New Naphthalene Derivatives from the Bulbs of Eleutherine americana with Their Protective Effect on the Injury of HUVECs

Abstract

Five new naphthalene derivatives, named Eleutherols A–C (1–3) and Eleuthinones B–C (4,5), together with three known compounds were isolated from the bulbs of Eleutherine americana. Their structures were elucidated on the basis of spectroscopic analysis including HR-ESI-MS, 1D and 2D NMR techniques. These compounds exhibited a potent effect against the injury of human umbilical vein endothelial cell (HUVECs) induced by high concentrations of glucose in vitro.

1. Introduction

Hong-Cong (Eleutherine americana L. Merr.), a small plant that belongs to the Iridaceae family, is mainly distributed in South America, South Africa, and Southeast Asia [1,2]. The red bulbs of this plant (Hong-Cong in Chinese) have been long used as a folk medicine for the treatment of cardiac diseases, diabetes, breast cancer, stroke, hypertension, and sexual disorders, especially coronary disorder in the Hainan Island of South China [3,4,5,6]. Literature reported that the bulbs of Hong-Cong contained anthraquinones, naphthoquinones, and naphthalene derivatives and some ofthem displayed important biological activities, such as coronary vasodilating, prothrombin decreasing, antifertility, wound healing, topoisomerase II inhibitory, HIV inhibitory, antifungal, and anticancer activities [7,8,9,10,11,12]. As part of our ongoing investigation on the discovery of naturally occurring bioactive agents from medicinal plant, we examined the methanol extract of this plant and isolated five new naphthalene derivatives, named eleutherols A–C (1–3) and Eleuthinones B,C (4,5), together with three known compounds (6–8) (Figure 1). Herein, we report the isolation and structural elucidation of the isolatedones, as well as their protective effect on the injury of HUVECs (human umbilical vein endothelial cells) induced by high concentrations of glucose in vitro.

2. Results

Compound 1 was obtained as yellow powder. Its molecular formula was deduced as C₁₄H₁₄O₃ by the HR-ESI-MS data m/z 253.0824 [M + Na]⁺ (calcd. for 253.0841 C₁₄H₁₄NaO₃). The UV spectrum showed absorption maxima at 220, 245, 265, and 420 nm. The IR spectrum exhibited the presence of hydroxyl group(s) at 3420 cm⁻¹, aromatic ring(s) at 3060, 3015, 3005, 1615, 1578 cm⁻¹ and ether linkage at 1240 and 1115 cm⁻¹. In the ¹H-NMR spectrum (

Table 1. ¹H (600 MHz) and ¹³C-NMR (150 MHz) assignments of compounds 1–3 (CDCl₃).

No.	1		2		3
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	79.5	5.53, q, 6.0	69.6	4.71, q, 6.6	78.1	5.74, q, 6.0
3	72.0	5.10, d, 12.6 5.23, d, 12.6	67.3	5.50, q, 6.6	170.6
3a	141.0		120.9		127.9
4	110.1	7.12, s	154.4		116.6	7.91, s
4a	137.5		126.0		137.2
5	122.0	7.36, d, 7.8	118.1	8.04, d, 8.4	123.7	7.60, d, 7.8
6	125.6	7.28, t, 7.8	125.3	7.39, t, 8.4	126.6	7.41, t, 7.8
7	103.7	6.75, d, 7.8	109.1	7.02, d, 8.4	106.3	6.94, d, 7.8
8	156.8		155.7		156.6
8a	114.7		107.7		117.5
9	148.4		139.4		149.2
9a	125.2		119.6		125.9
CH3-1	20.6	1.61, d, 6.6	16.1	1.53, d, 6.6	19.2	1.74, d, 6.6
CH3-3			17.4	1.64, d, 6.6
OCH3-8	56.3	4.06, s	56.2	4.07, s
OCH3-9					56.4	4.12, s
4-OH				8.99, s
8-OH						9.67, s
9-OH		9.44, s		12.83, s

) of compound 1, one methyl signal at δ_H 1.61 (3H, d, J = 6.6 Hz), one methoxy signal at δ_H 4.06 (3H, s) and two mutual coupled oxygenated protons at δ_H 5.10 (1H, d, J = 12.6 Hz, Ha), 5.23 (1H, d, J = 12.6 Hz, Hb) were observed. The ¹H-NMR spectrum also showed the resonances of three aromatic protons consistent with an ABM pattern at δ_H 6.75 (d, J = 7.8 Hz, H-7), 7.28 (t, J = 7.8 Hz, H-6) and 7.36 (d, J = 7.8 Hz, H-5). The ¹³C APT NMR spectrum (Table 1) of 1 exhibited 14 carbon signals including tenaromatic carbons (δ_C 103.7, 110.1, 114.7, 122.0, 125.2, 125.6, 137.5, 141.0, 148.4, and 156.8), two oxygenated carbons (δ_C 72.0, 79.5), one methoxy group at δ_C 56.3, one methylsignal at δ_C 20.6. The UV and IR patterns as well as the NMR data indicated compound 1 is a substituted naphthanolderivative which was further confirmed by 2D NMR spectra (See Supplementary) [13]. The connectivities of compound 1 were established mainly by HMBC correlations, shown in Figure 2. The-OCH₃ group were assigned to C-8 judging from the downfield chemical shifts of C-8 (δ_C 156.8) and the HMBC correlations from the signal of δ_H 4.06 (3H, s, -OCH₃) to C-8 (δ_C 156.8). The hydroxyl group was attached to C-9 on the basis of HMBC correlations from δ_H 9.44 to δ_C 148.4 together with the molecular formula C₁₄H₁₄O₃ above. The HMBC correlations from the methyl protons at δ_H 1.61to C-1 (δ_C 79.5) and C-9a (δ_C 125.2) proved the presence of a methyl group located at C-1. In fact, the structure of 1 were similarly to the known compound Eleutherol [3,13], except for the absence of the carbonyl group at C-3. The absolute configuration of C-1 was established by ECD spectrum. In this experiment, the ECD spectrum (See Supplementary) of 1 showed a positive Cotton effect around 310 nm, suggested the configurationat C-1 to be R [14] [(2S) dihydroeleutherinol-8-O-β-d-glucopyranoside, CD λ_max 319 (−0.46)]. In addition, the optical rotation of 1 (${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ = +12.5) also suggested a stereochemistry at C-1 to be R by comparing the optical rotations of (S) isoeleutherol (${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ = −60.5) [13] and (R) (+)-Dihydroeleutherinol (${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = +8.8) [6]. Thus, the structure of 1 was elucidated as shown and named Eleutherol A.

Compound 2 was isolated as a yellow solid. HR-ESI-MS gave a quasi-molecular ion peak at m/z 283.0912 in the positive mode (Calcd. for 283.0946). Taking together with the analysis of ¹H and ¹³C APT NMR spectra, the molecular formula of 2 was deduced as C₁₅H₁₆O₄. The IR spectrum exhibited the presence of hydroxyl group(s) at 3415 cm⁻¹, aromatic ring(s) at 3065, 3015, 3005, 1625, 1575 cm⁻¹ and ether linkage at 1235 and 1105 cm⁻¹. A detailed comparison of the NMR data between 2 and 1 revealed that there were one additional hydroxyl and methyl signals in 2. These findings were fully supported by 2D NMR spectra (See Supplementary). In the HMBC spectrum, the correlations from δ_H 1.64 (3H, d, J = 6.6 Hz) to δ_C 67.3 (C-3), δ_H 12.83 (1H, s) to δ_C 154.4 (C-4) suggested the extra methyl and hydroxyl signals were attached to C-3 and C-4, respectively. The relative configuration of compound 2 was established by NOESY spectrum. In the experiment, the NOE enhancements from H-1 to H-3 indicated the synperiplanar position of CH₃-1 and CH₃-3. The similar ECD spectra between 2 and 1 exhibited the R configuration of C-1 in 2. Therefore, compound 2 was determined as shown and named Eleutherol B.

Compound 3 was a brown amorphous powder. Its molecular formula of C₁₄H₁₂O₄ was in agreement with its HR-ESI-MS mass spectrum [M + Na]⁺ m/z 267.0679 (calcd. for 267.0633, C₁₄H₁₂NaO₄). Its UV absorptions at 225, 265, and 345 nm indicated the presence of benzene ring(s). The IR spectrum showed absorption bands of one or more hydroxyl groups at 3371 cm⁻¹ and ester carbonyl functionality at 1738⁻¹. The ¹H-NMR spectrum (Table 1) of 3 displayed one methyl at δ_H 1.74 (d, J = 6.6 Hz), one methoxy at δ_H 4.12 (s), one hydroxyl at δ_H 9.67 (s) and the resonances of aromatic protons H-5, H-6 and H-7 as an ABM system at δ_H 7.60 (d, J = 7.8 Hz), 7.41 (t, J = 7.8 Hz) and 6.94 (d, J = 7.8 Hz), respectively. The ¹H-NMR data seemed identical to those of compound 1. However, the ¹³C-NMR spectrum of 3 displayed one more carbonyl group at δ_C 170.6, which indicated the methylene at C-3 in 1 was oxygenated and formed to carbonyl group in 3. Further analysis its HSQC data (See Supplementary) displayed that δ_H 4.12 (3H, s, -OCH₃) had direct correlation with δ_C 56.4, δ_H 7.60 (1H, d, J = 7.8 Hz, H-5) with δ_C 123.7, δ_H 7.41 (1H, t, J = 7.8 Hz, H-6) with δ_C 126.6, δ_H 6.94 (1H, d, J = 7.8 Hz, H-7) with δ_C 106.3 and δ_H 7.91 (1H, s, H-4) with δ_C 116.6 and the HMBC spectrum (See Supplementary) exhibited the correlations from δ_H 4.12 (3H, s, -OCH₃) to δ_C 149.2 (C-9) and δ_H 9.67 (1H, s, -OH) to δ_C 156.6 (C-8), respectively. All the 2D NMR above suggested that the methoxygroup was located at C-9 and hydroxyl group at C-8. Taken together with the similar ECD spectrum (See Supplementary), the structure of compound 3 was established as depicted and named Eleutherol C.

Compound 4 was obtained as a yellow-brown solid. Its molecular formula of C₁₈H₁₈O₆ was established on the basis of its mass spectrum, [M + Na]⁺ m/z 353.1014 (calcd. for 353.1001, C₁₈H₁₈NaO₆). The¹HNMR spectrum (

Table 2. ¹H (600 MHz) and ¹³C-NMR (150 MHz) assignments of compounds 4,5 (CDCl₃).

No.	4		5
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	182.8		179.6
2	143.8		154.8
3	140.4		118.7
4	184.4		185.7
4a	140.0		135.0
5	119.4	7.74, dd, 8.4, 1.2	119.6	7.80, d, 8.4
6	135.0	7.67, t, 8.4	136.2	7.72, t, 8.4
7	117.9	7.29, d, 8.4	117.0	7.27, d, 8.4
8	159.8		160.2
8a	119.8		116.8
1′	33.2	3.62, s	33.0	2.81, dd, 13.2, 3.6 2.76, dd, 13.2, 7.8
2′	169.5		67.6	4.12, m
3′	41.8	3.78, s
4′	203.1
2′-OCH2CH3	61.4	4.14, m
2′-OCH2CH3	14.1	1.26, t, 6.0
2′-CH3			23.6	1.26, d, 6.6
4′-CH3	30.1	2.30, s
8-OCH3	56.5	4.00, s	56.7	4.04, s

) displayed an ABM aromatic pattern at δ_H 7.74 (dd, J = 8.4, 1.2 Hz, H-5), 7.67 (t, J = 8.4 Hz, H-6) and 7.29 (d, J = 8.4 Hz, H-7) together with ten downfield carbons (δ_C 182.8, 143.8, 140.4, 184.4, 140.0, 119.4, 135.0, 117.9, 159.8, and 119.8) indicated the presence of naphthaquninone moiety. The resonances of a methylene protons at δ_H 3.78 (2H, s) and methyl protons δ_H 2.30 (s) as well as three carbons (δ_C 41.8, 30.1, 203.1) suggested the existence of 3-one-propyl side chain. Furthermore, the resonances of a methylene protons at δ_H 3.62 (2H, s) and ethyl protons δ_H 1.26 (3H, t, J = 6.0), 4.14 (2H, m) as well as four carbons (δ_C 33.2, 169.5, 14.1, 61.4) displayed the presence of ethylethanoylunit. A methoxy group that resonated at δ_H 4.00 was placed at C-8 according to the HMBC experiment (See Supplementary) (Figure 2). The 3-one-propyl chain was proposed to be at the C-3 position due to the HMBC correlations of H-3′ to C-3 and C-4. The existence of anethylethanoyl sidechain (-CH₂CO₂CH₂CH₃) was located at the C-2 position according to the HMBC correlation of the methylene protons H-1′ to C-1 and C-2. Therefore, the structure 4 was assigned as shown and named as Eleuthinone B.

Compound 5 was isolated as an orange amorphous powder and was determined as C₁₄H₁₄O₅ by the HR-ESI-MS data m/z 285.0732 [M + Na]⁺, (calcd. for 285.0739, C₁₄H₁₄NaO₅). The strong absorption bands at 1655 and 1715 cm⁻¹ in the IR spectrum and absorption maxima at 220, 245, 265, 270, and 405 nm in the UV spectrum suggested the presence of a p-quinone moiety [5,6]. An examination of the ¹H and ¹³C APT NMR (Table 2) showed the structure of 5 to be similar to that of 4. Further analysis of the NMR data (See Supplementary) (Figure 2) of 5 indicated that the 3-one-propyl and ethylethanoyl groups in 4 were replaced by 2-hydroxyl-propyl and hydroxyl group, respectively in 5. The downfield chemical shift of C-2 (δ_C 154.8) and upfield chemical shift of C-2′ (δ_C 67.6) in 5 together with the molecular formula C₁₄H₁₄O₅aboveconfirmed these differences. However, the absence of proper model compounds to use as references made the assignment of the absolute configuration at C-2′ unreliable. As a result, the structure of 5 was deduced as shown and named as Eleuthinone C.

The known compounds were identified as hongconin (6) [10], Karwinaphthol A (7) [15] and dihydroisoeleutherin (8) [16], by comparing their ¹H and ¹³C-NMR data with the reported literature.

Studies showed that the injury of HUVECs induced by high glucose was closely related to the cardiovascular disease [17,18]. Considering the civil applications of this medicinal herb, the isolated compounds 1–8 were studied for their protective effect on the injury of HUVECs induced by high glucose, in vitro (Figure 3).

3. Discussion

There are three main skeletons naphthalene, anthraquinone, and naphtoquinone, which have been isolated from E. americana [2].These structures, with the characteristics of a naphthalene ring and a furan ring or six-membered ring, were isolated from the red bulbs of E. americana, which are widely distributed in genus of Eleutherine. Ethnobotanically, the bulbs of plant are known for treating coronary abnormality. As a result, we investigated all the compounds for their protective effect on the injury of HUVECs activity. Comparing with the glucose group, all compounds displayed protective effect on the injury of HUVECs induced by high concentrations of glucose in vitro. Further analysis the data showed that compounds 2–6 exhibited better protective effect than other compounds, which indicated that the carbonyl group at furan or pyran ring in naphthalene skeleton may affect the pharmacological activity regarding HUVECs damage induced by glucose. With our knowledge, the evaluation of protective effect on the injury of HUVECs activity induced by glucose was the first time in vitro.

4. Materials and Methods

4.1. General Experimental Procedures

Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, Waltham, MA, USA). UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometer (Shimadzu, Kyoto, Japan), respectively. ECD spectra were obtained using a JASCO J-815 spectro polarimeter. NMR spectra were obtained with a Bruker AV 600 NMR spectrometer (chemical shift are presented as δ values with TMS as the internal standard) (Bruker, Billerica, Germany). HR-ESI-MS were performed on a Q-tof spectrometer (Waters, Milford, MA, USA). Preparative HPLC was performed on an analytic LC equipped with a pump of P230, a DAD detector of 230+ (Ellte, Dalian, China), semi-preparative column. C18 ODS-A (50 μm, YMC, Kyoto, Japan) and Silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China) was used for column chromatography. TLC analyses were carried out on Silica gel GF254 pre-coated plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, China) with detection accomplished by spraying with 5% H₂SO₄ followed by heating at 100 °C. HUVECs cell was purchased at Shanghai cell bank of Chinese academy of sciences. All solvents used were of analytical grade (Beijing Chemical Works, Beijing, China).

4.2. Plant Material

The bulbs of Eleutherine americana L. Merr. were collected in November 2016 from Hechi, Guangxi Province and identified by Prof. Rong-Tao Li, Hainan Branch Institute of Medicinal Plant Development (Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine), where the voucher specimens were conserved (No. 20161125GXEA). Plant drug was shade dried (<40 °C), coarsely powdered and stored in air tight container.

4.3. Extraction and Isolation

The dried and powdered bulbs of E. americana (5.0 kg) were extracted with methanol (50 L × 3) at room temperature for 24 h. The methanol extract was evaporated to dryness under reduced pressure to yield the extract (812.0 g). The residue was suspended to H₂O (2.0 L) and partitioned with petroleum ether (3 × 2 L, MSO), CH₂Cl₂ (3 × 2 L), EtOAc (3 × 2 L) and n-BuOH (3 × 2 L), successively.

Fraction of the MSO (60.8 g) was subjected to column chromatography over silica gel (100–200 mesh) and eluted with MSO-CH₂Cl₂ (100:1) in increasing polarity. Then the fraction of MSO-CH₂Cl₂ (30:1) was subjected to column chromatography on silica gel and eluted in MSO-CH₂Cl₂ in gradient manner (from 80:1 to 0:100). Thin layer chromatography permitted to combine the resulted fractions which have the same R_f values into 6 series, Fr.A–F. Fr.C (0.75 g) was purified by HPLC with a gradient of 75% MeOH-H₂O on an Agilent SB-Phenyl column to get compounds 1 (8.4 mg) in R_t 12.5 min, 2 (8.7 mg) in R_t 18.2 min, compound 7 (7.8 mg) in R_t 22.6 min and compound 8 (6.8 mg) in R_t 30.5 min. Fr.E (0.36 g) was separated by semi-preparative liquid chromatography using a MeOH-H₂O (72:28) isocratic to yield 3 (9.6 mg, R_t 17.6 min), 6 (8.1 mg, R_t 23.4 min), 8 (10.2 mg, R_t 16.5 min) and 9 (8.6 mg, R_t 20.8 min). The entire detection was under UV 254 nm and the flow rate was 2 mL/min.

The structures of compounds 1–8 were determined by UV, IR, ¹H-NMR, ¹³C-NMR , ¹H-¹H COSY, HSQC, HMBC, NOESY and HR-ESI-MS.

Eleutherol A (1). C₁₄H₁₄O₃, yellow powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 12.5 (c 0.1, MeOH); UV λ_max (CHCl₃) nm (log ε): 220, 245, 265, 420; IR (KBr) ν_max cm⁻¹: 1115, 1240, 1578, 1615, 3005, 3015, 3060, 3420; CD (MeOH, Δε) λ_max 310 (+0.24); HR-ESI-MS m/z 253.0824 [M + Na]⁺ (calcd. 253.0841); ¹H and ¹³C-NMR spectra data, see Table 1.

Eleutherol B (2). C₁₅H₁₆O₄, yellow brown solid; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 36.7 (c 0.1, MeOH); UV λ_max (CHCl₃) nm (log ε): 225, 240, 265, 275, 415; IR (KBr) ν_max cm⁻¹: 1105, 1235, 1575, 1625, 3005, 3015, 3065, 3415; CD (MeOH, Δε) λ_max 325 (+0.22); HR-ESI-MS m/z 283.0912 [M + Na]⁺ (calcd. 283.0946); ¹H and ¹³C-NMR spectra data, see Table 1.

Eleutherol C (3). C₁₄H₁₂O₄, brown amorphous; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 24.8 (c 0.1, MeOH); UV λ_max (CHCl₃) nm (log ε): 225, 265, 345; IR (KBr) ν_max cm⁻¹: 1108, 1235, 1575, 1738, 3015, 3371, 3680; CD (MeOH, Δε) λ_max 310 (+0.64), 255 (+2.25); HR-ESI-MS m/z 267.0679 [M + Na]⁺ (calcd. 267.0633); ¹H and ¹³C-NMR spectra data, see Table 1.

Eleuthinone B (4). C₁₆H₁₄O₄, yellow brown solid; UV λ_max (CHCl₃) nm (log ε): 228, 243, 268, 272 and 405; IR (KBr) ν_max cm⁻¹: 1355, 1458, 1585, 1635, 1735, 2848, 2940; HR-ESI-MS m/z 353.1014 [M + Na]⁺ (calcd. 353.1001); ¹H and ¹³C-NMR spectra data, see Table 2.

Eleuthinone C (5). C₁₄H₁₄O₅, orange amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ − 6.7 (c 0.1, MeOH); UV λ_max (CHCl₃) nm (log ε): 224, 248, 268, 272 and 405; IR (KBr) ν_max cm⁻¹: 1355, 1466, 1585, 1605, 1720, 2845, 2940, 3320, 3630; HR-ESI-MS m/z 285.0732 [M + Na]⁺ (calcd. 285.0739); ¹H and ¹³C-NMR spectra data, see Table 2.

4.4. Activity Assay

Compounds 1–8 were screened protective effect against the injury of HUVECs induced by high concentrations of glucose using the MTT method as described in the previous published literature [19] with appropriate modifications. Briefly, the cells were cultured in DMEM medium with 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator for 24 h. 100 μL of adherent cells was seeded on 96-well microtiter plate and allowed to adhere for 12 h. Then, the cells were treated with the test compounds at various concentrations (1 μM, 5 μM and 10 μM) for 2 h in triplicate. After 2 h of treatment, with the final concentration of 30 mM glucose was added directly into all the appropriate wells for 24 h. Absorbance were measured by the absorbance at 490 nm using a multiwell spectrophotometer.