1. Introduction
Morocco is a Mediterranean country characterized by diverse biotopes (desert, mountains, coastal areas, etc.) and a complete range of Mediterranean bioclimates (from humid to Saharan), due to its middle position between two seas (
Figure 1) [
1,
2]. These peculiar ecological conditions provide huge and varied plant biodiversity, among which there are species with potential medical interest [
3]. Such species have been classified into 150 families and 940 genera, with around 900 endemic plants [
1,
2]. These features make Morocco an actual plant genetic reserve and a reservoir of novel bioactive compounds for drug discovery [
2]. At the international level, the production of Moroccan medicinal plants and their preparations (such as essential oils or dried extracts) is destined almost entirely for export, with an increased interest in the years due to the opening of novel markets [
3].
Moroccan medicinal plants have received a great deal of attention in the field of ethnopharmacology, being exploited as inexpensive and available sources of drugs by the local population, especially in rural areas, for primary health care; knowledge of medicinal plants and their properties are transmitted from generation to generation, being the basis of the old culture of Moroccan people [
4]. As highlighted by several ethnobotanical and ethnopharmacological surveys, ancestral medical practices, especially phytotherapy traditions, are well preserved by Moroccan people and are even active today; this widespread use is a result of the accumulation of knowledge from various sources and different ethnic traditions, coupled with the long exposure to, and experience of, these people with natural resources [
3]. Medicinal plants have traditionally been exploited by Moroccan people to fight several diseases, such as bacterial and viral infections, gastro-intestinal disorders, diabetes, hypertension, and skin affections, likely due to a richness in phytochemicals, such as polyphenols, saponins, and essential oils, whose bioactivities (e.g., anti-infective, antioxidant, antitumor, and antiviral ones) are known [
1,
4,
5,
6]. Moreover, surveys have shown that about 70% of Moroccan people currently use medicinal plants. For example,
Artemisia herba-alba,
Carum carvi, and
Nigella sativa are exploited as antidiabetic and antihypertensive remedies [
2], while some species from the Lamiaceae and Asteraceae families are utilized for digestive and genito-urinary disorders [
5]; in particular,
Mentha spp. are used to relieve respiratory and gastro-intestinal disorders [
6]. Along with traditional knowledge, a lack of health facilities, scanty accessibility to conventional medicine, and vulnerability of sociocultural status support the rush toward medicinal plants as essential resources for the primary care of poor Moroccan people, especially those living in rural areas [
6]. However, the pharmacological basis and phytochemical requirements for the bioactivities remain to be defined. This strengthens the importance of studying Moroccan medicinal plants, not only to valorize their biodiversity and support further developments in pharmaceutical, nutraceutical, and cosmetic fields, but also to give a scientific basis to ethnobotanical traditions, which can lead to their rational and safe use by local people.
Among the endemic plants from Morocco,
Anacyclus species, belonging to the Asteraceae family, widely occur in this Mediterranean country, with about 12 species distributed in North Africa, South Europe, and the Middle East [
7]. These plants are characterized by a close morphological flower similarity, thus being commonly associated with chamomile; indeed,
Anacyclus pyrethrum DC. is also known as “Spanish chamomile” [
8], while
Anacyclus clavatus (Desf.) Pers. (syn.
Anthemis tomentosa Gouan) is known as “wooly chamomile” [
9]. After all, a common phylogenetic origin of
Anacyclus and
Matricaria genera has been highlighted [
10]. Many
Anacyclus species, including
Anacyclus pyrethrum (L.) Lag.,
Anacyclus radiatus Loisel,
Anacyclus valentinus L., and
Anacyclus clavatus (Desf.), have been used traditionally to treat different ailments, such as digestive disorders, pain, and infections, likely due to their antioxidant, anti-inflammatory, analgesic, and antimicrobial properties [
11,
12,
13,
14]. In particular, extracts from
A. pyrethrum root are the most studied, and their benefits have been associated to the presence of alkaloids (e.g., pyrethrine), fatty acids, and unsaturated amides (e.g., pellitorine, anacycline, and sesamin), along with polyphenolic compounds; they have been recommended for treating toothache, digestive problems, infertility, and used as an aphrodisiac, analgesic, nervous system tonic, anti-arthritic, and eupeptic remedies [
15,
16,
17,
18]. Furthermore, the aerial parts of
A. pyrethrum were found to be endowed with antimicrobial and antioxidant properties, likely ascribable to the presence of polyphenolic compounds [
13]; similarly, polyphenols, tannins, and flavonoids seem to be responsible for the antioxidant, antimicrobial, and antinflammatory properties of
Anacyclus clavatus (Desf.) aerial parts, which are recognized to possess multiple beneficial properties [
19,
20]. Finally, the essential oil from aerial parts of
Anacyclus valentinus L. has shown antifungal properties [
21].
In the present study,
Anacyclus maroccanus Ball (AR;
Figure 2A,B) and
Anacyclus radiatus Loisel (AR;
Figure 2C,D) ecotypes, collected in the Demnate and Essaouira regions of Morocco, respectively (
Figure 1), were studied for their phytochemical composition and bioactivities to valorize their biodiversity and highlight any possible future pharmacological interests. While
A. maroccanus is not mentioned in traditional medicine, infusions of
A. radiatus flowers are used to treat stomach upsets and microbial infections [
22].
To perform the study, methanolic and ethyl acetate extracts from flowering aerial parts of each ecotype were prepared by fractionation (
Figure 1E); next, the polyphenolic composition was evaluated by a multimethodological spectrophotometric and chromatographic analysis. We focused on the flowering aerial parts, which are rich sources of polyphenols and recognized to possess multiple beneficial properties, making them interesting candidates for pharmaceutical and nutraceutical applications. In particular, the dietary polyphenols showed antidiabetic effects, likely due to their ability to affect carbohydrate metabolism, and cytoprotection against hypoglycemia-induced oxidative stress [
23].
In line with this evidence, the ability of the extracts to affect the enzymes involved in carbohydrate metabolism and hyperglycemia-associated stress (oxidative stress, formation of advanced glycation end products) have been evaluated. In particular, the antioxidant power and carbohydrate enzyme inhibition, along with the antiglycative and chelating properties, have been studied. In addition, considering the traditional use of AR aerial parts, the antimicrobial properties of the extracts against different bacterial, fungal, and dermatophytic species, along with a bioinformatic evaluation of the possible interactions accounting for antimicrobial activity, have also been assessed.
3. Discussion
In the present study, methanolic and ethyl acetate extracts from the flowers of Anacyclus maroccanus Ball and Anacyclus radiatus Loisel, two Moroccan ecotypes, have been characterized for their polyphenolic composition and for the polyphenol-associated bioactivities, especially the ability to affect the enzymes involved in carbohydrate metabolism and hyperglycemia-associated stress (oxidative stress, formation of advanced glycation end products), through antioxidant, antiglycative, and chelating properties. In line with the traditional use of AR aerial part infuses, the antimicrobial activity of the extracts has been evaluated too.
Anacyclus species are widely exploited in folk medicine of North Africa and other Mediterranean countries, due to their recognized therapeutic properties ascribed to the presence of different compounds, including flavonoids, terpenoids, and alkaloids [
7]. Although
A. pyrethrum DC. is the most studied variety, other endemic species are used as remedies for primary care by local people, who have limited access to conventional medicine [
19,
20,
21]; however, the mechanisms accounting for its bioactivities, along with the composition of bioactive compounds, remain to be defined. This strengthens the need to promote pharmacognostic research on these species in order to not only to valorize their biodiversity by highlighting their relevance as sources for drug discovery and industrial fields, but also to support their traditional use through the clarification of the pharmacological basis and the phytochemical features required to achieve the expected effect. This point is also relevant to limit the problem of the invalid metabolic panaceas (IMPs), which often occurs due to the poorly characterized biological activity profile of extracts or pure compounds [
24].
Concerning the extracts from
A. maroccanus and
A. radiatus, a phytochemical analysis revealed that methanolic extracts from both species, especially that from AM, were richer in polyphenols, tannins, flavonoids, and carotenoids than the ethyl acetate extracts; conversely, the ethyl acetate extracts contained higher amounts of total chlorophylls. Although the AM and AR species have been scantly investigated, the results obtained in this study agree with the composition of aerial parts of other
Anacyclus species. Indeed, Selles et al. [
13] reported that a methanolic extract from the aerial parts of
A. pyrethrum contained the highest amount in total polyphenols with respect to the aqueous and chloroform extracts; likewise, a methanolic extract from
A. clavatus was richer in total polyphenols than the aqueous one [
17]. The tannin content of the AM methanolic extract was similar to that of a methanolic extract from
A. clavatus aerial parts (39.21 mg TAE/g) [
13], while the amount from the respective AR sample was higher by about 1.4-fold. Regarding flavonoids, the methanolic extracts from the AM and AR samples contained levels about 1.5- and 3-fold lower than that of
A. pyrethrum aerial parts (92.50 mg QE/g), respectively [
13]. Moreover, the amount in the AM methanolic extract was almost doubled with respect to that of
A. clavatus (9.96 mg QE/g)
, despite a very similar level in the AR sample [
17]. These data highlight that our AM and AR species, albeit producing slight differences due to the species, cultivation condition, habitat, and extraction method, represent interesting sources of polyphenols, just like the most studied
Anacyclus species.
The polyphenol composition of
Anacyclus spp. has not been thoroughly investigated. According to the compounds identified in the AM and AR methanolic samples, Bouriche et al. [
17] highlighted the presence of gentisic acid (2,5-dihydroxybenzoic acid), chlorogenic acid, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, vanillic acid, and rutin in the methanolic extract from
A. clavatus aerial parts. Of note, compounds never before identified in these species, to the best of our knowledge, are meglutol, epicatechin, and ferulic acid. Altogether, these data constitute the first complete phytochemical characterization of AM and AR species and suggest a possible involvement of identified compounds in the bioactivities of the extracts, which deserves further study.
When assayed for antioxidant activity, all of the samples displayed a similar efficacy towards the ABTS radical, while methanolic and ethyl acetate AR extracts were found to be more potent than their respective AM extracts against the DPPH radical. In line with our data, Selles et al. [
13] reported that a methanolic extract from
A. pyrethrum aerial parts was more potent than water and chloroform extracts in scavenging DPPH, with an IC
50 value (56 μg/mL) comparable to that of ascorbic acid (48 μg/mL), which was used as positive control. Likewise, the methanolic extract from
A. clavatus strongly inhibited DPPH, with a IC
50 value of 28.30. Based on our results, the presence of DPPH-radical scavenger compounds in all the extracts from the AM and AR species can be hypothesized; moreover, fractionation by ethyl acetate seems to concentrate the bioactive compounds, thus leading to an IC
50 lower than that of the methanolic extract and of the positive control trolox.
DPPH and ABTS are two synthetic radicals that can be scavenged by electron- or hydrogen-transfer mechanisms, and that have different specificities and kinetics: DPPH is more selective for small molecules, being limited sterically with regard to access to the radical site, while ABTS reacts with both lipophilic and hydrophilic compounds with a poor selectivity in the reaction with hydrogen-atom donors [
25]. Indeed, ABTS is bleached by different antioxidants in fruits and vegetables while DPPH seems to not react with carotenoids [
25].
Based on this evidence, the ABTS-scavenging abilities of AM and AR extracts could be ascribable to the presence of both hydrophilic and lipophilic phytochemicals, such as polyphenols, tannins, and carotenoids; conversely, the higher potency of AR samples suggests the presence of specific, or more concentrated, scavenging compounds, such as phenolic acids and flavonoids. However, further studies are required to clarify their involvement in the observed radical scavenging activity.
The extracts have also been evaluated for their ability to chelate ferrous ions and to inhibit the production of advance glycation end-products (AGEs), which are toxic metabolites accumulated under different pathologies, and are responsible for inflammation and oxidative stress [
26]. Along with AGE, alterations in iron homeostasis are typical of diabetes, and have been found to be associated with an increased production of reactive oxygen species (ROS) and hyperglycemia complications [
27]; therefore, the metal chelating ability and AGE inhibition can provide benefits in hyperglycemia conditions, blocking both oxidative and inflammatory damage.
In the present study, the AM and AR ethyl acetate extracts showed the most effective antiglycative capacity and the most potent chelating activity. Evidence from the literature has highlighted the ability of different phenolic compounds, among which are rutin and catechin, to exert similar properties; moreover, gallic acid and catechin prevent AGE formation by trapping α-dicarbonyl compounds or by inhibiting the formation of Amadori products [
28,
29]. The presence of these compounds in the tested extracts can support our hypothesis about their involvement in the extract bioactivities.
In line with the evidence of the antiglycative and chelating properties, in order to better disclose the possible interest in the tested extract in the management of glucidic metabolism disfunction, such as hyperglycemia and diabetes, their ability to interfere with the activity of key enzymes involved in carbohydrate metabolism to glucose, i.e., α-amylase and α-glucosidase, has been studied. In particular, α-amylase is a salivary and pancreatic enzyme that catalyzes the endo-hydrolysis of α-1,4-glucosidic linkages of amylose, while α-glucosidases further breaks down α-1,4-glucosidic linkages in the small intestine, allowing glucose release and absorption [
30].
Under our experimental conditions, both methanolic and ethyl acetate extracts from AM species significantly inhibited α-amylase, despite a weak effect of the AR samples. Conversely, α-glucosidase inhibition was mainly induced by ethyl acetate extracts, which agrees with their chelating and antiglycative properties. Despite the differences in the ability of the tested extracts to mainly affect glycolytic enzyme activity, present data agree with previous evidence about the ability of extracts from
A. pyrethrum root and
A. valentinus aerial parts to counteract hyperglycemia in in vivo models of diabetes [
14,
31]; however, the bioactive compounds, and their true mechanisms of action, remain to be elucidated.
Multiple sources of evidence have highlighted a role of dietary phenolics in the regulation of carbohydrate metabolism, particularly as inhibitors of α-amylase and α-glucosidase enzymes [
30]. Among them, the flavonoid rutin has been reported to possess antiglycative properties and to deactivate α-amylase and α-glucosidase by forming an inactive complex [
32,
33]. In the ethyl acetate extracts, rutin is the prominent phenolic compound, and the in silico assessment indicated putative submicromolar affinities of rutin towards both α-amylase and α-glucosidase; this further indicates that this phytochemical plays a key role in the enzyme inhibition by the ethyl acetate extracts.
Moreover, phenolic acids, such as gallic acid and ferulic acid, are known to inhibit the glycolytic α-amylase and α-glucosidase enzymes [
34,
35]. Compounds containing more than one hydroxyl group, such as caffeic and protocatechuic acids, potently inhibit α-glucosidase [
33,
34]; conversely, chlorogenic acid seems to mainly block α-amylase [
36]. In silico studies have also highlighted the α-glucosidase inhibitory properties of catechin [
37]. Further attention can be devoted to meglutol or 3-hydroxy-3-methyl glutaric acid, which is known to induce hypoglycemia and to regulate lipid metabolism [
38,
39].
Finally, the extracts were found to possess antimicrobial properties, being able to counteract the growth of different bacteria, fungi, and dermatophytes. Both AM and AR ethyl acetate extracts were effective against
E. coli and
T. rubrum strains, with a lower potency of methanolic extracts against
E. coli, especially that from AR, being observed. This last sample also showed a moderate inhibition against
Bacillus cereus,
Pseudomonas aeruginosa,
Staphylococcus aureus, and
Candida albicans, despite a weak activity for the other samples. The antimicrobial properties of AM and AR samples agrees with the results of Selles et al. [
13], which found that a methanolic extract from
A. pyrethrum aerial parts was able to inhibit
S. aureus and
B. cereus, despite slightly affecting
E. coli and
Pseudomonas aeruginosa; however, the tested extracts showed lower potency, in terms of MIC values (µg/mL), compared to the reference drugs ciprofloxacin, fluconazole, and griseofulvin for the antibacterial and antifungal effects [
40]. In fact, the MIC values of ciprofloxacin, fluconazole, and griseofulvin were always ≤4 µg/mL towards the tested bacterial and fungal strains [
40]. We have to consider that the extracts’ MIC values for the ethyl acetate samples were of the same order compared to the reference drugs, with values < 10 µg/mL, with regard to growth inhibitory effects against
E. coli and
T. rubrum species. Additionally, considering the content in polyphenolic compounds of the extracts, and according to what reported in previous studies [
41], we could hypothesize that these phytochemicals could influence, albeit in part, the observed bacteriostatic and mycostatic effects. Present data are also consistent with the antimicrobial properties of herbal extracts containing benzoic acid, cinnamic acid, and quercetin [
42,
43,
44,
45], and collectively suggest a rationale for the use of
Anacyclus extracts as sources of biomolecules with antimicrobial effects.
Despite this evidence, the true bioactive compounds of Anacyclus maroccanus and Anacyclus radiatus remain to be clarified and the possible involvement of the tangled interactions among these phytochemicals cannot be excluded.