5.1 Plant materials

The ultra-low nicotine N. tabacum was provided by Dr. Xue-Yi Sui [11].

5.2 General experimental procedures

Nuclear magnetic resonance (NMR) spectra, including 1H, 13C, DEPT, 1H-1H COSY, HSQC, HMBC, and ROESY, were recorded using a Bruker Avance III 500 spectrometer (Bruker, Zurich, Switzerland), employing tetramethylsilane (TMS) as the internal standard. Infrared (IR) measurements were obtained with a Bruker Tensor-27 spectrometer (Bruker, Germany). High-resolution electrospray ionization mass spectrometry (HRESIMS) analyses were conducted on an Agilent Q-TOF mass spectrometer (Agilent, Redwood City, CA, USA). Ultraviolet (UV) data was collected using a thermal multimode microplate reader (Waltham, MA, USA). Column chromatography was carried out using MCI gel (CHP-20P, Mitsubishi Chemical Industries Co., Ltd., Japan), silica gel (200-300 mesh, Qingdao Marine Chemical Plant, Qingdao, China), and reversed-phase C18 silica gel (YMC Group). Analytical high-performance liquid chromatography (HPLC) was performed on Waters 2695 (Waters Associates, Massachusetts, USA) and Shimadzu LC-20 (Shimadzu, Japan) HPLC systems. The MAO-B inhibition assay was conducted by a Thermo Scientific Varioskan Flash (Waltham, MA, USA). The instruments, reagents, and general experimental procedures used in this study were the same as those detailed in our previous reports [38, 39].

5.3 Extraction and isolation

The dried leaves of the N. tabacum were ground into a powder weighing 28 kg. This powder underwent extraction three times with 50 L of methanol at ambient temperature. After extraction, the filtrates from each round were combined and evaporated under reduced pressure, resulting in 10 kg of extract. The extract was processed using silica gel column chromatography (CC) with CH₂Cl₂/MeOH eluent (1:0, 500:1, 200:1, 50:1, 20:1, 5:1, 0:1, v/v), producing seven distinct fractions (Fr. 1–Fr. 7).

Fr. 3, with a mass of 146.4 g, was further partitioned using MCI gel CC employing a MeOH/H₂O eluent (from 3:10 to 1:0, v/v), producing eight subfractions (Fr. 3.1–Fr. 3.8). Sub-fraction Fr. 3.6 (5.6 g) underwent chromatographic separation on Sephadex LH-20 with a mobile phase CH₂Cl₂/MeOH (1:1) and then purified by semi-preparative HPLC using MeCN/H₂O (6:10, v/v, 2 mL/min). This procedure resulted in the isolation of compounds 8 (8.6 mg, tR 23 min), 9 (4.3 mg, tR 28 min), and 22 (12.3 mg, tR 32 min).

Compound 3 was directly crystallized from Fr. 4. The supernatant of Fr. 4, weighing 455.7 g, was further fractionated using MCI gel CC with MeOH/H₂O (from 3:10 to 1:0, v/v), giving eight fractions (Fr. 4.1–Fr. 4.8). Sub-fraction Fr. 4.4 was partitioned using Sephadex LH-20 with MeOH and then further purified by semi-preparative HPLC with a solvent system of n-Hexane/isopropanol (1:10, v/v, 2 mL/min), yielding compounds 4 (8.4 mg, tR 18 min) and 5 (5.5 mg, tR 19 min). Subfraction Fr. 4.6 was chromatographed on silica gel CC with CH₂Cl₂/MeOH (from 200:1 to 0:1, v/v), resulting in eight subfractions (Fr. 4.6.1–Fr. 4.6.8). Fr. 4.6.4 was fractionated on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) and then purified by semi-preparative HPLC with MeCN/H₂O (30:70, v/v, 2 mL/min), yielding compounds 1 (5.6 mg, tR 15 min), 14 (6.5 mg, tR 21 min), and 15 (11.1 mg, tR 8 min).

Fr. 5, weighing 137.0 g, was further fractionated into five subfractions (Fr. 5.1–Fr. 5.5) using MCI gel CC with MeOH/H₂O (from 6:10 to 1:0, v/v). The subfraction Fr. 5.2, weighing 12.6 g, was first purified by silica gel CC with CH₂Cl₂/MeOH (20:1, v/v) and then by semi-preparative HPLC with MeCN/H₂O (6:10, v/v, 2 mL/min). This purification process led to the isolation of compounds 2 (23.0 mg, tR 45 min), 6 (32.6 mg, tR 26 min), 7 (30.6 mg, tR 18 min), 10/11 (2.6 mg, tR 22 min), 12/13 (2.5 mg, tR 24 min), 16 (8.9 mg, tR 23 min), 17 (5.2 mg, tR 25 min), 18 (3.3 mg, tR 20 min), 19 (12.6 mg, tR 28 min), 20 (15.6 mg, tR 32 min), and 21 (2.0 mg, tR 35 min).

5.3.1 Nicotiazanorpenoid A (1)

Colorless oil; IR (KBr) νmax 3446, 3437, 2959, 2921, 2853, 1637, 1543, 1456, 1435, 1420, 1384, 1316, 1261, 1164, 1095, 1047, 1030, 877, 860, 800, 675, 661, 619, and 421 cm− 1; UV (MeOH) λmax (log ε) 196 (0.50) nm; 1H and 13C NMR (CDCl3) spectra data see Table 3; HRESIMS: m/z 381.2768 [M + Na]+ (calcd for C24H38O2Na, 381.2764).

Table 3 1H (500 MHz) and 13C (125 MHz) NMR data of compound 1 in CDCl35.4 MAO-B assays

The MAO-B inhibition assay was performed on 96-well microtiter plates according to the procedure detailed in our prior report [40]. In preparing the microplates, 50 μL of MAO-B (2.5 U/mL) was combined with 100 μL of the test compounds at different concentrations. This mixture was incubated at 37 °C for 10 min. DMSO was the negative control, while safinamide was the positive control. Next, 50 μL of kynuramine (0.2 mM) sourced from Macklin in Shanghai, China, was added to the mixture. The resulting mixture was incubated at 37 °C for an additional 30 min. The reaction was halted by adding 80 μL of 2 N NaOH. Ultimately, the enzyme activity was measured using a microplate reader, with the excitation and emission wavelengths set at 310 nm and 400 nm, respectively.

5.5 Kinetic studies of MAO-B inhibition

The kinetic study of MAO-B inhibition was the Lineweaver–Burk plot, which was performed using the Lineweaver–Burk curve method. Compounds 3 and 10/11 with six different concentrations (0, 1/4 × IC50, 1/2 × IC50, 3/4 × IC50, 1 × IC50, and 5/4 × IC50) were added into the assay solution with a series of increasing concentrations of kynuramine. Kinetic characterization of their MAO-B inhibition was recorded 20 min after initiation. Constants Kis and Ki were calculated using the Lineweaver–Burk plots.

5.6 Docking studies

Docking studies were performed to explore the interactions between compounds 3 and 10/11 with MAO-B [41]. Among them, compounds 10 and 11 are a pair of enantiomers. Compounds 3, 10 and 11 were constructed in ChemDraw 4.5, and their three-dimensional (3D) structures were optimized by Chem3D 4.5 for molecular energy minimization using the MM2 force field. Structural data for MAO-B (PDB ID: 6YT2) was obtained from the Protein Data Bank (https://www.rcsb.org/structure). Molecular docking was conducted using the Glide functionalities within Schrödinger Maestro software.

5.7 Molecular simulation studies

Molecular dynamics (MD) simulations were performed using Desmond 2020.1 (Schrödinger) to investigate the interactions of compounds 3, 10, and 11 with MAO-B [42]. The OPLS-2005 force field and TIP3P explicit solvent model were applied within a periodic boundary solvation box (10 × 10 × 10 Å). Protein–ligand complexes underwent structural optimization and energy minimization via the Protein Preparation Wizard, followed by system assembly using the System Builder tool. Equilibration involved a 10 ns NVT ensemble phase for conformational stabilization and a subsequent 12 ns NPT ensemble phase for pressure–temperature equilibration, with temperature (300 K) and pressure (1 atm) regulated by the Nose–Hoover thermostat. Long-range electrostatic interactions were computed using the Particle Mesh Ewald (PME) method (9 Å cutoff), while pressure control employed the Martyna-Tuckerman-Klein scheme with a 2-fs timestep. A 100 ns production simulation was conducted, with system stability assessed through root mean square deviation (RMSD), radius of gyration (Rg), residue-specific root mean square fluctuation (RMSF), and hydrogen bond occupancy analysis, enabling comprehensive evaluation of ligand binding dynamics and structural integrity.

5.8 Cell viability assays

Two-dimensional (2D) cell culture models were used for performing cell viability assays [38]. Approximately 10,000 PC12 cells (Fenghui, Changsha, China) were seeded into each well of a 96-well plate. The cells underwent pre-treatment with the test compounds for 2 h at 37 °C. After this pre-treatment step, the cells were subjected to 300 μM 6-OHDA (Aladdin, Shanghai, China) for 24 h. The plate was incubated at 37 °C for 1 h after adding 10 μL of CCK-8 solution (ProteinTech, Chicago, USA) to each well. Then, each well's optical density (OD) was measured at 450 nm using a multimode microplate reader (Waltham, MA, USA).

5.9 Antioxidative activity

The antioxidative capacity of the obtained compounds was carried out through DPPH radical scavenging assays and ABTS assay (Macklin, Shanghai, China) with some modifications, and Vitamin C was used as the positive control [43,44,45]. In the DPPH assay, 60 μL of the isolates at varying concentrations were mixed with 100 μL of 100 μM DPPH solution in ethanol in a 96-well microplate. The mixture was shaken for 10 s and then allowed to sit in darkness at 30 °C for 5 min. Subsequently, the absorbance was determined using a microplate reader at a wavelength of 517 nm. The radical-scavenging activity of the compounds was assessed using the formula RSA (%) = [(AB-AA)/AB] × 100%, where AA was the absorbance of the sample and AB was the absorbance of the blank sample. The IC50 value was calculated with GraphPad Prism 7.0, and all experiments were conducted in triplicate.

In the ABTS+ assay, the ABTS solution was diluted using 95% methanol until it had an absorbance of 0.7 ± 0.02 at 734 nm. Various concentrations of the compounds (60 μL each) were then combined with 150 μL of the diluted ABTS+ solution. The reaction was maintained in the dark at 30 °C for 6 min. Afterwards, the absorbance was determined at 734 nm with a microplate reader. The calculation of ABTS radical scavenging activity followed the same method as for DPPH radical scavenging activity. IC50 values were determined using GraphPad Prism 7.0, and all tests were conducted in triplicate.

5.10 Chromatographic and mass spectrometric conditions

The dried leaves of the flue-cured Yunyan 87 and Nic1-2Nic2-2 were freeze-dried and subjected to ultra-fine grinding. 10.0 g aliquot of the N. tabacum leaf powder was accurately weighed, and methanol solvent was added at a solid-to-liquid ratio of 1:5 (w/v). Ultrasonic-assisted extraction was performed for 30 min per cycle. After filtration through a membrane filter, the extraction process was repeated three times. The three extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain the methanolic extract paste.

The chromatographic column was Luna Omega 3 μm Polar C18 100 Å 100 × 2.1 mm. The flow rate was 0.3 ml/min, the column temperature was 25 °C, the injection volume was 1 μL, the mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. The gradient elution conditions were as follows: 0–15 min (5–95% B), 15–20 min (95% B), 20–25 min (95–5% B), 25–30 min (5% B).

The mass spectrometry conditions were as follows: electrospray ionization source (ESI) positive ion mode, de-custering potential (DP): 80 V; collision energy (CE): 40 ± 10 eV; curtain gas (CUR): 35 psi; ion source gas 1 (GS1): 55 psi; ion source gas 2 (GS2): 55 psi; ion source temperature: 500 °C; ion spray voltage floating (SVF): 5500; primary scan mode: Full MS; scan range: 100–2000 m/z; secondary scan mode: Full MS/dd-MS2; scan range: 50–2000 m/z.

5.11 Statistical analysis

One-way analysis of variance (ANOVA) was used to evaluate statistical significance, followed by Dunnett's multiple comparisons test, and data analysis was performed using GraphPad Prism 7.0 software. The levels of significance are represented as follows: *p < 0.05, **p < 0.001, and ***p < 0.0001.