Evaluation of Biological Activities of Twenty Flavones and In Silico Docking Study

This work aimed to evaluate the biological activities of 20 flavones (M1 to M20) and discuss their structure–activity relationships. In vitro assays were established to assess their numerous biological activities (anti-α-amylase, anti-acetylcholinesterase, anti-xanthine oxidase, anti-superoxide dismutase, and anticancer cell lines (HCT-116, MCF7, OVCAR-3, IGROV-1, and SKOV-3 cells lines)). An in silico docking study was also established in order to find the relationship between the chemical structure and the biological activities. In vitro tests revealed that M5 and M13 were the most active in terms of anti-α-amylase activity (IC50 = 1.2 and 1.4 µM, respectively). M17 was an inhibitor of xanthine oxidase (XOD) and performed better than the reference (allopurinol), at IC50 = 0.9 µM. M7 presented interesting anti-inflammatory (IC50 = 38.5 µM), anti-supriode dismutase (anti-SOD) (IC50 = 31.5 µM), and anti-acetylcholinesterase (IC50 = 10.2 µM) activities. Those abilities were in concordance with its high scavenging activity in antioxidant ABTS and DPPH assays, at IC50 = 6.3 and 5.2 µM, respectively. Selectivity was detected regarding cytotoxic activity for those flavones. M1 (IC50 = 35.9 µM) was a specific inhibitor to the MCF7 cancer cell lines. M3 (IC50 = 44.7 µM) and M15 (IC50 = 45.6 µM) were particularly potent for the OVCAR-3 cell line. M14 (IC50 = 4.6 µM) contributed more clearly to inhibiting the colon cancer cell line (HCT116). M7 (IC50 = 15.6 µM) was especially active against the ovarian SKOV human cancer cell line. The results of the biological activities were supported by means of in silico molecular docking calculations. This investigation analyzed the contribution of the structure–activity of natural flavones in terms of their biological properties, which is important for their future application against diseases.


Introduction
Flavonoids contribute to numerous biological activities through their antioxidant, antimicrobial, anti-allergenic, anti-viral, and anti-inflammatory properties [1]. Their applicability to cancer and Alzheimer's disease treatments is also being considered [2]. Numerous anti-hyperglycemic drugs are used in monotherapy or in combination therapy, which commonly involves α-amylase inhibitors (AAIs) [3]. Using those AAIs is, however, highly expensive and also has side effects on the organism. Indeed, the inhibitory action of acarbose against pancreatic α-amylase-bound starch digestion causes unpleasant gastrointestinal consequences, such as the increased production of gases, flatulence, abdominal pain, and diarrhea. These discomforts can cause the suspension of AAI therapy by patients [3,4]. Therefore, several studies are now focusing on natural substances from plants that are used in traditional medicine for anti-hyperglycemic treatment [5,6]. Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid, which is excreted by the kidneys. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia [7]. The inhibition of XOD activity reduces both vascular oxidative stress and the circulating levels of uric acid. Some natural compounds that are present in plants as phenolics have been reported as XOD inhibitors [7,8]). Alzheimer's disease (AD) is the most common single cause of dementia in our aging society. A total understanding of the mechanism of this disease remains elusive. In the literature, it has been suggested that the β-amyloid protein, abnormal tau protein, or probably both are key factors in the development of AD. At present, the only group of drugs currently authorized for AD treatment is acetylcholinesterase inhibitors. This enzyme controls acetylcholine levels in the neuronal synapses. It has also been also demonstrated that acetylcholinesterase (AChE) is involved in the development of amyloid plaques. Progress in AChE inhibitor drugs followed the finding that cholinergic pathways in the cerebral cortex and basal forebrain are conceded in AD [9].
Owing to toxicity and the severe damage produced in cancer therapy when using non-natural agents, many studies are now focusing on natural compounds with anticancer potential [10,11]. Several important anticancer bio-substances (e.g., paclitaxel, docetaxel; vinblastine, vincristine, topotecan, irinotecan, etoposide, etc.) are being used to treat cancer [12][13][14]. With their anti-cancer specificity, flavonoids have great potential in terms of treatment, and they do not exhibit any considerable post-toxicity consequences [15].

Antioxidant Activity
In this part of the analysis, DPPH radical and ABTS radical cation scavenging activity was measured to assess the antioxidant activity of the 20 flavones (Tables 1 and 2). For the DPPH test, only M7 presented an interesting scavenging activity, at 96.4 ± 0.1% at 100 µM; the IC 50 value was equal to 5.2 ± 0.2 µM. This result was very interesting compared to vitamin C, which expressed a value of IC 50 = 2.3 ± 0.2. In a previous study, M7 showed scavenging ability via a DPPH assay at IC 50 = 21.2 µM [16]. M18 and M8 presented moderate activity (40.8 ± 0.6 and 20.3 ± 4.1% at 100 µM). All the other molecules were inactive at 100 µM. In the ABTS assay, M1, M2, M3, M6, M7, M8, M13, M16, M17, M18, and M19 presented more than 50% inhibition activity (Table 2). M2, M3, M6, M7, M8, and M13 showed the best IC 50 values at 20.0, 24.0, 24.5, 6.3, 7.3, and 24.5 µM, respectively. M7 presented the most important IC 50 value, as seen from the DPPH assay. In the literature, M2 has expressed a similarly low antioxidant activity of 3.8 ± 1.0% for the DPPH assay and 14.6 ± 0.1% for the ABTS assay when tested at 147.78 µM [17]. The rest of these molecules have never been tested for antioxidant activity. The antioxidant activity of phenolic compounds depends on their structure, in particular the number and the positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M7 expressed the best IC 50 value for the DPPH and ABTS assays, compared to M1, which was not active in the DPPH assay and moderately active in the ABTS assay. M7 presented a supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl group position contributes highly to antioxidant activity. The presence of two hydroxyl groups (C-3 and C-5 ) in the B-ring of M8 increased the antioxidant activity in the ABTS assay compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only a single hydroxyl group in the B-ring (C-3 ). M13 and M14 also have two hydroxyl groups, (C-7; C-3 ) and (C-5; C-3 ), respectively. M13 and M14 were less active compared to M8, in which the hydroxyl group is located at C-3 -C-5 . By looking at the structures of the three most strongly antioxidant compounds (M7, M8, and M18), the only common point between these compounds is the presence of the OH group, which is found in three completely different positions. This highlights the finding that activity is also dependent on the general structure of each molecule (conjugated system, accessibility of OH, disposition in space, etc.). positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M7 expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which was not active in the DPPH assay and moderately active in the ABTS assay. M7 presented a supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl group position contributes highly to antioxidant activity. The presence of two hydroxyl groups (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS assay compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only a single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl groups, (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, in which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the three most strongly antioxidant compounds (M7, M8, and M18), the only common point between these compounds is the presence of the OH group, which is found in three completely different positions. This highlights the finding that activity is also dependent on the general structure of each molecule (conjugated system, accessibility of OH, disposition in space, etc.). positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which w not active in the DPPH assay and moderately active in the ABTS assay. M7 presented supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl gro position contributes highly to antioxidant activity. The presence of two hydroxyl grou (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS ass compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl grou (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the thr most strongly antioxidant compounds (M7, M8, and M18), the only common po between these compounds is the presence of the OH group, which is found in thr completely different positions. This highlights the finding that activity is also depende on the general structure of each molecule (conjugated system, accessibility of O disposition in space, etc.). positions of the hydroxyl groups and the nature of substitutions on the aromatic rin expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, wh not active in the DPPH assay and moderately active in the ABTS assay. M7 pres supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxy position contributes highly to antioxidant activity. The presence of two hydroxyl (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABT compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of th most strongly antioxidant compounds (M7, M8, and M18), the only common between these compounds is the presence of the OH group, which is found i completely different positions. This highlights the finding that activity is also dep on the general structure of each molecule (conjugated system, accessibility disposition in space, etc.). positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which w not active in the DPPH assay and moderately active in the ABTS assay. M7 presented supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl gro position contributes highly to antioxidant activity. The presence of two hydroxyl grou (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS ass compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl grou (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the thr most strongly antioxidant compounds (M7, M8, and M18), the only common po between these compounds is the presence of the OH group, which is found in thr completely different positions. This highlights the finding that activity is also depende on the general structure of each molecule (conjugated system, accessibility of O disposition in space, etc.).  positions of the hydroxyl groups and the nature of substitutions on the aromatic rin expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, wh not active in the DPPH assay and moderately active in the ABTS assay. M7 pres supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxy position contributes highly to antioxidant activity. The presence of two hydroxyl (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABT compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of th most strongly antioxidant compounds (M7, M8, and M18), the only common between these compounds is the presence of the OH group, which is found i completely different positions. This highlights the finding that activity is also dep on the general structure of each molecule (conjugated system, accessibility disposition in space, etc.).  positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which w not active in the DPPH assay and moderately active in the ABTS assay. M7 presented supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl gro position contributes highly to antioxidant activity. The presence of two hydroxyl grou (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS ass compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl grou (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the thr most strongly antioxidant compounds (M7, M8, and M18), the only common po between these compounds is the presence of the OH group, which is found in thr completely different positions. This highlights the finding that activity is also depende on the general structure of each molecule (conjugated system, accessibility of O disposition in space, etc.).  positions of the hydroxyl groups and the nature of substitutions on the aromatic ri expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, wh not active in the DPPH assay and moderately active in the ABTS assay. M7 pres supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxy position contributes highly to antioxidant activity. The presence of two hydroxyl (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABT compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of th most strongly antioxidant compounds (M7, M8, and M18), the only commo between these compounds is the presence of the OH group, which is found i completely different positions. This highlights the finding that activity is also dep on the general structure of each molecule (conjugated system, accessibility disposition in space, etc.).
positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which w not active in the DPPH assay and moderately active in the ABTS assay. M7 presented supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl gro position contributes highly to antioxidant activity. The presence of two hydroxyl grou (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS ass compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and onl single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl grou (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8 which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the th most strongly antioxidant compounds (M7, M8, and M18), the only common po between these compounds is the presence of the OH group, which is found in th completely different positions. This highlights the finding that activity is also depende on the general structure of each molecule (conjugated system, accessibility of O disposition in space, etc.).
positions of the hydroxyl groups and the nature of substitutions on the aromatic rin expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, wh not active in the DPPH assay and moderately active in the ABTS assay. M7 pres supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxy position contributes highly to antioxidant activity. The presence of two hydroxyl (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABT compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of th most strongly antioxidant compounds (M7, M8, and M18), the only commo between these compounds is the presence of the OH group, which is found i completely different positions. This highlights the finding that activity is also dep on the general structure of each molecule (conjugated system, accessibility disposition in space, etc.).
positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. M expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, which w not active in the DPPH assay and moderately active in the ABTS assay. M7 presented supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxyl gro position contributes highly to antioxidant activity. The presence of two hydroxyl grou (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABTS ass compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and only single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl grou (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the thr most strongly antioxidant compounds (M7, M8, and M18), the only common po between these compounds is the presence of the OH group, which is found in thr completely different positions. This highlights the finding that activity is also depende on the general structure of each molecule (conjugated system, accessibility of O disposition in space, etc.).  positions of the hydroxyl groups and the nature of substitutions on the aromatic ri expressed the best IC50 value for the DPPH and ABTS assays, compared to M1, wh not active in the DPPH assay and moderately active in the ABTS assay. M7 pres supplemented hydroxyl group in the A-ring at position C-6. This specific hydroxy position contributes highly to antioxidant activity. The presence of two hydroxyl (C-3′ and C-5′) in the B-ring of M8 increased the antioxidant activity in the ABT compared to that of M16. M16 presented methyl groups in C-6 for the A-ring and single hydroxyl group in the B-ring (C-3′). M13 and M14 also have two hydroxyl (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of th most strongly antioxidant compounds (M7, M8, and M18), the only commo between these compounds is the presence of the OH group, which is found completely different positions. This highlights the finding that activity is also dep on the general structure of each molecule (conjugated system, accessibility disposition in space, etc.).   (C-7; C-3′) and (C-5; C-3′), respectively. M13 and M14 were less active compared to M8, in which the hydroxyl group is located at C-3′-C-5′. By looking at the structures of the three most strongly antioxidant compounds (M7, M8, and M18), the only common point between these compounds is the presence of the OH group, which is found in three completely different positions. This highlights the finding that activity is also dependent on the general structure of each molecule (conjugated system, accessibility of OH, disposition in space, etc.).   In total, 14 flavonoids of the 20 that were tested (Table 3) presented interesting antiα-amylase activity (1.2-37.9 µM). M5 and M13 were the most effective flavonoids with IC 50 = 1.2 ± 0.1 and 1.4 ± 0.1 µM, respectively better than acarbose IC 50 = 1.5 ± 0.1 µM. Those molecules presented similar chemical structures, differing just with a hydroxyl group (C-3 ) for M13. M7, M9, M10, and M19 showed a good level of activity. M9 and M10 presented analogous IC 50 = 2.0 ± 0.2 and 2.3 ± 0.1 µM at one-to-one. Those two flavones diverged only in the position of the phenyl group attached to the flavone A-ring (C-5 and C-6, along with C-7 and C-8, corresponding to M9 and M10, respectively). M7 showed a value of IC 50 = 3.4 ± 0.1 µM; however, M1, with a value of IC 50 = 36.6 ± 0.2 µM, presented the nearest chemical structure but was 10 times less active. This C-6 hydroxyl group contributes highly to anti-α-amylase activity. M3, M4, and M5, showed the capacity to inhibit α-amylase to the order of 90.3 ± 3.2, 58.4 ± 1.4, and 90.3 ± 3.2%, respectively, at 100 µM. M5 was tested in a previous study by different protocols [18] at 1 mM and demonstrated porcine pancreas α-amylase inhibition at 4.8 ± 3.6%. However, M3 was less active [18] at 4.5 ± 1.1%, whereas M4 was ineffective at 1 mM. M7 demonstrated a value of IC 50 = 3.4 ± 0.1 µM. In a previous study, M7 displayed 13.2 ± 2.4% of inhibition at 1 mM when α-amylase activity was assayed using the chromogenic substrate, p-nitrophenyl-α-D-maltopentaoside [18]. M13 and M14 having just some differences in hydroxyl-group positions (C-7 and C-5, respectively), were effective against α-amylase (IC 50 = 1.4 ± 0.1 and 6.7 ± 0.9 µM, respectively), but M13 was four times more active. The C-7 hydroxyl position showed more potential in terms of this activity. Two proposed interactions can be attributed to the inhibitory activity of flavonoids, compared to acarbose activity. The first one involves the hydroxyl groups linked to a flavonoid chemical structure that can form a hydrogen bond with the OH groups present in the enzyme's active side chains. The second way consists of a conjugated π-arrangement, which can probably form between the A-C ring of the flavonoids and the active site present in α-amylase. Those two interactions can encumber the reaction between α-amylase and starch and, thus, inhibit starch degradation [19].

Effects of Flavonoids on the Inhibition of SOD and XOD
M8, M12, M13, M14, M16, and M17 (Table 3) were very active against XOD (IC 50 = 0.8-2.3 µM). This flavone series presented similar structures, with two hydroxyl groups differing only in their positions (except in the case of M16 and M17). M12, M13, and M17 showed the best values of IC 50 = 1.2 ± 0.1, 0.9 ± 0.2, and 0.8 ± 0.1 µM, respectively. Those results were better than the reference, allopurinol (IC 50 = 1.3 ± 0.1 µM). M8 and M16 displayed similarities in terms of C-3 hydroxyl position, contributing to enhancing this activity for both flavones. M12 was more effective than M11, differing in hydroxyl number and position and in methoxy group position. The C-4 hydroxyl group present in M12 and/or C-7 methoxy, which is absent in M11, enhanced this activity. M13 and M14, showing both C-3 hydroxyl groups, were both active. In M13, the C-7 hydroxyl group helped to improve the IC 50 value by about four times. M17, with a methoxy group in C-4 compared to M3, was not active. The methoxy group's presence in the B-ring diminished the anti-XOD activity. The results for this flavone group were established for the first time in terms of XOD inhibitory activity. The position and number of the hydroxyl group or others function in the flavones to influence anti-XOD activity. This series of molecules was effective in terms of in vitro anti-XOD activity, but this requires supplementary in vivo experiments to confirm their bio-viability, the mechanism involved, their distribution, and if they have health-related side effects or not. In terms of SOD inhibitory activity, only 9 flavonoids were effective; the expressed percentage varied from 52.3 to 80.9% at 100 µM. M7 and M5 revealed the most remarkable values for IC 50 = 36.5 ± 1.2 and 31.5 ± 0.5 µM, respectively; they presented a common C-7 hydroxyl group. M5 displayed the best results at 74.3 ± 3.1%, whereas M3 and M4 were less effective at 54.8 ± 3.2 and 25.9 ± 2.4%, respectively. The hydroxyl group in the C-7 position contributed markedly to enhancing this activity, compared to the other positions for M3 and M4 in C-6 and C-5. M5, compared to M1, presented a common hydroxyl group in C-7. M1 contained a hydroxyl group in C-5; this group is also present in M15 and negatively affects anti-SOD activity. M17 expressed a higher percentage of inhibition at 80.9 ± 0.2% in 100 µM and IC 50 > 50 µM. M3 also presented a similar structure (without C-4 ), showing 54.8 ± 3.2% of inhibition at 100 µM. Finally, the hydroxyl in the C-7 position is very important for the flavones' anti-SOD activity.

Anti-AChE Activity
In total, 10 molecules were active against AChE (50. .9%) at 100 µM (Table 3). M7 was the most potent in terms of this activity, with a value of IC 50 = 10.2 ± 3.0 µM. M7 presented a supplemented hydroxyl group in position C-6, compared to the less active M1 (45.8 ± 1.3% at 100 µM). The group of M3, M4, and M5 exhibited similar inhibition activities against that enzyme and varied from 40.1 ± 2.8 to 47.7 ± 0.7% at 100 µM. Each hydroxyl, when present on its own, did not allow sufficient activity; however, M7 presented three hydroxyls in C-5-6-7 that contributed to improving anti-AChE activity. Previous in vivo tests on M7 suggested that this flavonoid perfected Aβ1-40-induced dementia in rats and may be a new and promising drug for the treatment of AD [20].

Anti-Inflammatory Activity
Of all the tested molecules, only M7 was effective against the 15-lipoxygenase enzyme (IC 50 = 38.5 ± 2.7 µM). None of the other flavones exhibited activity at 100 µM. All of the tested flavones (except M7) have never been tested for their anti-inflammatory (15lipoxygenase) activity in the literature. M7 presented a three-hydroxyl group in C-5-6-7 that contributed to improving anti-AChE activity, compared to the most similar mono or dihydroxyl flavones (M1, M3, M4, and M5), which were ineffective. M7 displayed a specific chemical structure, with two different groups in the A-ring making it highly polar, and B was clearly apolar. M7 is a natural product extracted from Scutellaria baicalensis and is also present in herbs used in traditional Chinese medicine against inflammation, hypertension, cardiovascular diseases, and bacterial and viral infection treatments [21]. M7 inhibited 12-lipoxygenase activity (IC 50 = 1 µM) in a previous report, which may contribute to Alzheimer's disease prevention [22].

In Silico Docking Study
To investigate the possible binding interactions of the flavonoids M1-M20 with the catalytic site of the targets and to elucidate the related inhibitory effect, we performed molecular docking, which is regarded as a powerful computational technique in structurebased drug design. The flavonoids M1-M20 showed different binding energy values against acetylcholinesterase (4EY7), superoxide dismutase (1CB4), xanthine oxidase (1FIQ), 15-lipoxygenase (3V99), and α-amylase (5e0F) ( Table 5). We calculated the superimposed RMSD for the docking of the co-crystallized ligand. The results showed good values of 0.951, 0.528, 0.595, and 1.178, respectively, for 4EY7, 5E09, 3V99, and 1FIQ (PDB 1CB4 does not have a co-crystallized ligand). In the current paper, we investigated the interaction modes of the most potent compounds. For acetylcholinesterase, the most potent compound, M7 presented suitable interactions with the different acid residues and the results are shown in Figure 3. The reported results (Table 5) confirmed that compound M7, displaying potent inhibitory effects, fits perfectly at the active site of acetylcholinesterase, with a binding energy of −10.6 kcal mol −1 . Compound M7 shows one H-bonding between the oxygen (OH group) of the ligand and Ser203 (2.18 Å) (Figure 3). The rings of the ligand show π-π interactions with Tyr341, Trp286, and Phe338. Donepezil, [24] a reference drug, shows interactions with Phe295, Trp86, Trp286, Tyr337, Phe338, and Tyr341. Our results indicated that compound M7 forms interactions with Trp286, Phe338, Tyr341, Phe295, and Tyr337. These interactions are involved in complex stabilization and contribute to its inhibitory effect. For 4EY7, we noted that most compounds have a lower docking score than M7. For M9, M10, M15, M16, and M19, which have a higher docking score, we noted that they have fewer interactions with the key amino acids than M7. Regarding superoxide dismutase, the most potent compound is also M7. Figure 4 presents the numerous interactions of M7 with the protein. The corresponding binding energy is −7.4 kcal mol −1 . There are two hydrogen bonds formed between the (OH group) of the ligand and Ser105 (2.56 Å and 2.15 Å). There is one hydrogen bond between the oxygen of the carbonyl group and Ala1 (2.15 Å). We also found a π-alkyl interaction with Ile111 and a carbon-hydrogen interaction with Ser105. It was shown that their compounds demonstrate numerous activities with SOD and that they also have interactions with Ser109, Arg113, Tyr108, and Glu 107 [25]. Our compound presented interactions with similar amino acids: Ser109, Arg113, and Tyr108. In the case of 1CB4, M3, M5, M7, M9, and M19, these fitted well into the catalytic site. M7 has more interactions with the catalytic sites than the remaining compounds in this group. The best compound for xanthine oxidase activity was M17. The interactions of M17 with xanthine oxidase are presented in Figure 5. The binding energy is −9.3 kcal mol −1 . M17 has two hydrogen bonds with Glu1261 (2.54 Å) and Thr1010 (1.75 Å). It also has π-π interactions with Phe914 and Phe1009, along with Pi Alkyl interactions with Leu873, Val1011, Leu648, and Ala1078, as well as Pi sigma interactions with Ala1079 and Leu1014. Joshi et al. [26] found that their two best-performing compounds have interactions with Phe914 and Phe1009 and that a similar type of π-π interaction was observed in the xanthine oxidase active site with allopurinol and salicylic acid [26]. These same interactions are found in our compound with the protein and contribute to its inhibitory effect. In the case of 1FIQ, M8, M12, M13, M14, M16, and M17, which demonstrate good in vitro activity, fit very well with the catalytic site, while M17 presents more interactions than the other compounds. The remaining compounds have different orientations when compared to M17. M7 is the most active compound, with 15-lipoxygenase. It has a binding energy of −7.5 kcal mol −1 . M7 interactions with the protein are shown in Figure 6. M7 has two hydrogen bonds with Asn554 (2.12 and 2.00 Å) and one hydrogen bond with Val671 (2.35 Å). It has π-π interactions with Phe177 and His372, as well as π-sigma interaction with Ala410. Chaudhry et al. studied the interactions of the compound they developed and found that M7 was the most active LOX inhibitor [27]. They observed that it interacted with the Fe atom and also noticed its interactions with Leu368, Ala672, and Ala410. Our compound showed interactions with similar amino acids: Ala410 and Ala672. It also demonstrates an interaction with the Fe atom. In the case of 3V99, among all the compounds, only M7 fits very well in the active site and has three hydrogen bonds with key amino acids. This condition is not found in the other compounds; only M7 shows biological activity for this protein. M5 is the most active compound for α-amylase. It has a binding energy equal to −8.5 kcal mol −1 . Figure 7 presents the amino acids of the α-amylase surrounding M5. There is one hydrogen bond between the oxygen of the carbonyl group of the ligand and Gln63 (2.35 Å). There are π-π interactions between the compound and Trp59 and Tyr62.

Preparation of Molecules
All samples of the molecules (Table 1) were prepared and dissolved in DMSO (2.5%) to a final solution of 100 µM and were aliquoted at the appropriate volumes for each assay,

Antioxidant Activity and Biological Activities
All tests were performed in triplicate according to the methods specified by El Euch et al. [30].

Spectrophotometry
All optical density assays were performed using a Thermo Scientific Multiskan GO UV/Vis spectrophotometer microplate, allowing wavelength selection for 96-well plates.
Free Radical Scavenging Activity DPPH• (1-1-diphenyl 2-picryl hydrazyl) assay This method is based on the spectrophotometric assay using the stable radical, DPPH•, as a reagent. First, 20 µL of each sample (4 mg/mL) was mixed with 180 µL of 0.1 mM DPPH• in methanol, in plates, after an incubation of 25 min in the dark. Absorbance was measured at 517 nm. Ascorbic acid was used as the reference compound. The radicalscavenging activities of molecules, expressed as a percentage inhibition of DPPH•, were calculated according to the following formula: where A is the absorbance determined at 517 nm. The absorbance of the solvent and DPPH• radical without the molecule was measured as the control.
ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonate) assay The radical scavenging capacity of antioxidants for the ABTS radical cation was evaluated following this method: ABTS was prepared by adding 7 mM solution of ABTS at pH 7.4 (5 mM NaH 2 PO 4 , 5 mM Na 2 HPO 4 , and 154 mM NaCl) with 2.5 mM potassium persulfate. The mixture was stored at room temperature for 16 h in the dark before use and prepared the evening of the assay. Once diluted with persulfate buffer, the absorbance of the mixture yielded an absorbance value of 0.70 ± 0.05 units at 734 nm. Then, 20 µL of each tested sample was mixed with fresh ABTS solution (180 µL), then the absorbance was measured 6 min after the initial mixing. Ascorbic acid was used as a standard. The capacity of free radical scavenging was expressed as a percentage of the inhibition values. The percentage of inhibition is calculated using the same expression employed in the DPPH assay.

Anti-α-Amylase Activity
The α-amylase inhibitory activity (%) value was defined as the percentage decrease in the maltose production rate over the control. Fifty microliters of each sample (at different concentrations of 25, 50, and 100 µg/mL) was tested and added to 50 µL of α-amylase enzyme (1U/mL), then the mixture was incubated for 15 min at 25 • C. Then, we added 100 µL of the substrate starch solution (at 1%, prepared in the same buffer solution under heating at 60 • C and agitation). Finally, we added 100 µL of DNS color reagent solution (96 mM 3,5-dinitrosalicylic acid and 5.31 M sodium potassium tartrate in 2M NaOH) in an Eppendorf tube, putting this in a water bath heated to 100 • C. After 15 min, this mixture was removed from the water bath and diluted with 900 µL of distilled water. The anti-α-amylase activity was determined by measuring the absorbance at 540 nm. Blank incubations were determined as the absorbance produced by samples where the enzyme was replaced with a buffer solution. The total enzyme activity (100%) was assayed with the same procedure, supplementing the sample with DMSO (at 2.5% in the final reaction mixture). Acarbose was used as the positive control. The IC 50 values were calculated from the mean values of the percentage of α-amylase inhibition data, determined in triplicate.

Anti-Xanthine Oxidase Activity
Anti-xanthine oxidase activity was assayed spectrophotometrically under aerobic conditions. First, 50 µL (50 µg/mL) of samples were added to 60 µL of phosphate buffer (pH 7.5) and 30 µL of xanthine oxidase enzyme solution (0.01 units/mL in phosphate buffer; pH 7.5). After pre-incubation at 25 • C for 15 min, the reaction was initiated by the addition of 60 µL of xanthine substrate solution (prepared in the same buffer). The assay mixture was incubated at 25 • C for 5 min. The absorbance was measured at 295 nm. The assay was performed in triplicate. Allopurinol is an effective inhibitor of the enzyme xanthine oxidase (XOD), used here as the positive control. Tests were carried out in triplicate.

Anti-Superoxide Dismutase Activity
Pyrogallol auto-oxidation due to oxygen in the air can be inhibited by an enzyme that is naturally present in the body: superoxide dismutase (SOD). Furthermore, variations in the pyrogallol auto-oxidation rate in the presence of SOD were evaluated. Tests were carried out in triplicate. The objective was to find an inhibitor of the auto-oxidation of pyrogallol in the presence of a SOD enzyme solution. Samples were tested at a concentration of 50 µg/mL, followed by incubation for 4 min at 25 • C. The reaction was initiated by the addition of 30 µL of pyrogallol (30 mM) and the absorbance was measured at 325 nm over 4 min. The self-oxidation of pyrogallol was evaluated in the absence of the enzyme. For this, 30 µL of pyrogallol was added to 120 µL of tris buffer (in place of the SOD) (pH 8.5), then followed by the same steps of incubating a positive control (without the molecule); this was performed in the presence of 5% of DMSO to evaluate the percentage of SOD inhibition activity on the auto-oxidation of pyrogallol.

Anti-Cholinesterase Activity
The enzymatic activity was assessed via a modified colorimetric Ellman s method. First, 50 µL of Tris-HCl buffer (pH 8), 25µL of a buffer solution of the sample (50 µg/mL), and 25 µL of an acetylcholinesterase (AChE) enzyme solution containing 2.8 U/mL were mixed. A reaction was then initiated via the addition of 125 µL of 3 mM 5-5 -thiobis-2-nitrobenzoic acid. After incubation for 15 min at 25 • C, 25 µL of a solution of iodide acetylthiocholine15 mM was added to a 96-well microplate. The absorbance of the mixture was measured at 412 nm after 10 min. A control mixture was prepared in a similar way but using DMSO instead of the sample. Inhibition (%) was calculated in the following way: where (A sample) is the absorbance of the molecule containing the reaction and (A control) is the absorbance of the reaction control. Tests were carried out in triplicate.

Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated via the spectrophotometric measurement of the conjugated diene obtained via the oxidation of linoleic acid by 20 µL of the 5lipoxygenease enzyme (5-Lox). First, 20 µL of the sample was tested individually with sodium phosphate buffer (pH 7.4) containing 5-Lox (500 U) and 60 µL of linoleic acid (3.5 mM), and 170 µL of potassium phosphate buffer solution (0.1 M, pH 7.4). The mixture was then incubated at 25 • C for 10 min and the absorbance was determined at 234 nm. We determined the percentage of inhibition for each sample. Nordihydroguaiaretic acid (NDGA) was used as a positive control. All tests were carried out in triplicate. The percentage inhibition of enzyme activity was calculated using the same method of anticholinesterase activity.

Cytotoxic Activity
Cell lines The cancer cell lines used in this study were HCT116 (colorectal carcinoma), IGROV1 (ovarian cancer), OVCAR3 (ovarian cancer), and MCF7 (mammary gland/breast cancer). The first three lines were maintained in RPMI-1640 medium supplemented with 10% FCS, with 2 mM L-glutamine as a complete growth medium, while the base medium for the MCF7 cell line was DMEM (Dulbecco's Modified Eagle's medium), with phenol red and 10% fetal bovine serum. Cell lines were maintained in culture and were incubated at 37 • C in an incubator with 5% CO 2 in a humidified atmosphere. All tests were established at 80% of confluence, and viability was estimated with a trypan blue exclusion assay.
Cytotoxic Activity Evaluation with an MTT Assay The cytotoxic activities of molecules against cancer cell lines were evaluated using an MTT assay. The MTT colorimetric assay was performed using 96-well plates. Cells were seeded in a 96-well plate, at a concentration of 10 × 10 3 cells/well for HCT116 and 12 × 10 3 cells/well for MCF7, IGROV1, and OVAR. Adherent cells were dispatched and incubated at 37 • C overnight in a 5% CO 2 -enriched atmosphere. Cells in the exponential growth phase were incubated at 37 • C for 72 h, with each tested sample at 50 µg/mL. After that, the medium was removed, and cells were treated with 50 µL of 3-(4,5-dimethylthazolk-2yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (3 mg/mL in PBS) at 37 • C for 20 to 40 min. To dissolve the cells' mitochondria and thereby precipitate the violet formazan, we added 80 µL of 100% DMSO. Optical density was measured at 540 nm. All tests were established in triplicate. The anti-cancer effect of samples was estimated in terms of growth inhibition percentage. We utilized tamoxifen as an anti-cancer drug solution reference.

Statistical Analysis
All data were analyzed using Microsoft Excel for calculating the means, standard deviation, and correlation coefficient (R 2 ).

Conclusions
The biological activities of the 20 flavones tested herein demonstrated the important role of hydroxyl group positions and numbers in improving their inhibitory activities (antioxidant, anti-SOD, anti-XOD, anti-AChE, anti-inflammatory, and anti-cancer activities against different cancer cell lines). Several molecules display interesting capabilities, depending on the enzyme or cancer cell lines. We can attest that M7 displayed higher capability in the in vitro tests for antioxidant, anti-inflammatory, and anti-Alzheimer's activity. Studies by means of molecular docking supported the inhibitory potential of the most potent compound for each biological activity. The study of bioactive molecules is a very important point when assessing future in vivo activities.