Chemical Constituents and Their Activities From the Seeds of Ormosia hosiei

Ormosia hosiei Hemsl. et Wils. (Leguminosae), as an ornamental plant, is mainly distributed in the Zhejiang, Fujian, and Sichuan Provinces of China. ¹ Its seeds, known as “hongdou,” are used for the treatment of hernia, bellyache, and amenorrhea. ² The previous phytochemical studies of Ormosia species have resulted in the isolation of alkaloids, ^{3 -5} triterpenoids, ⁶ and isoflavonoids, ^{7 -9} especially the alkaloids, known as “Ormosia alkaloids,” ⁵ which showed excellent biological activities. However, few phytochemistry studies have been carried out on O. hosiei, and only 4 cytisine-like alkaloids have been isolated from its roots and stems. ¹⁰ In order to study any novel alkaloids from O. hosiei, we conducted a phytochemical study of its seeds which resulted in the isolation of 3 alkaloids (1-3, Figure 1 ), one of which is a new compound, named hosimonoal (1), and 3 known lignans (4-6). Herein, we reported the extraction, separation, purification, and structural identification of these compounds.

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Figure 1

The structures of compounds 1 to 6.

Hosimonoal (1) was obtained as yellow gum, soluble in methanol. Its molecular formula was determined to be C₂₅H₃₂O₄N₂ based on the high resolution electrospray ionization mass spectroscopy (HRESIMS) ion at m/z 425.24258 [M+H]⁺ (calcd for C₂₅H₃₃O₄N₂, 425.24348), requiring 11 degrees of unsaturation. Its ¹H nuclear magnetic resonance (NMR) spectrum showed 6 aromatic protons [δ_H 7.48 (1H, dd, J = 8.9, 7.0 Hz, H-4), 6.44 (1H, dd, J = 8.9, 1.3 Hz, H-3), 6.27 (1H, dd, J = 7.0, 1.3 Hz, H-5), 7.19 (1H, d, J = 7.8 Hz, H-3′), 6.92 (1H, m, overlapped, H-4′), and 6.93 (1H, m, overlapped, H-5′)] in the downfield region and 3 methyl groups [δ_H 2.12 (3H, s, H₃-17) and 1.49 (6H, each s, H₃-9′ and 10′)] in the upfield region. The ¹³C NMR data displayed 25 carbons, comprising 3 methyls, 5 methylenes, 11 methines, and 6 quaternary on the basis of the distortionless enhancement by polarization transfer and ¹H detected heteronuclear single quantum coherence spectra. The structure of 1 (Table 1) seemed to be made up of 2 known structural units, hosieine B ¹⁰ and 2-hydroxymethyl-5-(2-hydroxypropan-2-yl)phenol, ¹¹ by comparing their ¹H and ¹³C NMR spectral data; this was further confirmed by the 2D NMR spectra (Figures 2 and 3).

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Table 1

¹H and¹³C NMR Spectroscopic Data for Compound 1.

Position	δC a	δH b	Position	δC a	δH b
2	165.5		16	66.0	3.47, m
3	116.7	6.44, dd (8.9, 1.4)	17	43.1	2.12, s
4	141.5	7.48, dd (8.9, 7.0)	1′	156.0
5	107.7	6.27, dd (7.0, 1.4)	2′	126.5
6	153.9		3′	129.0	7.19, d (7.8)
7	41.2	2.81, td (4.2, 2.1)	4′	116.6	6.92, overlapped
8	44.4	2.16, m	5′	151.5
9	37.8	2.45, m	6′	112.5	6.93, overlapped
10	45.5	4.05, m	7′	60.9	4.62, s
11	67.7	3.12, t (4.9)	8′	72.8
13	56.1	2.66, dd (11.7, 4.2); 2.50, d (11.7)	9′	31.9	1.49, s
14	25.3	2.09, t (7.0); 1.16, m	10′	31.9	1.49, s
15	35.3	2.18, t (4.5)

^aIn methanol-d ₄ (100 MHz).

^bIn methanol-d ₄ (400 MHz).

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Figure 2

The key ¹H,¹H COSY and HMBC correlations of compound 1.

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Figure 3

The nuclear overhauser effect spctroscopy correlations of compound 1.

The hosieine B unit was determined by ¹H, ¹H-correlation spectroscopy (¹H COSY) correlations of H-3/H-4/H-5, H-7/H-13, H-7/H-8/H-9/H-10, H-8/H-15, and H-9/H-11/H-14/H-15/H-16; and 1H detected heteronuclear multiple bond correlation (HMBC) correlations from H-3 to C-2 (δ_C 165.5) and C-5 (δ_C 107.7), from H-4 to C-2 and C-4 (δ_C 141.5), from H-5 to C-3 (δ_C 116.7), C-6 (δ_C 153.9), and C-7 (δ_C 41.2), from H-10 [δ_H 4.05 (1H, m)] to C-2, C-9 (δ_C 37.8), and C-11 (δ_C 67.7), from H-11 [δ_H 3.12 (1H, t, J = 4.9 Hz)] to C-9, C-13 (δ_C 56.1), and C-15 (δ_C 35.3), and from H-17 to C-11 and C-13 (Figure 2). The 2-hydroxymethyl-5-(2-hydroxypropan-2-yl)phenol unit was confirmed on the basis of HMBC correlations (Figure 2) from H-7′ [δ_H 4.62 (2H, s)] to C-1′ (δ_C 156.0), C-2′ (δ_C 26.5), and C-3′ (δ_C 129.0), from H-3′ to C-1′, C-4′ (δ_C 116.6), and C-5′ (δ_C 151.5), from H₃-9′(10′) to C-5′ and C-8′ (δ_C 72.8); ¹H,¹H COSY correlation from H-3′ to H-4′. The connection of the 2 structural units was 16-O-7′, which could be deduced by the HMBC correlation from H-16 [δ_H 3.47 (1H, m)] to C-7′ (δ_C 60.9).

The relative configuration could be established by the nuclear overhauser effect spctroscopy correlations of H-8/H₂-16/H-9/H₂-10 and H-7/H-15 (Figure 3). The absolute configuration of 1 was confirmed from the electronic circular dichroism spectrum. The Cotton effect at λ _max (Δε) 233.5 (+4.31) and 308.5 (−3.14) was similar to that of the known compounds hosieines A-D, ¹⁰ which showed that compound 1 had an identical configuration. Thus, compound 1 was assigned as a new cytisine-like alkaloid and given the name hosimonoal (Figure 1).

In addition to hosimonoal (1), a new cytisine-like alkaloid with a fused monoterpene group, and hosieine A (2) and B (3), ¹⁰ 3 lignans , (+)-lariciresinol (4), ¹² 5′-methoxy lariciresinol (5), ¹³ and maackoline (6), ¹⁴ were isolated, and their structures confirmed by comparison of their ESIMS, and ¹H and ¹³C NMR spectroscopic data with that in the literature.

All the compounds were evaluated for their in vitro tumor cytotoxic activities against A2780, A-549, HepG2, and MCF-7 cell lines (Table 2). Compounds 1 to 3 showed moderate activities against the HepG2 cell line. Compound 1 also showed activities against A-2780 and HepG2, but not against A-549 and MCF-7. Compound 3 showed activities against A-549 and HepG2, but not against A-2780 and MCF-7. These results implied that compounds 1 and 3 displayed selective tumor cytotoxic activities against the cancer cell lines. Compound 6 also showed moderate activities against A-2780 and HepG2.

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Table 2

Tumor Cytotoxic Activities of Compounds 1 to 3 ^a (IC₅₀ ^b ).

Compounds	A-2780	A-549	HepG2	MCF-7
1	24.2 ± 1.6	>50	36.5 ± 1.5	>50
2	15.2 ± 0.5	5.7 ± 0.2	17.8 ± 1.0	10.0 ± 1.1
3	>50	17.3 ± 1.5	23.5 ± 1.3	>50
6	9.2 ± 1.4	>50	10.3 ± 0.9	>50
5-Fluorouracil	1.7 ± 0.3	3.2 ± 0.5	9.5 ± 0.4	8.2 ± 0.4

^aAll results are expressed as mean ± SD.

^bIC₅₀ means 50% inhibitory concentration.

Experimental

General Experimental Procedures

A JASCO P-2000 polarimeter (JASCO, Easton, MD, United States) and JASCO V650 (Thermo Scientific, Waltham, MA, United States) spectrophotometer were used to collect optical rotations and UV spectra, respectively. A Bruker-400 spectrometer (Bruker-Biospin, Billerica, MA, United States) was used to measure the 1D and 2D NMR spectra. An Agilent 6520 High Performance Liquid Chromatography-Quadrupole-Time-of- flight mass spectrometry (Agilent, Waldbronn, Germany) was used to obtain the HRESIMS. High Performance Liquid Chromatography-diode array detector analysis was performed using a Waters 2690/5-W2998 series system with a Dikma Diamonsil (C18 250 × 4.6 mm, 5 µm). Preparative High Performance Liquid Chromatography (HPLC) was performed on a Shimadzu LC-8AD instrument with a SPD-20A detector (Shimadzu Corp., Tokyo, Japan), using 2 types of YMC-Pack ODS-A column (20 × 250 mm, 5 µm and 10 × 250 mm, 5 µm). Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), D101 macroporous resin (Changfeng Technology Co. Ltd, Jiangsu, People’s Republic of China), and Sephadex LH-20 (GE, United States).

Plant Material

The seeds of O. hosiei Hemsl. et Wils. were gathered from 3 trees in the Jin’an District, Fuzhou, Fujian Province, on April 15, 2017. The trees were more than 100 years old and identified by Professor Bizhu He and Shasha Wu from Fujian Agriculture and Forestry University (FAFU), People’s Republic of China.

Extraction and Isolation

The air-dried and powdered seeds of O. hosiei (600 g) were subjected to reflux extraction with 70% EtOH (2 × 6 L) at 85°C and the solution was evaporated under reduced pressure in a rotary evaporator at 50°C to yield 100 g extract. This was fractionated by PRP-512A macroporous resin CC, using a gradient solvent system of ethanol-H₂O (10%, 30%, 50%, 70%, and 100%; v/v; 2.0 L for each gradient) to yield 5 fractions (A1-A5). Fraction A2 (26.2 g) was subjected to silica gel CC using a gradient solvent system of CHCl₃-MeOH (1:0-0:1) to afford fractions B1-B5. Fraction B1 (3.5 g) was subjected to Sephadex LH-20 CC eluting with CHCl₃:MeOH (1:1) to give fractions C1-C7. Fraction C4 (170.8 mg) was further purified by preparative HPLC [MeOH-H₂O-TEA (25:75:0.05), v/v, 8.0 mL/min, 210 nm] to yield compound 1 (5.6 mg). Fraction C5 (237 mg) was purified by preparative HPLC [MeOH-H₂O-TEA (23:77:0.05), v/v, 8.0 mL/min, 210 nm] to yield compounds 2 (83.2 mg) and 3 (12.6 mg). Fraction C5 (42.3 mg) was further purified by preparative HPLC [MeOH-H₂O (46:60), v/v, 8.0 mL/min, 210 nm] to yield compound 4 (6.3 mg). Fraction C5 (53.2 mg) was purified by preparative HPLC [MeOH-H₂O (40:60), v/v, 8.0 mL/min, 210 nm] to yield compounds 5 (7.4 mg) and 6 (11.1 mg).

Biological Activities

A2780 (ovarian neoplasm cell line), A-549 (lung cancer cell line), HepG2 (liver cancer cell line), and MCF-7 (breast cancer cell line) were used to test for cytotoxic activities. The cytotoxicity assay was carried out using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide method, as previously described. ^15,16 5-Fluorouracil (Sigma, St. Louis, MO, United States) was used as a positive control, and the IC₅₀ values were calculated by Graphpad prism 7.0.

Hosimonoal (1)

Yellow gum.

[ α ] D 20 : –52 (c 0.097 MeOH).

IR (Microscope Transmission): 3353, 2932, 1647, 1547, 1420, 1364, 1176, 1148, 1026, 804, 654 cm⁻¹.

UV (CH₃OH) λ_max (log ε): 203.0 (4.07), 282.0 (3.31), 310.0 (3.41).

CD (MeOH) λ_max (Δε): 233.5 (+4.31), 308.5 (−3.14);

¹H NMR (methanol-d ₄, 400 MHz) and ¹³C NMR (methanol-d ₄, 100 MHz): see Table 1.

HRESIMS m/z: 425.24258 [M+H]⁺ (calcd for C₂₅H₃₃O₈N₂, 425.24348).

Supplemental Material

Supplemental data - Supplemental material for Chemical Constituents and Their Activities From the Seeds of Ormosia hosiei

Supplemental material, Supplemental data, for Chemical Constituents and Their Activities From the Seeds of Ormosia hosiei by Huiyou Xu, Yatie Qiu, Jingxin Chen, Yan Shi, Xiaoqin Zhang, Qiang Song, Mingqing Huang, Zujian Wu, and Lin Ni in Natural Product Communications