2.2. Identification of Secondary Metabolites by Reverse Phase Ultra-High Performance Liquid Chromatography Mass Spectrometry (RP-UHPLC-QTOF-MS)
The methanolic extract of
H. crispum was subjected to a UHPLC-MS (positive and negative ionization mode) screening, which resulted in the possible identification of 50 secondary metabolites (
Table 2 and
Table 3). These secondary metabolites belong to different classes such as irodide, pyrrolizidine alkaloids, peptides, pyruvic and coumaric acids, polyphenolic compounds, and long-chain fatty acids. Among the irodides identified through the negative mode, nepetaside (
1) was found to be a known phytochemical [
16], while the unknown compound
2 was tentatively identified as a derivative of
1 (
Scheme 1). UHPLC-MS analysis depicted the molecular formula of compound
1 as C
16H
26O
8 with four double bond equivalents (DBE), while the unknown compound
2 (C
17H
30O
8 with three DBE) was elucidated as though the carbonyl moiety of the ester function might have been reduced to an ether linkage. Further, the mass analysis showed a difference of 16 amu between
1 and
2, which also confirmed the methylation along with a reduction of
1 into
2. This kind of methylation is a common biosynthetic process found in natural products, specifically in irodide classes [
16].
The tentatively identified plant pyrrolizidine alkaloids (in positive ionization mode) were supinine (
3), europine
(4), and heliotrine
(5) [
14]. These pyrrolizidine alkaloids (
Scheme 2) are known phytochemicals from various
Heliotropium species. Pyrrolizidine alkaloid
6 was found to be a new derivative of
4 (C
16H
27NO
6), since the UHPLC-MS data displayed molecular ions for
4 and
6 at
m/z 329.1847 (C
16H
27NO
6) and 331.2003 (C
16H
29NO
6), respectively, which indicated that compound
6 is a reduced form of
4. Thus, on the basis of the molecular formula and the number of DBE, two structures,
6a and
6b are possible; however, structure
6a is more suitable based on its stability factor and the fact that both saturated and unsaturated pyrrolidine rings are reported in pyrrolizidine alkaloids from various
Heliotropium species [
5]. Further, a similarity with structure
b (reduced ester linkage) was not reported in the literature. Therefore, compound
6a is identified as new pyrrolidine alkaloids form this plant. Further compounds
3, 4, 5, and
6a are biosynthetic analogs, as compound
3 is converted into
4 and
5 by simple methylation and hydroxylation, and these reactions are very common in the biosynthesis of alkaloids. Similarly, compound
4 on dehydrogenation gives compound
6a.
A total of 13 known nitrogen-containing secondary metabolites were tentatively identified through negative ionization mode, including mostly cyclic and acyclic peptides derivatives; 5-acetylamino-6-formylamino-3-methyluracil (
7), 1-methylxanthine (
8), and hypoxanthine (
9) of the xanthine family compounds [
17].
N-Acryloylglycine (
10) and orysastrobin (
11) are strobilurins derivatives [
18], which are widely used as a fungicide [
19]. Other identified compounds, including mecarbinzid (
12), the oxidized compound oplophorus luciferin (
13), and biotin-X-NHS (
14), are important secondary metabolites. Carbinoxamine (
15), a natural product derived from carbamic acid, was also found in the extract; however, these compounds are very rare in nature, especially from plant sources. In the past, natural products derived from carbamic acid have been isolated from the Taiwanese plant
Magnolia kachirachirai [
20] and the Pakistani plant
Vincetoxicum stocksii [
21]. The nitrogen-based compounds which were identified through the positive mode of UHPLC-MS analysis include tranexamic acid (
16), 5-pentyloxazole (
17), homoarecoline (
18),
N-(3-oxododecanoyl) homoserine lactone (
19), alizapride (
20),
L-cladinose (
21), N1, N10-dicoumaroylspermidine (
22), 9-acetoxyfukinanolide (
23), and dodemorph (
24).
Among other metabolites, nine secondary metabolites that were tentatively identified from the negative ionization mode of screening were from pyruvic acid derivatives such as
β-hydroxypyruvic acid (
25) and nonic acid (
26). Further coumaric acid derivatives include compounds isobergaptene (
27), 2-hydroxy-3,4-dimethoxybenzoic acid (
28), 3-methoxymandelic acid-4-
O-sulfate (
29),
p-salicylic acid (
30), cis-ferulic acid [arabinosyl-(1→3)-[glucosyl-(1→6)]-glucosyl] ester (
31), and ferulic acid (
32). These important phenolic compounds are known for various potent antioxidant and enzyme inhibitory activities [
22]. The tentatively identified polyphenolic compounds included 2-hydroxy-3,4-dimethoxy-6-methyl-5-(sulfooxy) (
33), 1-hexanol arabinosylglucoside (
34), (S)-chiral alcohol, and (3S)-4-(3-acetyl-5-hydroxy-4-oxo-1,2,3,4-tetra hydronaphthalen-2-yl)-3-hydroxy butanoic acid (
35). Flavonoids which were identified included cimifugin (
36), rivenprost (
37), eupatin 3-
O-sulfate (
38), tamadone (
39), wightin (
40), salvianolic acid A (
41), and lithospermic acid (
42). In addition, eight long-chain fatty acids were also tentatively identified through the negative ionization mode of UHPLC-MS analysis, which included 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid (
43), 5,8,12-trihydroxy-9-octadecenoic acid (
44), 9,16-dihydroxy-palmitic acid (
45), 9,10-epoxy-18-hydroxystearate (
46), α-9(10)-EpODE (
47), 12-oxo-10Z-octadecenoic acid (
48), 9Z,12Z,15E-octadecatrienoic acid (
49), and 6E,9E-octadecadienoic acid (
50).
2.4. Enzyme Inhibition Potential
Interest is increasing in the use of natural enzyme inhibitors to combat global health problems including Alzheimer’s disease, Diabetes mellitus, hyperpigmentation, and hypertension. The prevalence of these diseases is critically increasing worldwide, and thus, effective strategies are required to control these diseases. With this in mind, the discovery of natural and safe enzyme inhibitors is one of the most investigated subjects in science [
24]. Previously, the methanol extract of
H. strigosum showed an amylase inhibition potential with an IC
50 value of 9.97 + 0.01 μg/mL. Heliotropamide, a new alkaloid with a novel oxopyrrolidine-3-carboxamide central moiety, has been isolated as the major product of the dichloromethane extract of
H. ovalifolium aerial parts, which showed inhibitory potential toward acetylcholinesterase [
23]. The whole herb of
H. digynum was tested against a glucosidase enzyme at 25 ppm and showed a weak percentage inhibition [
25]. In the present study, we tested the enzyme inhibitory effects of the
H. crispum extract against cholinesterases, tyrosinase, α-amylase, and α-glucosidase enzymes. The results are illustrated in
Table 4. The extract of
H. crispum possesses the highest activity against AChE (3.80 mg GALAE/g extract) and BChE (3.44 mg GALAE/g extract), while in the α-glucosidase assay, it showed a 1.86 mmol ACAE/g extract inhibitory activity; however, in the α-amylase inhibition assay, the extract was found comparatively less active, with a value of 0.57 mmol ACAE/g extract. The same extract showed the promising tyrosinase inhibition with a 129.65 mg KAE/g extract inhibition potential.
2.5. Docking Results
Docking calculations offer essential quantitative results. The binding affinity and the inhibition constant are among these results. As shown in
Table 5, the binding affinity or the docking free energy along with the inhibition constants are listed for the eight selected compounds. Interestingly, lithospermic acid (
42) and salvianolic acid A (
41) have shown the best binding affinity against the five enzymes which may indicate a potential bioactive role for these compounds.
Figure 1 gives a better insight into the interactions of these compounds at the active site and offers explanation for the relative high binding affinity. Strong non-bonding interactions were found between these compounds and the active site, such as hydrogen bonds, π–π interactions, and electrostatic forces.
In order to investigate the interactions of the highest affinity compounds, lithospermic acid (
42) and salvianolic acid A (
41) were further studied; the affinity of these compounds against the studied enzymes, along with their interactions with the active site of the corresponding enzymes, are shown in
Table 6. Hydrogen bonds and the π–π interactions may represent the strongest non-bonded interactions. Although the highlighted two compounds have shown a similar binding affinity, they may interact with similar or different amino acids at the active site of the enzyme. For example, lithospermic acid (
42) was involved with Asn298, Asp297, Arg204, Leu232, and Gly230 of the α-amylase enzyme, while in the case of salvianolic acid A (
41), the formed hydrogen bonds are with Tyr155, Asp206, Asp340, Glu35, and Tyr79 amino acids. This difference is attributed to the difference in the docked conformations of these compounds, as shown in
Figure 1. The same two compounds have shown different behavior in the case of AChE, in which they have formed hydrogen bonds with the same amino acids, such as Phe295, Phe383, Tyr124, and Ser293. In the case of BChE, α-glucosidase, and tyrosinase enzymes, these two compounds have interacted with different amino acids. Similarly, the π–π interactions of the selected compounds did not follow a specific trend. For example, both compounds formed a π–π interaction with the Phe601 and Pro201 of the active site of α-glucosidase and tyrosinase enzymes, respectively, and with different amino acids of the rest of the enzymes.