Phytochemical analysis
The phytochemical composition of Trichilia catigua barks is well described in the literature. The flavalignans (flavanols substituted with phenylpropanoids) cinchonains IIa, Ia and Ib and proanthocyanidins were isolated in extracts obtained from barks of T. catigua [8,9,10]. 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 antioxidant and anticholinesterase activities, but the hydroalcoholic extract was the most active, which is consistent 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 m/z 341, which after MS/MS experiments produced base peak ion at m/z 179 (deprotonated caffeic acid). In high-resolution q-Tof mass spectrometer, the HR-ESI molecular ion [M – H]− was detected at m/z 341.0909, which produced a fragmental cleavage at m/z 179.0463. Based on MS data reported by Gouveia and Castilho [30], compound 1 was assigned as 6-O-caffeoyl glucoside. For compound 6, the HR-ESI molecular ion [M – H]− was detected at m/z 353.0692, which produced a fragmental cleavage at m/z 191.0454. Compound 6, found only in aqueous extract, was assigned as 3-O-cafeoylquinic acid.
Exact mass can distinguish isobar molecules, which exhibit the same integer mass but different molecular formula. Isomeric structures, compounds with the same atoms, but different arrangements, cannot be separated by exact mass [31]. Compounds 7 and 12 were detected in the four extracts and could be distinguished through elution order. They exhibited the same [M − H]− signal at m/z 451 in negative ESI-MS of low resolution. After MS/MS experiments produced base peak at m/z 341 (Table 1), corresponding to the loss of 110 Da, which was attributed to 3,4-dihydroxyphenyl moiety (C6H6O2), characteristic of cinchonains [17, 18]. Cinchonains Ia and Ib are isomers, that possess molecular formula C24H20O9, average mass – 452.410 g/mol and monoisotopic mass – 452.1107 g/mol. The molecular formula for compounds 7 and 12 were determined from the HR-ESI-MS molecular ion [M − H]− detected at m/z 451.0788 (C24H20O9). The fragmental cleavage profile from the HR-ESI-MS spectrum for compound 7, which produced base peak at m/z 341.0481, was very similar to that produced for compound 12, providing further evidence that these compounds are isomers with the same skeleton, but different arrangements. The peak m/z 903.1652 corresponds to the dimmer of cinchonains (Figs. 7 and 12 of Additional file 1). Based on HR-ESI-MS spectra and also MS data reported by Fasciotti et al. [17] and Gu et al. [18], compounds 7 and 12 were assigned as cinchonain Ia and cinchonain Ib, respectively.
Compound 4 detected in the four extracts exhibited [M − H]− signal at m/z 739, which after MS/MS experiments exhibited base peak ion at m/z 587 and a fragment ion at m/z 451. According to MS data reported by Resende et al. [8] and Fasciotti et al. [17] compound 4 was identified as cinchonain IIa. Compounds 9 and 10 detected only in chloroform extract exhibited the same deprotonated molecule at m/z 901. As can be seen in Table 1, the MS/MS spectrum of compound 9 exhibited base peak at m/z 791 derived from the loss of 3,4-dihydroxyphenyl moiety, characteristic of cinchonains. The MS/MS spectrum of compound 10 produced base base peak at m/z 597, derived from the neutral loss of (C16O6H16) 304 Da, but also produced fragments ions characteristic of cinchonains at m/z 791, m/z 451 and 341 [17, 18]. Compound 9 was tentatively identified as cinchonain IIa glucoside and compound 10 as cinchonain IIb glucoside. Curiously, cinchonain Ia, Ib, cinchonain IIa and cinchonain IIa glucoside were also found in bark from Erythroxylum vaccinifolium Mart., a medicinal plant also known as catuaba in the northeast of Brazil [32].
Compounds 8 and 11 detected in chloroform, hydroalcoholic and aqueous extract, showed the same [M − H]− signal at m/z 613. The MS/MS spectrum of compound 8 produced base peak at m/z 503 and fragment ion at m/z 393, both derived from the neutral loss of 3,4-dihydroxyphenyl moiety (110 Da), which indicated the presence of two dihydroxyphenyl groups. The fragment ion at m/z 451 was derived from the neutral loss of C9H6O3, while the fragment ion at m/z 341 was derived from the neutral loss of C6H6O2 + C9H6O3 (Table 1). Since the fragment ions of compound 8 match the ones reported by Gu et al. [18], it was assigned as a bis-(3,4-dihydroxyphenylpropanoid)-substituted catechin. The ESI-MS spectrum of compound 11, also exhibited a base peak at m/z 503 resulting from the neutral loss of 3,4-dihydroxyphenyl moiety (110 Da), but the relative intensities of fragments ions at m/z 451 and m/z 341 are very low. According to mass spectral database HMDB [24], compound 11 was tentatively assigned as cinchonain Id-7- glucoside.
Proanthocyanidins are polymeric flavonoids based on flavan-3-ols (oligomers of catechin and/or epicatechin and their gallic acid esters). Compounds 2 and 3 also exhibited UV maximum absorption at 280 nm and are proposed to be type B dimmer proanthocyanidins. These compounds were detected only in hydroalcoholic extract and exhibited [M − H]− signals at m/z 723 and 577, respectively. The MS/MS spectra of compounds 2 and 3 produced fragment ions at m/z 425, 407 and 289 (Table 1) characteristic for procyanidin B-type dimmers and a fragment ion at m/z 289 (catechin). Retro-Diels-Alder reaction of the heterocyclic ring system of the flavan-3-ol subunits gave rise to a fragment of m/z 425. The ion at m/z 425 eliminates water, probably from ring C at position C3/C4, resulting in a fragment ion of m/z 407 [19, 20]. Based on MS data reported by Kicel et al. [20], compound 3 was identified as type B dimmer of proanthocyanidin (epi) catechin - (epi) catechin, known as procyanidin B2. The MS/MS spectrum of compound 2 also exhibited abundant fragment ion at m/z 433, resulting from the loss of 290 Da (catechin). Based on mass spectral database [24], compound 2 was tentatively identified as procyanidin B2–8-C-rhamnoside. Compound 5 detected only in chloroform extract, exhibited [M − H]− signal at m/z 467, which after MS/MS experiments produced base peak at m/z 449, resulting from the loss of water. Compound 5 was tentatively assigned as apocynin E, which was also detected by Resende et al. [8] in T. catigua bark.
The presence of procyanidins and cinchonains in T. catigua bark was corroborated by the studies of Truiti et al. [16] and Resende et al. [8] that obtained a similar chemical profile. On the other hand, flavonoids rutin and quercetin identified by Kamdem et al. [15] and catiguanin A and catiguanin B (phenylpropanoid-substituted epicatechins) isolated from the bark of T. catigua by Tang et al. [33] were not present in our extracts in detectable amounts. However, as far as we know, a bis-(3,4-dihydroxyphenylpropanoid)-substituted catechin (8), cinchonain Id-7- glucoside (11) and cinchonain II glucosides (9 and 10) were detected for the first time in the species. This difference in chemical composition could be explained leading in consideration that the chemical constituents can vary in their structure and concentration depending on the region and season of collection, genetic variability, as well as the extraction method.
Biological activity
Several studies have shown the antioxidant activity of different catuaba (T. catigua) extracts [8, 33,34,35]. The data of our study confirm the antioxidant effect of T. catigua on DPPH assay for the four extracts analyzed. The most potent effect was achieved with the hydroalcoholic extract (EC50 = 43 μg/ml), with potency similar to rutin (EC50 = 44 μg/ml), the positive control, followed by aqueous, hexane and chloroform extracts. Lonni et al. [35] compared the antioxidant capacity (DPPH assay) of T. catigua extracted with different solvents and found the best result with ethanol, followed by acetone, water and methanol. In other study, Kamdem et al. [34] found that the content of total phenolics was higher in ethyl acetate extract, but the best effect on DPPH assay was obtained for the ethanolic extract. Using compounds isolated from T. catigua bark, Resende et al. [8] observed the most potent antioxidant activity with procyanidin C1, cinchonain IIb and cinchonain IIa, while Tang et al. [33] found the best results with the cinchonains Id, Ic and Ib. In our study, the hydroalcoholic extract containing cinchonains and procyanidins also exhibited the most potent antioxidant activity.
The neuroprotective activity of T. catigua is mainly attributed to its antioxidant activity. The 70% ethanolic extract of catuaba at concentrations from 10 to 100 μg/ml protected hippocampal neurons in vitro from oxidative stress and increased the survival after ischemia and reperfusion [15] or in the presence of hydrogen peroxide, sodium nitroprusside and nitropropionic acid [6]. The crude extract (acetone:water 7:3) and its semipurified fraction (partitioned with ethyl acetate), rich in epicatechin and procyanidin B2, were administered to mice in doses of 200 to 800 mg/kg for 7 days before the animals were submitted to a bilateral occlusion of the carotid. The treatment improved the performance of the animals in the Morris water-maze and protected hippocampal neurons [16]. These effects were mainly assigned to flavonoids and polyphenols present in these extracts, due to their antioxidant activity.
Other effects, as antinociceptive and antidepressant-like effect, seem to be related to a dopaminergic action [12, 13]. Neurochemical studies showed that the ethanolic extract of T. catigua inhibited dopamine and serotonin uptake and increased the release of these neurotransmitters, with more potent activity to dopamine. The antidepressant-like effect was evaluated in animals treated with doses of 200 and 400 mg/kg in the forced swimming test and tail suspension test. The extract induced antidepressant-like effect, which was blocked by haloperidol and chlorpromazine, anti-dopaminergic agents [13]. Another study using the ethyl acetate fraction of T. catigua showed antidepressant-like effect and increased cellular proliferation in the hippocampus [14].
The central cholinergic system is involved in the regulation of many cognitive functions and cholinergic alterations that occur during aging are associated with learning and memory deficits. Acetylcholinesterase hydrolyzes the acetylcholine released on central nervous system synapses regulating its concentration and effect. However, there is a progressive loss of cholinergic neurons that innervate hippocampus and the neocortex in Alzheimer’s disease and some other dementias resulting on cholinergic hypofunction. AChE inhibitors are used clinically on the treatment of Alzheimer’s disease, because they increase the availability of acetylcholine present in cholinergic synapses, enhancing the cholinergic functions. Drugs as rivastigmine (used as positive control in our study), galantamine and huperzine A (active principles isolated from medicinal plants) are AChE inhibitors employed in the treatment of Alzheimer’s disease [36].
In the current study, the effect of T. catigua extracts on cholinergic system was evaluated for the first time. All extracts tested inhibited the activity of acetylcholinesterase in vitro, and the most potent effect was obtained for the hydroalcoholic extract (IC50 = 142 μg/ml), followed by chloroform, aqueous and hexane extracts, with IC50 ranging from 313 to 346 μg/ml. The inhibition of AChE demonstrated for the four extracts may be due to the presence of high contents of cinchonains IIa, Ia and Ib, which are flavalignans - flavanols substituted with phenylpropanoids. Flavonoids that possess a free OH-group at C3 position showed major activity when compared to their C3 − OH glycosylated counterparts and those having no C3 − OH group, such as luteolin and apigenin [37, 38]. The major inhibition observed for the hydroalcoholic extract can be explained by the presence of procyanidins B2 found only in this extract. Proanthocyanidins exhibited a potent role in enhancing cognition in older rats, which was attributed to an increase in the acetylcholine concentration with a moderate reduction in AChE activity [39]. Proanthocyanidins exhibited ameliorative effects on learning and memory impairment of mice in scopolamine-induced amnesia test, showing protection against memory deficit [40].
The anticholinesterase effect found in our study can support the promnesic effect observed by Chassot et al. [5] for the crude extract and ethyl-acetate fraction of T. catigua. However, the hydroalcoholic extract of catuaba in doses of 50 and 300 mg/kg in our study did not promote memory improvement in mice treated with scopolamine, a competitive antagonist of muscarinic receptors. The inhibition of AChE causes an increase of concentration and time of acetylcholine on synaptic cleft, facilitating the cholinergic transmission. However, it is not possible to know in this study whether the in vitro anticholinesterase effect is also present in vivo. Or perhaps, the increase in acetylcholine concentration may not be enough to displace the scopolamine from the receptor and avoid its amnesic effect.
Kamdem et al. [15] discuss that T. catigua ethanolic extract seems to have preventive, but not curative effect on experimental ischemia, since the in vitro treatment of hippocampal slices after the protocol of ischemia and reperfusion did not protect the neurons. This prophylactic profile corroborates with the expected effect of an adaptogen, which is used chronically to avoid or diminish damages from stress and aging. In fact, the folk use of catuaba is similar to what we would expect for a typical adaptogen: the plant is used chronically to prevention and treatment of neurasthenia, fatigue, stress, impotence and memory deficits [1].
This is the first study evaluating the effect of T. catigua on stress and fatigue. We employed the hydroalcoholic extract of catuaba, which corresponds to the form popularly used and that showed the best results in our in vitro tests. The doses employed were comparable with those of previous in vivo studies and they did not interfere with the locomotor activity and motor coordination on rotarod, suggesting they were safe. The treatment with catuaba at doses of 25 and 250 mg/kg p.o. (starting 7 days before the repeated stress protocol) did not protect the animals from ulceration, neither prevented corticosterone and ACTH increase or thymus and spleen atrophy induced by stress. Adaptogens can lightly raise the basal level of corticosteroids, nevertheless adaptogens prevent the overwhelming increase of cortisol induced by stress [41]. The protocol of cold and immobilization causes an intense stress on the animal, seeing that the levels of ACTH and corticosterone increased tenfold in control-stressed rats when compared with non-stressed controls. Catuaba is widely used against fatigue and stress, but as far as we known, it is not used to treat or prevent gastric ulcers.
In order to evaluate whether T. catigua has an antifatigue effect, mice were chronically treated with hydroalcoholic extract at doses of 25, 100 and 250 mg/kg (p.o.) and submitted to forced exercise on a treadmill in three phases: before the treatment (basal performance) and after 21 and 49 days of treatment. The administration of catuaba did not alter the fatigue time, nor the lactate levels measured immediately after the exercise. However, mice treated with the highest dose showed increased spontaneous locomotor activity after the forced exercise on the 21th day. This result suggests that the treatment with catuaba may decrease the recovery time after an exhaustion protocol. Moreover, catuaba treatment for 49 days at the highest dose was able to diminish the impact of the forced exercise on the animals’ strength since the impairment on grip strength after the exercise was shortened at day 49 compared with the basal performance (difference on grip strength after fatigue between days 49 and basal). Even modest, these results suggest that the hydroalcoholic extract of catuaba may have beneficial effects on fatigue, at least shortening the recovery time after exhaustion. Stress-protective and antifatigue effects have been described for some adaptogens, as Rhodiola rosea L., Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. and Panax ginseng C.A. Meyer and several clinical trials were already conducted [41]. The importance of antioxidants on physical exercise and to prolong endurance and reduce fatigue has been evaluated. An extract of Polygonatum altelobatum Hayata rich in polyphenols and polysaccharides increased the endurance running time to exhaustion and the antioxidant ability in rats’ blood [42]. A supplementation with Chaenomeles speciosa (Sweet) Nakai fruit prolonged the exhaustive swimming time of rats and raised antioxidant enzymes levels, possibly by modulating the Nrf2 pathway [43].
Panax ginseng and other adaptogens are chronically used for several purposes – to increase stress resistance and physical capacity, to improve memory and other cognitive functions and as neuroprotective agents [44]. Ginseng acts by multiple mechanisms of action: it reduces the oxidative stress and excitotoxicity, modulates cholinergic neurotransmission, and increases dopamine and noradrenaline in the cerebral cortex [44]. It is likely that both the acetylcholinesterase inhibition and the antioxidant effect of T. catigua may contribute to its neuroprotective and pro-cognitive effects, as well as its dopaminergic and serotonergic effects are important for its antinociceptive and antidepressant effects. The antioxidant effect of different extracts or isolated constituents of catuaba was well evaluated. Several studies confirm that ethanolic or hydroalcoholic extracts of catuaba seems to have the most potent antioxidant effect [6, 33,34,35], but the proportion of water and ethanol can be better explored. Another alternative should be the use of special extracts prepared by extraction with different solvents, as suggested by Lonni et al. [35].