A New Pyrrolidone Alkaloid and Other Constituents from <i>Rourea oligophlebia</i> Stems

Ngoc Dinh, Thuc; Vu Thi Ha, Mai; Ngo Xuan, Luong; Trinh Thi, Huan; Nguyen Thi, Huong; Nguyen Thi Ngoc, Mai; Tran Huu, Giap; Vu Thi Kim, Oanh; Le Nguyen, Thanh

doi:https://doi.org/10.1155/2021/6659106

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2021 | Article ID 6659106 | https://doi.org/10.1155/2021/6659106

A New Pyrrolidone Alkaloid and Other Constituents from Rourea oligophlebia Stems

Thuc Ngoc Dinh,¹Mai Vu Thi Ha,¹Luong Ngo Xuan,²Huan Trinh Thi,²Huong Nguyen Thi,²Mai Nguyen Thi Ngoc,²Giap Tran Huu,³Oanh Vu Thi Kim,³and Thanh Le Nguyen³

Academic Editor: Angela Patti

Received24 Dec 2020

Accepted06 Aug 2021

Published18 Aug 2021

Abstract

Phytochemical study of Rourea oligophlebia stems led to the isolation of a new 2-pyrrolidone alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1), together with 14 known compounds including friedelin (2), friedanol (3), taraxerol (4), vanillin (5), coniferyl aldehyde (6), apigenin (7), 7α-hydroxy-3β-sitosterol (8), coniferyl alcohol (9), scopoletin (10), emodin (11), protocatechuic acid (12), catechin (13), procyanidin A1 (14), and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15). Several isolated compounds were evaluated for cytotoxicity and antimicrobial activity. Compound 11 exhibited good antimicrobial activity on Gram (+) strains and moderate cytotoxicity against KB, Hep-G2, and LU cancer cell lines. Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 over KB and LU cancer cell lines, while compound 7 exhibited similar effects on KB, HepG-2, and MCF-7 cell lines with IC₅₀ values of 36.46 ± 0.81, 32.00 ± 0.58, and 32.03 ± 0.61 µg/mL, respectively.

1. Introduction

The genus of Rourea is a group of climbing shrubs belonging to the family of Connaraceae which are widely distributed in the Amazon, Pacific region, Africa, and Asia [1]. Chemical investigations have revealed that Rourea species contain a substantial number of biologically active constituents, including flavonoids, phytosteroids, triterpenes, lipids, phenolic acids, and coumarins [1–6]. The Rourea plant extracts and their active components exhibited interesting biological activities such as hypoglycemic activity [1, 5, 7], antibacterial activity [1, 8], antinociceptive activity [9], antiplasmodial activity [3, 10], and antioxidant activity [1, 4, 8]. Rourea oligophlebia Merr. is a species found on mountains of central provinces of Vietnam. According to folk medicine, R. oligophlebia stems have been used for the treatment of bleeding and bone fractures [11]. A preliminary study of R. oligophlebia revealed the presence of triterpene, sterol, and phenolic constituents [12]. In this study, we described the isolation and identification of a new pyrrolidone alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1) (Figure 1) and fourteen known compounds including friedelin (2), friedanol (3), taraxerol (4), vanillin (5), coniferyl aldehyde (6), apigenin (7), 7α-hydroxy-3β-sitosterol (8), coniferyl alcohol (9), scopoletin (10), emodin (11), protocatechuic acid (12), catechin (13), procyanidin A1 (14), and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15) from the stems of R. oligophlebia (Figure S1). The isolated compounds were evaluated for antimicrobial activity and cytotoxicity.

2. Materials and Methods

2.1. General Experimental Procedures

The NMR data including ¹H-NMR, ¹³C-NMR, ¹⁵N-NMR, HSQC, and HMBC spectra were recorded by a Bruker AM500 FT-NMR spectrometer using TMS as an internal standard. The HR-ESI-MS was obtained using an Agilent 6530 Accurate Mass Q-TOF LC/MS system. Column chromatography (CC) was performed on silica gel (Merck, 230–400 mesh) or Sephadex LH-20 (Sigma Aldrich). Thin layer chromatography used precoated silica gel plates (Merck 60 F₂₅₄). Compounds were visualized by spraying with 10% aqueous H₂SO₄.

2.2. Plant Materials

The plant stems were collected from the Ben En National Park, Thanh Hoa province, Vietnam, in 2018. The plant was identified by Dr. Do Ngoc Dai, Nghe An University of Economics, as Rourea oligophlebia Merr. (Connaraceae). A voucher specimen (MT-202) was deposited at Hong Duc University, Faculty of Natural Sciences.

2.3. Extraction and Isolation

The stem powder (5.3 kg) of Rourea oligophlebia was extracted successively with n-hexane, ethyl acetate, and methanol (12 L × 3 times, 24 hours/time) at room temperature. The combined extracts were evaporated in vacuo to obtain n-hexane residue (17.6 g), ethyl acetate residue (10 g), and MeOH residue (300 g), respectively.

The ethyl acetate residue (10 g) was separated on the silica gel CC, eluted with n-hexane/ethyl acetate gradient (0–100% ethyl acetate) to afford 15 fractions E1–E15. Compounds 2 (15 mg), 3 (5.5 mg), and 4 (4.5 mg) were obtained from E1 fraction (0.2 g), E3 fraction (0.12 g), and E4 fraction (0.15 g), respectively, by crystallization in n-hexane. Fraction E10 (0.5 g) was fractionated by Sephadex CC, eluted with CH₂Cl₂/MeOH (1/9, v/v) to afford 4 subfractions E10.1–E10.4. Subfraction E10.2 (17 mg) was purified by preparative TLC using CH₂Cl₂/MeOH (95/5, v/v) as eluent to yield 5 (2 mg) and 6 (2.5 mg). Fraction E10.3 (25 mg) was purified by silica gel CC, eluted with CH₂Cl₂/MeOH (95/5, v/v) to obtain compound 7 (15 mg). Fraction E12 (0.9 g) was fractionated by Sephadex CC, eluted with CH₂Cl₂/MeOH (1/9, v/v) to give 3 subfractions E12.1–12.3. Compound 8 (6.1 mg) was obtained from E12.1 fraction (85 mg) by crystallization in n-hexane. Fraction E12.2 (30 mg) was chromatographed by silica gel CC, eluted with CH₂Cl₂/MeOH (95/5, v/v) to yield 9 (15 mg). Fraction E13 (0.7 g) was purified by Sephadex CC, eluted with CH₂Cl₂/MeOH (1/9), to give two subfractions E13.1–E13.2. Subfraction E13.2 (20 mg) was purified by preparative TLC using CH₂Cl₂/MeOH (95/5, v/v) as an eluant to afford compound 10 (3 mg). The MeOH extract (300 g) was chromatographed on a silica gel CC and eluted with a gradient solvent system of n-hexane-ethyl acetate (100 : 1–0 : 1, v/v) to afford 14 fractions M1–M14, respectively. Fraction M6 (0.8 g) was separated by Sephadex LH-20 CC eluting with CH₂Cl₂/MeOH (1/9, v/v) to yield compound 11 (7 mg). Fraction M12 (0.6 g) was purified by Sephadex LH-20 CC and eluted with CH₂Cl₂/MeOH (1/9, v/v) to afford compound 12 (6 mg). Compound 13 (12 mg) was obtained from M13 fraction (1.1 g) by purification on Sephadex LH-20 CC, eluted with CH₂Cl₂/MeOH (1/9, v/v). Fraction M14 was purified by silica gel CC and eluted with CH₂Cl₂/acetone (9 : 1, v/v) to give 3 fractions M14.1–M14.3. Fraction M14.1 (0.5 g) was subjected to a Sephadex LH-20 CC and eluted with CH₂Cl₂/MeOH (1/9, v/v) to yield 14 (5.7 mg). Fraction M14.2 (2 g) was separated by Sephadex LH-20 CC and eluted with CH₂Cl₂/MeOH (1/9, v/v) to give 3 fractions M14.2.1–M14.2.3. Fraction M14.2.1 (0.45 g) was purified by Sephadex LH-20 CC, followed by separation with silica gel CC, eluted with CH₂Cl₂/MeOH (9/1, v/v) to yield compound 1 (20 mg). Fraction M14.2 (0.28 g) was subjected to silica gel CC eluted with CH₂Cl₂/MeOH (9/1, v/v) to yield 2 subfractions M14.2.2.1-M14.2.2.2. Fraction M14.2.2.2 (84 mg) was purified by Sephadex LH-20 CC, eluting with CH₂Cl₂/MeOH (1/9, v/v) to yield compound 15 (7 mg).

(R,S)-N-(5-Hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1): colorless oil; ∼0 (c 0.4, MeOH); IR ν_max (KBr) 3239, 3022, 1706, 1664, 1211 cm⁻¹; HR-ESI-MS (positive-ion mode) m/z 141.0673 [(M-H₂O) + H]⁺ (calcd. for C₆H₉N₂O₂⁺, 141.0659); ¹H and ¹³C-NMR data (Table 1).

2.4. Cytotoxicity Assay

The cytotoxicity assays were carried out in triplicate against KB, HepG-2, LU, and MCF-7 cell lines (American Type and Culture Collection, ATCC). Cells were maintained in Dulbecco’s D-MEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100 μg/mL), and gentamicin (10 μg/mL). Stock solutions of compounds were prepared in DMSO/H₂O (1/9), and the cytotoxicity assays were carried out against cancer cell lines (3 × 10³ cells/mL) using a modification of the published method [13]. After 72 h of incubation at 37°C in air/CO₂ (95 : 5) with or without test compounds, cell growth was estimated by colorimetric measurement at 540 nm with a Titertek Multiskan photometer. Ellipticine was used as a positive compound.

2.5. Antimicrobial Assay

The antimicrobial activity was evaluated by the microdilution method previously described by Hadacek [14] and expressed as IC₅₀ (50% inhibitory concentration) values. Six bacterial strains and a fungus were used for the test: 3 strains of Gram (+) bacteria: Staphylococcus aureus ATCC 13709, Bacillus subtilis ATCC 6633, and Lactobacillus fermentum N4; 3 strains of Gram (−) bacteria: Salmonella enterica ATCC12228, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 15442; fungus: Candida albicans ATCC 10231. Bacterial solutions with a concentration of 5 × 10⁵ CFU/ml and a fungal solution with a concentration of 1 × 10³ CFU/ml were prepared. The isolated compounds were diluted in DMSO at the following dilution concentrations: 128, 64, 32, 16, 8, and 4 μg/mL. The positive controls were ampicillin for Gram (+) bacterial strains, cefotaxime for Gram (−) bacterial strains, and nystatin for fungus. IC₅₀ values were determined based on the measured turbidity by optical Biotech spectra and raw data software.

3. Results and Discussion

3.1. Structure Elucidation

Compound 1 was isolated as colorless oil. Its molecular formula of 1 was deduced as C₆H₁₀N₂O₃ from the [(M-H₂O)+H]⁺ peak at m/z 141.0673 (C₆H₉N₂O₂, calcd. for 141.0659) in the positive-ion HR-ESI-MS spectrum (Figure S2). The molecular formula of C₆H₁₀N₂O₃ was further confirmed by combined ¹H, ¹³C, and ¹⁵N-NMR spectra as ten protons, six carbons, and two signals of nitrogen (δ_N 199.99 and 165.86) were observed (Figure S3). The IR spectrum suggested that 1 contained carbonyl amide groups (1706, 1664 cm⁻¹) and hydroxyl and amine groups (3239 cm⁻¹) (Figure S4), which appeared in the ¹H-NMR spectrum (in acetone-d₆) at δ_H 5.47 (br d, J = 5.5 Hz) and 9.16 (br s), respectively (Figure S5). The ¹H-NMR spectrum (in MeOH-d₄) showed signals of an oxymethine group at δ_H 5.22 (1H, dd, J = 3.5, 6.5 Hz, H-5), two methylene groups (H-3, H-4), and a methyl singlet of an acetyl group at δ_H 2.06 (3H, s, H-8) (Figure S6). The ¹³C-NMR spectrum of 1 displayed six carbon signals including two signals of carbonyl group at δ_C 175.0 (C-2) and 172.1 (C-7), an oxymethine group at δ_C 84.2 (C-5), two methylene groups at δ_C 27.1 (C-3) and 27.8 (C-4), and a methyl signal at δ_C 20.5 (C-8) (Figures S7−S10). The HMBC cross-peaks (in acetone-d₆) of the hydroxyl group (δ_H 5.47) to C-5 (δ_C 84.2) confirmed the free hydroxyl group was placed at C-5 position. In the HMBC spectrum (in MeOH-d₄), the correlations of H-3 (δ_H 2.59–2.50 and 2.38–2.32), H-4 (δ_H 2.27–2.21 and 1.88–1.81), and H-5 (δ_H 5.47) to C-2 (δ_C 175.0) suggested a 5-hydroxy-pyrrolidin-2-one moiety in the structure of 1(Figures S11 and S12). In addition, methyl protons H-8 (δ_H 2.06) have only correlated with the carbonyl carbon C-7 (δ_C 172.1) (Figure 2). Based on the optical rotation value of nearly zero and the CD spectrum (Figure S13), compound 1 may be regarded as a racemate. Therefore, the structure of 1 was assigned as (R, S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide. It is noted that alkaloids containing 5-hydroxy-2-pyrrolidone fragment were rarely found in nature. To our knowledge, only few examples are brachystemidine D isolated from Brachystemma calycinum [15], longistrobin and isolongistrobin from Macrorungia longistrabus [16], and lepiota and (R)-5-hydroxypyrrolidin-2-one from the mushroom Macrolepiota neomastoidea [17].

The known compounds were elucidated as friedelin (2), friedanol (3) [12], taraxerol (4) [18], vanillin (5), coniferyl aldehyde (6) [19], apigenin (7) [20], 7α-hydroxy-3β-sitosterol (8) [21], coniferyl alcohol (9) [22], scopoletin (10) [23], emodin (11) [24], protocatechuic acid (12), catechin (13) [25], procyanidin A1 (14) [26], and (E)-2,3,5,4’-tetrahydroxystilbene-2-β-D-glucoside (15) [27]. Compounds 2-3, 6–9, 11-12, and 15 were reported for the first time from Rourea genus.

3.2. Biological Activities

Several isolated compounds were evaluated for antimicrobial activity and cytotoxicity. Only emodin (11) showed good antimicrobial activity against Gram (+) strains S. aureus, B. subtilis, and L. fermentum with IC₅₀ values of 4.51, 15.83, and 28.94 µg/mL, respectively, but exhibited no activity against Gram (−) strains and fungus at the concentration of 128 µg/mL. Other compounds were also inactive (IC₅₀ > 128 µg/mL) in antimicrobial activity test. The cytotoxicity of isolated compounds was evaluated against KB, HepG-2, LU, and MCF-7 cancer cell lines (Table S1). Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 with IC₅₀ ranging from 18.73 ± 0.40 µg/mL to 43.63 ± 0.90 µg/mL over KB and LU cancer cell lines. Compound 7 exhibited similar effect on KB, HepG-2 and MCF-7 cell lines. Among the tested compounds, emodin (11) had the best cytotoxicity on LU cancer cell line with IC₅₀ values of 21.04±0.52 µg/mL, respectively.

4. Conclusions

In conclusion, fifteen compounds were isolated from the stems of Rourea oligophlebia, among which a new alkaloid (R,S)-N-(5-hydroxyl-pyrrolidin-2-one-1-yl)acetamide (1) was identified along with fourteen known compounds 2–15. Compound 11 showed good antimicrobial activity on Gram (+) strains and moderate cytotoxicity against KB, Hep-G2, and LU-1 cell lines. Compounds 6 and 8–10 showed selective activity on HepG-2 and MCF-7 over KB and LU cancer cell lines, while compound 7 was moderately active on KB, HepG-2, and MCF-7 cell lines.

Data Availability

The data used to support the findings of this study are included within the supplementary materials.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2018.08.

Supplementary Materials

HR-ESI-MS, IR, CD, and NMR spectra of compound 1 associated with this article (). (Supplementary Materials)

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