avoidance test (IAT)
This experiment is used to measure long-term memory. In the present study, all nine group of animals were submitted to an IAT as previously performed (Izquierdo and Dias 1983) with some modifications. Mice were trained and tested in an apparatus consisted of a 50 cm long, 25 cm wide, 25 cm high plywood box with a glass frontal wall and floor made of steel bars stainless steel parallel with caliper of 0.1 cm and to 1 cm of distance of each other, with a 2.5 cm high, 7.0 cm wide, 2.5 long platform. A 15 W lamp lit up the apparatus, while the room remains dark. On the training trial, animals were gently placed and held on the platform facing the rear left corner. Their latency to step down (placing the four paws on the grid) was timed, and immediately after a 0.4 mA foot shock was delivered for 2 s.The test session was conducted 24 h later. The procedure was identical to training session with omission of the foot shock. Their latency to step down was also measured up to a limit of 180 s.
Preparation of brain homogenate
After completion of experiments animals were killed and, whole intact brain was carefully removed and rinsed with 0.9% NaCl solution for cleaning, and weighed. Braintissue was homogenized in a phosphate buffer (pH 7.6), centrifuged at 20,000×g, at 4 °C for 10 min. An aliquot of supernatant was collected and stored at -20 °C for furtherbiochemical tests. Protein concentrations were determined by the Bradford assay with Bovine serum albumin as standard (0.05–1.00 mg/mL).
Assessment of brain antioxidant markers
Catalase (CAT) activity
CAT activity was determined by the protocol of Maehly and Chance (1954) with slight modifications.CAT reaction solution consists of 625 μL of 50 mM of potassium phosphate buffer (pH 5), 100 μL of 5.9 mM H2O2 and 35 μL enzyme extract. Changes in the absorbance of the reaction solution was noted after 1 min at 240 nm. An absorbance change of 0.01 as units/min denotes one unit of catalase activity (Maehly and Chance 1954).
Superoxide dismutase (SOD) activity
In this experiment, Kakkar et al. (1984) method was utilized. Buffers were exploited for the assessment of SOD activity. Centrifugation of tissue homogenate was done at 1500×g for 10 min and then at 10,000×g for 15 min. Supernatant was collected and 150 μL of it was added to the aliquot containing 600 μL of 0.052 mM sodium pyrophosphate buffer (pH 7.0) and 186 mM of phenazine methosulphate (50 μL). 100 μL of 780 μM NADH was added to initiate enzymatic reaction. After 1 min, glacial acetic acid (500 μL) was added to stop the reaction. At 560 nm optical density was determined to enumerate the color intensity. Results were evaluated in units/mg protein.
Glutathione-S-transferase (GST) assay
Scheme of Habig et al. (1974) protocol was strictly followed for the estimation of GST potency. 150 μL aliquot of tissue homogenate was added to 720 μL of sodium phosphate buffer together with 100 μL of reduced glutathione (1 mM) and 12.5 μL of 1-chloro-2,4-dinitrobenzene (CDNB) 1 mM. Changes in absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M-1 cm-1.
Glutathione reductase (GR) assay
GR activity in tissue samples was analyzed as described by Carlberg eand Mannervik (1975) with minor modifications.The reaction reagent 2 mL was made of of 1.65 mL phosphate buffer: (0.1 M; pH 7.6), 100 μL EDTA (0.5 mM), 50 μL oxidized glutathione (1 mM), 100 μL NADPH (0.1 mM) and 100 μL of homogenate. Activity of enzyme was monitored by recording the absorbance of the vanishing of NADPH at 340 nm at 25 °C. Estimated of enzyme level was accomplished as nM NADPH oxidized/min/mg protein by employing molar extinction coefficient of 6.22 × 103/M/ cm.
Glutathione peroxidase (GPx) assay
GPx activity was assessed as described elsewhere (Jollow et al. 1974). Entire volume of 2 mL reaction solution comprised of 1 mM EDTA (100 μL), 0.1 M phosphate buffer(1.49 ml; pH 7.4), 1 mM sodium azide (100 μL), 1 IU/mLGR (50 μL), 1 mM reduced glutathione (GSH) (50 μL), 0.2 mM NADPH (100 μL), 0.25 mM H2O2 (10 μL) and tissue homogenate (100 μL). The loss of NADPH was recorded at 340 nm at room temperature. Enzyme level was estimated as nM NADPH oxidized/min/mg protein employing 6.22 × 103/M/cm molar extinction coefficient.
Reduced glutathione (GSH) assay
For this assays, GSH activity was checked as described by Jollow et al. (1974) using Ellman's reagent (DTNB) as a substrate. The yellow color developed was read immediately at 412 nm and expressed as μmoL GSH/g tissue.
Assessment of oxidative stress markers
Estimation of Malondialdehyde (MDA)
This assay was carried out following the previous protocol (Wills 1966) with minor modifications. MDA is one of lipid peroxidation product that can be used as a marker of oxidative stress.The reaction mixture in a total volume of 1.0 mL contained 0.58 mL phosphate buffer (0.1 mol; pH 7.4), 0.2 mL homogenate sample, 0.2 mL ascorbic acid (100 mmoL), and 0.02 mL ferric chloride (100 mmol). The reaction mixture was incubated at 37 °C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 mL 10% trichloroacetic acid. Following addition of 1.0 mL 0.67% thiobarbituric acid, all tubes were placed in boiling water bath for 20 min and then shifted to ice bath before centrifuging at 2500×g for 10 min. The amount of thiobarbituric acid reactive substances (TBARS) formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using a spectrophotometer against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37 °C using a molar extinction coefficient of 1.56 × 105 M-1 cm-1.
The results were submitted to one-way analysis of variance (ANOVA), followed by multiplepost hoc comparison test using the software GraphPad InStat®. The results are expressed as mean ± standard error values of the mean and p lower than 0.05 (p <0.05) were considered statistically significant.