3.1. Phytochemical Profile Determination
In this work, standard in vitro spectrophotometric analyses was used to determine the total phenolic and flavonoid contents of the ethyl acetate, methanolic, and aqueous extracts of
F. clypeata. The results are presented in
Table 1.
As can be observed from the table, the highest phenolic (33.42 mg GAE/g) and flavonoid (21.58 mg RE/g) contents were obtained when considering the methanolic extract. Besides, a strong correlation coefficient (
p < 0.01) of 0.809 was found between the two different assays (
Supplementary Material Table S1). In our previous study on
F. eriocarpa [
16], the highest total phenolic level was determined in ethyl acetate extract (41.87 mg GAE/g), followed methanol (35.42 mg GAE/g) and water (33.63 mg GAE/g). Similar to current results, methanol extract (24 mg RE/g) of
F. eriocarpa contained the highest level of flavonoids. In accordance with our results, several researchers reported that methanol was one of the best solvents to extract flavonoids [
17,
18,
19]. However, spectrophotometric analyses are mainly able to provide an insight into the content of the bioactive compound of herbal extracts without providing detailed phytochemical composition. Besides, a possible interference of non-phenolic compounds characterizing the extracts has been reported yielding false-positive results [
12]. Therefore, in the present study, high performance liquid chromatography coupled to mass spectrometry was used to assess the detailed profile of
F. clypeata extracts. The identification of compounds was performed by HPLC-ESI-QTOF mass spectrometry in negative ionization mode. The tentatively identified compounds from the extracts were summarized in
Table 2, and chromatograms were reported in
Figure 1.
Overall, in our experimental conditions,
51 compounds were tentatively identified, mainly consisting of polyphenols and phenolic derivatives, such as flavonoids and phenolic acids. Interestingly, we also found two glucosinolates (i.e., compounds
4 and
5), namely
p-Methoxy-2-hydroxy-2-phenylethyl glucosinolate and
p-Methoxy-2-hydroxy-2-phenylethyl glucosinolate—desulfo, previously described in crucifer seeds [
22]. In addition,
1,
43, and
57, corresponding to a caffeic acid-
O-hexoside derivative, a caffeyl alcohol
O-glucopyranoside, and a ferulic acid derivative, respectively. These latter were tentatively identified in all the extracts tested. In addition, compounds
6 and
7,
8 and
9, exhibited the same fragmentation patterns and were characterized from the water extract as dihydroxybenzoic acid hexosides and
p-coumaric acid ethyl ester derivatives. The fragmentation of compound
16 corresponded to
p-coumaric acid hexoside, which was tentatively identified from the ethyl acetate extract. Compound
31, with [M-H]- at
m/
z 193, was identified as ferulic acid. Moreover, several sesquiterpenes (i.e., compounds
44,
46, and
51) were also tentatively characterized (
Table 2).
3.2. Enzyme Inhibition Activity
Enzyme inhibitors hold a significant share in clinically approved drugs, and their attractiveness has been associated with their specific roles in several metabolic pathways. Drug discovery and development focus on identifying and optimizing lead compounds that act on specific enzymes [
26]. In the present study, the ability of
F. clypeata extracts to inhibit key enzymes targeted in the management of Alzheimer’s disease, skin hyperpigmentation, and type II diabetes was assessed. About 50 million people worldwide have dementia and Alzheimer’s disease, the most common form of dementia accounts for 60–70% of the cases [
27]. Based on the cholinergic hypothesis, the lack of neurotransmitters hinders connections between neurons and thus affects the brain’s neuronal circuits [
28]. The inhibition of acetylcholinesterase has been advocated in the management of mild cognitive impairment due to Alzheimer’s disease. Clinically approved drugs, such as donepezil, galantamine, and rivastigmine, containing acetylcholinesterase inhibitor as an active ingredient, are currently used in the management of Alzheimer’s disease [
29]. Later, the function of another cholinesterase enzyme, namely, butyrylcholinesterase, has also been evoked. The increased activity of butyrylcholinesterase, up to 120%, in the late stage of Alzheimer’s disease has been related to the wasting away of the brain and aggravation of behavioral and cognitive dysfunction in Alzheimer’s disease patients [
30]. Besides, it has been reported that butyrylcholinesterase could compensate a lack of acetylcholinesterase in the acetylcholinesterase knockout mice model [
31]. These facts have brought the role and need for butyrylcholinesterase inhibition into the limelight. Finding a novel candidate showing both acetylcholinesterase and butyrylcholinesterase inhibitory activity represents an interesting therapeutic strategy for the management of Alzheimer’s disease. However, as reported in the literature, further ad-hoc studies are strongly required to find also possible markers of the neurodegenerative disease [
32]. As can be observed from
Table 3, the ethyl acetate and methanol extracts of
F. clypeata showed inhibitory action against both cholinesterase enzymes.
The methanolic extract (4.87 mg GALAE/g) was most active against acetylcholinesterase. Both the ethyl acetate (3.54 mg GALAE/g) and methanol (3.52 mg GALAE/g) extracts showed comparable inhibition against butyrylcholinesterase. In our previous study, the best cholinesterase inhibition abilities of
F. eriocarpa were reported for ethyl acetate (2.12 mg GALAE/g for AChE and 2.01 mg GAELAE/g for BChE) and methanol (1.83 mg GALAE/g for AChE and 1.08 mg GALAE/g for BChE) extracts [
16]. Based on these values, the ethyl acetate and methanol extracts of
F. clypeata were stronger than
F. eriocarpa. In the literature survey, several researchers reported the significant cholinesterase inhibitory abilities for other solvents (ethyl acetate, chloroform, and methanol, etc.) rather than water [
33,
34,
35]. This fact could be explained to extract low polarity compounds such as alkaloids and terpenoids with these solvents, and they could be more active on cholinesterases.
Likewise, the methanolic and ethyl acetate extracts of
F. clypeata showed potent inhibition against tyrosinase. The inhibition of tyrosinase is crucial in the management of skin hyperpigmentation conditions, such as melasma and freckles. Inhibiting tyrosinase is directly related to the reduced production of the dark brown melanin. Moreover, the increased public interest towards naturally derived products, including cosmetic and dermatological products, has fueled the need for natural tyrosinase inhibitors. Another species of the
Fibigia genus, namely
F. eriocarpa, was previously reported to inhibit tyrosinase [
16]. In contrast to our present findings, the ethyl acetate and methanol extracts of
F. eriocarpa were not active on tyrosinase. This fact could be explained with the differences of chemical profiles in these extracts and the complex interactions of phytochemicals. However, methanolic extracts exhibited stronger actions on tyrosinase in earlier studies conducted by some researchers [
36,
37].
Monitoring hyperglycemia, the hallmark of type II diabetes, is playing a pivotal role in the management of the disease. Apart from dietary modifications, the inhibition of carbohydrate hydrolyzing enzymes assists in maintaining a normal glycemic level. The inhibition of α-amylase, which is situated in the upper gastrointestinal tract and catalyzes the initial breakdown of ingested polysaccharides into oligosaccharides and the inhibition of α-glucosidase, which is situated in the brush border of the small intestine and catalyzes the last step of carbohydrate digestion, significantly prevents glycemic peaks. However, prominent α-amylase inhibition has been associated with gastrointestinal discomforts caused by the fermentation of undigested carbohydrates in the colon. In the present study, a low α-amylase inhibition was observed while the ethyl acetate (22.32 mmol ACAE/g) and methanol (24.68 mmol ACAE/g) extracts of
F. clypeata exhibited potent α-glucosidase inhibitory action. It was noted that, in general, the water extract of
F. clypeata exhibited the lowest enzymatic inhibition. The higher activity of the ethyl acetate and methanol extracts might be related to the synergistic action of the different bioactive compounds present in these extracts. Anti-diabetic properties of the ethyl acetate and methanol extracts of
F. clypeata were stronger than those of
F. eriocarpa (amylase: 0.44 mmol ACAE/g for ethyl acetate and 0.43 mmol ACAE/g for methanol; glucosidase: 5.01 mmol ACAE/g for ethyl acetate and 1.57 mmol ACAE/g for methanol) in our previous paper. Additionally, it is worth mentioning the level of the different bioactive compounds might have also affected enzyme activity. The alkaloidal amine, sinapine, tentatively characterized from the ethyl acetate extract of
F. clypeata was previously reported to inhibit acetylcholinesterase isolated from rat cerebral homogenate with an IC50 of 3.66 µmol/L [
38]. Regarding correlations between total phenolic-flavonoid content with enzymatic assays, we found strong correlation coefficients when considering only TFC (total flavonoid content) values. In this regard, total flavonoids were strongly correlated to AChE (0.870;
p < 0.01), tyrosinase (0.795;
p < 0.05), α-glucosidase (0.771;
p < 0.05), and BChE (0.705;
p < 0.05) inhibition values (
Table S1).
3.3. In Vitro Antioxidant Activity of the Tested Extracts
The role of oxidative stress in the onset and/or progression of human ailments supports the systematic antioxidant evaluation of studied plant extracts. Antioxidants can act by different mechanisms, namely, hydrogen atom transfer, single electron transfer, or transition metal chelation [
39]. In this study, multiple antioxidant assays were used to obtain a comprehensive understanding of the antioxidant properties of the
F. clypeata extracts. As presented in
Table 4, the methanolic extract (96.52 and 109.10 mg TE/g, for DPPH and ABTS, respectively) of
F. clypeata showed potent radical scavenging properties.
Likewise, the methanolic extract exhibited the highest reducing potent in the CUPRAC (154.02 mg TE/g) and FRAP (104.85 mg TE/g) assays. Obtained results could be compared with earlier studies on other
Fibigia or Brassicaceae species. For example, the antioxidant properties in
F. eriocarpa extracts can be ranked methanol > ethyl acetate > water. However, the antioxidant abilities of the water extract of
F. clypeata were stronger than that of
F. eriocarpa. [
16] Besides, different levels for antioxidant properties in some Brassicaceae species have been reported in the literature [
40,
41,
42]. At this point, the chemical profiles of the extracts were closely related to their antioxidant properties. In this sense, several compounds present in
F. clypeata methanolic extract might be responsible for the observed activity. Sinapic acid was reported to exhibit radical scavenging potential [
43]. Caffeic acid has been reported to be an effective radical scavenger and reducing agent as well as a metal chelator [
44]. In terms of metal chelating, the aqueous extract of
F. clypeata showed the highest activity. These findings suggest that bioactive compounds present in the methanolic extract exhibited mostly radical scavenging and reducing potential, while compounds present in the aqueous extract were potential metal chelators. Regarding the potential correlation between TPC and TFC values, we found strong correlation coefficients between TPC and some antioxidant assays, such as DPPH (0.943;
p < 0.01), CUPRAC (0.954;
p < 0.01), FRAP (0.874;
p < 0.01), and ABTS (0.790;
p < 0.05) values. In addition, total flavonoids were inversely correlated to chelating activity (−0.902;
p < 0.01) and strongly correlated to DPPH values (0.876;
p < 0.01) (
Table S1).
3.4. Cell Assays
To assess the anti-cancer potential of
Fibigia clypeata extracts, their cytotoxicity was measured on two cancer cell lines belonging to the group of head and neck squamous cell carcinomas (HNSCC), i.e., squamous cell carcinoma of the pharynx (FaDu) and squamous cell carcinoma of the tongue (SCC-25) and compared with the results obtained for the normal VERO cell line. The results of cytotoxicity evaluation are presented in
Table 5.
In the case of VERO cells, no cytotoxicity was observed for methanolic and aqueous extracts. The ethyl acetate extract showed a cytotoxic effect in concentrations above 125 µg/mL, but it was impossible to calculate the CC50 value because even in the highest tested concentration of 1000 µg/mL the viability of VERO cells was above 50% (
Figure 2).
Similarly, it was impossible to calculate CC50 for aqueous extract on FaDu cells. Both ethyl acetate and methanolic extracts showed selective toxicity towards FaDu cells. In the case of SCC-25 cell line, selective toxicity was observed for all tested extracts. The CC50 and CC10 values of ethyl acetate extract on both cancer cell lines showed a statistically important difference (
p < 0.001). In the case of methanolic extract tested on FaDu and SCC-25, the CC50 values were similar, but a significant difference was found for the CC10 values (
p < 0.001) (
Figure 3 and
Figure 4).
The criteria of classification of plant extract cytotoxicity according to the protocols suggested by the National Cancer Institute (NCI) [
45] and selected literature are shown in
Table 6 [
45,
46,
47,
48].
According to this criterion, all tested extracts can be classified as non-cytotoxic towards the VERO cell line. In the case of cancer cell lines, ethyl acetate and methanolic extracts tested on FaDu and methanolic and aqueous extracts tested on SCC-25 can be classified as weakly cytotoxic. However, the selective activity towards cancer cell lines encourages further investigations focused on the isolation of bioactive constituents and testing their anti-cancer potential. Di Giorgio et al. [
11] studied the immunomodulatory and anti-leishmanial activities of Lebanese plants and reported the cytotoxicity of
F. clypeata extracts assessed on the THP1 human monocyte cell line by colorimetric determination of cell viability using the oxidation–reduction indicator Alamar Blue. The aqueous and dichloromethane extracts showed CC50 of 124.9 and 123.6 µg/mL, whereas in the case of methanol extract, it was above 250 µg/mL.