Phytochemistry and Biological Activities of Amburana cearensis (Allemão) ACSm

Amburana cearensis (Allemão) ACSm. belongs to the Fabaceae family and occurs in the Brazilian semiarid, Argentina, Paraguay, Bolivia, and Peru. Numerous studies that portray its ethnobotany, use in popular medicine, chemical composition, and biological activities exist in the literature. This review aimed to provide an overview of the chemical composition, ethnopharmacology, and biological activities associated with A. cearensis and its isolated constituents. Information was collected from internet searches in the Scopus, Medline, PubMed, Google Scholar, and ScienceDirect databases were performed covering publications from 1997–2020. An ethnopharmacological literature analysis revealed that A. cearensis is used to treat a wide range of respiratory disorders in addition to intestinal, circulatory, and inflammatory problems. Coumarins, flavonoids, phenolic glycosides, phenolic acids, phenylpropanoid derivatives, and triterpenoids, among others, have been reported as active compounds, with High-Performance Liquid Chromatography (HPLC) being the main analytical technique used. The A. cearensis extracts and compounds presented several biological activities, including antimicrobial, antinociceptive, anti-inflammatory, antioxidant, neuroprotective, and myorelaxant activities, among others. This review provides a useful bibliography for future investigations and A. cearensis applications; however, future studies should focus on its toxic effects and the mechanisms of action of its extracts and isolated constituents to guide clinical applications.


Introduction
Since the beginning of human civilization, natural products such as plants have been used to treat and cure various diseases [1]. According to data from the World Health Organization (WHO), roughly 80% of the population in developing countries use phytotherapics to meet needs associated with primary health care [2]. Thus, investment in research that focuses on investigating the bioactivity of phytotherapic products has grown in recent decades. The majority of the interest lies within the field of active constituents, which are present in the composition of these products and can be effective sources for brown colored stems that can reach 10 to 12 m in height [9]. A. cearensis has unique fruits, including Papilionoideae, that vary from green with yellowish spots to dark brown spots when dry has a wrinkled and dehiscent aspect, with winged seeds that are inside a thin membranous endocarp envelope and have an anemochory dispersion [11,12]. A. cearensis seeds are stenospermic, with a shape that varies between elliptical, oblong, and ovoid, with an average biometry of 14.4 mm in height, 10.4 mm in width, and 4.7 mm in thickness. A. cearensis seed integuments have a woody texture with a brown and opaque color and a very visible hilum that is elliptical in shape [13,14]. Its leaves are composed of 7 to 15 leaflets, which are elliptical to oval in shape, with an obtuse apex and rounded base. The inflorescences are between 2 and 5 cm long [6].

Uses in Popular Medicine
Traditional plants present a huge diversity of phytocompounds with possible applications against several diseases due the possible discovery of many medicines for clinical use [15]. Traditionally, Amburana cearensis is used by the quilombola and indigenous communities in Bolivia and northeastern Brazil for medicinal purposes [16][17][18]. The Chacobo Bolivian ethnic group (Department of Beni), who live in an endemic malaria region, use an aqueous decoction from the A. cearensis bark to combat fevers [19], whereas the Tocana ethnic group (Department of La Paz, Bolivia) uses the bark in the form of decoctions to treat abdominal and kidney pain and in the form of baths to treat rickets.
A. cearensis has extensive usage in popular medicine, where its bark, seeds, and fruits have been used, especially by populations in the semiarid regions of Brazil, for the treatment of respiratory system diseases such as bronchitis, asthma, rhinitis, flu, cough, and sinusitis and as an expectorant in the forms of a decoction, oral ingestion, and gargle [8,[20][21][22]. The plant is also highly indicated for intestinal problems, colic, diarrhea, placental delivery, headaches, whooping cough, thrombosis, hypertension, heartburn, skin ulcers, urinary infections, dehydration, emesis, joint pain, vertigo, and fever and has been used as a depurative and diuretic to treat kidney infections as well as an antispasmodic, cicatrizing agent, tonic, and anti-anemic for fungal dermatitis, and as an antiophidic in the event of snake bites and sores [23][24][25][26][27].
Reports on the use of its bark together with other medicinal herbs in the form of syrups and macerations against fatigue and as an expectorant can also be found in the literature [28,29]. An immersion of bark in water is used in the form of baths to combat skin, throat, and gynecological inflammations [30]. The literature also reports the use of the bark and seed oil against parasitic worms, general pain, circulation problems, and wound healing [7].

Phytochemistry
Phytochemical investigations are important for the discovery of new bioactive phytocompounds and for the exploration of a plant's compounds for the treatment of diseases [31]. The phytochemical characterization of different A. cearensis parts has been carried out by several groups in recent decades by using different analytical techniques. The isolated chemical components are mainly coumarins, flavonoids, phenolic acids, glycosylated phenols, phenylpropanoid derivatives, and triterpenoids. A summary of the phytochemical data is shown in Table 1.

Leaves
Gouveia et al. [41] identified the presence of phenolic compounds such as protocatechuic acid, epicatechin, p-coumaric acid, gallic acid, and kaempferol in A. cearensis leaf ethanolic extract through HPLC analysis.

Cultivated Young Plants
Canuto et al. [43] investigated the phytochemical profile of cultivated young A. cearensis specimens, where coumarins, isokaempferidium, amburoside A and B, vanillic acid, p-hydroxybenzoic and aiapin acid, glycosylated (E/Z)-o-coumaric acid, and protocatechuic acid were identified in the ethanolic extract from young plants. Leal et al. [35] also detected vanillic acid and coumarins as constituents of the aerial parts in their study.

Bioactivities Attributed to Amburana Cearensis
Many of the medicinal properties of A. cearensis, such as anti-inflammatory, antinociceptive, antimicrobial, and others, have scientific support through in vivo and in vitro assays, as evidenced in this review, which justifies the application of this plant for medicinal purposes. Moreover, new evidence about other bioactivities as acaricidal, repellent, photoprotective, among others, have been observed. The major part of these studies were performed using bark extracts and fractions, with only nine compounds being evaluated for their pharmacological effects ( Figure 1). The list of the ethnomedicinal scientific results available for this species are listed below. The biological properties of the crude extracts are summarized in Table 2, while their isolated phytochemicals have been compiled in Table 3.
The antibacterial activity of the leaf ethanolic extract was also demonstrated against the S. aureus SA-ATCC 25923 strain with a minimum inhibitory concentration (MIC) value of 512 µg/mL. Despite direct antibacterial activity against E. coli (MIC ≥ 1024) not being observed, a modulation of the aminoglycosides gentamicin and amikacin activity was noted, with decreases in MIC ranging from 64 to 4 µg/mL and from 128 to 8 µg/mL, respectively, against E. coli 27 being recorded [45].
In their study, Silva et al. [46] also found antibacterial activity for the hydroalcoholic extract against Acinetobacter spp., with a mean MBC of 9.375 µg/mL. The ethanolic extract, however, presented an average MBC value of 390.6 µg/mL against Enterobacter spp.; 1302 µg/mL against E. coli; and 911.4 µg/mL for the Klebsiella spp., genus. Similar results were found in assays performed by Peixoto et al. [47], where the A. cearensis bark ethanolic extract demonstrated an antibacterial potential with a mean MBC value 12,500 µg/mL against Staphylococcus spp. For the stem bark chloroform extract, inhibitory effects against P. aeruginosa and Bacillus cereus were also demonstrated, with MIC values of 6900 µg/mL being obtained for both strains [36].
In terms of the antibacterial activity of the seed extracts, the ethanolic extract was effective against E. coli (25,922), S. aureus (29,213), P. aeruginosa (27,853), Listeria monocytogenes (35,152), and Shigella flexneri (700,930), with MIC values of 1000, 250, 250, 500, and 250 µg/mL, respectively. An antibacterial effect for the seed aqueous extract against the same E. coli, S. aureus, P. aeruginosa, L. monocytogenes, and S. flexneri strains was also observed with MIC values of 1000, 1000, 500, 500, and 500 µg/mL, respectively [38]. Oliveira et al. [48] evaluated the antibacterial activity of the A. cearensis seed crude extract and its fractions (fraction 1-0 to 30%; fraction 2-30-60%; fraction 3-60-90%, of ammonium sulfate saturation). The samples did not show direct antibacterial activity against the S. aureus (SA10) and E. coli (EC06) strains, both with MIC values ≥ 1024 µg/mL. However, the A. cearensis seed crude extract and its fractions acted as antibiotic activity modifying agents, with a synergism being observed in the association between gentamicin and the crude extract against E. coli and S. aureus. Moreover, norfloxacin combined with the crude extract and fraction 1 also presented a significant synergism against E. coli and S. aureus. Likewise, gentamicin in combination with fractions 1 and 3 exhibited synergism against S. aureus. In addition, the combined effect of the crude extract and its fractions with penicillin demonstrated synergisms against S. aureus, while a synergism against E. coli was observed with the association between penicillin and the crude extract.
Many natural compounds with traditional usage have been investigated for their antimicrobial effects [49]. Regarding A. cearensis, its antibacterial potential is the strongest evidence of its traditional usage in treating gastrointestinal problems and infections of the urinary system [23].
The antifungal activity of the extracts and compounds isolated from A. cearensis is not well documented. However, the protein fraction from the seeds exhibited antifungal activity against different fungal species, presenting an inhibitory growth effect of up to 60% of the filamentous fungi Fusarium solani, Fusarium oxysporum, and Colletotrichum lindemuthianum at a concentration of 160 µg/mL. The 80 and 160 µg/mL concentrations also significantly inhibited the growth of Saccharomyces cerevisiae and Candida albicans yeasts, showing inhibition of up to 80% at the 160 µg/mL concentration [50]. The good effect against fungi demonstrated the traditional usage in the tretatment of fungal dermatitis [26].
Although the action mechanisms of the extracts and their compounds against bacteria have not been evidenced in the studies analyzed, these effects are possibly due to interferences in lipid bilayers inducing bacterial membrane rupture and the inhibition of processes such as biofilm formation, cell envelope synthesis, nucleic acid synthesis, electron transport chain, and ATP synthesis in these microorganisms performed by secondary metabolites such as flavonoids, which have been considered to be promising sources for antimicrobial therapy [51].

Anti-Inflammatory, Antinociceptive and Antiedematogenic Activities
The Torresea cearensis (synonym of A. cearensis) bark hydroalcoholic extract reduced the nociception produced by acetic acid and formalin in a dose-dependent manner by 21 and 30% at the 100 and 200 mg/kg concentrations, respectively. The 200 mg/kg concentration also significantly inhibited carrageenan-induced paw edema by 30 and 48% within 3 and 4 h, respectively [52]. The reduction of the nociceptive effect induced by acetic acid indicated the effect on the peripheral analgesy mechanisms [53]. Similar results were observed by Leal et al. [54], where the oral administration of the hydroalcoholic extract at 100 and 200 mg/kg inhibited the contortions induced by acetic acid by 21 and 49%, respectively, in Wistar rats. The same concentrations also inhibited nociception by 19 and 60.1%, respectively, during the 2nd stage of the formalin test. The 200 mg/kg concentration also reduced carrageenan-induced paw edema by 48% within 4 h. Likewise, bark hydroalcoholic extract, when administered orally (400 mg/kg) or intraperitoneally (200 mg/kg), in BALB/c mice significantly inhibited antigen-induced paw edema and the production of immunoglobulin-specific ovalbumin by 50 to 80% [55]. As shown in the study by Leal et al. [56], the stem bark hydroalcoholic extract at concentrations of 200 and 400 mg/kg (administered orally) inhibited leukocyte and neutrophil migration by 37 and 42%, respectively.
According to a study by Oliveira et al. [57] the stem bark ethanolic extract demonstrated antinociceptive activity at the 200 and 400 mg/kg doses. In addition, the 100, 200, and 400 mg/kg doses decreased nociception during the 2nd stage of the formalin test by 77.5, 79.7, and 91.3%, respectively. The suppression of the pain in the second phase of the formalin assay indicated an antihyperalgesic and peripheric effect, demonstrating an anti-inflammatory effect [58].
In a comparative study using cultivated and wild A. cearensis species, the administration of 100-400 mg/kg of the ethanolic extract from specimens with 4, 7, or 9 months of cultivation presented antinociceptive activity, inhibiting both stages of the formalin test in mice by 32 to 64%. Similar results were observed for the bark ethanolic extract of wild species. Both extracts also reduced carrageenan-induced paw edema (Cg) at the 100-400 mg/kg concentrations [35]. Lima et al. [59] also demonstrated the antiedematogenic effects of the A. cearensis seed aqueous extract, where concentrations of 10 and 20% weight/volume significantly reduced carrageenan-induced paw edema in rats, with effects starting 2 h after administration of the phlogistic agent. A number of constituents isolated from A. cearensis also had their effects evaluated, where coumarin was one of these constituents, which at concentrations of 5-20 mg/kg, reduced, in a dose-dependent manner, the nociception produced by acetic acid and formalin [52].
Leal et al. [54] also demonstrated the antinociceptive effects of coumarin (1) in the inhibition of contortions induced by acetic acid and the formalin test at the 5, 10, and 20 mg/kg concentrations. The 10 and 20 mg/kg concentrations also inhibited carrageenaninduced paw edema in a similar manner to the aforementioned study. In the study by Leal et al. [54], orally administered coumarin (1) (20 and 40 mg/kg) significantly inhibited leukocyte and neutrophil migration in the peritoneal cavity of rats. Moreover, as demonstrated by Marinho et al. [55], coumarin (1) (10 and 20 mg/mL) reduced paw edema in animals sensitized with ovalbumin and challenged with the antigen, where coumarin, at the stated doses, induced the inhibition of anti-ovalbumin antibody titers in a similar way to dexamethasone.
The administration of vanillic acid (6) at the concentrations of 25 and 50 mg/kg showed antinociceptive effects by significantly reducing the two phases of the formalin test. In addition, the compound inhibited paw edema at the 25 and 50 mg/kg concentrations and leukocyte migration in the rat peritoneal cavity induced by carrageenan at the 50 mg/kg concentration [35]. The afrormosin (7) isoflavone inhibited the degranulation of the neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA), with LC 50 values of 66.70 µM and 0.374 µM, respectively. The 3.35 and 167.6 µM concentrations inhibited myeloperoxidase activity by 39 and 76%, respectively. Cellular treatment with afrormosin at 16.76-335.2 µM concentrations reduced TNF-α release by 44% [62].

Myorelaxant Activity
The A. cearensis bark hydroalcoholic extract caused concentration-dependent relaxations in the trachea of guinea pigs pretreated with 0.3 µM carbacol, obtaining an EC 50 value of 3.64 ± 0.4 mg/mL [54]. In the study by Leal et al. [56] the same extract evoked a concentration dependent relaxation in guinea pig trachea with an IC 50 value of 3.16 ± 0.55 mg/mL in the presence of three antagonists.

Antioxidant Activity
The leaf ethanolic extract as well as the ethyl acetate and methanolic fractions, showed antioxidant activity when evaluated by the DPPH assay at the 0.1 mg/mL concentration, with an antioxidant potential of 93.63, 83.21 and 93.01%, respectively. The bark ethanolic extract, on the other hand, presented an antioxidant potential of 94.55% at the 1 mg/mL concentration [50]. Antioxidant activity for the seed ethanolic extract at 30, 60, and 120 µg/mL concentrations with an IC 50 value of 17.95 µg/mL was also observed [38].
Antioxidative agents have a role in inhibiting oxidative stress that is induced due to free radicals, protecting the cells. Medicinal plants present a huge variety of compounds with antioxidative properties, with this effect being mediated by different mechanisms [63]. The antioxidant potential of the Fabaceae family has been demonstrated by several studies, most of which have been associated with the occurrence of flavonoids and highlighting the isoflavone class, which are considered to be the most bioavailable flavonoids and are restricted to some legume subfamilies [51].

Neuroprotective Activity
Several studies have shown evidence that natural products can reduce neuronal damage, making them a possible source for the development of new drugs and therapeutic strategies for the treatment of these diseases [64]. The dry extract, phenol fraction and amburoside A (2), obtained from A. cearensis demonstrated neuroprotection over microglial cells at the 5-200 mg/mL concentration [65]. The ethanolic, hexane, dichloromethane, and ethyl acetate extracts obtained from A. cearensis seeds presented a neuroprotective potential over PC12 neural cells exposed to glutamate-induced neuronal damage (1 mM) at concentrations of 0.1-1000 µg/mL [39]. The ethanol and dichloromethane extracts from the seeds also demonstrated the neuroprotective effects against the neural damage induced by glutamate in rat cerebellar cultures at a concentration of 0.1 µg/mL, with this effect being possibly associated with the increased expression of the astrocytic enzyme glutamine synthetase from the glial fibrillary acidic protein (GFAP) and β-III tubulin, indicating glial and neuronal preservation, respectively [40].
The neuroprotective effects observed are possibly due to the metabolites present in A. cearensis with antioxidant and anti-inflammatory properties that are able to regulate the intracellular signaling cascades of glutamate excitotoxicity, which is a critical component in the development of neurological and neurodegenerative diseases, and this regulation may minimize the main causes of neuronal death [40].
The compounds kaempferol (9), isokaempferide (3), amburoside A (2), and protocatechuic acid (8) [67]. Based on these facts, extracts and compounds from A. cearensis demonstrated activity against several cancer cell lines. However, the mechanisms involved in these activities were not elucidated, making more studies necessary.

Allelopathic Activity
Allelopathy is a defense mechanism observed on several organisms that are mainly present in plants and involves the production and liberation of bioactive compounds in the environment. On plants, these compounds affect the growth and the development of neighbouring plants [68]. Previous reports have demonstrated the allelopathic effects on the germination of Lactuca sativa seeds that have received aqueous and methanolic treatments at concentrations 5, 10, and 15 g/L and 1, 5, 10, and 15 g/L, respectively, of the A. cearensis ground seed. Both extracts also showed an allelopathic activity on the germination of Raphanus sativus L. seeds treated with 15 g/L of A. cearensis extract [69]. The leaf aqueous extract almost completely prevented the germination of Amaranthus deflexus at the 50 and 100 g/L doses, with a 99.3 and 99.5% decrease in germination percentage and germination velocity index, respectively, at the highest concentration [70].
The seed hydroalcoholic extract prevented the germination of Cucumis melo L. seeds and the emergence of seedlings at the 1, 0.5, and 0.25% weight/volume concentrations. However, the leaf extract evaluated in the same study did not prevent seed germination, despite the seedlings presenting abnormalities that exceeded 25% at the highest concentration [71]. Similar results were observed in the study by Oliveira et al. [48], where the hexane and dichloromethane fractions of the seed extract affected the emergence and initial growth processes of Cucumis melo L. at 1 and 0.5% weight/volume concentrations.
The allelochemicals belong to several different classes of secondary metabollites, including phenols, benzoic acid, and derivates of cinnamic acid, terpenoids, glicosides, among others [72]. However, the inhibition of germination in both weed seedlings and in Amaranthus deflexus as well as in edible plants such as Lactuca sativa, Raphanus sativus and Cucumis melo indicates a possible non-biosselectivity, which could result in a threat to non-target organisms. Therefore, this highlights the need for studies to systematically determine the bioselectivity and biosafety of the allelochemicals present in A. cearensis, which will guide the use of this species in practical applications.

Other Bioactivities
In addition to the aforementioned bioactivities, A. cearensis has other properties that have been described in the literature. According to the study carried out by Trevisan et al. (2003) [73], the ethanolic extract of the stem bark caused 100% inhibition of acetylcholinesterase (AChE) enzyme activity at a concentration of 2.3 mg/mL. Meanwhile the hydroalcoholic extract obtained from the stem powder caused 83% inhibition, with an IC 50 value of 0.3789 mg/mL [66].
The A. cearensis leaf ethanolic extract at a concentration of 0.2 mg/mL increased the preservation of pre-antral goat follicles for up to 6 h [41,74]. At a concentration of 0.1 mg/mL, the same extract also had a significant effect on the survival and in vitro development of secondary follicles isolated from sheep (Ovis aries) [75]. Similar results were observed in the study by Menezes et al. [76], where the A. cearensis leaf ethanolic extract at a concentration of 0.2 mg/mL maintained follicular survival and DNA integrity in a similar manner to those observed for the minimum essential medium (MEM) over 24 h.
The seed aqueous extract exhibited marked larvicidal activity against the Aedes aegypti mosquito, with an LC 50 value of 8.10 ± 0.27 mg/mL within 24 h of exposure [77]. The leaf and stem bark powder (2 g) presented repellent activity against the bean weevil Callosobruchus maculatus, obtaining repellency rates of 0.46 ± 0.05 and 0.42 ± 0.04, respectively [78]. In addition, the hexane fraction from the leaf ethanolic extract at a concentration of 25 mg/mL showed in vitro acaricidal activity against engorged Rhipicephalus (Boophilus) microplus females, reaching oviposition and hatching inhibition values of 52.7% and 39.0%, respectively [79]. In the study by Lima et al. [59], the A. cearensis seed aqueous extract demonstrated mutagenic activity over Allium cepa at concentrations of 0.1 and 0.5 mg/mL. An in vitro photoprotective activity for the A. cearensis leaf ethanolic extract has also been described, with UVB radiation absorption efficiency at wavelengths being in the 280 to 320 nm region, with a sun protection factor (SPF) equal to 17.60 at a concentration of 0.2 mg/mL [80].
Treatment with amburoside A (2) at 25 and 50 mg/kg doses significantly inhibited the increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels in animals poisoned by carbon tetrachloride (CCl 4 ). In liver tissues, the compound inhibited the formation of CCl 4 -induced thiobarbituric acid reactive substances (TBARS) at both doses, indicating a CCl 4 -induced lipid peroxidation blockade [82].
Although the extracts and phytocompounds isolated from A. cearensis present a wide range of therapeutic properties that have already supported by experimental results, some medicinal properties such as depurative, diuretic, antispasmodic, and antivenom reported for this species remain unexplored, highlighting important areas of research with the opportunity for chemical and biological exploration to carry out in vivo and in vitro assays as well as for the evaluation of the mechanisms of action of the crude extracts and isolated compounds.

Conclusions
Phytochemical, ethnobiological, and pharmacological studies of Amburana cearensis are well reported. Many of these activitites were elucidated based on studies with different extracts and phytocompounds that had been isolated, with its traditional usages being supported by these bioactivities. For example, all of the antibacterial, antifungal, antiinflammatory, antinociceptive, antioxidant, and myorelaxant activities studied can be directly associated with traditional usages against infections caused by bacteria and fungi, against respiratory and gastrointestinal illnesses, fever, malaria, among others. New biological activities such as acaricidal, larvicide, repellent, photoprotective, antileishmanial, activities were observed, among others. However, there are some activities that are unable to be associated with traditional usage, as depurative, diuretic, antispasmodic, antivenom, activities, among others. The extensive use of this plant suggests relative security against acute toxicity. More studies determining the general toxicity of this plant are necessary.