Sesquiterpenoids From the Leaves of Datura metel L.

Abstract

In the course of chemical investigations of Datura metel L., a new sesquiterpenoid, dmetelin J (1) was isolated from the leaves, along with the 4 known compounds apocynol A (2), blumenol B (3), megastigmane (4), and ( + )-dehydrovomifoliol (5). Structure elucidation was performed by spectroscopic techniques, including 1- and 2-dimensional NMR spectroscopy and HRESIMS. Compounds 2, 3, and 5 were isolated from the Solanaceae for the first time. All isolated compounds were subjected to anti-inflammatory activity evaluation.

Introduction

Datura metel L., family Solanaceae, is distributed mainly in Guangdong, Guangxi, and Sichuan, most frequently on hillsides and grasslands. ¹ Clinically, the plant is often used for asthmatic cough, epigastric cold pain, rheumatic arthralgia, slow pediatric shock, and surgical anesthesia. ² Many chemical constituents have been isolated from D metel, including sesquiterpenoids, flavonoids, withanolides, and alkaloids.^3–7 D metel has remarkable biological activity such as hypoglycemia, cell protection, immunosuppression, sedation, and anesthetic,^8–11 which is attributed to its rich chemical components. In particular, compounds in D metel show good anti-tumor and anti-inflammatory activity,^12–14 and leaf sesquiterpenoids show good anti-inflammatory properties. ¹⁵ To discover further new sesquiterpenoids, our research focused on the chemical constituents of D metel leaves. In this study, a new sesquiterpenoid, dmetelin J (1), and 4 known sesquiterpenoids (2-5) (Figure 1) were separated from D metel leaves. All 5 compounds were tested for anti-inflammatory activity.

Figure 1.

The structures of compounds 1-6.

Results and Discussion

Compound 1 had the molecular formula of C₂₁H₃₄O_7, based on the HR-ESI-MS ion at m/z 399.2385 [M + H]⁺ (calculated to be C₂₁H₃₅O₇, 399.2383). The¹H-NMR spectrum (Table 1) revealed 4 methyl signals at δ_H 1.27 (3H, s, H-12), 1.29 (3H, s, H-13), 2.03 (3H, d, J = 1.0, H-14), and 1.04 (3H, d, J = 6.8, H-15), 1 olefinic hydrogen signal at δ_H 5.73 (1H, d, J = 1.0, H-7), and a terminal stromal signal of sugar at δ_H 4.86 (1H, d, J = 7.8, H-1′). The ¹³C-NMR spectrum of compound 1 showed that it had 21 carbon signals, 4 methyl carbons at δ_C 24.9 (C-12), 26.3 (C-13), 21.2 (C-14), and 16.7 (C-15), 4 methylene carbons at δ_C 32.6 (C-1), 28.7 (C-3), 35.9 (C-4), and 43.6 (C-9), and a β-D-allopyranoside moiety at δ_C 96.0, 72.5, 73.3, 69.1, 75.1, 63.3. In the ¹H-¹H COSY correlations of compound 1 (Figure 2), the fragment of -CH₂-CH-CH₂-CH₂- was determined by the correlation of H₂-1/H-2/H₂-3/H₂-4. The correlation of the -CH₂-CH-CH₃- fragment with H₂-9/H-10/H₃-15 was determined. The HMBC correlations of H-7 to C-5, 9, and 14, and H-9 to C-8, 10, and 5 indicated the presence of cyclohexanone. An isopropyl group was confirmed to be located at C-2 by the HMBC correlations of H-12 to C-2 and 11, and H-13 to C-2 and 11. The correlation of H-1′ (δ_H 4.86) with C-11 (δ_C 79.2) in the HMBC spectrum (Figure 2) showed that the C-11 position of the aglycone was connected with β-D-allopyranosyl through a glycosidic bond. The main difference from the known compound daturaterpenoid D (6) was that the sugar group contained in it had changed. ¹⁶ Additionally, the DEPT, HSQC, ¹H-¹H COSY, and HMBC spectral data further confirmed the structure of compound 1. In the NOESY spectrum, because there was no obvious signal, we made an NMR calculation. Therefore, an NMR calculation with DP4⁺ analysis was used to identify the relative configuration of compound 1. The shielding tensors of all 8 possible isomers 1-I (2R,5S,10R), 1-II (2R,5R,10R), 1-III (2S,5S,10R), 1-IV (2S,5R,10R), 1-V (2R,5S,10S), 1-IV (2R,5R,10S), 1-VII (2S,5S,10S), and 1-VIII (2S,5R,10S) were predicted using the gauge-independent atomic orbital (GIAO) method at the mPW1PW91/ 6-31G*//B3LYP/6-31G* (in gas phase) and mPW1PW91/6-31G**//B3LYP/6-31G* (in methanol) levels. The results showed that 1-V (2R,5S,10S) was the most likely candidate structure, with a 100% DP4⁺ (all data) probability (Figure 3, Supplemental Figures S17-18). The absolute configuration of the β-allose was further determined to be D after acid hydrolysis. ¹⁷ Based on the above deduction, 1 was elucidated as a new compound named dmetelin J.

Figure 2.

Key HMBC and ¹H-¹H COSY correlations of compound 1.

Figure 3.

Linear correlation plots of calculated and experimental ¹³C NMR data of compound 1.

Table 1.

¹H and ¹³C-NMR Spectroscopic Data of 1 (400 MHz in ¹H NMR and 150 MHz in ¹³C NMR, in CD₃OD).

No.	δC	δH	No.	δC	δH
1	32.6	1.97 (2H, m) 1.66 (2H, dd, J = 13.4, 12.0)	12	24.9	1.27 (3H, s)
2	52.2	2.17 (1H, m)	13	26.3	1.29 (3H, s)
3	28.7	1.85 (2H, m)	14	21.2	2.03 (3H, d, J = 1.0)
4	35.9	1.84 (2H, m)	15	16.7	1.04 (3H, d, J = 6.8)
5	51.8	-	1’	96.0	4.86 (1H, d, J = 7.8)
6	173.1	-	2’	72.5	3.30 (1H, m)
7	125.9	5.73 (1H, d, J = 1.0)	3’	73.3	4.06 (1H, t, J = 3.0)
8	202.3	-	4’	69.1	3.49 (1H, m)
9	43.6	2.42 (2H, m) 2.24 (2H, m)	5’	75.1	3.67 (1H, m)
10	38.5	2.10 (1H, m)	6’	63.3	3.85 (2H, m) 3.67 (2H, m)
11	79.2	-

The 4 known compounds were identified by comparison of their spectroscopic data with those reported in the literature as apocynol A (2), ¹⁸ blumenol B (3), ¹⁹ megastigmane (4), ²⁰ and ( + )-dehydrovomifoliol (5). ²¹ Compounds 2, 3, and 5 were isolated from Solanaceae for the first time. None of the compounds (1-5) exhibited inhibitory activity (IC₅₀ values > 50 μmol/L) on NO production.

Materials and Methods

General Experimental Procedures

A Bruker-400 Superconducting NMR Spectrometer (Bruker Corporation) was used for obtaining the ¹H and ¹³C NMR spectra. HPLC was conducted on a Shimadzu CBM-20A (Shimadzu Corporation). The HR-ESI-MS data were acquired on a Q-TOF (ESI) high-resolution mass spectrometer (Waters Corporation). TLC was performed on a silica gel plate (Silicagel 60 F254, Merck Corporation) and a reverse phase plate (Rp-18, Merck Corporation).

Plant Material

The plant material was gathered in September 2017 from Shuang Chen District, Heilongjiang Province, China. Prof. Rui-Feng Fan identified the plant as D metel leaves. The voucher specimen (No. 20170901) has been preserved at Heilongjiang University of Chinese Medicine.

Extraction and Isolation

Ten kilogram of dried D metel leaves was heated and refluxed with 70% EtOH (3 × 20 L, each 2 h), filtered, and the solvent recovered to obtain 2.41 kg of residue. This was uniformly suspended in water, extracted with light petroleum, EtOAc, and n-BuOH, and the extracts of each part were concentrated to obtain 465.2 g of light petroleum extract, 502.1 g of ethyl acetate extract, and 443.5 g of n-butyl alcohol extract. The EtOAc extract (200 g) was subjected to silica gel column chromatography eluted with a CH₂Cl_2/CH₃OH gradient (100:0→0:1, v/v) to acquire 18 fractions namely Fr. A–Fr. R. Fr. N was subjected to ODS chromatography (MeOH and H₂O, 1:5 to 1:0, v/v) to give 9 fractions (Fr. N-1∼Fr. N-9). Fr. N-6 was separated by semi-preparative HPLC (MeOH-H₂O, 46:54) to obtain compounds 1 (1.4 mg) and 2 (5.4 mg). Fr. H was subjected to ODS chromatography (MeOH and H₂O, 1:5 to 1:0, v/v) to give 20 fractions (Fr. H-1∼Fr. H-20). Fr. H-4 was separated by semi-preparative HPLC (MeOH-H₂O, 51:49) to obtain compounds 3 (2.1 mg) and 4 (3.4 mg). Fr. J was subjected to ODS chromatography (MeOH and H₂O, 1:5 to 1:0, v/v) to give 10 fractions (Fr. J-1∼Fr. J-10). Fr. J-7 was separated by ODS reversed-phase column chromatography and then semi-preparative HPLC to obtain compound 5 (1.5 mg).

Dmetelin J (1): White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{20}$  = −10.2 (c = 0.1, MeOH); UV (MeOH), λ_max (logε) 245.4 (3.89) nm; IR (KBr) v_max 3421, 2938, 1693, 1583, 1437, 1374, 1078 cm⁻¹; HR-ESI-MS: m/z 399.2385 [M + H]⁺ (calculated to be C₂₁H₃₅O₇, 399.2383); ¹H and ¹³C NMR data, see Table 1.

Apocynol A (2): White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{20}$  =  + 17.8 (c = 0.1, MeOH); IR (KBr) v_max 3470, 3414, 2969, 1721, 1639, 1619, 1371, 1227, 1166, 1040, 618, 479, 425 cm⁻¹; HR-ESI-MS: m/z 225.1440 [M + H]⁺ (calculated to be C₁₃H₂₀O₃, 225.1446).

Blumenol B (3): White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{20}$  =  + 6.7 (c = 0.1, MeOH); IR (KBr) v_max 3417, 2966, 2928, 1632, 1437, 1373, 1275, 1222, 1076, 981, 905, 836, 563, 466 cm⁻¹; HR-ESI-MS: m/z 249.1460 [M + Na]⁺ (calculated to be C₁₃H₂₂O₃, 249.1467).

Megastigmane (4): White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{20}$  = −3.4 (c = 0.1, MeOH); IR (KBr) v_max 3416, 2924, 2026, 1632, 1367, 1270, 1077, 775, 563, 479 cm⁻¹; HR-ESI-MS: m/z 267.1564 [M + Na]⁺ (calculated to be 267.1572).

( + )-Dehydrovomifoliol (5): White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{20}$  =  + 32.9 (c = 0.1, MeOH); IR (KBr) v_max 3439, 2968, 1668, 1632, 1436, 1366, 1265, 1179, 1128, 993 cm⁻¹; HR-ESI-MS: m/z 245.1145 [M + Na]⁺ (calculated to be 245.1154).

Quantum Chemistry Calculations

All theoretical calculations were performed using Gaussian 09. ²² A conformation search was initially performed using the program Conflex 7 Rev. C.

NMR calculation of dmetelin J (1) was carried out at the levels of mPW1PW91/6-31G*//B3LYP/6-31G* (in gas phase) and mPW1PW91/6-31G**// B3LYP/6-31G* by the GIAO method (in methanol with PCM model). By using DP4⁺ probability, ²³ the obtained shielding constants (including ¹³C and ¹H) were directly statistically analyzed by using experimental chemical shifts.

Acid Hydrolysis and GC Analysis

Dmetelin J (1) was acid hydrolyzed according to the previous publication. ¹⁴ Analysis of the n-hexane layer (reaction product) was determined using an Agilent 7890 A GC system with a DM-5 column. The absolute configuration was determined by comparing the retention time with the derivative prepared from the standard sugar in the same way. The absolute configuration of the β-allose was further determined to be D after acid hydrolysis.

Anti-Inflammatory Activity Assay

The cytotoxicity of compounds 1-5 to RAW264.7 cells was evaluated by MTT assay. Results showed that none of the compounds had a cytotoxic effect on RAW264.7 cells at the concentration of 1 to 50 μmol/L. Therefore, different concentrations (5, 10, 20, 30, 40, 50 μmol/L) were selected for testing in each group.

RAW264.7 cells were cultured in 96 well culture plates in DMEM in an incubator (37 °C, 5% CO₂). After the cells had adhered completely, 100 μL DMEM medium containing 1 μg/mL LPS was added to each group. After incubation for 24 h, 100 μL DMEM medium was added to the blank group and LPS model group, and 100 μL DMEM medium containing different concentrations of drugs was added to the other groups. The positive control was NG-monomethyl-L-arginine (L-NMMA). A microplate reader measured absorbances at 540 nm, and the IC₅₀ values were calculated by nonlinear regression using GraphPad Prism 7.0. The results suggested that none of the compounds (1-5) exhibited inhibitory activity (IC₅₀ values > 50 μmol/L) on NO production.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221128097 - Supplemental material for Sesquiterpenoids From the Leaves of Datura metel L.

Supplemental material, sj-docx-1-npx-10.1177_1934578X221128097 for Sesquiterpenoids From the Leaves of Datura metel L. by Yan Liu, Leixin Zhuang, Jinyan Tan, Yuanyuan Zhou, Peng Jiang, Ruifeng Fan, Haixue Kuang and Bingyou Yang in Natural Product Communications