Cembranoids From the Leaves of Nicotiana tabacum and Their Neuroprotective Activities

Abstract

A new cembrane-type diterpenoid (1) and 9 known cembranoids (2-10) were isolated from the leaves of Nicotiana tabacum. The structure of the new metabolite was confirmed by extensive analyses of their spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D- and 2D nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy (IR). All of the isolated compounds were tested for their protective effects against oxygen-glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells. The new compound 1 exhibited a moderate protective effect against OGD-induced neurotoxicity at 10 μM.

Introduction

Cembrane-type diterpenoids (cembranoid), comprising 4 isoprene units, are a kind of 14-member macrocyclic diterpene that have been isolated and identified mainly from marine organisms (eg soft corals and gorgonians) and terrestrial plants (eg tobacco, Pinus, myrrh, and frankincense).^1,5 As reported previously, 105 cembranoids have been obtained from Nicotiana tabacum, which possess a wide range of biological functions, ¹ including antitumor, anti-inflammatory, antibacterial, anti-metastatic, anti-pathogenic, neuroprotective, anti-fouling, and anti-osteoporotic properties. However, studies of bioactivities of those metabolites to date have mainly focused on α-CBT-diol and β-CBT-diol. ⁶ In our current study, we report the isolation, structure identification, and neuroprotective effects of a new cembranoid (1), along with 9 known ones (2-10), from the leaves of N tabacum. The structure of compound 1 was evaluated as (1S,2S,3E,6R,7E,11E)-3,7,11-cembratriene-2,6-diol by spectroscopic methods (Figure 1), including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), infrared spectroscpy (IR), 1D- and 2D nuclear magnetic resonance (NMR). All of the compounds were tested for their neuroprotective activities against oxygen-glucose deprivation (OGD)-induced cell injury in SH-SY5Y cells. Among these compounds, 1, 2, 5, and 7 at 10 μM attenuated SH-SY5Y cell damage induced by OGD. Compound 1 exhibited neuroprotective effects with a cell viability of 69.6% compared with that of the positive control L-3-n-butylphthalide (L-NBP), with a cell viability of 72.3%.

Figure 1.

Cembranoids 1 to 10 isolated from the leaves of Nicotiana tabacum.

Results and Discussion

Compound 1, isolated as a colorless oil, has a molecular formula of C₂₀H₃₄O₂, which was determined by HR-ESI-MS (m/z 329.2458 [M + Na]⁺, calculated for C₂₀H₃₄O₂Na, 329.2457), corresponding to 4 indices of hydrogen deficiency. The IR spectrum (Supplemental Material) displayed characteristic absorptions of hydroxy groups (3314 cm⁻¹). The ¹H-NMR data (Table 1) of 1 showed 3 olefinic protons at δ_H 5.38 (1H, d, J = 8.4 Hz, H-3), 5.07 (1H, d, J = 9.0 Hz, H-7), and 4.95 (1H, m, and H-11) and 2 oxymethine protons at δ_H 4.56 (2H, m, H-2, and H-6). In the upfield region, 2 doublet methyl protons at δ_H 0.97 (3H, d, J = 7.2 Hz, H-17) and 0.94 (3H, d, J = 7.2 Hz, H-16) and 3 singlet methyl protons at δ_H 1.62 (3H, s, H-20), 1.61 (3H, s, H-19), and 1.60 (3H, s, H-18) were also observed. In the ¹³C-NMR spectrum, 20 carbon resonances were observed and classified by HSQC experiments as 5 methyl carbons [δ_C 21.9 (C-16), 19.0 (C-17), 16.4 (C-18), 16.9 (C-19), 17.9 (C-20)], 5 methylene carbons [δ_C 48.9 (C-5), 38.7 (C-9), 23.9 (C-10), 35.2 (C-13), 22.2 (C-14)], 2 methine carbons [δ_C 46.2 (C-1), 29.5 (C-15)], and 2 oxygenated methine carbons [δ_C 68.4 (C-2), 66.8 (C-6)]. The resonances at δ_C 131.3 (C-3), 127.2 (C-7), and 123.4 (C-11) were attributed to 3 olefinic methine carbons, and 3 signals at δ_C 131.5 (C-4), 138.8 (C-8), and 134.0 (C-12) were assigned to 3 olefinic quaternary carbons. In combination with the aforementioned NMR data, 1 was determined as a cembranoid containing 3,7,11-triene-2,6-diol. The assignments for the cembrane-type diterpenoid skeleton were confirmed by heteronuclear multiple bond correlation (HMBC) and ¹H − ¹H correlation spectroscopy (COSY) experiments (Figure 2a). The ¹H − ¹H COSY correlations of H-3/H-2/H-1/H-14/H-13, H-1/H-15, and H-16/H-15/H-17 confirmed the presence of structural fragment A (C-3 − C-2 − C-1 − (C-15) − C-14 − C-13) and B (C-16 − C-15 − C-17). The ¹H − ¹H COSY cross-peaks of H-5/H-6/H-7 implied the fragment C (C-5 − C-6 − C-7), and the existence of fragment D (C-9 − C-10 − C-11) was confirmed by the spin-coupling sequence of H-9/H-10/H-11. Furthermore, in the HMBC spectrum, correlations from H-3 to C-5 and H-5 to C-4 demonstrated that fragments A and C were linked via C-4, while cross-peaks from H-7 to C-9, and H-10 to C-8 suggested the linkage of fragments C and D via C-8. Thus, the planar structure of compound 1 was established as 3,7,11-cembratriene-2,6-diol. The rotating frame overhauser effect spectroscopy (ROESY) correlations (Figure 2) from H-3 to H-5, H-7 to H-9, and H-11 to H-13, along with the chemical shift values at δ_C 16.4 (C-18), 16.9 (C-19), and 17.9 (C-20), determined the E configuration for the 3 trisubstituted olefin. ⁷ The ROESY cross-peak (Figure 2b) between H-2 and H-17 revealed that H-2 and the isopropyl group at C-1 were cofacial. As all cembranoids from the Nicotiana genus have a (1S,6R) configuration, the absolute configuration of compound 1 was tentatively determined as (1S,2S,3E,6R,7E,11E)- 3,7,11-cembratriene-2,6-diol from its biogenesis.

Figure 2.

(a) ¹H − ¹H COSY and key HMBC correlations of compound 1. (b) Selected ROESY correlations of compound 1.

Table 1.

¹H and ¹³C-NMR Spectroscopic Data of Compound 1 in CDCl₃-d (δ, ppm; J, Hz).

Position	δH (J, Hz)	δ C	Position	δH (J, Hz)	δ C
1	1.25 (1H, m)	46.2	11	4.95 (1H, m Hz)	123.4
2	4.56 (1H, m)	68.4	12		134.0
3	5.38 (1H, d, J = 8.4 Hz)	131.3	13	1.92 (1H, m); 2.09 (1H, m)	35.2
4		131.5	14	1.52 (1H, m); 1.67 (1H, m)	22.2
5	2.49 (1H, dd, J = 12.0, 3.0 Hz) 2.14 (1H, m)	48.9	15	1.78 (1H, m)	29.5
6	4.56 (1H, m)	66.8	16	0.94 (1H, d, J = 7.2 Hz)	21.9
7	5.07 (1H, d, J = 9.0 Hz)	127.2	17	0.97 (1H, d, J = 7.2 Hz)	19.0
8		138.8	18	1.60 (3H, s)	16.4
9	2.11 (2H, m)	38.7	19	1.61 (3H, s)	16.9
10	2.27(1H, m); 2.11 (1H, m)	23.9	20	1.62 (3H, s)	17.9

Abbreviation: NMR, nuclear magnetic resonance.

By comparing their observed and reported spectroscopic data, 9 known compounds were determined as (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (2, α-CBT-diol), ⁸ (1S,2E,4R, 6E,8S,11E)-2,6,11-cembratriene-4,8-diol (3), ⁹ (1S,2E,4S,6E, 8S,11E)-2,6,11-cembratriene-4,8-diol (4), ⁹ 4-O-methyl-(1S,2E,4R,6E,11E)-2,7,11-cembratriene-4,6-diol (5), ¹⁰ (1S,2E,4S,6E,8R,11E)-2,6, 11-cembratriene-4,8-diol (6), ⁹ (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (7, β-CBT-diol), ⁸ (1S,2E,4R,6E, 8R,11S,12E)-8,11-epoxy-2,6,12-cembratriene-4-ol (8), ¹¹ (1S,2E,4S, 6R,7E,10S, 11S)-10, 11-epoxy-2,7,12(20)-cembratriene-4,6-diol (9), ¹² and (1S,2E,4S,6R,7E,10R,11R)-10,11-epoxy-2,7,12(20)-cembratriene-4,6-diol (10). ¹²

To the best of our knowledge, studies of the neuroprotective activities of cembranoids from tobacco to date have mainly focused on α-CBT-diol and β-CBT-diol. Thus, all of our isolates were evaluated for their neuroprotective effects against OGD-induced cell injury in SH-SY5Y cells. Compounds 1, 2, 5, and 7 at 10 μM attenuated SH-SY5Y cell damage (Figure 3). Among them, compound 1 exhibited neuroprotective effects with a cell viability of 69.6% compared with that of the positive control L- NBP (72.3%).

Figure 3.

Neuroprotective effect of compounds 1, 2, 5, and 7 against oxygen-glucose deprivation (OGD)-induced toxicity in SH-SY5Y cells.

Experimental

General Experimental Procedures

IR spectra were measured on a Thermo Scientific Nicolet 5700 spectrometer, NMR spectra using a Bruker ascend 600 MHz spectrometer, and HR-ESI-MS on an AB SCIEX Triple TOF 6600 mass spectrometer. Preparative high-performance liquid chromatography (HPLC) was carried out on a Waters Prep 150 LC system using an YMC-Pack ODS column (250 × 10 mm, 5 μM). Column chromatography was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd ) and Sephadex LH-20 (Pharmacia Fine Chemicals) columns. Neuroprotective activity was performed in a super clean workbench (Suzhou Antai Airtech Co., Ltd). Cells were cultured in a Thermo Scientific Steri-Cycle CO₂ incubator. Cell counting was performed under an inverted microscope (Sunny Optical Technology Co., Ltd). The absorbance was detected with a microplate reader (Bio-Rad Laboratories, Inc) at 490 nm wavelength. All of the other chemicals and solvents were of analytical grade.

Plant Material

The leaves of N tabacum L. were collected from Sanmenxia city (Henan province, China) in August 2018 and authenticated by associate professor, Shen Huang, from Zhengzhou University of Light Industry. A voucher specimen (20180024) was stored in the laboratory of the Duobin Mao research group.

Extraction and Isolation

The fresh green leaves of N tabacum (960 kg) were rinsed 4 times with CH₂Cl₂. After the CH₂Cl₂ had been evaporated under reduced pressure, dark yellow residues (516 g) were obtained. The obtained extracts were subjected to chromatography on a silica gel column (2000 × 50 cm i.d.) and eluted successively with light petroleum/ethyl acetate (100:0, 50:1, 30:1, 10:1, 5:1, 3:1, 1:1, and 0:1, v/v) as the mobile phase, and finally washed with MeOH to yield 9 fractions (fractions A-I). Fraction D (34 g) was separated on a Sephadex LH-20 column (120 × 10 cm i.d.) and eluted with a MeOH/CH₂Cl₂ (1:1, v/v) mixture using liquid chromatography with a photodiode array detector to give fractions D1-D9. Fraction D5 was further chromatographed on a silica gel column and separated by light petroleum/ethyl acetate (10:1, 7:1, 5:1, 3:1, and 1:1) to generate fractions D5-1 to D5-12, Fraction D5-9 was purified by reverse preparative HPLC using MeCN/H₂O (55:45, v/v) as the mobile phase at 5 mL/min to yield compounds 1 (10 mg), 3 (14 mg), and 4 (8 mg), while fraction D5-6 was fractionated by preparative HPLC using MeCN/H₂O (65:35, v/v) at 5 mL/min to produce compounds 5 (6 mg) and 6 (8 mg). Fraction G (89 g) was chromatographed on a Sephadex LH-20 column (120 × 10 cm i.d.) and eluted with a MeOH/CH₂Cl₂ (1:1, v/v) mixture to give fractions G1 to G13. Compound 2 (58 mg) was collected from fraction G4, and compound 7 (12 mg) was isolated by preparative HPLC from fraction G5 using MeCN/H₂O (65:35, v/v) as the mobile phase. Fraction G8 was fractionated by silica gel CC using CH₂Cl₂/ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, and 1:1) to generate fractions G8-1 to G8-9. Compounds 8 (4 mg), 9 (3 mg), and 10 (5 mg) were obtained from fraction C8-5 by preparative HPLC using MeCN/H₂O (45:65, v/v) as the mobile phase at 5 mL/min. All of the isolates were dried under reduced pressure and their spectroscopic data were collected.

Neuroprotective Activity Assays

Human neuroblastoma SH-SY5Y cells were maintained in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C under a water-saturated atmosphere of 5% CO₂ and 95% air. Cells, in the log growth phase, were seeded in 96-well culture plates (1 × 10⁵ cells/well in 100 µL) until 80% to 90% confluent for the next experiments. After pretreatment with compounds at 10⁻⁶ M for 2 h, SH-SY5Y cells were exposed to glucose-free DMEM in a hypoxia incubation chamber with 95% N_2% and 5% CO₂ for 1 h and then cultured for another 24 h under normal conditions. These cells were included in the OGD/R group compared with SH-SY5Y cells cultured with complete medium under normal conditions as the control group. Cell viability was evaluated by incubating with 1 mg/mL 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37 °C in an incubator. Media were then replaced with 100 μL DMSO and absorbance was read at 490 nm. Data were analyzed by one-way analysis of variance (ANOVA) and expressed as means ± SD with P <.05 regarded as significant.

Conclusion

A new cembranoid, (1S,2S,3E,6R,7E,11E)-3,7,11-cembratriene-2,6-diol (1), was isolated from the leaves of N tabacum, along with 9 known ones (2-10). To the best of our knowledge, this is the second tobacco cembranoid that has been isolated with the E-configuration of a 3,4-double bond in its structure. ⁶ Compound 1 exhibited neuroprotective effects against OGD-induced toxicity, with a cell viability of 69.6% compared with that of the positive control L-NBP (72.3%).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221094107 - Supplemental material for Cembranoids From the Leaves of Nicotiana tabacum and Their Neuroprotective Activities

Supplemental material, sj-docx-1-npx-10.1177_1934578X221094107 for Cembranoids From the Leaves of Nicotiana tabacum and Their Neuroprotective Activities by Peng-fei Yang, Mei-yu Chen, Jian-chun Yu, Jing Yang and Qiong-bo Wang, Duo-bin Mao in Natural Product Communications