Chemical Constituents Isolated from the Leaves of Toricellia angulata Oliv. var. intermedia (Harms.) Hu

Abstract

In our survey on the chemical composition of Chinese folk medicines, nine compounds were isolated from methanol extract of the leaves of Toricellia angulata Oliv. var. intermedia (Harms.) Hu (Corniaceae). The structures of these compounds were elucidated on the basis of NMR data analysis, which were identified as dimethyl 2-(hydroxymethyl)-5-oxocyclohexane-1,4-dicarboxylate (1), methyl succinate (2), 5-hydroxymethyl-2-furfuraldehyde (3), 7-hydroxy-6-methoxycoumarin (4), loliolide (5), (8S)-deca-2-trans-2,9-diene-4,6-diyn-1,8-diol (6), methyl malate (7), griselinoside (8), and methyl linoleate (9), respectively. Among them, compound 1 is a new cyclohexanone derivative and given a trivial name torriangulate A, while others are categorized to be organic acids (2, 7, and 9), a coumarin derivative (4), a terpene lactone (5), a polyacetylene (6), and an iridoid glycoside (8). Compounds 3–5 were isolated from this genus for the first time and compound 9 was first identified from this species. The discovered compounds with novel or known structures further reveal the chemical basis of T. angulata var. intermedia, which lays a foundation for the development of T. angulata var. intermedia used as a traditionally folk medicine.

Introduction

Chinese herbal medicine is a unique medicine, which has been used for thousands of years in China. Because plant medicine accounts for the majority of traditional Chinese medicine, traditional Chinese medicine is also called Chinese herbal medicine. At present, there are over 5000 kinds of traditional Chinese medicines used in various places, and there are countless prescriptions formed by the combination of various kinds of traditional Chinese medicines, which play an important role in the fight against various diseases, such as Corona Virus Disease 2019 (COVID-19).^1,2 Chinese herbal medicine is an important part of medicine. Although herbal medicine plays an important role in the treatment of various diseases, it is difficult to widely accept Chinese herbal medicine because of its complex chemical composition, unclear pharmacodynamic components and mechanism. Thus, it is necessary to elucidate the chemical constituents and investigate action mechanism of Chinese herbal medicines.

Among numerous Chinese herbal medicines, Toricellia angulata Oliv. var. intermedia (Harms.) Hu is a traditionally folk medicine widely used in Guizhou province of China. T. angulata var. intermedia, a member of the Corniaceae plant family, is distributed in the Himalayas and Yunnan, Guizhou, and Sichuan provinces of China. ³ As a historically folk medicine, the leaves or roots (cortex) were documented to possess the functions of promoting blood circulation, removing blood stasis, and connecting bones and tendons, which has been used traditionally to treat various diseases such as rheumatoid arthritis pain, postpartum low back pain, chronic enteritis, diarrhea, according to the description in the book “Chinese Materia Medica”. ⁴ The extensive efficacy of T. angulata var. intermedia has evoked the attention on its chemical constituents. In the previous chemical studies, nearly 33 compounds have isolated and identified from the roots of this plant.^5-8 The leaves of T. angulata Oliv. var. intermedia (Harms) Hu are also widely used for various medical indications in ethnic minority areas or National medical institutions. ⁹ However, there have been few reports on the chemical constituents of the leaves of T. angulata var. intermedia.

The current study aims to investigate the chemical constituents in the leaves of T. angulata var. intermedia. Herein, the isolation and structural identification of these chemical constituents are described.

Results and Discussion

After extraction, the petroleum ether- and ethyl acetate-soluble fractions of the methanol extract derived from the leaves of T. angulata var. intermedia were fractionated by column chromatography and MPLC, and purified further by HPLC to afford nine pure compounds. Using NMR experiments, the structures of isolated compounds were elucidated as follows.

Compound 1 was isolated as colorless oil. It had a molecular formula of C₁₁H₁₆O₆ as determined by HRESIMS and ¹³C NMR data analysis, which indicated four indices of hydrogen deficiency for this compound. The ¹H NMR spectrum of 1 exhibited typical proton signals assignable to two methoxy groups [δ_H 3.75 (3H, s, 7-OCH₃) and 3.70 (9-OCH₃)] and a set of oxymethylene protons [δ_H 3.84 (1H, dd, J = 11.8, 3.2 Hz, H-8a) and 3.72 (1H, m, H-8b)]. In addition, numerous aliphatic proton signals in the high-field were observed. The ¹³C NMR spectrum of 1 showed 11 resonances including one ketone carbonyl [δ_C 214.4 (C-5)], two ester carbonyl carbons [δ_C 174.4 (C-7) and 173.1 (C-9)], two methoxy cabons [δ_C 52.3 (7-OCH₃) and 52.2 (9-OCH₃)], and one oxymethylene carbon [δ_C 61.0 (C-8)]. There were five aliphatic carbon signals left in the high-filed, which comprised two methylenes [δ_C 40.6 (C-3) and 32.2 (C-6)], three methines [δ_C 47.0 (C-1), 40.5 (C-2), and 47.5 (C-4)] with the aid of DEPT and HQMC spectra. The molecular formula of this compound required 4 unsaturation degree, which suggested one ring present in the structure according to the presence of three carbonyl carbons. The ring was deduced by the 2D NMR data, especially the HMBC and ¹H-¹H COSY spectra. In the HMBC spectrum, the proton signal H-1 (δ_H 2.60) showed long-range correlations with C-2, C-3, C-5, C-6, and C-9, H-4 (δ_H 2.33) was correlated with C-2, C-3, C-5, C-6, and C-7, and H₂-6 (2.86 and 2.57) was found to be correlated with C-1, C-2, C-4, C-5, and C-9. The evidence of HMBC correlations described above, together with the ¹H-¹H COSY couplings of H₂-6/H-1/H-2/H₂-3/H-4 (Figure 1), supported the construction of a six-membered ring consisting of C-1–C-6, in which the ketone carbonyl was assigned to C-5 (δ_C 214.4), three methines existed at C-1 (δ_C 47.5), C-2 (δ_C 40.5), and C-4 (δ_C 47.0), and two methylenes were assignable to C-3 (δ_C 40.6) and C-6 (δ_C 32.2). The substituent groups were positioned according to the HMBC correlations (Figure 1), which demonstrated that the hydroxymethyl group was attached at C-2 and two methoxycarbonyl groups were located at C-1 and C-4, respectively. The 2D structure of compound 1 was thus defined as shown in Figure 2.

Figure 1.

¹H-¹H COSY and key HMBC and NOESY correlations of compound 1.

Figure 2.

Structures for compounds 1–9.

The configuration of 1 was elucidated by NOESY spectrum and Chem3D modeling, as well as TDDFT ECD calculations. From the NOESY spectrum, the NOE effects between H-2/H-4, H-2/H-6α, H-4/H-6α, indicated these protons were cofacial and assigned randomly in α-positions. While, H-1 was assigned being in a β-position according to the NOE interaction of H-1/H-3β. The absolute configuration of 1 was defined via comparison of the experimental and calculated ECD data. Through systematic conformational search and geometry optimizations by MOE and Gaussian 09,^10-12 the ECD calculations at the B3LYP/SVP level in acetonitrile were accomplished. The calculated ECD spectrum of (1R,2R,4S)-1 coincided well with the experimental data (Figure 3). The structure of compound 1 was therefore elucidated as shown, which was given a trivial name torriangulate A (Figure 2).

Figure 3.

The ECD spectra of compound 1.

In addition to a new compound, torriangulate A, eight known compounds were also isolated from the leaves of T. angulata var. intermedia. Using 1D (¹H and ¹³C) and/or 2D NMR experiments and comparing their NMR data with the chemical shift data reported, the known compounds were identified as methyl succinate (2), ¹³ 5-hydroxymethyl-2-furfuraldehyde (3), ¹⁴ 7-hydroxy-6-methoxycoumarin (4), ⁶ loliolide (5), ¹⁵ (8S)-deca-2-trans-2,9-diene-4,6-diyn-1,8-diol (6), ¹⁶ methyl malate (7), ¹⁷ griselinoside (8), ¹⁸ and methyl linoleate (9). ¹⁹ Among them, compounds 3–5 were isolated from this genus for the first time and compound 9 was isolated first from this species (Figure 2).

In conclusion, the current chemical composition survey on the traditionally folk medicine led to the isolation of nine chemical constituents from the leaves of T. angulata var. intermedia. Through NMR, HRESIMS data analysis, and ECD calculations, the structures of nine compounds were determined, which included one new compound and four ones isolated for the first time from this genus or species. The current investigation revealed the chemical composition of the leaves, which lays a foundation for the development of T. angulata var. intermedia used as a traditionally folk medicine.

Experimental

General

Optical rotations were measured by InsMark IP120 automatic optical rotator (InsMark Co.Ltd, Shanghai, China). ECD data were measured by JASCO J-715 CD spectrometer (JASCO Co.Ltd, Japan). IR spectrum was recorded on a Bruker Tensor 37 FT-IR spectrometer with KBr discs (Bruker Co. Ltd, Germany). All NMR tests were performed on a Bruker AV 400 instrument (Bruker Co. Ltd, Germany) with TMS as an internal standard at room temperature. High resolution spectra obtained on IonSpec 7.0T FTICR (IonSpec Co. Ltd, Canada) mass spectrometer. HPLC separations were conducted on a CXTH system, equipped with a Shodex RI-102 detector (Showa Denko Co., Ltd, Tokyo, Japan) and a YMC-pack ODS-AM (20 × 250 mm) column (YMC Co. Ltd, Kyoto, Japan). Medium pressure liquid chromatography (MPLC) was run on a P0100 pump with an ultraviolet (UV) detector (Huideyi Co., Beijing, People's Republic of China) and a column (40 × 400 mm) filled by octadecylsilyl (ODS, 50 μm, YMC Co., Ltd). Silica gel (200–300 mesh) used for column chromatography was purchased from Qingdao Haiyang Chemical Group Co., Ltd (Qingdao, People's Republic of China). Chemical reagents (analytical grade) for isolation were of analytical grade and were provided by Tianjin Chemical Reagent Co. (Tianjin, China) and Sigma Co., respectively.

Plant Material

The leaves of T. angulata var. intermedia were collected from Guizhou Province (N 26.262822°, E 107.514536°, altitude 807 m), China, in May 2020. The botanical identification was made by Zhongyao Han (Dept. of Pharmacy, Qiannan Medical College for Nationalities, China), and a voucher specimen (No.20200518) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China.

Extraction and Isolation

The air-dried leaves (6.0 kg) of T. angulata var. intermedia were extracted with methanol under reflux for three times (2.5 h, 1.5 h, and 1.5 h, each time). The organic solvent was evaporated under vacuum to give a crude methanol extract (2.1 kg), which was suspended in H₂O (2.1 L) and then partitioned successively with petroleum ether and ethyl acetate to give the petroleum ether-soluble portion (350 g) and ethyl acetate-soluble portion (80 g).

The ethyl acetate-soluble portion was fractionated by silica gel column chromatography (silica gel, 700 g; column, 7  ×  50 cm), using a gradient solvent system of petroleum ether-acetone (100: 0, 100: 2, 100: 4, 100: 6, 100: 9, 100: 13, 100: 19, 100: 27, 100: 35, 21 L for each gradient elution), to afford nine fractions (F₁−F₉) according to the TLC analysis. F₅, F₆, and F₇ were subjected to MPLC and eluted with a step gradient of 64 − 92% MeOH in H₂O to give subfractions F_5-1−F_5-6, F_6-1−F_6-8, F_7-1−F_7-8. Subsequenly, HPLC separations were carried out for some of these subfractions. The purification of F_6-1 (45% MeOH in H₂O) led to the isolation of compounds 1 (t_R = 15 min, 10.2 mg), 3 (t_R = 21 min, 11.4 mg), 4 (t_R = 22 min, 8.7 mg), 5 (t_R = 31 min, 11.0 mg), 6 (t_R = 39 min, 13.8 mg). Compound 2 (t_R = 11.5 min, 17.2 mg) was obtained from F_5-1 (55% MeOH in H₂O). The fractionation of F_7-1 (43% MeOH in H₂O) yielded compound 7 (t_R = 10.8 min, 82.3 mg).

The petroleum ether-soluble portion was subjected to silica gel column chromatography (silica gel, 700 g; column, 7  ×  50 cm), using ethyl acetate as eluent to give one fraction A₁. Then, A₁ was separated by MPLC to obtain nine subfractions A_1-1−A_1-9. The purification of A_1-1 (70% MeOH in H₂O) resulted in the obtainment of compounds 8 (t_R = 9.5 min, 54.8 mg) and 9 (t_R = 40.5 min, 10.7 mg).

Dimethyl 2-(hydroxymethyl)-5-oxocyclohexane-1,4-dicarboxylate (1): colorless oil; $[\alpha ]\frac{20}{\mathrm{D}}$  + 86 (c 0.4, CH₂Cl₂); ECD (CH₃CN) 214 (Δε +0.44), 244 (Δε +0.05), 296 (Δε +2.03) nm; IR (film) ν_max 3446, 2956, 2924, 1736, 1439, 1369, 1243, 1203, 1168, 1052 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ_H 3.84 (1H, dd, J = 11.8, 3.2 Hz, H-8a), 3.75 (3H, s, 7-OCH₃), 3.72 (1H, m, H-8b), 3.70 (9-OCH₃), 3.09 (1H, dd, J = 19.7, 10.5 Hz, H-3β), 2.86 (1H, dd, J = 12.2, 5.7 Hz, H-6α), 2.69 (1H, m, H-3α), 2.60 (1H, m, H-1), 2.60 (1H, m, H-2), 2.57 (1H, dd, J = 12.2, 6.5 Hz, H-6β), 2.33 (1H, m, H-4); ¹³C NMR (100 MHz, CDCl₃): δ_C 214.4 (C-5), 174.4 (C-7), 173.1 (C-9), 61.0 (C-8), 52.3 (7-OCH₃), 52.2 (9-OCH₃), 47.5 (C-4), 47.0 (C-1), 40.5 (C-2), 40.6 (C-3), 32.2 (C-6). HRESIMS m/z 267.0840 [M + Na]⁺ (calcd for C₁₁H₁₆O₆Na, 267.0845).

Methyl succinate (2): colorless oil; ¹H NMR (400 MHz, CDCl₃): δ_H 3.71 (3H, s, 4-OCH₃), 2.69 (2H, d, J = 5.2 Hz, H-2), 2.64 (2H, d, J = 5.4 Hz, H-3); ¹³C NMR (100 MHz, CDCl₃): δ_C 177.9 (COOH), 172.9 (4-CO), 52.1 (–OCH₃), 28.9 (C-2), 28.6 (C-3).

5-Hydroxymethyl-2-furfuraldehyde (3): yellow oil; ¹H NMR (400 MHz, CDCl₃): δ_H 9.58 (1H, s, 2-CHO), 7.22 (1H, d, J = 3.5 Hz, H-3), 6.52 (1H, d, J = 3.5 Hz, H-4), 4.72 (1H, s, 5-CH₂OH); ¹³C NMR (100 MHz, CDCl₃): δ_C 177.0 (2-CHO), 160.8 (C-5), 152.3 (C-2), 123.0 (C-3), 110.0 (C-4), 57.5 (5-CH₂OH).

7-Hydroxy-6-methoxycoumarin (4): light yellow needle crystal (MeOH); ¹H NMR (400 MHz, CDCl₃): δ_H 7.60 (1H, d, J = 9.5 Hz, H-4), 6.97 (1H, s, H-5), 6.83 (1H, s, H-8), 6.29 (1H, d, J = 9.5 Hz, H-3), 3.98 (3H, s, 6-OCH₃); ¹³C NMR (100 MHz, CDCl₃): δ_C 161.6(C-2), 150.1 (C-6), 149.2 (C-9), 143.5 (C-4), 142.7 (C-7), 113.8 (C-3), 112.2 (C-10), 111.1 (C-5), 99.3 (C-8), 56.1 (6-OCH₃).

Loliolide (5): white amorphous powder; ¹H NMR (400 MHz, CDCl₃): δ_H 5.70 (1H, s, H-7), 4.34 (1H, m, H-3), 2.47 (1H, dt, J = 13.9, 2.4 Hz, H-4a), 2.00 (1H, dt, J = 14.6, 2.4 Hz, H-2a), 1.79 (3H, s, H-11), 1.77 (1H, dd, J = 13.9, 3.7 Hz, H-4b), 1.53 (1H, dd, J = 14.6, 3.7 Hz, H-2b), 1.47 (3H, s, H-13), 1.27 (3H, s, H-12); ¹³C NMR (100 MHz, CDCl₃): δ_C 182.8 (C-6), 172.2 (C-8), 112.8 (C-7), 86.9 (C-5), 66.7 (C-3), 47.2 (C-2), 45.6 (C-4), 36.0 (C-1), 30.7 (C-12), 27.0 (C-11), 26.5 (C-13).

(8S)-Deca-2-trans-2,9-diene-4,6-diyn-1,8-diol (6): colorless needle crystal (petroleum ether/ EtOAC); ¹H NMR (400 MHz, CDCl₃): δ_H 6.43 (1H, dt, J = 16.0, 4.7 Hz, H-2), 5.95 (1H, ddd, J = 17.0, 10.1, 5.4 Hz, H-9), 5.83 (1H, d, J = 16.0 Hz, H-3), 5.47 (1H, br. d, J = 17.0 Hz, H-10), 5.26 (1H, br. d, J = 10.1 Hz, H-10), 4.97 (1H, d, J = 5.0 Hz, H-8), 4.24 (2H, br. s, H-1);¹³C NMR (100 MHz, CDCl₃): δ_C 146.0 (C-2), 135.9 (C-9), 117.2 (C-10), 108.5 (C-3), 80.4 (C-7), 77.4 (C-4), 73.6 (C-5), 70.8 (C-6), 63.5 (C-8), 62.5 (C-1).

Methyl malate (7): yellow needle crystal (CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ_H 4.54 (1H, dd, J = 6.0, 4.3 Hz, H-3), 3.80 (3H, s, –OCH₃), 2.92 (2H, m, H-2); ¹³C NMR (100 MHz, CDCl₃): δ_C 175.0 (C-4), 173.8 (C-1), 67.1 (C-3), 52.9 (–OCH₃), 38.4 (C-2).

Griselinoside (8): white powder (MeOH); ¹H NMR(CD₃OD, 400 MHz): δ_H 7.49 (1H, s, H-3), 5.14 (1H, d, J = 8.5 Hz, H-1), 4.67 (1H, d, J = 7.8 Hz, H-1′), 3.88 (1H, d, J = 11.8 Hz, H-6a′), 3.76 (3H, s, 10-OCH₃), 3.73 (3H, s, 11-OCH₃), 3.64 (1H, dd, J = 11.8, 5.5 Hz, H-6b′), 3.54 (1H, d, J = 7.8 Hz, H-9), 3.38 (1H, m, H-8), 3.35 (1H, s, H-5), 3.20∼3.30 (H-2′∼H-5′), 2.70 (1H, dd, J = 19.8, 4.6 Hz, H-7a), 2.64 (1H, dd, J = 19.8, 10.1 Hz, H-7b);¹³C NMR(CD₃OD, 100 MHz): δ_C 212.0 (C-6), 174.3 (C-10), 167.1 (C-11), 152.7 (C-3), 103.7 (C-4), 99.7 (C-1′), 96.0 (C-1), 76.9 (C-5′), 76.5 (C-3′), 73.1 (C-2′), 70.1 (C-4′), 61.3 (C-6′), 51.6 (10-OCH₃), 50.7 (11-OCH₃), 48.5 (C-5), 43.1 (C-9), 38.7 (C-8), 36.3 (C-7).

Methyl linoleate (9): colorless oil; ¹H NMR (400 MHz, CDCl₃): δ_H 5.40 (1H, m, H-13), 5.35 (1H, m, H-12), 5.34 (1H, m, H-9), 5.30 (1H, m, H-10), 3.67 (3H, s, 1-OCH₃), 0.89 (3H, t, J = 6.1 Hz, 18-CH₃); ¹³C NMR (100 MHz, CDCl₃): δ_C 174.4 (C-1), 130.2 (C-9), 130.0 (C-13), 128.0 (C-10), 127.9 (C-12), 51.4 (1-OCH₃), 34.1 (C-2), 31.5 (C-16), 29.6 (C-15), 29.4 (C-6), 29.2 (C-5), 29.1 (C-4), 29.1 (C-7), 27.2 (C-8), 27.2 (C-14), 25.6 (C-11), 24.9 (C-3), 22.6 (C-17), 14.1 (C-18).