Chemical Constituents from the Leaves of Pachyrhizus erosus Collected in Vietnam

Abstract

A phytochemical investigation of methanol extract from leaves of Pachyrhizus erosus (L.) Urban, a leguminous shrub distributed in Vietnam and other tropical and subtropical countries led to the isolation of a new prenylated chalcone, erosusone (1) and a new megastigmane glycoside epimer, 3-episedumoside F₁ (9), together with thirteen known compounds including flavonoids (2–6), a 3-benzoxepine lactone (7), a pyridine-4,5-diol derivative (8), megastigmanes and megastigmane glycosides (10–15). Their structures were elucidated by means of high resolution-electrospray ionization (HR-ESI)-MS, one dimensional (1D) and two-dimensional NMR (2D-NMR) spectroscopy as well as comparison with the data reported in the literature. The cytotoxic effects on LU-1 (lung carcinoma), HepG2 (hepatocellular carcinoma), and MCF7 (breast carcinoma) cell lines were assessed. Prenylated chalcones 1–2 and isoflavone 3 exhibited cytotoxicity against all tested cell lines with IC₅₀ values ranging from 22.04 to 45.03 µM.

Introduction

Pachyrhizus erosus (L.) Urban (Fabaceae), commonly called yam bean, is a vigorous climbing herb which is indigenous to Central America (from South Mexico to Nicaragua and Costa Rica) but has a widespread distribution in Vietnam and other tropical and subtropical countries.^1,2) This plant is an important crop with the crispy tuber, which contains starch and sugars, used as a food ingredient.³⁾ The seeds and leaves of this plant are known to be highly toxic to insect pests, mites, and fish, but not to mammals. The pulverized seeds have been traditionally used as fish poison and pesticides in Vietnam and other tropical countries.⁴⁾ Previous phytochemical investigations focused on the seeds of P. erosus and revealed the presence of rotenone and rotenoid derivatives, isoflavonoids, phenylfuranocoumarins, pterocarpans, triterpenoids, and pachyerosin.^5–10) Some of these compounds exhibited promising bioactivities including antiviral,⁷⁾ antifungal,⁸⁾ antibacterial and cytotoxic,^10–12) antioxidant and tyrosinase inhibition.¹³⁾ It was also reported that extracts from the roots and seeds of P. erosus exhibit various biological activities such as α-glucosidase inhibitory,¹⁴⁾ insecticidal,¹⁵⁾ and immunomodulatory effects.¹⁶⁾ To date, no phytochemical and biological investigations had been reported on the leaves of P. erosus. In our preliminary screening, the EtOAc soluble fraction of methanol extract of P. erosus leaves was found to exhibit cytotoxicity against three tested cell lines HepG2 (hepatocellular carcinoma), MCF7 (breast carcinoma), and LU-1 (lung carcinoma) with the IC₅₀ values of 60.52, 60.96, and 89.16 µg/mL, respectively. The chemical investigation on this extract led to the isolation of fifteen compounds including a new prenylated chalcone (1), a new megastigmane glycoside (9), together with thirteen known ones: flavonoids (2–6), a 3-benzoxepine lactone (7), a pyridine-4,5-diol derivative (8), megastigmanes and megastigmane glycosides (10–15). Herein, we report the isolation and structure elucidation of the fifteen compounds, as well as the cytotoxicity of six flavonoids (1–6) against three tested cell lines as before.

Results and Discussion

The EtOAc soluble fraction of the methanol extract of P. erosus leaves was subjected to multiple chromatographic separations to afford fifteen compounds (1–15, Fig. 1). The known compounds, isobavachalcone (2),¹⁷⁾ wighteone (3),¹⁸⁾ prunin (4),¹⁹⁾ vitexin (5),²⁰⁾ orientin (6),²¹⁾ 1,4-methano-3-benzo[d]oxepin-2(1H)-one (7),²²⁾ 3-(2′,3′-dihydroxy-4′-hydroxymethyl-tetrahydrofuran-1′-yl)pyridine-4,5-diol (8),²³⁾ sedumoside F₁ (10),²⁴⁾ (3S,5R,6S,9R)-3,6-dihydroxy-5,6-dihydro-β-ionol (11),²⁵⁾ 4,5-dihydroblumenol A (12) and ampelopsisionoside (13),²⁶⁾ simplicifloranoside (14),²⁷⁾ and (6S,9R)-roseoside (15),²⁸⁾ were identified by comparing the NMR and optical rotation data with those reported in the literature. All of these compounds have not been reported before from P. erosus. According to the literature,²²⁾ the NMR data of compound 7 has not been found. In this work, we assigned for the first time the ¹H- and ¹³C-NMR data of 7 by detailed analysis of the two dimensional (2D) NMR spectral data (see Supplementary materials).

Fig. 1. Chemical Structures of Compounds 1–15 from P. erosus

Compound 1, named erosusone, was isolated as a lemon-yellow powder. The molecular formula of 1 was determined as C₂₀H₁₈O₅ on the basis of a pseudo-molecular ion peak at m/z 361.1055 [M + Na]⁺ (calcd. 361.1046) in its high resolution-electrospray ionization (HR-ESI)-MS. Compound 1 has a ¹H-NMR spectrum compatible with a chalcone skeleton that reveals two doublets corresponding to olefinic trans-coupling protons (J = 16.0 Hz) at δ_H 7.66 (H-α) and 7.83 (H-β), two doublets corresponding to aromatic ortho-coupling protons (J = 9.0 Hz) of ring A at δ_H 7.96 (H-6′) and 6.51 (H-5′), and an A₂B₂ system of a para-substituted B-ring at δ_H 7.65 (2H, d, J = 8.5 Hz, H-2, 6) and 6.87 (2H, d, J = 8.5 Hz, H-3, 5). The ¹H-NMR of 1 also revealed a 2-oxo-3-methylbut-3-enyl group at δ_H 6.24 (1H, br s, H-4″a), 5.88 (1H, br s, H-4″b), 4.09 (2H, s, H₂-1″), and 1.91 (3H, s, H₃-5″). The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 displayed 18 carbon resonances comprising 2 carbonyl carbons at δ_C 201.9 and 193.7, 14 aromatic or olefinic carbons, a methylene group at δ_C 33.3, and a methyl group at δ_C 17.9. This spectroscopic evidence suggested that 1 possesses the tri-oxygenated chalcone skeleton with a 2-oxo-3-methylbut-3-enyl group. The heteronuclear multiple bond connectivity (HMBC) correlations (Fig. 2) from H₂-1″ to C-2′, C-4′, and C-3′; from H-4″a and H-4″b to C-2″, C-3″, and C-5″; from H₃-5″ to C-2″, C-3″, and C-4″ confirmed that the location of the 2-oxo-3-methylbut-3-enyl unit was at C-3′ position. Comparative analysis of the ¹H- and ¹³C-NMR spectroscopic data (Table S1) of 1 with those of xanthoangelol K²⁹⁾ revealed that both structures are closely related except for the only difference being at C-4′ position, where a hydroxyl group replaced the methoxy group in xanthoangelol K. Thus, the structure of the new compound 1 was determined to be 2′,4′,4-trihydroxy-3′-[2″-oxo-3″-methylbut-3″-enyl]chalcone.

Fig. 2. Key HMBC, ¹H–¹H COSY, NOESY Correlations of Compounds 1, 9, and 10

Compound 9, named 3-episedumoside F₁, was obtained as a white powder ([α]²⁵_D − 7.6 in MeOH). It has the same molecular formula (C₁₉H₃₄O₇) as sedumoside F₁ (10)²⁴⁾ base on the HR-ESI-MS ion at m/z 397.2187 [M + Na]⁺ (Calcd. for C₁₉H₃₄O₇Na, 397.21967), inferring three degrees of unsaturation. Its ¹H- and ¹³C-NMR spectra, in combination with 2D-NMR experiments, indicated the presence of a hexose unit together with 13 carbon signals of a megastigmane skeleton. The sugar unit was identified as β-D-glucopyranose based on the chemical shifts of carbons at δ_C 102.2, 78.1, 77.9, 75.4, 71.4, 62.5, and the anomeric proton signal at δ_H 4.38 (d, J = 8.0 Hz).²⁴⁾ The presence of D-glucose in the acid hydrolysis products of compound 9 was confirmed by TLC and by comparison of its optical rotation with that of authentic D-glucose. The NMR data of compound 9 were found to be similar to those of sedumoside F₁ (10), previously isolated from Sedum sarmentosum,²⁴⁾ suggesting their planar structural resemblance. However, ¹H- and ¹³C-NMR analysis of compounds 9 and 10 (Table S3) revealed some chemical shift differences of cyclohexanol ring which suggested that compounds 9 and 10 differ in the configuration of one or more asymmetric centers of the ring. Thus, the nuclear Overhauser effect (NOE) correlations between H-6 and H-8 and between H-7 and H-5 (E-configuration of double bond at C-7) presented in both compounds 9 and 10 indicated the same relative configuration at C-6 and C-5. However, the 1,3-diaxial interaction of H-3 and H-5, H-11 presented only in nuclear Overhauser effect spectroscopy (NOESY) spectrum of compound 10 and absent in NOESY spectrum of compound 9 clearly showed the different relative configuration at C-3 of the two compounds. The equatorial configuration of H-3 of 9 also can be confirmed through small coupling constants between H-3 and both protons H-2 (J = 2.5 and 3.0 Hz). The difference of 9 and 10 additionally confirmed by the up-field shifts of carbons at C-1 (δ_C 34.3), C-2 (δ_C 47.3), C-4 (δ_C 42.8) and C-5 (δ_C 27.3) of 9 in comparison with those of 10.³⁰⁾ Based on above analysis, compound 9 was found to be 3-epimer of sedumoside F₁ (10). The structure of 9 was established as shown in Fig. 1.

Six flavonoids 1–6 were evaluated in vitro for their cytotoxicity against the LU-1 (lung carcinoma), MCF7 (breast carcinoma), and Hep-G2 (hepatocellular carcinoma) cancer cell lines using sulforhodamine B (SRB) method with ellipticine as the positive control.³¹⁾ As the result (Table 1), among the compounds tested, prenylated chalcones (1–2) and isoflavone (3) exhibited moderate cytotoxicity against all the tested cancer cell lines with IC₅₀ values ranging from 22.04 to 45.03 µM. The other flavonoids (4–6) were inactive in this test (IC₅₀ > 100 µM).

Table 1. Cytotoxicity of EtOAc Extract and Flavonoids 1–6

Compound	IC50 (µM)
	LU-1	HepG2	MCF7
EtOAc extract	89.16 µg/mL	60.52 µg/mL	60.96 µg/mL
1	45.03	37.34	38.99
2	35.24	41.64	29.04
3	22.04	32.81	23.34
4	>100	>100	>100
5	>100	>100	>100
6	>100	>100	>100
EC	1.51	1.34	1.67

EC: ellipticine, positive control.

Experimental

General Experimental Procedures

Optical rotations were measured on a JASCO P-2000 polarimeter (Hachioji, Japan). HR-ESI-MS were obtained on X500 QTOF mass spectrometer system (MA, U.S.A.). NMR spectra were recorded on Bruker Avance 500 and 600 MHz spectrometers with tetramethylsilane (TMS) as internal standard. For column chromatography (CC), silica gel 60 (0.04–0.063 mm, Merck, Germany), RP-C₁₈ silica gel (150 µm, YMC, Japan), and Sephadex LH-20 (25–100 µm, Sigma-Aldrich, MO, U.S.A.) were used. The TLC was performed on Merck precoated TLC DC-Alufolien silica gel 60F₂₅₄ and RP-18F_254S. Compounds were detected by UV fluorescence at 254 nm and after spraying with 1% vanillin-H₂SO₄ in MeOH, followed by heating at 100 °C for 1–2 min.

Plant Material

The leaves of P. erosus (Fabaceae) were collected in 2020 from Hai Duong province in Northern Vietnam. The plant material was identified by Dr. Nguyen Thi Phuong Anh, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology (VAST). A voucher specimen (HUST.U04) was deposited at the Herbarium of the Department of Organic Chemistry, School of Chemical Engineering, Hanoi University of Science and Technology (HUST), Vietnam.

Extraction and Isolation

The air-dried leaves of P. erosus (1 kg) were powdered and extracted with MeOH (5 × 5 L) at 45 °C for 1 h under sonication and then concentrated to 1 L in vacuo. The extract was partitioned with n-hexane (5 × 1 L, 51.5 g) and then the MeOH layer was concentrated under reduced pressure. The obtained residue (213 g) was suspended in MeOH/H₂O (1 L, 1 : 2, v/v) and further extracted with EtOAc (5 × 1 L) to obtain EtOAc (38 g) and water (87 g) residues, after removal of the solvents. The EtOAc soluble fraction (38 g) was subjected to silica gel CC, eluting with a CH₂Cl₂/acetone (100 : 1 to 1 : 1, v/v) gradient, followed by a CH₂Cl₂/MeOH (10 : 1 to 1 : 1, v/v) gradient to afford nine fractions (P-1 to P-9). Fraction P-1 (600 mg) was fractionated on an RP-C₁₈ silica gel CC, eluting with MeOH/H₂O (3 : 1, v/v) and three subfractions (P-1.1 to P-1.3) were obtained. Subfractions P-1.1 and P-1.3 were further purified by preparative TLC using a solvent system of n-hexane/acetone (2 : 1, v/v) to yield compounds 1 (2.0 mg) and 2 (34.3 mg). Subfraction P-1.2 was chromatographed repeatedly on a silica gel column, eluting with n-hexane/acetone (5 : 1, v/v) to yield compound 3 (29.5 mg). Fractions P-2 (370 mg), P-3 (940 mg), and P-4 (170 mg) were purified by Sephadex LH-20 CC with MeOH, followed by RP-C₁₈ silica gel CC with MeOH/H₂O (1 : 1, v/v) to yield compounds 12 (60.6 mg), 11 (30.4 mg), and 14 (32.4 mg). Fraction P-5 (630 mg) was subjected to Sephadex LH-20 CC, eluting with MeOH to give two subfractions (P-5.1 and P-5.2). Subfraction P-5.1 (320 mg) was further separated by RP-C₁₈ silica gel CC, eluting with MeOH/H₂O (1 : 2, v/v) to obtain compounds 9 (20.3 mg), 10 (12.5 mg), 13 (70 mg), and 15 (45.2 mg). Compound 4 (2.7 mg) was obtained from the subfraction P-5.2 by RP-C₁₈ silica gel CC, eluting with MeOH/H₂O (2 : 3, v/v). Fraction P-6 (450 mg) was purified by Sephadex LH-20 CC with MeOH and then by RP-C₁₈ silica gel CC with MeOH/H₂O (1 : 1.5, v/v) to obtain compounds 5 (70.8 mg) and 6 (50 mg). Fraction P-8 (520 mg) was purified by Sephadex LH-20 CC with MeOH, followed by RP-C₁₈ silica gel CC with MeOH/H₂O (1 : 3, v/v) to yield compounds 7 (9 mg) and 8 (7.2 mg).

Erosusone (1): Lemon-yellow powder, HR-ESI-MS m/z: 361.1055 [M + Na]⁺ (Calcd for C₂₀H₁₈O₅Na: 361.1046); ¹H (500 MHz, CD₃OD) δ: 7.96 (1H, d, J = 9.0 Hz, H-6′), 7.83 (1H, d, J = 16.0 Hz, H-β), 7.66 (1H, d, J = 16.0 Hz, H-α), 7.65 (2H, d, J = 8.5 Hz, H-2, 6), 6.87 (2H, d, J = 8.5 Hz, H-3, 5), 6.51 (1H, d, J = 9.0 Hz, H-5′), 6.24 and 5.88 (1H each, both br s, H-4″a and H-4″b), 4.09 (2H, s, H₂-1″), 1.91 (3H, s, H₃-5″); ¹³C-NMR (125 MHz, CD₃OD) δ: 201.9 (C-2″), 193.7 (C-β′), 165.3 (C-4′), 164.0 (C-2′), 161.5 (C-4), 145.6 (C-β), 145.5 (C-3″), 131.8 (C-2, C-6), 131.5 (C-6′), 127.9 (C-1), 125.6 (C-4″), 118.4 (C-α), 116.9 (C-3, C-5), 114.5 (C-1′), 111.1 (C-3′), 108.1 (C-5′), 33.3 (C-1″), 17.9 (C-5″).

3-Episedumoside F₁ (9): White powder, [α]²⁵_D − 7.6 (c 0.1, MeOH); HR-ESI-MS m/z: 397.2187 [M + Na]⁺ (Calcd for C₁₉H₃₄O₇Na: 397.21967); ¹H (500 MHz, CD₃OD) δ: 5.54 (1H, dd, J = 6.5, 15.5 Hz, H-8), 5.46 (1H, dd, J = 10.0, 15.5 Hz, H-7), 4.38 (1H, d, J = 8.0 Hz, H-1′), 4.37 (1H, m, H-9), 4.06 (1H, m, H-3), 3.82 (1H, dd, J = 3.0, 12.0 Hz, H-6′a), 3.71 (1H, dd, J = 4.5, 12.0 Hz, H-6′b), 3.36 (2H, m, H-3′, 4′), 3.22 (2H, m, H-2′, 5′), 1.88 (1H, m, H-5), 1.83 (1H, m, H-4β), 1.68 (1H, dt, J = 2.5, 14.5 Hz, H-2β), 1.41 (1H, t, J = 10.0 Hz, H-6), 1.38 (1H, dd, J = 3.0, 14.5 Hz, H-2α), 1.31 (3H, d, J = 6.5 Hz, H₃-10), 1.22 (1H, m, H-4α), 1.07 (3H, s, H₃-11), 0.86 (3H, s, H₃-12), 0.81 (3H, d, J = 6.5 Hz, H₃-13); ¹³C-NMR (125 MHz, CD₃OD) δ: 135.7 (C-8), 133.8 (C-7), 102.2 (C-1′), 78.1 (C-3′), 78.0 (C-9), 77.9 (C-5′), 75.4 (C-2′), 71.4 (C-4′), 68.1 (C-3), 62.5 (C-6′), 59.5 (C-6), 47.3 (C-2), 42.8 (C-4), 34.3 (C-1), 33.1 (C-12), 27.3 (C-5), 24.0 (C-11), 21.7 (C-13), 21.4 (C-10).

Cytotoxic Activity Assay

see Supplementary materials.

Acknowledgments

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant number 104.01-2018.36.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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