Background: Trichilia catigua A. Juss. (Meliaceae) is a species known as catuaba and used in folk medicine for the treatment of fatigue, stress, impotence and memory deficit. The main phytochemical compounds identified in the barks of T. catigua are flavalignans, flavan-3-ols and flavonoids which are associated with its antioxidant activity. Pre-clinical studies with T. catigua extracts have identified many pharmacological properties, such as anti-inflammatory, antidepressant, antinociceptive, pro-memory and neuroprotective against ischemia and oxidative stress. This study was designed in order to compare the chemical composition and in vitro antioxidant and anticholinesterase activity of four different polarity extracts and selected the one most active for in vivo studies in rodent models of stress, fatigue and memory. Methods: Hexane, chloroform, hydroalcoholic and aqueous extracts from bark of Trichilia catigua were analyzed by RPHPLC-DAD-ESI-MS/MS. Antioxidant activity was assessed by 2,2-diphenyl-1-picryl hydrazyl (DPPH) assay and acetylcholinesterase inhibition by Ellman’s modified method. In vivo studies (stress, fatigue and memory) were carried out with adult male mice and rats treated with hydroalcoholic extract in doses of 25–300 mg/kg (p.o.). Results: We confirmed the presence of cinchonain IIa, Ia and Ib, as main constituents in the four extracts, while procyanidins were detected only in hydroalcoholic extract. Antioxidant and anticholinesterase activity were observed for all extracts, with most potent activity found on the hydroalcoholic extract (EC =43 μg/mL and IC =142 μg/mL 50 50 for DPPH scavenger and acetylcholinesterase inhibition, respectively). The treatment of laboratory animals with hydroalcoholic extract did not protect rats from cold immobilization stress and did not prevent the scopolamine- induced amnesia in mice. However, the treatment of mice with the hydroalcoholic extract partially reduced the fatigue induced by treadmill, since the highest dose increased the spontaneous locomotor activity and reduced the deficit on grip strength after the forced exercise (p < 0.05), in some observation times. Conclusions: These data suggest the hydroalcoholic extract as the most suitable for plant extraction and partially support the folk use of T. catigua as antifatigue drug. Keywords: Trichilia catigua, Adaptogen, Antifatigue, Acetylcholinesterase inhibition, Antioxidant, Phenylpropanoids, Cinchonains, Procyanidins * Correspondence: firstname.lastname@example.org Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Arcturus, 03, São Bernardo do Campo, SP CEP 09210-180, Brazil Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 2 of 13 Background order to compare the chemical composition and in vitro There are several examples of plants used to keep a good antioxidant and anticholinesterase activity of four health status and to reduce the cognitive deficits that extracts with different polarity and select the one most result from aging, such as memory deficit, fatigue and active for in vivo studies in rodent models of stress, general weakness. Guarana (Paullinia cupana Kunth), fatigue and memory. muirapuama (Ptychopetalum olacoides Benth.), nó-de- cachorro (Heteropterys tomentosa A. Juss.), damiana Methods (Turnera diffusa Willd. ex Schult.) and catuaba (Trichi- Plant material and extracts preparation lia catigua A. Juss.) are species widely used for these Ground barks of T. catigua were obtained from Santos purposes in Brazil . The literature has many examples Flora with quality control assurance. The extracts were of active principles with remarkable antioxidant action, prepared using 10% of botanical material in PA grade especially polyphenols. The cholinergic system and the solvents (Synth, Diadema, Brazil). The aqueous extract enzyme acetylcholinesterase (AChE) are other important was prepared by decoction (50 g of plant in 500 mL of targets for nootropics and cognitive enhancing drugs. In boiling water); the hydroalcoholic extract was prepared fact, the inhibition of AchE was described for muira- by turbolysis (100 g of barks in 1 L of ethanol: water puama and guarana [2, 3], two Brazilian species used for 50% under vigorous agitation); the chloroform and n- the improvement of cognitive functions similarly to the hexane extracts were prepared by macerating 25 g of folk use of Trichilia catigua. plant with 250 mL of solvent for four days at room Trichilia catigua (Meliaceae) is a species of South temperature, followed by 50 min in ultrasound. The ex- America, known as catuaba, tatuaba and catiguá, and tracts were filtered, concentrated in a rota-evaporator used in folk medicine as a tonic for the treatment of and then dried in a fume hood (chloroform and hexane fatigue, stress, impotence and memory deficit [1, 4–6]. extracts) or lyophilized (aqueous and hydroalcoholic These popular uses are typical of an adaptogen, which is extracts). The percent yields of extractions were 15.25 supposed to decrease the consequences of stress and (hydroalcoholic), 13.52 (aqueous), 1.98 (chloroform) and improve physical and cognitive performances both in 1.76 (hexane). All extracts were analyzed by HPLC- healthy and ill patients . The most common popular DAD-ESI-MS/MS in order to obtain their respective form of preparation is as “garrafada” (the maceration of phytochemical profile. the barks in alcoholic drinks, usually 38–48% alcohol). Several other species are also known as catuaba and are Phytochemical analysis used for similar purposes, but most of the available com- Thin-layer chromatography (TLC) mercial products use barks of T. catigua . The four extracts were examined by TLC using silica gel Flavonoids, tannins, alkaloids, saponins, among other plates (200 μm layer thickness, Merck). The extracts phytochemical classes were identified in the barks of T. were dissolved in a mixture of methanol and chloroform catigua . The bark contains high concentrations of (1:1) and the TLC was developed with chloroform: polyphenols including flavan-3-ols (procyanidin B2, epi- methanol:water (65:35:10, v/v/v) as the mobile phase. catechin, catechin), flavalignans (cinchonains Ia, Ib, IIa, The plates were visualized by UV at 254 and 365 nm IIb) and phenylpropanoid derivatives (chlorogenic acid) and by spraying with a 5% vanillin solution in 10% HCl [7–10]. The main constituents of T. catigua exhibited in ethanol (Synth, Diadema, Brazil) (v/v), followed by potent antioxidant activity, which is important in the pre- heating the plate. Flavanols (condensed tannins, mono- vention of cellular damage triggered by oxidative stress in mers, dimers) react with vanillin in acidic medium to acute and chronic neuropathological conditions . yield a red adduct. Compounds were also revealed by Pre-clinical studies with T. catigua extracts have spraying 1% ethanolic FeCl solution (Synth, Diadema, shown many pharmacological properties, such as anti- Brazil). inflammatory , antinociceptive , antidepressant [5, 13, 14], pro-memory  and neuroprotective against RPHPLC-DAD-ESI-MS/MS analyses ischemia and oxidative stress [6, 15, 16]. The antinoci- The RPHPLC-DAD-ESI-MS/MS ion trap analysis was ceptive and antidepressant effects are attributed mainly conducted in the DADSPD-M10AVP Shimadzu system to dopaminergic action [12, 13] and were also described equipped with a photodiode array detector coupled to for a commercial preparation containing T. catigua, Amazon Speed ETD, Bruker Daltonics, which consisted Paullinia cupana, Ptychopetalum olacoides and Zingiber of two LC-20 AD pumps, SPD-20A diode array detector, officinale Roscoe . CTO-20A column oven and SIL 20 AC auto injector Even though many biological activities have been (Shimadzu Corporation, Kyoto, Japan). The mass de- reported to T. catigua, its adaptogen-like effect was not tector was a quadrupole ion trap equipped with atmos- fully evaluated. Thus, the present study was designed in pheric pressure ionization source through electrospray Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 3 of 13 ionization interface, which was operated in the full scan Phenomenex – C18 RP-18 column (4.6 × 250 mm, 5 μm, MS/MS mode. All the operations, acquisitions and data Hewlett Packard) connected to a guard column and a mo- analysis were controlled by the Shimadzu CBM-20A bile phase composed by eluent A (0.1% aq. formic acid) and system controller. HPLC grade water was prepared with eluent B (methanol) at the constant flow rate 1.0 mL/min distilled water using a Milli-Q system (Millipore, Waters, and constant temperature of the oven at 40 °C. The same Milford, MA, USA). chromatography conditions were used in the analyses by Q- The extracts (3.33 mg/mL) were dissolved in a mixture ToF. Calculations were performed using the high precision of water milli-Q and methanol (1:1), filtered by a 0.45 μm calibration quadratic algorithm. PFTE filter and then an aliquot of 30 μL was injected into the HPLC system. Spectral UV data from all peaks were In vitro tests collected at the range 240–400 nm and chromatograms of DPPH assay flavanols were recorded at 280 nm. Separation of the mix- The test was based on the protocol of Duarte-Almeida ture of the constituents was performed in reverse phase et al. , with some modifications. An ethanolic solu- Luna Phenomenex – C18 RP-18 column (4.6 × 250 mm, tion of 2,2-diphenyl-1-picryl hydrazyl (DPPH) (Sigma, 5 μm, Hewlett Packard) connected to a guard column. St. Louis, MO, USA) was prepared in order to produce The mobile phase was composed by eluent A (0.1% aq. an absorbance between 0.8 and 0.99 at 517 nm. One formic acid) and eluent B (methanol) (Merck, Darmstadt, hundred microliters of each extract diluted in ethanol Germany) at the constant flow rate 1.0 mL/min and (Synth, Diadema, Brazil) at initial concentrations of constant temperature of the oven at 40 °C. The following 0.004, 0.01, 0.04, 0.1, 0.4 and 1.0 mg/mL were pipet- elution program was used: 0 min – (20% B), 10 min – ted in a cuvette and after the addition of 900 μLof (30% B), 20 min – (50% B), 30 min – (70% B), 40 min– DPPH the cuvette was placed in a spectrophotometer (90% B), 45 min – (40% B), and finally returned to the (PG Instruments LTD, Leicestershire, United Kingdom) initial conditions (20% B) to re-equilibrate the column and read during 2 min at 517 nm. Ethanol (vehicle) was prior to the next run. used as control and rutin (Acros Organics, New Jersey, The parameters were set as follows: electrospray USA) as positive control. The percentage of DPPH scaven- voltage of the ion source at − 38 V, capillary voltage at ger (S) was calculated by the formula: S = [(A -A )/ DPPH c s 4500 V, end plate set at 500 V and capillary temperature A ] × 100, where A = absorbance for the control and A = c c s of 300 °C. Helium was used as the collision gas and absorbance for the sample. The concentration of each nitrogen as the nebulizing gas. Nebulization was added extractthatquenches50% of DPPH (EC ) was calculated with coaxial nitrogen sheath gas at the pressure of by linear regression (% of scavenge vs final concentration) 40 psi. Desolvation was facilitated using a counter- using the mean of 4 assays. current nitrogen (dry gas) flow set at 9.0 L/min. The spectra were acquired over a mass-to-charge (m/z) ran- Acetylcholinesterase activity ging between 100 and 1200 mass units with resolution The inhibition of AChE activity was determined spectro- setat30,000using thenormalscanratetubelens − 110.0 V. photometrically based on Ellman’s method, as previously The constituents were fragmented using the auto MS/MS reported by Padilla et al. , with minor modifications. mode. All collision-induced dissociation mass spectra were In microplate, 20 μLof T. catigua extracts at different obtained using helium as the collision gas at the fragmenta- concentrations (0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mg/mL), tion voltage from 0.5 up to 1.3 V. Each generated mass 10 μL of acetylcholinesterase (1 U/mL) (Sigma, St. Louis, spectrum was based on an average of 10 scans. The MO, USA) and 160 μL of 5,5-dithiobis-2-nitrobenzoic proposed structure was based on the characteristic acid (DTNB, Ellman’s reagent) 0.33 mM (Sigma, St. fragmentation patterns and comparison with MS data Louis, MO, USA) in phosphate buffer (0.1 M, pH 8.0) reported in previous studies with the species [17–20] were pipetted in triplicate and incubated at room and also by searching the following mass spectral data- temperature for 10 min. Then, 10 μL of acetyltiocholine bases: SciFinder Scholar , Phenol-Explorer , iodide (20 mM) (Sigma, St. Louis, MO, USA) was added ChemSpider  and HMDB . and the plate was immediately placed in a microplate HR-ESI-TOF-MS (high resolution electrospray ionization- reader (BioTek Instruments, Inc., Winooski, VT, USA). time-of-flight-mass spectroscopy) analyses were carried out The hydrolysis of acetyltiocholine iodide leads to pro- to determine the exact molecular mass and identify the duction of acetic acid and thiocholine, that reacts with elemental composition of constituents. The Q-ToF spec- DTNB producing the anion 5-thio-2-nitrobenzoic acid, trometer MAXIS 3G – Bruker Daltonics consisted of ESI which was monitored at 412 nm during 20 min. operating at 4500 V, nebulization with nitrogen at 4 Bar and Rivastigmine (Exelon®) from Novartis Farmacêutica SA dry gas flow of 8 L/min at temperature of 200 °C. Separation (Barberà del Vallès, Spain) was used as a positive con- of the constituents was performed in reverse phase Luna trol. The percentage of inhibition (I) of AChE was Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 4 of 13 calculated by the formula: I = [(A -A )/A ] × 100, began on the eighth day. The animals were restricted AChE c s c where A = absorbance for the control and A = absorb- inside acrylic animal restrainer for 2 h in the morning c s ance for the sample. The concentration of each extract (8-10 h) and later placed in a cold chamber (10–13 °C) that inhibits 50% of AChE activity (IC ) was calculated for 2 h in the afternoon (16-18 h) from the 8th to 13th by linear regression (% of inhibition vs final concentra- day. The animals were then fasted for 20 h and on the tion) using the mean of 3–4 assays. 14th day they were immobilized in a wire screen and placed in the cold chamber for 2 h as described by Behavioral studies with the hydroalcoholic extract Mendes et al. . After that, the animals were killed by Animals decapitation and their blood collected for plasma meas- Male albino Swiss mice (30-50 g) and male Wistar urement of adrenocorticotropic hormone (ACTH) in a rats (300-450 g), 3–4 months old, from our vivarium clinical analysis laboratory, and corticosterone by radio- (Department of Psychobiology, UNIFESP) were housed in immunoassay according to the manufacturer’s instruc- rooms with 12 h light-dark circle, controlled temperature tions (MP Biomedicals, Santa Ana, CA, USA). The (21 ± 2 °C), with filtered tap water and food (Nuvilab, stomachs were immediately removed and the degree and Brazil) ad libitum, except during the experiments. The an- index of ulceration were evaluated according to the imals were kept in in polypropylene cages (4–5animals scales previously described . Simultaneously, the each) with pine shavings as bedding material. The animals adrenal glands, thymus and spleen were dissected and were randomly divided into the different groups and were weighted in an analytical balance scale. An extra group treated by gavage (oral administration, p.o.) with of rats not subjected to the stress (non-stressed control) water (controls) or hydroalcoholic extract dissolved in received water for the same period, and was used to ob- water, receiving 0.1 mL per 10 g body weight (mice) tain the normal levels of hormones and tissue weights. or 0.1 mL/100 g (rats). The animals were euthanized in CO chamber or by decapitation (in the case of Forced treadmill exercise stress by immobilization test). The protocols followed The physical resistance and fatigue were evaluated in an the International Guiding Principles for Biomedical Exer 3/6 treadmill (Columbus Instruments, Columbus, Research Involving Animals and were approved by OH, USA) using the following protocol: 3 min running UNIFESP ethics committee (Comissão de Ética no at 5 m/min for warm-up, and then increasing the speed Uso de Animais, from Universidade Federal de São 3 m/min each minute until 20 m/min. After that, the Paulo, São Paulo, Brazil) - protocol #0752/07. speed was increased in 2 m/min each minute until reach 26 m/min, and then increased in 1 m/min each minute. Evaluation of motor activity The animal was considered exhausted when it refused to The effect of the hydroalcoholic extract of T. catigua at run even when challenged with tactile stimuli . Mice doses of 50 and 500 mg/kg (p.o.) on animals’ motor ac- that failed to reach the speed of 24 m/min were discarded tivity was evaluated on the rotarod and in activity cages from the study. in order to check whether the extracts induce any inco- After the basal evaluation, the animals were divided in ordination or locomotor alteration in high doses. Groups groups (n =8–10) with similar performance and orally of 10 mice each were placed in plexiglas cages measur- treated with T. catigua hydroalcoholic extract (25, 100 ing 47.5 cm × 25.7 cm × 20.5 cm equipped with 16 pairs and 250 mg/kg) or water (exercised control) for 7 weeks of photoelectric beams distributed in the horizontal axis and submitted to the treadmill at the 3rd and 7th week (Opto-Varimex, Columbus Instruments, Columbus, OH) (days 21 and 49) when the maximum speed was regis- and the ambulation was detected by subsequent inter- tered for each animal. Immediately after reaching ex- ruptions of adjacent photo beams every 30 min for a haustion, each mouse was removed from the apparatus total of 120 min, as described by Bezerra et al. . The and 25 μL of blood was collect from its tail for lactate motor coordination was evaluated in a rotarod apparatus quantification in a L-lactate analyzer (YSI 2300 Stat Plus, (AVS Projects, São Paulo, Brazil) at 12 RPM before the YSI Life Science, Yellow Springs, OH, USA). After that, treatment (basal) and 30, 60 and 120 min after the ad- the animal was submitted to the grip strength meter ministration. We used mice pre-selected 24 h before by (AVS Projects, São Paulo, Brazil) and then placed in a eliminating those that could not stay on the bar for at plexiglas cage for measurement of the spontaneous loco- least 60s . motor activity for 1 h, as previously described. The grip strength was measured before (basal) and after the exer- Stress by immobilization and cold cise (post-fatigue) in order to determine how the forced Groups of 9–10 rats were orally treated with T. catigua exercise affect the strength of each animal. The protocol hydroalcoholic extract (25 and 250 mg/kg) or water consisted in placing the animal on a grid and measuring (stressed control) for 14 days and the stress protocol the resistance presented by the mice when it was pulled Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 5 of 13 by the tail, recording the highest figure of three (12) were detected in the four extracts, including the measurements . An extra group of animals treated apolar hexane and chloroform extracts. Apocynin E (5), with water not submitted to the treadmill (non-exercised cinchonain IIa glucoside (9) and cinchonain IIb gluco- control) was used to measure the lactate normal level side (10) were detected only in chloroform extract, while and spontaneous locomotor activity. the procyanidin B2–8-C-rhamnoside (2) and procyani- din B2 - (epi)-catechin – (epi)-catechin (3) were de- Classical fear conditioning tected only in hydroalcoholic extract. In the aqueous Groups of 9–10 mice were orally treated with T. catigua extract were detected the same constituents found in hydroalcoholic extract (50 and 300 mg/kg) or water hydroalcoholic extract, except procyanidins. The HPLC- (controls) for 3 weeks (21 days). On the training day, the ESI-MS/MS spectra of the compounds 1–12 are pro- animals (except the negative control group) received vided as Additional file 1. scopolamine (2 mg/kg, i.p. - Sigma, St. Louis, MO, USA) in 0.9% saline 30 min after the extract treatment and DPPH assay 30 min later were placed individually in the context cage The four extracts of T. catigua showed DPPH scavenger dotted with some visual cues. After 2 min of habituation, activity (Table 2). The most potent effect was found for three electrical shocks (0.3 mA, 1 s duration and interval the hydroalcoholic extract (EC =43 μg/ml) with of 10s) were delivered to the paws of the animal potency similar to rutin, the positive control. (adapted from Soeiro et al.) . The mice were returned to their home cages and after 24 h placed in the context cage again, without delivery any shock, being Acetylcholinesterase activity the freezing time registered during 5 min. The extracts of T. catigua showed in vitro AChE inhibi- tory activity, with the hydroalcoholic extract being the Statistical analysis most potent with IC = 142 μg/mL (Table 2). Rivastig- The statistical analysis was carried out using the Statistica® mine, the positive control, presented IC =18 μg/mL. and Graph Pad Prism® software. The EC for DPPH and IC for acetylcholinesterase were calculated by linear re- gression using the mean of scavenge or inhibition for each Motor activity concentration of the tested drugs. ANOVA followed by The acute treatment of mice with hydroalcoholic extract Duncan post-hoc test was used for parametric analysis. of T. catigua (50 and 500 mg/kg, p.o.) did not alter the The Kruskall Wallis test followed by the Mann-Whitney locomotor activity (p > 0.05) during the 120 min of ob- test was used to compare the groups on rotarod test due servation (Fig. 1). Similarly, the treatment with the same to non-parametric distribution. We adopted a p value of doses did not alter the motor coordination on rotarod 0.05 as statistically significant. (p > 0.05, Additional file 2), indicating that these doses do not disturb the animals’ motor activity. Results Phytochemical analysis Vanillin reacted with flavanols to yield a red adduct, Stress by immobilization and cold while the reaction with ferric chloride in TLC developed The stress by immobilization and cold was effective a brownish grey color, both characteristic for flavan-3- to induce increased levels of ACTH [F(3,35) = 40.05; ols, confirming the presence of these constituents, which p < 0.001] and corticosterone [F(3,35) = 103.81; p <0.001] was corroborated by the ultraviolet spectra of constitu- when comparing the stressed and non-stressed control ents with maximum absorption at 278 nm in the HPLC- groups (Table 3). Similarly, the stress induced gastric DAD analyses. ulceration [F(3,35) = 8.10 (index) and 22.11 (degree); Table 1 summarizes the mass spectral (MS) data p < 0.001], as well as thymus and spleen atrophy obtained in HPLC-ESI-MS/MS analyses of hexane, [F(3,35) = 7.29 (thymus) and 30.02 (spleen); p < 0.001] chloroform, hydroalcoholic and aqueous extracts from (Table 3). However, we did not observe alteration on bark of Trichilia catigua. The mass spectral data adrenals weight for the stressed control group. The obtained by high resolution tandem mass spectrometry oral treatment of rats with T. catigua at doses of 25 is described in discussion. The identification of constitu- and 250 mg/kg was not able to prevent the alter- ents was achieved based on the MS data obtained and ations induced by cold immobilization stress. More- taking into account the compounds and MS data previ- over, the adrenals weight of rats treated with the dose ously reported for this species by several authors, as de- 250 mg/kg was statistically higher (p < 0.05) than that tailed in the discussion. As can be seen in Table 1, of the stressed control group, indicating a tissue cinchonain IIa (4), cinchonain Ia (7) and cinchonain Ib hypertrophy. Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 6 of 13 Table 1 Summary of the MS data obtained in the analysis of the hexane (HEX), chloroform (CLO), hydroalcoholic (HA) and aqueous (AQ) extracts from barks of Trichilia catigua through HPLC-DAD-ESI-MS/MS Rt HPLC/(−)ESI-MS/MS Proposed structure HEX CLO HA AQ m/z (%base peak) 1 4.0 [M – H] - 341 6-O-caffeoyl glucoside – XX X MS/MS - 179 2 14.1 [M – H] - 723 procyanidin B2–8-C-rhamnoside –– X – MS/MS - 597 (20), 433 (80) 425 (90), 407 (100), 289 (20) 3 15.3 [M – H] - 577 procyanidin B2 (epi)-catechin – –– X – MS/MS - 559 (20), 451 (30), (epi)-catechin 425 (100), 407 (80), 289 (20) 4 17.2 [M – H] - 739 cinchonain IIa X X X X MS/MS - 587 (100), 451 (40) 5 18.0 [M – H] - 467 apocynin E – X –– MS/MS - 449 6 19.0 [M – H] - 353 3-O-cafeoylquinic acid –– – X MS/MS - 191 7 19.6 [M – H] - 451 cinchonain Ia X X X X MS/MS - 341 8 21.4 [M – H] - 613 bis-(3,4-dihydroxyphenylpropanoid)- – XX X MS/MS - 503 (100), 451 (50), substituted catechin 393 (20), 341 (20) 9 22.2 [M – H] - 901 cinchonain IIa glucoside – X –– MS/MS - 791 (100), 597 (70), 451 (60), 341 (20) 10 24.4 [M – H] - 901 cinchonain IIb glucoside – X –– MS/MS - 791 (60), 597 (100), 451 (40), 341 (50) 11 24.8 [M – H] - 613 cinchonain Id-7- glucoside –– XX MS/MS - 503 (100), 451 (10), 341 (10) 12 26.4 [M – H] - 451 cinchonain Ib X X X X MS/MS - 341 Forced treadmill exercise [F(4,40) = 9.05; p < 0.001 at day 21] compared to that of Mice were submitted to the forced exercise on treadmill mice not submitted to the treadmill (non-exercised con- before (basal) and after 21 and 49 days of treatment (3rd trol), but no difference was observed among the control and 7th week) with the hydroalcoholic extract of T. cati- and the groups treated with T. catigua (Fig. 2b). gua. There was no difference in fatigue time among the The spontaneous locomotor activity of non-exercised groups (p > 0.05) in the three time points (Fig. 2a). The mice was higher than other groups on the test carried plasmatic level of lactate after the exercise was increased out at 21 days of treatment [F(4,40) = 10.65; p < 0.001], but there was no difference among the groups when the same procedure was repeated after 49 days of treatment Table 2 DPPH radical scavenging and acetylcholinesterase [F(4,39) = 1.09; p > 0.05]. On the other hand, the group inhibition by different extracts of Trichilia catigua treated with 250 mg/kg of T. catigua also differed from Extract or positive control DPPH scavenging AChE inhibition the exercised control at 21 days, showing increased EC (μg/mL) IC (μg/mL) 50 50 locomotor activity (p < 0.05) after the fatigue protocol Hexane 53 (28–136) 346 (270–502) (Fig. 2c). Chloroform 60 (35–145) 313 (193–1128) The evaluation of mice grip strength after the fatigue Hydroalcoholic 43 (7–210) 142 (112–196) shows that there was no difference among the groups Aqueous 52 (24–150) 315 (230–527) (p > 0.05) in the test carried out at 21 or 49 days of treat- Rutin 44 (9–216) – ment (Fig. 3a). The impairment of grip strength induced by exhaustion (difference on grip strength before and after Rivastigmine – 18 (0–136) the exercise) also did not change (Fig. 3b). On the other The EC ,IC and 95% confidence interval (in parenthesis) were calculated by 50 50 linear regression using the mean of 3–4 assays for each concentration hand, when we compared the post-exercise performance Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 7 of 13 in extracts obtained from barks of T. catigua [8–10]. In our study, we intended to identify the compounds extracted from the barks of T. catigua with different solvents in order to correlate the phytochemical profile with the in vitro activities. All extracts exhibited antioxi- 800 control dant and anticholinesterase activities, but the hydroalco- TC 50 mg/kg holic extract was the most active, which is consistent TC 500 mg/kg with the high reactivity of hydroxyl and carbonyl groups contained in their constituents. Compound 1, found in chloroform, hydroalcoholic and aqueous extract exhibited deprotonated molecule at 30 min 60 min 90 min 120 min m/z 341, which after MS/MS experiments produced Time of observation base peak ion at m/z 179 (deprotonated caffeic acid). In Fig. 1 Spontaneous locomotor activity of mice treated acutely with high-resolution q-Tof mass spectrometer, the HR-ESI Trichilia catigua (TC) hydroalcoholic extract at doses of 50 and 500 mg/kg (p.o.). The columns and bars represent the means ± SEM molecular ion [M – H] was detected at m/z 341.0909, (n = 10). ANOVA, n.s which produced a fragmental cleavage at m/z 179.0463. Based on MS data reported by Gouveia and Castilho in different moments, we observed that the group treated , compound 1 was assigned as 6-O-caffeoyl glucoside. with T. catigua at dose of 250 mg/kg presented better per- For compound 6, the HR-ESI molecular ion [M – H] was formance [F(3,30) = 3.61; p < 0.05] than the other groups detected at m/z 353.0692, which produced a fragmental (difference on grip strength after fatigue between days 49 cleavage at m/z 191.0454. Compound 6, found only in and basal) meaning that the animals recovered faster from aqueous extract, was assigned as 3-O-cafeoylquinic acid. the exercise on day 49 if compared with the basal evalu- Exact mass can distinguish isobar molecules, which ation (Fig. 3c). exhibit the same integer mass but different molecular formula. Isomeric structures, compounds with the same Classical fear conditioning atoms, but different arrangements, cannot be separated Although the pre-treatment of mice with scopolamine by exact mass . Compounds 7 and 12 were detected reduced the freezing time by 40.9% compared with con- in the four extracts and could be distinguished through trol mice that did not receive scopolamine, there was no elution order. They exhibited the same [M − H] signal statistically significant difference among the groups at m/z 451 in negative ESI-MS of low resolution. After [F(3,35) = 1.45; p > 0.05], possibly due the high variability MS/MS experiments produced base peak at m/z 341 observed. In spite of that, the freezing time of mice treated (Table 1), corresponding to the loss of 110 Da, which with T. catigua at doses of 50 and 300 mg/kg (p.o.) was was attributed to 3,4-dihydroxyphenyl moiety (C H O ), 6 6 2 similar to that of the scopolamine control group, indicat- characteristic of cinchonains [17, 18]. Cinchonains Ia ing that the pre-treatment with the extract did not im- and Ib are isomers, that possess molecular formula prove the mice memory (Fig. 4). C H O , average mass – 452.410 g/mol and monoiso- 24 20 9 topic mass – 452.1107 g/mol. The molecular formula for Discussion compounds 7 and 12 were determined from the HR- Phytochemical analysis ESI-MS molecular ion [M − H] detected at m/z 451.0788 The phytochemical composition of Trichilia catigua (C H O ). The fragmental cleavage profile from the HR- 24 20 9 barks is well described in the literature. The flavalignans ESI-MS spectrum for compound 7, which produced base (flavanols substituted with phenylpropanoids) cincho- peak at m/z 341.0481, was very similar to that produced nains IIa, Ia and Ib and proanthocyanidins were isolated for compound 12, providing further evidence that these Table 3 Effect of treatment of rats with Trichilia catigua (TC) hydroalcoholic extract (25 and 250 mg/kg, p.o.) for 14 days on degree and index of ulceration induced by stress; on adrenal, thymus and spleen weights; and on ACTH and corticosterone plasmatic levels. The data express the mean ± SEM (n =9–10) Group Degree of Index of Adrenal weight Thymus weight Spleen weight ACTH Corticosterone ulceration ulceration (mg) (mg) (mg) (pg/mL) (μg/dL) control 3.2 ± 0.3 15.3 ± 3.1 54.4 ± 3.8 326.8 ± 21.1 809.8 ± 48.3 812 ± 31 980 ± 62 non-stressed 0.3 ± 0.2* 0.6 ± 0.3* 52.8 ± 2.6 434.2 ± 22.9* 1470.1 ± 72.6* 71 ± 27* 88 ± 35* # # # # # # TC 25 mg/kg 3.7 ± 0.4 19.4 ± 4.4 60.1 ± 2.0 286.0 ± 29.0 840.6 ± 49.5 735 ± 70 1012 ± 44 # # # # # # # TC 250 mg/kg 3.3 ± 0.4 13.2 ± 2.3 62.3 ± 2.7 301.3 ± 26.4 791.3 ± 66.0 833 ± 84 1080 ± 40 (*) p < 0.05: statistically different of control (stressed) group; (#) p < 0.05: statistically different of non-stressed group. ANOVA followed by Duncan Ambulation Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 8 of 13 a a control 25 control TC 25 mg/kg TC 25 mg/kg 150 TC 100 mg/kg TC 100 mg/kg TC 250 mg/kg TC 250 mg/kg 21 days 49 days Basal 21 days 49 days b 7 # 50 control control 4 non-exercised 30 TC 25 mg/kg * TC 25 mg/kg TC 100 mg/kg 20 TC 100 mg/kg TC 250 mg/kg TC 250 mg/kg 21 days 49 days -10 21 days 49 days -20 control *,# n.s. non-exercised # TC 25 mg/kg TC 100 mg/kg TC 250 mg/kg -20 -40 21 days 49 days -60 Fig. 2 a Fatigue time (maximum speed), b plasmatic lactate level control TC 25 mg/kg TC 100 mg/kg TC 250 mg/kg and (c) spontaneous locomotor activity of mice chronically treated Fig. 3 a Post exercise grip strength, b difference of grip strength with Trichilia catigua (TC) hydroalcoholic extract (25, 100 and (before - post-exercise) and (c) performance change (post-exercise 250 mg/kg, p.o.) and subjected to the forced exercise on treadmill. grip strength 49d - basal) of mice chronically treated with Trichilia The columns and bars express the mean ± SEM (n = 8–10). (*) catigua (TC) hydroalcoholic extract (25, 100 and 250 mg/kg, p.o.) and p < 0.05: statistically different of control exercised group; (#) p < 0.05: subjected to the forced exercise on treadmill. The columns and bars statistically different of non-exercised control group. ANOVA express the mean ± SEM (n =8–10). (*) p < 0.05: statistically different followed by Duncan of control group. ANOVA followed by Duncan compounds are isomers with the same skeleton, but differ- ent arrangements. The peak m/z 903.1652 corresponds to detected only in chloroform extract exhibited the same the dimmer of cinchonains (Figs. 7 and 12 of Additional deprotonated molecule at m/z 901. As can be seen in file 1). Based on HR-ESI-MS spectra and also MS data re- Table 1, the MS/MS spectrum of compound 9 exhibited ported by Fasciotti et al.  and Gu et al. , com- base peak at m/z 791 derived from the loss of 3,4- pounds 7 and 12 were assigned as cinchonain Ia and dihydroxyphenyl moiety, characteristic of cinchonains. cinchonain Ib, respectively. The MS/MS spectrum of compound 10 produced base Compound 4 detected in the four extracts exhibited base peak at m/z 597, derived from the neutral loss of [M − H] signal at m/z 739, which after MS/MS (C O H ) 304 Da, but also produced fragments ions 16 6 16 experiments exhibited base peak ion at m/z 587 and a characteristic of cinchonains at m/z 791, m/z 451 and fragment ion at m/z 451. According to MS data reported 341 [17, 18]. Compound 9 was tentatively identified as by Resende et al.  and Fasciotti et al.  compound cinchonain IIa glucoside and compound 10 as cinchonain 4 was identified as cinchonain IIa. Compounds 9 and 10 IIb glucoside. Curiously, cinchonain Ia, Ib, cinchonain IIa Ambulation Maximum speed (m/min) lactate (mmol/L) grip strength (post - pre-exercise) change on grip strength grip strength (gf) (49d-basal post-exercise) Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 9 of 13 system of the flavan-3-ol subunits gave rise to a frag- ment of m/z 425. The ion at m/z 425 eliminates 120 water, probably from ring C at position C3/C4, result- ing in a fragment ion of m/z 407 [19, 20]. Based on MS data reported by Kicel et al. , compound 3 80 was identified as type B dimmer of proanthocyanidin (epi) catechin - (epi) catechin, known as procyanidin B2. The MS/MS spectrum of compound 2 also exhib- ited abundant fragment ion at m/z 433, resulting from the loss of 290 Da (catechin). Based on mass spectral database , compound 2 was tentatively identified as procyanidin B2–8-C-rhamnoside. Com- control NC TC 50 mg/kg TC 300 mg/kg pound 5 detected only in chloroform extract, exhib- Fig. 4 Effect of treatment with Trichilia catigua (TC) hydroalcoholic ited [M − H] signal at m/z 467, which after MS/MS extract (50 and 300 mg/kg, p.o.) for 21 days on the amnesia induced experiments produced base peak at m/z 449, resulting by scopolamine (2 mg/kg, i.p.) on classical fear conditioning in mice. from the loss of water. Compound 5 was tentatively NC = negative control (did not receive scopolamine). The columns assigned as apocynin E, which was also detected by and bars express the mean ± SEM (n =9–10). ANOVA, n.s Resende et al. in T. catigua bark. The presence of procyanidins and cinchonains in T. and cinchonain IIa glucoside were also found in bark from catigua bark was corroborated by the studies of Truiti et Erythroxylum vaccinifolium Mart., a medicinal plant also al.  and Resende et al.  that obtained a similar known as catuaba in the northeast of Brazil . chemical profile. On the other hand, flavonoids rutin Compounds 8 and 11 detected in chloroform, hydroal- and quercetin identified by Kamdem et al.  and cati- coholic and aqueous extract, showed the same [M − H] guanin A and catiguanin B (phenylpropanoid-substituted signal at m/z 613. The MS/MS spectrum of compound 8 epicatechins) isolated from the bark of T. catigua by produced base peak at m/z 503 and fragment ion at m/z Tang et al.  were not present in our extracts in 393, both derived from the neutral loss of 3,4- detectable amounts. However, as far as we know, a bis- dihydroxyphenyl moiety (110 Da), which indicated the (3,4-dihydroxyphenylpropanoid)-substituted catechin (8), presence of two dihydroxyphenyl groups. The fragment cinchonain Id-7- glucoside (11) and cinchonain II gluco- ion at m/z 451 was derived from the neutral loss of sides (9 and 10) were detected for the first time in the C H O , while the fragment ion at m/z 341 was derived species. This difference in chemical composition could be 9 6 3 from the neutral loss of C H O +C H O (Table 1). explained leading in consideration that the chemical 6 6 2 9 6 3 Since the fragment ions of compound 8 match the ones constituents can vary in their structure and concentration reported by Gu et al. ,itwasassignedasabis-(3,4- depending on the region and season of collection, genetic dihydroxyphenylpropanoid)-substituted catechin. The variability, as well as the extraction method. ESI-MS spectrum of compound 11, also exhibited a base peak at m/z 503 resulting from the neutral loss of Biological activity 3,4-dihydroxyphenyl moiety (110 Da), but the relative Several studies have shown the antioxidant activity of intensities of fragments ions at m/z 451 and m/z 341 different catuaba (T. catigua) extracts [8, 33–35]. The are very low. According to mass spectral database data of our study confirm the antioxidant effect of T. HMDB , compound 11 was tentatively assigned as catigua on DPPH assay for the four extracts analyzed. cinchonain Id-7- glucoside. The most potent effect was achieved with the hydroalco- Proanthocyanidins are polymeric flavonoids based on holic extract (EC =43 μg/ml), with potency similar to flavan-3-ols (oligomers of catechin and/or epicatechin rutin (EC =44 μg/ml), the positive control, followed by and their gallic acid esters). Compounds 2 and 3 also aqueous, hexane and chloroform extracts. Lonni et al. exhibited UV maximum absorption at 280 nm and are  compared the antioxidant capacity (DPPH assay) of proposed to be type B dimmer proanthocyanidins. These T. catigua extracted with different solvents and found compounds were detected only in hydroalcoholic extract the best result with ethanol, followed by acetone, water and exhibited [M − H] signals at m/z 723 and 577, and methanol. In other study, Kamdem et al.  found respectively. The MS/MS spectra of compounds 2 and that the content of total phenolics was higher in ethyl 3 produced fragment ions at m/z 425, 407 and 289 acetate extract, but the best effect on DPPH assay was (Table 1) characteristic for procyanidin B-type dim- obtained for the ethanolic extract. Using compounds iso- mers and a fragment ion at m/z 289 (catechin). lated from T. catigua bark, Resende et al.  observed Retro-Diels-Alder reaction of the heterocyclic ring the most potent antioxidant activity with procyanidin Freezing (sec) Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 10 of 13 C1, cinchonain IIb and cinchonain IIa, while Tang et al. In the current study, the effect of T. catigua extracts  found the best results with the cinchonains Id, Ic on cholinergic system was evaluated for the first time. and Ib. In our study, the hydroalcoholic extract contain- All extracts tested inhibited the activity of acetylcholin- ing cinchonains and procyanidins also exhibited the esterase in vitro, and the most potent effect was ob- most potent antioxidant activity. tained for the hydroalcoholic extract (IC = 142 μg/ml), The neuroprotective activity of T. catigua is mainly at- followed by chloroform, aqueous and hexane extracts, tributed to its antioxidant activity. The 70% ethanolic ex- with IC ranging from 313 to 346 μg/ml. The inhibition tract of catuaba at concentrations from 10 to 100 μg/ml of AChE demonstrated for the four extracts may be due protected hippocampal neurons in vitro from oxidative to the presence of high contents of cinchonains IIa, Ia stress and increased the survival after ischemia and re- and Ib, which are flavalignans - flavanols substituted perfusion  or in the presence of hydrogen peroxide, with phenylpropanoids. Flavonoids that possess a free sodium nitroprusside and nitropropionic acid . The OH-group at C3 position showed major activity when crude extract (acetone:water 7:3) and its semipurified compared to their C3 − OH glycosylated counterparts fraction (partitioned with ethyl acetate), rich in epicate- and those having no C3 − OH group, such as luteolin chin and procyanidin B2, were administered to mice in and apigenin [37, 38]. The major inhibition observed for doses of 200 to 800 mg/kg for 7 days before the animals the hydroalcoholic extract can be explained by the pres- were submitted to a bilateral occlusion of the carotid. ence of procyanidins B2 found only in this extract. The treatment improved the performance of the animals Proanthocyanidins exhibited a potent role in enhancing in the Morris water-maze and protected hippocampal cognition in older rats, which was attributed to an in- neurons . These effects were mainly assigned to fla- crease in the acetylcholine concentration with a moder- vonoids and polyphenols present in these extracts, due ate reduction in AChE activity . Proanthocyanidins to their antioxidant activity. exhibited ameliorative effects on learning and memory Other effects, as antinociceptive and antidepressant- impairment of mice in scopolamine-induced amnesia like effect, seem to be related to a dopaminergic action test, showing protection against memory deficit . [12, 13]. Neurochemical studies showed that the ethanolic The anticholinesterase effect found in our study can extract of T. catigua inhibited dopamine and serotonin support the promnesic effect observed by Chassot et al. uptake and increased the release of these neurotrans-  for the crude extract and ethyl-acetate fraction of T. mitters, with more potent activity to dopamine. The catigua. However, the hydroalcoholic extract of catuaba antidepressant-like effect was evaluated in animals treated in doses of 50 and 300 mg/kg in our study did not pro- with doses of 200 and 400 mg/kg in the forced swimming mote memory improvement in mice treated with scopol- test and tail suspension test. The extract induced amine, a competitive antagonist of muscarinic receptors. antidepressant-like effect, which was blocked by haloperi- The inhibition of AChE causes an increase of concentra- dol and chlorpromazine, anti-dopaminergic agents . tion and time of acetylcholine on synaptic cleft, facilitat- Another study using the ethyl acetate fraction of T. ing the cholinergic transmission. However, it is not catigua showed antidepressant-like effect and increased possible to know in this study whether the in vitro anti- cellular proliferation in the hippocampus . cholinesterase effect is also present in vivo. Or perhaps, The central cholinergic system is involved in the regu- the increase in acetylcholine concentration may not be lation of many cognitive functions and cholinergic alter- enough to displace the scopolamine from the receptor ations that occur during aging are associated with and avoid its amnesic effect. learning and memory deficits. Acetylcholinesterase hy- Kamdem et al.  discuss that T. catigua ethanolic drolyzes the acetylcholine released on central nervous extract seems to have preventive, but not curative effect system synapses regulating its concentration and effect. on experimental ischemia, since the in vitro treatment of However, there is a progressive loss of cholinergic hippocampal slices after the protocol of ischemia and neurons that innervate hippocampus and the neocortex reperfusion did not protect the neurons. This prophylac- in Alzheimer’s disease and some other dementias result- tic profile corroborates with the expected effect of an ing on cholinergic hypofunction. AChE inhibitors are adaptogen, which is used chronically to avoid or dimin- used clinically on the treatment of Alzheimer’s disease, ish damages from stress and aging. In fact, the folk use because they increase the availability of acetylcholine of catuaba is similar to what we would expect for a present in cholinergic synapses, enhancing the choliner- typical adaptogen: the plant is used chronically to pre- gic functions. Drugs as rivastigmine (used as positive vention and treatment of neurasthenia, fatigue, stress, control in our study), galantamine and huperzine A impotence and memory deficits . (active principles isolated from medicinal plants) are This is the first study evaluating the effect of T. catigua AChE inhibitors employed in the treatment of Alzhei- on stress and fatigue. We employed the hydroalcoholic ex- mer’s disease . tract of catuaba, which corresponds to the form popularly Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 11 of 13 used and that showed the best results in our in vitro tests. Panax ginseng and other adaptogens are chronically used The doses employed were comparable with those of previ- for several purposes – to increase stress resistance and ous in vivo studies and they did not interfere with the physical capacity, to improve memory and other cognitive locomotor activity and motor coordination on rotarod, functions and as neuroprotective agents . Ginseng acts suggesting they were safe. The treatment with catuaba at by multiple mechanisms of action: it reduces the oxidative doses of 25 and 250 mg/kg p.o. (starting 7 days before the stress and excitotoxicity, modulates cholinergic neuro- repeated stress protocol) did not protect the animals from transmission, and increases dopamine and noradrenaline ulceration, neither prevented corticosterone and ACTH in the cerebral cortex . It is likely that both the acetyl- increase or thymus and spleen atrophy induced by stress. cholinesterase inhibition and the antioxidant effect of T. Adaptogens can lightly raise the basal level of corticoste- catigua may contribute to its neuroprotective and pro- roids, nevertheless adaptogens prevent the overwhelming cognitive effects, as well as its dopaminergic and serotoner- increase of cortisol induced by stress . The protocol of gic effects are important for its antinociceptive and anti- cold and immobilization causes an intense stress on the depressant effects. The antioxidant effect of different animal, seeing that the levels of ACTH and corticosterone extracts or isolated constituents of catuaba was well evalu- increased tenfold in control-stressed rats when compared ated. Several studies confirm that ethanolic or hydroalco- with non-stressed controls. Catuaba is widely used against holic extracts of catuaba seems to have the most potent fatigue and stress, but as far as we known, it is not used to antioxidant effect [6, 33–35], but the proportion of water treat or prevent gastric ulcers. and ethanol can be better explored. Another alternative In order to evaluate whether T. catigua has an antifa- should be the use of special extracts prepared by extraction tigue effect, mice were chronically treated with hydroal- with different solvents, as suggested by Lonni et al. . coholic extract at doses of 25, 100 and 250 mg/kg (p.o.) and submitted to forced exercise on a treadmill in three Conclusions phases: before the treatment (basal performance) and In brief, we confirmed the presence of cinchonains and after 21 and 49 days of treatment. The administration of procyanidins in T. catigua and found the best antioxidant catuaba did not alter the fatigue time, nor the lactate and anticholinesterase activity for the hydroalcoholic ex- levels measured immediately after the exercise. However, tract. This extract did not avoid the damages induced by mice treated with the highest dose showed increased stress and did not prevent the amnesia induced by scopol- spontaneous locomotor activity after the forced exercise amine, but had a mild protective effect on forced exercise on the 21th day. This result suggests that the treatment and fatigue. These data suggest the hydroalcoholic extract with catuaba may decrease the recovery time after an as the most suitable for plant extraction and partially sup- exhaustion protocol. Moreover, catuaba treatment for port the folk use of T. catigua as antifatigue drug. 49 days at the highest dose was able to diminish the im- pact of the forced exercise on the animals’ strength since the impairment on grip strength after the exercise was Additional files shortened at day 49 compared with the basal perform- ance (difference on grip strength after fatigue between Additional file 1: HPLC-ESI-MS/MS spectra of the compounds 1–12. days 49 and basal). Even modest, these results suggest Total ion current chromatogram, ESI-MS/MS in negative mode and Q-Tof that the hydroalcoholic extract of catuaba may have – mass spectrometry of the main compounds found in the extracts. (PDF 175 kb) beneficial effects on fatigue, at least shortening the re- Additional file 2: Effect of acute treatment of mice with Trichilia catigua covery time after exhaustion. Stress-protective and anti- hydroalcoholic extract on rotarod performance. Table showing the mean fatigue effects have been described for some adaptogens, ± EPM of the control and experimental groups on rotarod. (PDF 13 kb) as Rhodiola rosea L., Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. and Panax ginseng C.A. Meyer and several clinical trials were already conducted . The Abbreviations AChE: Acetylcholinesterase; ACTH: Adrenocorticotropic hormone; DPPH: 2,2- importance of antioxidants on physical exercise and diphenyl-1-picryl hydrazyl; DTNB: 5,5-dithiobis-2-nitrobenzoic acid; Q-ToF: Hybrid to prolong endurance and reduce fatigue has been quadrupole orthogonal acceleration time-of-flight mass spectrometer; RPHPLC- evaluated. An extract of Polygonatum altelobatum DAD-ESI-MS/MS: Reverse phase high performance liquid chromatography-diode array-electrospray ionization- mass spectra-mass spectra Hayata rich in polyphenols and polysaccharides in- creased the endurance running time to exhaustion and the antioxidant ability in rats’ blood . A Acknowledgements supplementation with Chaenomeles speciosa (Sweet) We thank CNPq and CAPES for the scholarships, Associação Fundo de Incentivo à Psicobiologia (AFIP) for providing the animals and Núcleo de Cognição e Nakai fruit prolonged the exhaustive swimming time Sistemas Complexos (NCSC/UFABC) and Centro Brasileiro de Informações sobre of rats and raised antioxidant enzymes levels, possibly Drogas Psicotrópicas (CEBRID) for providing most of drugs and materials. We by modulating the Nrf2 pathway . also thank Prof. Allen Lockwood for the English review. Martins et al. BMC Complementary and Alternative Medicine (2018) 18:172 Page 12 of 13 Funding 10. Rabelo DS, Paula JR, Bara MTF. Quantificação de fenóis totais presentes nas NOM and IMB received scholarships from Conselho Nacional de cascas de Trichillia catigua A. Juss. (Meliaceae). Rev Bras Pl Med. 2013;15:230–6. Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de 11. Barbosa NR, Fischmann L, Talib LL, Gattaz WF. Inhibition of platelet Aperfeiçoamento de Pessoal de Nível Superior (CAPES, #11015912), respectively. phospholipase A2 activity by catuaba extract suggests antiinflammatory properties. Phytother Res. 2004;18:942–4. Availability of data and materials 12. Viana AF, Maciel IS, Motta EM, Leal PC, Pianowski L, Campos MM, Calixto JB. The raw data generated and/or analyzed during the current study are Antinociceptive activity of Trichilia catigua hydroalcoholic extract: new available from the corresponding author on reasonable request. evidence on its dopaminergic effects. Evidence-based Complement Altern Med. 2011; https://doi.org/10.1093/ecam/nep144. Authors’ contributions 13. Campos MM, Fernandes ES, Ferreira J, Santos ARS, Calixto JB. NOM, IMB and SSOA performed the in vitro and in vivo tests, while GN Antidepressant-like effects of Trichilia catigua (Catuaba) extract: evidence for carried out the phytochemical analysis. FRM and EAC wrote and managed dopaminergic-mediated mechanisms. Psychopharmacology (Berl). the project and helped on the experiments and statistical analysis. All the 2005;182:45–53. authors participated in the analysis of the data and have read and approved 14. Bonassoli VT, Chassot JM, Longhini R, Milani H, Mello JC, De Oliveira RMW. the final submitted manuscript. Subchronic administration of Trichilia catigua ethyl-acetate fraction promotes antidepressant-like effects and increases hippocampal cell Ethics approval and consent to participate proliferation in mice. J Ethnopharmacol. 2012;143:179–84. The project was approved by the Comissão de ética no uso de animais 15. Kamdem JP, Stefanello ST, Boligon AA, Wagner C, Kade IJ, Pereira RP, Preste (ethics committee) of UNIFESP (protocol #0752/07). The consent to ADS, Roos DH, Waczuk EP, Appel AS, et al. In vitro antioxidant activity of participate is not applicable. stem bark of Trichilia catigua Adr. Juss Acta Pharm. 2012;62:371–82. 16. 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Published: Jun 5, 2018