Antioxidant and anti-Alzheimer activities of M. lutea

Experimentally, LEE demonstrated stronger antioxidant activity than FEE in the employed assays, even when compared to the standard Trolox. (IC50 24.42 ± 0.87 µg/ml). The IC50 of LEE was 35.69 ± 1.02 µg/ml compared to 38.39 ± 1.02 µg/ml for flowers utilizing DPPH assay. The leaves had a greater antioxidant potential (16,694.4 ± 2526.7μM TE/mg extract and 70.7 ± 5.4 μM EDTA eq/mg extract) than the flowers (5213.3 ± 517.8 μM TE/mg extract and 7.8 ± 0.7 μM EDTA eq/mg extract), respectively (Table 1).

Table 1 DPPH, ORAC and ferrozine iron metal chelation antioxidant results for LEE and FEE

Similarly, LEE exhibited superior anti-Alzheimer action compared to FEE. LEE indicated a higher level of inhibition of acetylcholinesterase and butyrylcholine esterase with IC50 value 5.252 ± 0.28 ug/ml and 0.025 ± 0.008 ug/ml than that of FEE with IC50 value 17.69 ± 0.93 ug/ml and 0.112 ± 0.022 ug/ml compared to donepezil with IC50 value 2.031 ± 0.11 ug/ml and 0.026 ± 0.003 ug/ml as a positive control, respectively (Table 2), Figs. 2 and 3. In Aβ-Amyloid-42 inhibition assay, LEE showed a greater inhibition IC50 of 12.02 ± 0.56 ug/ml than FEE (IC5O 47.99 ± 2.25 ug/ml) and donepezil (IC50 40.59 ± 1.9 ug/ml) Fig. 4 and (Table 2).

Table 2 IC50 for acetylcholinesterase, butyryl cholinesterase and Aβ amyloid-42 inhibitory activities for FEE and LEEFig. 2

Acetyl cholinesterase inhibition activities of different extracts of M. lutea and donepezil

Fig. 3

Butyryl cholinesterase inhibition activities of LEE, FEE of M. lutea, donepezil and rivastigmine

Fig. 4

Aβ-amyloid-42 inhibition activities of LEE, FEE of M. lutea and donepezil

The data showed that LEE is the most promising drug for preventing Alzheimer’s disease and serving as an antioxidant, and that it should undergo further chemical investigation.

Chemical profiling of M. lutea

Analysis of total flavonoids and phenolics revealed that FEE had a greater total phenolic content (101.9 mg/g extract ± 3.5) than LEE (94.3 mg/g extract ± 5.9). Interestingly, the highest level of total flavonoids was detected in LEE (51.8 mg/g extract ± 2.8) than FEE with (31.7 mg/g extract ± 1) (Table 3). Several research works have demonstrated the diverse pharmacological properties of flavonoids, including their anti-inflammatory, hepatoprotective, antioxidant and anti-angiogenic properties [15]. The flavonoid content of LEE was analysed by UHPLC–ESI–TOF–MS to identify and correlate with the biological activities.

Table 3 Total phenolics and flavonoids contents for M. lutea LEE and FEE

Sixty-two phytoconstituents of different chemical classes were tentatively recognized in the LEE using positive and negative modes (Table 4, Figs. 5, 6). The successfully identified compounds were 36 flavonoids, 11 phenolic acids, 2 terpenoids, 2 phenylpropanoids, 6 polyphenols, 3 coumarins and 2 organic acids placed in order of retention duration (RT). Structures of some compounds identified in UPLC–ESI–TOF–MS are shown in Figs. 7 and 8. For more information about identified compound fragmentation, kindly check (Table 4) in supplementary data.

Table 4 Metabolites identified in M. luteaLEE using Q-TOF LC/MS/MSFig. 5

M. lutea LEE total ion chromatogram. a. Negative mode chromatogram, b. Positive mode chromatogram

Fig. 6

Phytochemical composition of M. lutea LEE determined using LC–MS/Q-TOF

Fig. 7

Selected compounds from different chemical classes identified in M. lutea leaves extract

Fig. 8

Terpenoids and phenylpropanoids identified in M. lutea leaves extract

Flavonoids

Most common compounds found in LEE were flavonoids, mostly identified as O-glycosides. The sugar residues hexose, deoxyhexose, pentose and rutinoside moieties were identified by the mass loss of 162, 146, 132 and 308 amu, respectively [16, 17]. Flavonoids had 36 peaks, belonging to different subclasses: flavanonols, flavonols, flavanones, flavones and isoflavonoids. Table 4 shows the tentatively identified flavonoids and their aglycones. The type of sugar in the glycosidic bond was identified by the mass loss that matched the eliminated sugar residues. (Peak 24) [M–H]− at m/z 623.1973 calculated for C28H32O16 and (peak 27) [M–H]− at m/z 477.1435 calculated for C22H22O12− were determined to be isorhamnetin rutinoside and isorhamnetin hexoside, respectively, using the negative mode of ionization, with characteristic fragments at m/z 315 counted for the aglycone [16, 17]. This isorhamnetin derivative was previously identified in family Bignoniaceae. Numerous pharmacological properties, including antioxidant, anti-inflammatory and anti-tumour properties, have been found for isorhamnetin [18]. Isorhamnetin glycosides could be considered a main reason for antioxidant activity reported in this study.

The presence of hydroxyl groups in the structure of flavonoids has previously been reported to exhibit antioxidant properties, especially flavonols, such as quercetin, which were reported to possess the highest IC50 values due to their higher hydroxyl groups [19]. Various quercetin derivatives (peaks 23, 41, 46) were also detected. The typical quercetin product ion was produced at m/z 301 or 303, respectively, when quercetin derivatives were ionized in a positive or negative mode [15]. As a matter of fact, quercetin hexoside (peak 23), quercetin (peak 41) and hyperoside (quercetin-O-hexoside) (peak 46) have previously been identified in the family [20, 21].

Kaempferol was reported to have a good antioxidant activity that help in myocardial ischaemia [22]. The product ion at m/z 285 that kaempferol glycosides displayed is only seen in negative mode of kaempferol aglycone [23]. For example, the molecular ion [M–H] at m/z 461.1685 and the product ion at m/z 285.04199 were used provisionally to identify kaempferol hexoside (peak 8).

In addition, apigenin and luteolin derivatives were identified. A variety of apigenin derivatives (peak 39) and (peak 48) were identified, as rhoifolin (apigenin-O-neohesperidoside) as previously reported in the family [24]. Apigenin was also previously identified in M. Platycalyx [25] at m/z 577.1915 and 269.0457, respectively [26], while (peak 33 and peak 59) showed molecular ions at m/z 433.1504 and 433.1094, respectively, suggesting that they are, apigenin-O-hexoside and apigenin-C-hexoside (vitexin), respectively. Following that, unique ion peak fragments of aglycone were identified at m/z 271 and 311 owing to the loss of O-glucoside and C-glycoside, respectively [27, 28]. Apigenin-O-hexoside and Apigenin-C-hexoside(vitexin) were identified for first time in this genus.

Also, luteolin-O-hexoside was identified previously in family Bignoniaceae [24]. Both luteolin-O-hexoside (peak 26) and luteolin-di-O-hexoside (peak 10) were identified at m/z value 447.09 and 609.13, with neutral ion loss of couple of hexose molecules (324 m/z) and the characteristic fragmentation for aglycone at 285 [26]. Earlier studies have identified luteolin and its derivatives from genus Markhamia, viz. M. tomentosa [29], M. platycalyx [25] and M. zanzibarica [30], while luteolin-di-O-hexoside has been identified in M. zanzibarica [30] and M. tomentosa [31]. Luteolin was reported to ameliorate oxidative and nitrosative stress and suppress the expression of NF-B, an inflammatory factor [32].

Phenolic acids

Plant phenolic acids are being studied for their potential to be anti-inflammatory, liver protective, antioxidant, anti-bacterial, cardioprotective, anti-diabetic, anticancer and neuroprotective properties [15]. Our study identified several phenolic acids, as well as their derivatives (11 phenolic acids in leaves), mainly hydroxy cinnamic acid and hydroxyl benzoic acid and their derivatives. A characteristic fragment ion peak at m/z 191.0546 corresponding to [C7H11O6]− residue and a molecular ion peak [M − H]− at m/z 353.0872 are detected for chlorogenic acid (peak 16) [18, 33]. Caffeic acid (peak 51) and protocatechuic acid (peak 4) were also identified at m/z 181.123 and m/z 153.0192, respectively. All of these peaks were accompanied by distinct fragments at m/z 109.0180, 135.0395, confirming a CO2 neutral loss [15]. Both caffeic and protocatechuic acid are previously identified in M. platycalyx [25]. Nonetheless, it is interesting to note that this is the first report of both acids in M. lutea. The molecular ion peak [M − H] − at m/z 359.0984 corresponds to rosmarinic acid [32], which was previously reported in M. tomantosa [31] but identified for first time in M. lutea. Peak 7 was determined to be coumaric acid, because it had the typical fragment of coumaric acid at m/z 119 and base peak 163.039 [34]. Coumaric acid was previously identified in family Bignoniaceae but identified for first time in M. lutea. Interestingly, this is the first study to detect phenolic acids in M. lutea detected using Q-TOF LC/MS/MS.

As an effective antioxidant, caffeic acid terminates the chain reaction of lipid peroxidation as well as minimizes its detrimental effects by quenching free radicals and inhibiting their formation. It was also discovered to boost the activities of antioxidant enzymes. Also, both rosmarinic acid and chlorogenic acid are reported to reduce oxidative stress [32].

Polyphenols

M. lutea LEE contained seven polyphenolic components, three of which were anthocyanidins (peaks 21, 34 and 47), one bioflavonoids (peak 19), one stilbene glycoside (peak 36) and Cyanidin-O-rutinoside known as antirrhinin (peak 21) exhibited a pseudo-molecular ion [M + H] + at m/z 595.1667, followed by a peak at m/z 287 due to cyanidin nucleus (− 308 Da). The molecular ion peak of cyanidin-O-hexoside (peak 34) was observed at 449.1086, while the main fragment ion was observed at 287 [(M + H)-162] [15]. Malvidin-hexoside (peak 47) produced a [M + H] + at m/z 493.1346; however, hexose loss caused the fragmentation to give a m/z 331 [35]. Procyanidin B2(peak 19), which was detected by a precursor ion at m/z 579.1451, was previously identified in M. tomantosa [31] and M. platycalyx [36], but fortunately it was first time to be identified in M. lutea. E-3,4,5′-trihydroxy-3′-glucopyranosyl stilbene (peak 36) was found to exhibit [M–H]− at m/z 405.1031, indicating the presence of stilbene derivatives. The polyphenolic components found in M. lutea leaf extract were identified for the first time in this study.

Coumarins

According to the study, three peaks were tentatively identified as coumarins (Peaks 13, 35 and 40) that are esculin, daphnetin [31] and scopoletin [37] depending on precise molecular weights and compared to literature. The compound daphnetin is dihydroxycoumarins that has been identified by MS/MS analysis at m/z 133, 149 and 177 [26]. A coumarin glycoside with MS/MS fragments at m/z 133 was identified as scopoletin [18]. All these coumarins have been identified in M. lutea for the first time.

Phenylpropanoids glycosides

Among the chemical classes identified in the genus Markhamia were phenylpropanoids glycosides. In M. lutea extract, two phenylpropanoids glycosides were tentatively detected as verbascosides (acetosides) and isoverbascosides (isoacteosides), which were assigned to (peaks 50 and 52), respectively. The two isomers are different in the caffeoyl moiety position isoverbascosides, and verbascosides showed the same [M−H]− ion at m/z 623.3208 with characteristic fragments ion peak at m/z 461 due to [C9H6O3]– residue and m/z 315 for further loss of rhamnose sugar moiety loss [38].

Terpenoids

One of the characteristic chemical classes in the genus Markhamia were terpenoids. In M. lutea extract, two terpenoids were tentatively detected as pomolic acid and oleanolic acid. Pomolic acid (m/z 471.3474) and oleanolic acid (m/z 455.3459) were attributed to peaks 61 and 62, respectively, and both showed characteristic fragments at m/z 453 and 407, respectively, due to loss of water (− 18 Da) and (− 60 Da) due to loss of acetate loss, respectively [39]. All these compounds have been isolated from M. lutea [6] but, interstitially, it is the first time to be identified by Q-TOF LC/MS/MS.

Organic acid

A product ion at m/z 115 due to water loss and a molecular ion [M-H] at m/z 133.0136 were found in (Peak 1), and these were tentatively identified as malic acid. The molecular ion of [M–H] at m/z 191.0562 was tentatively recognized as citric acid (peak 3) [40].

Top docking phytoconstituents interaction with AChE and BChE

Phytoconstituents identified by UHPLC–ESI–TOF–MS were screened against both AChE and BChE inhibition using molecular docking. Seven characteristic phytoconstituents showed a higher binding affinity to allosteric site of AChE and BChE with low binding energy compared to the binding energy of donepezil and rivastigmine as Isorhamnetin-O-rutinoside (− 22.0042 kcal/mol), sissotrin (− 20.7694 kcal/mol), 3,5,7-Trihydroxy-4′-methoxyflavone (diosmetin) (− 19.5477 kcal/mol), rosmarinic acid (− 19.3211 kcal/mol), kaempferol hexoside (− 19.0069 kcal/mol), kampferol-7-neohesperosides (− 16.9908 kcal/mol) and acacetin (− 16.4584 kcal/mol) for AChE and isorhamnetin-0-rutinoside (− 29.904 kcal/mol), kampferol-7-neohesperosides (− 23.5882 kcal/mol), rosmarinic acid (− 22.7783 kcal/mol), taxifolin (− 21.4458 kcal/mol), sissotrin (− 19.8249 kcal/mol), kaempferol hexoside (− 20.7694 kcal/mol) and apigenin-O-hexoside (− 19.3567 kcal/mol) for BChE versus donepezil (− 16.01522 kcal/mol), rivastigmine (− 15.38282 kcal/mol) for AChE and donepezil (− 28.6337 kcal/mol), rivastigmine (− 18.5409 kcal/mol) for BChE. The results for both molecular docking against both BChE and AChE are shown in Figs. 9 and 10, respectively. High binding affinity and selectivity were shown by these phytoconstituents with both AD treatment targets, making them a promising lead molecule for anti-AD effects.

Fig. 9

2D binding interaction of AChE with most promising compounds, A Isorhamnetin-o-rutinoside, B sissotrin, C 3,5,7-trihydroxy-4-methoxyflavone, D rosmarinic acid, E kaempferol-7-neohesprosides, F kaempferol hexoside, G acacetin

Fig. 10

2D binding interaction of BChE with most promising compounds, A Apigenin-o-glycoside, B taxifolin, C sissotrin, D kaempferol hexoside, E kaempferol-7-neohesprosides, F isorhamnetin-o-rutinoside, G rosmarinic acid