Phytochemical Identification of Major Compounds From Leaves of Nicotiana glauca Graham (Solanaceae) Extracts Using LC‐ESI‐MS/MS and Molecular Docking Analysis

ABSTRACT

Nicotiana glauca Graham (Solanaceae), also known as tree tobacco, naturally grows in dry, arid regions of North America, Africa, and Europe. It is recognized for both toxic and medicinal properties. This study aimed to analyze the metabolic profile of crude leaf extracts from N. glauca using liquid chromatography–electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS). A total of 16 different compounds were identified, including five alkaloids, two coumarins, two flavonoids, and two monoterpenes, as well as four hydroxycinnamic acid derivatives and one homoserine lactone. Some of these compounds were reported for the first time in N. glauca . According to the available literature, this is the first study to report the presence of puromycin in the genus Nicotiana. In addition, molecular docking studies were carried out to investigate the active site of human peroxiredoxin 5 (PDB ID: 1HD2), aiming to elucidate the potential interactions between the identified compounds and this receptor. These results suggest that N. glauca could be a good source of natural chemicals for future medical or nutritional research.

1. Introduction

The genus Nicotiana belongs to the family Solanaceae, which includes over 40 species. Among these, Nicotiana tabacum L. and Nicotiana rustica L. are the most commonly known species. The leaves of these species produce tobacco and are widely cultivated as crops [1, 2]. However, Nicotiana glauca is considered the most potent species within the genus. It has traditionally been used in various countries to treat burns, inflammatory diseases, and sore throats, often applied topically or used in the form of smoke for therapeutic or ethno‐veterinary purposes, including painful feet. Additionally, the plant has been employed as a natural insecticide [1, 2, 3]. N. glauca is known for its toxicity to both humans and animals. This toxicity is mainly due to the presence of two alkaloids: nicotine and anabasine, which have similar molecular structures [4].

High‐performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) has proven highly effective for studying and identifying bioactive compounds in medicinal plants. This method enables the detection of minor constituents, which cannot be identified by classical methods. Over the past two decades, LC/MS has been widely used to characterize various components, including terpenoids, alkaloids, saponins, organic acids, and flavonoids in plant species [5].

In previous research, the antioxidant activity of DCM, EA, and n‐BuOH extracts obtained from N. glauca leaves was evaluated [6]. Based on those findings, the present study aims to analyze the metabolite profile of N. glauca leaves using ultra‐high‐performance liquid chromatography ‐electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS). Moreover, in vitro molecular docking studies were conducted to investigate the interactions between selected compounds and the active site of human peroxiredoxin 5 (PDB ID: 1HD2), with the objective of exploring the therapeutic relevance of these phytoconstituents.

2. Results and Discussion

2.1. UHPLC‐ESI‐MS/MS and Data Analysis

To obtain the metabolite profile of the three extracts from N. glauca leaves, an analytical technique based on LC‐ESI‐MS/MS was used. The resulting LC‐ESI‐MS/MS revealed the presence of a large group of compounds related to the protonated molecular ions of various polyphenols. The chromatograms obtained from the analysis of N. glauca extracts in positive electrospray ionization mode are shown in Figure 1.

FIGURE 1.

LC/ESI‐MS/MS chromatogram (in positive ion mode) of N. glauca extracts. (a) DCM, (b) EA extract, and (c) n‐BuOH extract.

The LC/ESI‐MS/MS base peak chromatogram of N. glauca leaf extracts showed a relatively complex mixture of various compounds. In total, 16 compounds were identified in the three extracts (Table 1) by comparing their retention times and fragmentation patterns with reference standards or based on accurate mass measurement and characteristic fragmentation profiles. These compounds include five alkaloids, two coumarins, two flavonoids, two monoterpenes, four hydroxycinnamic acid derivatives, and one homoserine lactone. Most of these compounds were reported in N. glauca for the first time.

TABLE 1.

Characterization of major compounds in three extracts of N. glauca leaves using LC/ESI‐MS/MS in positive ion mode.

Peak label (Figure 1 )	Rt	[M + H]+	MS/MS fragments	Identified compound (NO)	DCM EA n‐BuOH
1(a), (b), (c)	0.75	163.1245	146.0923, 130.0538, 94.0639	Nicotine (1)	+ + +
2(a), (b), (c)	0.90	163.1245	146.0923, 130.0538, 94.0639	Anabasine (2)	+ + +
3(a)	1.20	191.1156	173.1069	ß‐Damascenone (3)	+ − −
3(c)	1.40	251.1316	163.0407	Caffeoyl putrescin (4)	− − +
4(c)	2.20	188.0753	146.0605, 118.0623, 170.0542	Indoleacrylic Acid (5)	− − +
3(b), 5(c)	3.25	163.0407	89.0398	3‐Hydroxycoumarin (6)	− + +
4(b), 6(c)	4.80	472.2303	222.0903, 163.0407	Puromycin (7)	− + +
6(b), 7(c)	5.51	613.2016	303.0413	Rutin (8)	− + +
4(a)	5.75	193.0487	1 780 255, 133.0275	Scopoletin (9)	+ − −
7(b)	5.85	550.1735	287.0530	Kaempferol‐3‐O‐rutinoside (10)	− + −
5(a)	6.10	179.1064	161.0981, 133.0981	Loliolide (11)	+ − −
8(b)	6.20		163.0407	3,4‐Dicaffeoyl‐1,5‐quinolactone (12)	− + −
8(c)	6.45	187.1231	187.1231, 84.0454	3‐oxo‐C4‐homoserine lactone (13)	− − +
6(a)	6.75	314.1398	177.0525, 145.0267	N‐trans‐feruloyltyramine (14)	+ − −
7(a)	7.05	314.1398	177.0583, 145.0267	N‐cisferuloyltyramine (15)	+ − −
8(a)	7.65	275.2137	149.1095	Octanoyl‐nornicotine (16)	+ − −

Note: Peak labels correspond to the chromatographic peaks shown in Figure 1; compound numbers indicate the order of identified compounds in Table 1 according to their retention times (RT)and do not necessarily correspond to the peak labels.

Abbreviations: (+), (−): indicates presence or absence of the compound; 5_(b) and 9_(b): not identified; DCM: dichloromethane extract; EA: ethyl acetate extract; n‐BuOH: butanolic extract; Rt: retention time (min).

2.2. Alkaloids

The employed methodology enabled the identification of five alkaloids.

Compounds 1 and 2 are isomers; they were eluted at 0.75 and 0.90 min, respectively. Furthermore, the MS/MS fragmentation patterns of both compounds are highly similar. The ions observed at m/z 146.0923 and 130.0538 were generated in correspondence to the loss of small molecular groups, such as CH₃, H₂, and NH₃, respectively, from the protonated ion at m/z 163.1245 (Figure S1), characteristic of cyclic amines [7]. Compared to the analytical standard, compound (1) was identified as nicotine and compound (2) as anabasine [8] and has been reported previously in this species [1]. The two alkaloids were seen to be present in all extracts.

Compound 5 was only detected in the n‐BuOH extract, showing a [M + H]⁺ peak at m/z 188.0753 with a retention time of 2.20 min. Compared to the analytical standard, compound (5) was identified as indolacrylic acid and has been detected for the first time in this species. Two major fragmentation products at m/z 146.0605 and 118.0623 (Figure S2) were formed after the successive loss of CH₂CO and CO, respectively. Along with this, the loss of a water molecule and the appearance of a peak at m/z 170.0542 were also observed [9].

Compound 7 was eluted at 4.8 min and showed a [M + H]⁺ peak at m/z 472.2303. A fragment ion was recorded at m/z 163.0407 (Figure S3), which is characteristic of cyclic amines [7]. Based on its MS fragmentation pathway, retention time, and comparison with the analytical standard, compound 7 was identified as (3′‐desoxy‐N,N‐dimethyl‐3′‐(O‐methyl‐L‐tyrosinamido)adenosin) or puromycin (Figure S3). This compound was detected for the first time in this genus and was present in both AE and n‐BuOH extracts.

Compound 16, representing the last peak detected in the DCM extract, showed a [M + H]⁺ peak at m/z 275.2137 and a retention time of 7.65 min. This compound corresponds to a nicotinic derivative, specifically octanoyl‐nornicotine, and is reported for the first time in this species. The fragment ion was observed at m/z 149.1095 (Figure S4), which is a characteristic of cyclic amines [7] and results from the loss of the octanoyl moiety.

2.3. Coumarins

Through this study, two coumarins were identified:

Compound 6 showed a [M + H]⁺ ion at m/z 163.0407 with a retention time of 3.25 min and was, therefore, identified as 3‐hydroxycoumarin. This was the first report of detecting this compound in this species. The major fragment observed at m/z 89.0398 was formed by the neutral loss of H₂O from the protonated ion at m/z 163.0407 (Figure S5) and successive loss of two CO moieties, respectively [10]. This coumarin was detected in both AE and n‐BuOH extracts.

Compound 9 was only detected in the DCM extract, eluted at 5.75 min and recorded at [M + H]⁺ m/z 193.0487. It was, therefore, identified as Scopoletin and has been reported previously in this species [10, 11]. Sequential loss of CH₃, CO, and OH groups led to the formation of fragment ions at m/z 178.0255, 150.0346, and 133.0275, respectively (Figure S6) [12, 13].

2.4. Flavonoids

Among the phenolic compounds, two flavonoids were identified.

Compound 8 was eluted at 5.51 min and was identified as quercetin‐3‐O‐rutinoside (rutin), a compound previously reported in this species [11]. A major fragment ion was generated at m/z 303.0413 (Figure S7), resulting from the loss of a rutinoside moiety from the parent ion [14, 15, 16]. This flavonoid was present in both the AE and n‐BuOH extracts.

Compound 10 was detected only in the AE extract and eluted at 5.85 min. It was identified as kaempferol‐3‐O‐rutinoside based on its accurate mass and MS/MS fragmentation pattern. A characteristic fragment ion at m/z 287.0530 (Figure S8) was generated, corresponding to the loss of the rutinoside moiety from the parent ion [14, 15, 16].

2.5. Monoterpenes

Two monoterpenes were exclusively detected and identified in the DCM extract.

The first compound 3 showed a [M + H]⁺ peak at m/z 191.1156 at a retention time of 1.20 min. Based on the analytical standard mass spectrum, it was identified as ß‐damascenone and has been previously reported in this species [11]. Successive losses of H₂O and CH2=CH2 fragments were observed, producing ions at m/z 173.1069 and 145.0688, respectively (Figure S9). The second one, compound 11, showed a [M + H]⁺peak at m/z 197.1169 with a retention time of 6.10 min. A major fragment ion was detected at m/z 179.1064, corresponding to the loss of an H₂O moiety [17]. The ions observed at m/z 161.0981 and 133.0981 (Figure S10) were generated and attributed to the successive losses of H₂O and CO groups, respectively, from the protonated ion at m/z 197.1169 [18]. Compound 11 was identified as loliolide and reported for the first time in this species.

2.6. Phenolic Acids

This class of compounds represents the second group, following the identification of four hydroxycinnamic acid derivatives:

Compounds 14 and 15, which are isomers, were only detected in the DCM extract, eluting at 6.75 and 7.05 min, respectively. The MS/MS spectra revealed a [M + H]⁺ m/z 314.1398, and the fragmentation pattern of both compounds showed predominant fragment ions at m/z 177.0525 and m/z 145.0267 (Figures S11–S12), characteristic of hydroxycinnamic acid amides [19]. Based on the MS fragmentation pattern, retention time, and the analytical standard, compound 14 was identified as N‐trans‐feruloyltyramine or moupine amid, and compound 15 was reported for the first time in this species.

Compound 4 represents the third detected amid, which was identified only in the n‐BuOH extract. Its mass spectrum showed a [M + H]⁺ ion at m/z 251.1316 (Figure S13) with a retention time of 1.40 min. In comparison with the analytical standard, compound (5) was identified as Caffeoyl putrescin and is reported for the first time in this species. A major fragment ion was observed at m/z 163.0407 (Figure S14), corresponding to an ion of formula C₉H₇O₃ ⁺ [20].

The last one in this group is compound 12, which was detected only in the AE extract and showed a [M + H]⁺ ion at m/z 499 and a retention time of 6.2 min, corresponding to a hydroxycinnamic acid derivative (3,4‐dicaffeoyl‐1,5‐quinolactone). It was identified for the first time in this species by comparing the retention time, MS, and MS/MS data with those of the reference compound. A major fragment peak was observed at m/z 163.0407 (Figure S14), corresponding to the ion with formula C₉H₇O₃ ⁺ [20].

2.7. Homoserin Lactone

Compound 13 represents the final peak detected in the n‐BuOH extract, showing a [M + H]⁺ ion at m/z 187.1231 and a retention time of 6.45 min. Based on the comparison of retention time, MS, and MS/MS data with those of a reference standard, the structure of compound (13) was confidently identified as 3‐oxo‐C4‐homoserine lactone. This compound has not previously been reported in this species, and this is its first report. A major fragment peak was observed at m/z 84.0454 (Figure S15) corresponding to the formula C₉H₇O₃ ⁺ [21].

A comparison of the three extracts revealed distinct phytochemical profiles, reflecting the influence of solvent polarity on metabolite extraction. The DCM extract was the only extract containing both identified monoterpenes (β‐damascenone and loliolide) and was characterized by the presence of hydroxycinnamic acid derivatives (N‐trans‐feruloyltyramine, N‐cis‐feruloyltyramine). These findings indicate that DCM preferentially extracted relatively non‐polar and moderately polar metabolites. The EA extract was distinguished by the exclusive detection of kaempferol‐3‐O‐rutinoside and 3,4‐dicaffeoyl‐1,5‐quinolactone, together with rutin, 3‐hydroxycoumarin, and puromycin. The n‐BuOH extract exhibited a distinctive phytochemical profile, characterized by the detection of indole acrylic acid, caffeoyl putrescin, and the exclusively detected 3‐oxo‐C4‐homoserine lactone. In addition, it contained puromycin, rutin, and 3‐hydroxycoumarin, suggesting that n‐BuOH was particularly effective in recovering relatively polar metabolites. Nicotine and anabasine were the only alkaloids detected in all three extracts. Overall, these findings demonstrate that the qualitative phytochemical composition of N. glauca leaf extracts was strongly influenced by solvent polarity.

2.8. Docking Simulation and ADME (Tox)

The primary objective of molecular docking studies is to identify the most favorable conformation between a protein and a ligand, where their spatial arrangement minimizes the system's total free energy. A lower predicted binding free energy implies stronger protein‐ligand interactions, indicating a potentially higher antioxidant capacity.

The molecular docking binding energy results for the 16 phytochemicals studied against the selected PDB ID: 1HD2 are presented in Table 2. As shown, the binding energies follow this trend: 3,4‐Dicaffeoyl‐1,5‐quinolactone (12) (−6.7 kcal mol⁻¹) < Rutin (8) (−6.2 kcal mol⁻¹) < N‐trans‐Feruloyltyramine (14) (−6.0 kcal mol⁻¹) < Puromycin (7) (−5.9 kcal mol⁻¹) < Kaempferol‐3‐O‐rutinoside (10) (−5.8 kcal mol⁻¹) < Loliolide (11) (−5.2 kcal mol⁻¹) < N‐cis‐Feruloyltyramine (15) (−5.2 kcal mol⁻¹) < Octanoyl‐nornicotine (16) (−5.2 kcal mol⁻¹) < Caffeoyl Putrescin (4) (−5.0 kcal mol⁻¹) < Scopoletin (9) (−5.0 kcal mol⁻¹) < 3‐Hydroxycoumarin (6) (−5.0 kcal mol⁻¹) < Indoleacrylic Acid (5) (−4.9 kcal mol⁻¹) < Anabasine (2) (−4.6 kcal mol⁻¹) < 3‐oxo‐C4‐homoserine lactone (13) (−4.4 kcal mol⁻¹) < β‐Damascenone (3) (−4.2 kcal mol⁻¹) < Nicotine (1) (−4.0 kcal mol⁻¹).

TABLE 2.

Molecular docking results of the studied phytochemicals against human peroxiredoxin 5 (PDB ID: 1HD2).

Compound (NO)	Binding affinity	Hydrogen bond	Hydrophobic interactions
3,4‐Dicaffeoyl‐1,5‐quinolactone (12)	−6.7	GLY46 (2.64)	PHE120 (5.48), LEU149 (5.42)
Rutin (8)	−6.2	GLY46, CYS47, THR147	PRO40, PRO45, ILE119
N‐trans‐Feruloyltyramine (14)	−6	ASN76, ARG124	PHE43, PRO45, ILE119, VAL80
Puromycin (7)	−5.9	ASN76, ILE119, PHE43	PHE43, VAL80
Kaempferol‐3‐O‐rutinoside (10)	−5.8	THR147	ILE119, PRO40
Loliolide (11)	−5.2	CYS47, ARG127
N‐cis‐feruloyltyramine (15)	−5.2	GLY46, THR147, CYS47	PRO45, PRO40
Octanoyl‐nornicotine (16)	−5.2	GLY46, ARG127, THR147	PRO40, PRO45, LEU149, LEU116, PHE120
Caffeoyl putrescin (4)	−5	ALA42, THR147	PHE120
Scopoletin (9)	−5	GLY46, CYS47, ARG127	LEU116, PRO40
3‐Hydroxycoumarin (6)	−5	GLY46, ARG127, THR44, CYS47	LEU116
Indoleacrylic acid (5)	−4.9	GLY46, ARG127, CYS47	PRO45
Anabasine (2)	−4.6	THR147, THR44	LEU149
3‐oxo‐C4‐homoserine lactone (13)	−4.4	GLY46, ARG127, CYS47
ß‐Damascenone (3)	−4.2	GLY46	LEU116, PRO45, PHE120
Nicotine (1)	−4	HR147	PRO40

Note: Binding affinity: kcal mol⁻¹.

3,4‐Dicaffeoyl‐1,5‐quinolactone exhibits the highest binding affinity among the studied phytochemicals (Table 2), with a value of −6.7 kcal mol⁻¹. As depicted in Figure 2, this strong interaction is mainly due to a hydrogen bond with GLY46 at the active site of the protein. Hydrophobic interactions with PHE120 and LEU149 further enhance the binding strength. Rutin had a binding affinity of −6.2 kcal mol⁻¹, indicating a robust interaction with the target protein. It forms hydrogen bonds with GLY46, CYS47, and THR147, which are critical for the binding stability. The hydrophobic interactions with PRO40, PRO45, and ILE119 further reinforce this binding. N‐trans‐Feruloyltyramine has a binding affinity of −6.0 kcal mol⁻¹, forming hydrogen bonds with ASN76 and ARG124. These hydrogen bonds contribute significantly to the binding stability. Additionally, hydrophobic interactions with PHE43, PRO45, ILE119, and VAL80 enhance the binding affinity. Puromycin shows a binding affinity of −5.9 kcal mol⁻¹, interacting via hydrogen bonds with ASN76, ILE119, and PHE43. Hydrophobic interactions with PHE43 and VAL80 further support its binding. Kaempferol‐3‐O‐rutinoside has a binding affinity of −5.8 kcal mol⁻¹. It forms a hydrogen bond with THR147, which helps stabilize the ligand within the binding site. Furthermore, it engages in hydrophobic interactions with ILE119 and PRO40. Loliolide exhibits a binding affinity of −5.2 kcal mol⁻¹. It forms hydrogen bonds with CYS47 and ARG127 but lacks significant hydrophobic interactions. N‐cis‐Feruloyltyramine also shows a binding affinity of −5.2 kcal mol⁻¹, interacting via hydrogen bonds with GLY46, THR147, and CYS47. Hydrophobic interactions with PRO45 and PRO40 further stabilize the binding. Octanoyl‐nornicotine exhibits a binding affinity of −5.2 kcal mol⁻¹. It forms hydrogen bonds with GLY46, ARG127, and THR147, while also engaging in extensive hydrophobic interactions with PRO40, PRO45, LEU149, LEU116, and PHE120. Caffeoyl putrescin shows a binding affinity of −5.0 kcal mol⁻¹, forming hydrogen bonds with ALA42 and THR147. It likewise engages in hydrophobic interactions with PHE120. Scopoletin has a binding affinity of −5.0 kcal mol⁻¹. It forms hydrogen bonds with GLY46, CYS47, and ARG127, and engages in hydrophobic interactions with LEU116 and PRO40. 3‐Hydroxycoumarin exhibits a binding affinity of −5.0 kcal mol⁻¹. It forms hydrogen bonds with GLY46, ARG127, THR44, and CYS47, whereas hydrophobic interactions are observed with LEU116. Indoleacrylic acid shows a binding affinity of −4.9 kcal mol⁻¹, forming hydrogen bonds with GLY46, ARG127, and CYS47. It also engages in hydrophobic interactions with PRO45. Anabasine exhibits a binding affinity of −4.6 kcal mol⁻¹, forming hydrogen bonds with THR147 and THR44. Hydrophobic interactions with LEU149 further stabilize the binding. 3‐oxo‐C4‐homoserine lactone shows a binding affinity of −4.4 kcal mol⁻¹. It forms hydrogen bonds with GLY46, ARG127, and CYS47 but lacks significant hydrophobic interactions. ß‐Damascenone has a binding affinity of −4.2 kcal mol⁻¹, forming a hydrogen bond with GLY46 and engaging in hydrophobic interactions with LEU116, PRO45, and PHE120. Nicotine exhibits the lowest binding affinity at −4.0 kcal mol⁻¹. It forms a hydrogen bond with THR147 and engages in hydrophobic interactions with PRO40. The minimal interaction profile suggests that nicotine does not fit as well within the binding site.

FIGURE 2.

3,4‐Dicaffeoyl‐1,5‐quinolactone/1HD2 complex and the surrounding residues.

Eventually, the ADME‐Tox (Adsorption, Digestion, Metabolism, Excretion and Toxicity) analysis of 3,4‐Dicaffeoyl‐1,5‐quinolactone, which has a molecular weight of 498.44 g/mol, presents a complex profile of pharmacological, toxicological, and physicochemical properties. While exhibiting favorable characteristics, for instance, high human intestinal absorption (0.9551) and affinity for various receptors, including estrogen (Estrogen receptor binding: 0.8110) and androgen receptors (Androgen receptor binding: 0.7716), it similarly poses significant challenges such as low oral bioavailability (0.6714) and potential toxicity towards aquatic organisms (Fish aquatic toxicity: 0.9913). Moreover, its ability to inhibit several transporters and enzymes (e.g., OATP1B1 inhibition: 0.9577, CYP3A4 substrate: 0.5790), coupled with indications of hepatotoxicity (0.5033) and skin corrosion (Skin corrosion: 0.9208), underscores the need for thorough evaluation of its safety and efficacy profiles. What is more, its non‐biodegradable nature (Biodegradation: 0.7000) and low water solubility (Water solubility: −2.651) raise concerns regarding its environmental impact.

Although 3,4‐Dicaffeoyl‐1,5‐quinolactone achieved the highest predicted binding affinity toward 1HD2 in the molecular docking analysis, the difference compared with rutin and other top‐ranked compounds was relatively small. Therefore, these docking results should not be interpreted as definitive evidence of superior biological activity. Rutin is a well‐characterized flavonol glycoside with extensively documented antioxidant activity, mainly attributed to its ability to scavenge free radicals, chelate transition metals, and modulate endogenous antioxidant defense systems [22, 23, 24]. Its antioxidant activity has been largely attributed to the presence of multiple hydroxyl groups capable of donating hydrogen atoms and stabilizing free radicals.

In contrast, experimental studies specifically investigating the antioxidant activity of purified 3,4‐Dicaffeoyl‐1,5‐quinolactone remain limited. Nevertheless, chlorogenic acid lactones and dicaffeoyl quinic acid derivatives have also been reported to possess considerable antioxidant capacity owing to their polyphenolic structure and radical‐scavenging properties [25, 26, 27, 28, 29], suggesting that this compound may also contribute to the antioxidant potential of N. glauca leaf extracts. Consequently, molecular docking analysis should be regarded as a computational approach for prioritizing promising candidates, whereas the actual antioxidant potential of individual compounds should be confirmed through standardized experimental assays.

2.9. Conclusion

The present study provides a comprehensive LC‐ESI‐MS/MS characterization of N. glauca leaf extracts obtained using solvents of different polarities. A total of 16 compounds were identified across the three extracts based on their retention times, accurate mass measurements, and MS/MS fragmentation patterns with reference standards. These compounds comprised: five alkaloids, two coumarins, two flavonoids, two monoterpenes, four hydroxycinnamic acid derivatives, and one homoserine lactone. Among these, several compounds are reported here for the first time in N. glauca leaves including three alkaloids (octanoyl‐nornicotine, indoleacrylic acid, and puromycin) from the three extracts, one coumarin (3‐hydroxycoumarin) detected in EA and n‐BuOH extracts, one monoterpene (loliolide) detected in DCM extract, three hydroxycinnamic acid derivatives (N‐trans‐feruloyltyramine, N‐cis‐feruloyltyramine and caffeoyl putrescin) detected in DCM and n‐BuOH extracts, and one homoserine lactone (3‐oxo‐C4‐homoserine lactone) detected in n‐BuOH extract. Based on the currently available literature, this appears to be the first report of puromycin (3′‐desoxy‐N,N‐dimethyl‐3′‐(O‐methyl‐L‐tyrosinamido)adenosin) in the genus Nicotiana.

Overall, only nicotine and anabasine were common to all three extracts, whereas each solvent yielded a distinct metabolite profile. The DCM extract was characterized by the richness in alkaloids, monoterpenes, and feruloyltyramine derivatives, the EA extract by its higher content of flavonoids, coumarins, and dicaffeoyl quinolactone derivatives, whereas the n‐BuOH extract preferentially recovered highly polar metabolites, including caffeoyl putrescine and homoserine lactone. These results clearly demonstrate that solvent polarity strongly influences the qualitative phytochemical composition of N. glauca leaf extracts. Collectively, the results confirmed that N. glauca leaves constitute a rich source of structurally diverse specialized metabolites, particularly alkaloids and phenolic compounds, with respect to the high expression of these compounds shown by the metabolic profiling analysis presented in this work.

In addition to the phytochemical characterization, the molecular docking analysis identified 3,4‐dicaffeoyl‐1,5‐quinolactone as the highest‐ranked ligand based on its predicted binding affinity toward the selected target protein. However, the differences in docking scores among the highest‐ranked compounds, particularly rutin and feruloyltyramine derivatives, were relatively small, indicating that several identified metabolites may collectively contribute to the predicted biological activity. The complementary ADME‐Tox analysis revealed favorable predicted pharmacokinetic properties together with potential toxicological liabilities for 3,4‐dicaffeoyl‐1,5‐quinolactone, emphasizing the importance of balancing efficacy with safety during candidate selection. Therefore, the docking and ADME‐Tox analyses should be regarded as complementary computational tools for prioritizing promising bioactive candidates rather than as definitive evidence of biological activity. Further in vitro and in vivo studies are required to validate these computational predictions.

3. Experimental Section

3.1. Plant Collection and Extraction

Leaves of N. glauca were collected from El‐Hodna (M'sila, central Algeria) during spring 2016. One kilogram of powdered N. glauca leaves was extracted three times using a methanol‐water mixture (MeOH: H2O) (70:30) at room temperature. The combined filtrates were collected, filtered, and concentrated under reduced pressure at 40°C. The aqueous extract was subjected to liquid‐liquid extraction using solvents of increasing polarity: dichloromethane (DCM), ethyl acetate (EA), and n‐butanol (n‐BuOH). These solvents were selected to separate the highest possible number of chemical constituents into three fractions based on their polarity‐ranging from non‐polar (dichloromethane) to polar (n‐butanol).

3.2. LC‐ESI‐MS/MS and Data Analysis

The three extracts analyzed were obtained using different organic solvents: Dichloromethane (DCM), ethyl acetate (EA), and n‐butanol (n‐BuOH). These extracts were dissolved in methanol at a concentration of 5 mg/mL for analysis by UHPLC‐ESI‐Q‐TOF‐HRMS/MS. A blank sample was also prepared under the same conditions to account for any impurities from the solvents used.

The extracts were separated on a T3 column (2.1‐mm internal diameter, 100‐mm length, 1.8‐μm particle size, from Waters), equipped with a 0.2‐μm prefilter, on a UHPLC system (Thermo Fisher Scientific). The mobile phases were composed of water and acetonitrile, each containing 0.1% formic acid. The LC system was coupled to a hybrid quadrupole‐time of flight (QTOF, Synapt G2 HDMS, Waters) mass spectrometer equipped with an electrospray ionization source. Ionization was performed in positive and negative modes consecutively. Tandem mass spectrometry experiments were realized in data‐dependent mode to obtain structural information on the metabolites detected. Mass measurement precision was guaranteed attributable to the internal calibration based on leucine‐enkephalin introduction with the Lockspray system. The mass range studied was from m/z 50 to m/z 1200.

All analyzes were processed manually using MassLynx, MZMine 2 software, and database searches (HMDB, MassBank, GNPS), as well as the study of MS/MS fragmentation in order to identify the compounds of interest for the study. Analyzes in negative ionization mode were also carried out; however, the chromatograms do not provide any additional information compared to the positive ionization results.

3.3. Docking Simulation and ADME (Tox)

This molecular docking study aimed to predict the antioxidant activity of 16 identified phytochemicals from N. glauca Graham (Solanaceae). Each reported structure was first prepared using the MMFF94 force field theory, and then optimized with the same level of theory in AVOGADRO software [30]. Subsequent to conformational analysis, the best conformer was selected and used as ligands in the molecular docking simulations.

To evaluate their potential antioxidant properties, the studied compounds were docked against the crystal structure of human peroxiredoxin 5 (PDB ID: 1HD2) [31]. The selected protein was corrected by Swiss‐Pdb viewer software after having been prepared by removing all solvent molecules and co‐crystallized ligands by AutoDockTools1.5.2 [32]. The molecular docking of each structure against (PDB ID: 1HD2) was performed by Autodock Vina as implemented in PyRx [33] in tin the following search center (BC) and the box dimensions (BD) in Angstroms: (1HD2; BC: X = 11.83, Y = 37.73, Z = 32.41; BD: X = 28.32, Y = 28.87, Z = 23.33).

The results were visualized with the Discovery Studio Visualizer software [34]. In the end, ADMET properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity) of the most promising compounds were performed using admetSAR 2.0 [35].

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: MS/MS spectrum of nicotine (A) and anabasine (B).

Figure S2: MS/MS spectrum of indoleacrylic acid.

Figure S3: MS/MS spectrum of puromycin.

Figure S4: MS/MS spectrum of octanoyl‐nornicotin.

Figure S5: MS/MS spectrum of 3‐hydroxycoumarin.

Figure S6: MS/MS spectrum of scopoletin.

Figure S7: MS/MS spectrum of rutin.

Figure S8: MS/MS spectrum of kaempferol‐3‐O‐rutinosid.

Figure S9: MS/MS spectrum of ß‐damascenone.

Figure S10: MS/MS spectrum of loliolide.

Figure S11: MS/MS spectrum of N‐cis‐feruloyltyramine.

Figure S12: MS/MS spectrum of N‐trans‐feruloyltyramine.

Figure S13: MS/MS spectrum of caffeoyl putrescin.

Figure S14: MS/MS spectrum of 3,4‐Dicaffeoyl‐1,5‐quinolacton.

Figure S15: MS/MS spectrum of 3‐oxo‐C4‐homoserine lactone.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.