Two New Iridoids from Verbena officinalis L.

Abstract

Two new iridoids, 3-(5-(methoxycarbonyl)-2-oxo-2H-pyran-3-yl)butanoic acid, named verbeofflin I (1), and 7-hydroxydehydrohastatoside (2), were isolated from the aerial part of Verbena officinalis L, along with three known iridoids, verbenalin (3), 3,4-dihydroverbenalin (4), hastatoside (5) by means of various column chromatography steps. The structures of these compounds were elucidated through analysis of their spectroscopic data obtained using 1D and 2D NMR and MS techniques. Verbeofflin I (1) is the new class of secoiridoid in the family Verbenaceae.

1. Introduction

Verbena officinalis L. is a medicinal herb widely distributed in the southern part of the Yellow River in China. It is used as a Chinese folk medicine for the treatment of rheumatism and bronchitis [1]. Several investigations showed that Verbena species contained flavonoids, terpenoids, phenyl propanoids and iridoids [2,3,4,5,6]. Iridoids isolated from Verbena species, for example, verbenalin and hastatoside, are characteristic constituents of V. officinalis, and they exhibit various biological activities including sleep-promoting [7], antioxidant [8] and hepatoprotective activity [9]. Moreover, the relative contents of verbenalin and hastatoside are higher than those of other characteristic iridoids in Herba Verbenae [10]. The two compounds are very fit as target constituents for quality control of Herba verbenae, so we have established a method of quality control of Herba Verbenae [10]. As a part of our ongoing study on quality control of the plant, we investigated the constituents of Herba verbenae, the present study dealt with the isolation and structural elucidation of two new iridoids, verbeofflin I (1) and 7-hydroxydehydrohastatoside (2), together with three known iridoids, verbenalin (3) [11,12], 3,4-dihydroverbenalin (4) [13], and hastatoside (5) [11,12,14]. To the best of our knowledge, compound 1 is the new class of secoiridoid. The structural characteristic of compound 1 is that the iridoid skeleton ring is opened between C-5 and C-6.

2. Results and Discussion

The methanol extract of V. officialis was stirred with diatomite and silica gel, and then extracted separately with petroleum ether, ethyl acetate and methanol. The ethyl acetate fraction was purified using a succession of silica gel and Sephadex LH-20 column chromatography and reversed-phase HPLC (C-18) to give a new iridoid (1) and a new iridoid glucoside (2), along with three known iridoid glucosides 3–5. The structures of compounds 1–5 are illustrated in Figure 1.

Figure 1. Structures of compounds 1–5.

Figure 1. Structures of compounds 1–5.

Compound 1 was obtained as brown amorphous powder. It gave a negative result for normal chromogenic agents like H₂SO₄/EtOH, H₃[P(Mo₃O₁₀)]₄/EtOH and I₂. It had a dark spot at λ_{254 nm} in the UV. Compound 1 showed in its HR-ESI-MS a [M+Na]⁺ molecular ion at m/z 263.0530, compatible with the molecular formula C₁₁H₁₂O₆Na, suggesting six degrees of unsaturation. In the analysis of the ¹H-NMR (Table 1) and HSQC spectrum, the signals of one olefinic protons (δ_H 8.68, d, J = 0.5 Hz, H-3), one methoxyl group (δ_H 3.83, s, -OCH₃), one methyl group (δ_H 1.25, d, J = 7.0 Hz, H-10), one methine proton (δ_H 1.25, m, H-8), along with protons of one methylene group (δ_H 2.71, dd, J = 6.5, 15.5 Hz, H-7a; 2.48, dd, J = 8.0, 15.5 Hz, H-7b) indicate compound 1 has an iridoid skeleton (Figure 1) [15], and this was supported by the ¹³C-NMR spectrum (10 carbon atoms in total excluding the methoxyl group (Table 1). The ¹³C-NMR spectrum of 1 showed three carbonyl c-atoms (δ_C 176.2, C-1; 175.9, C-6; 165.0, C-11), and by HMBC (Figure 2), the signal at δ_C 176.2 could be assigned to C-1 due to correlations with H-3 and H-5. Also, the second signal at low field (δ_C 175.2), could be shown to arise from C-6 from the HMBC spectrum, since a long-range connectivity ³J was observed between H-8 and C-6. The presence of only two doublets at δ_H 8.68 (d, J = 0.5 Hz, H-3) and 8.00 (d,J = 0.5 Hz, H-5) in ¹H-NMR, and the corresponding ¹³C-NMR signals at δ_C 154.7, 135.8 indicated that C-5 and C-9 possessed a double bond. This was also confirmed by the degrees of unsaturation and HMBC spectrum, H-5 showed cross-peaks with C-1, C-3, C-8 and C-11. Furthermore, H-3 and H-8 had correlation with C-5. From the above information, compound 1 was further identified as a secoiridoid, which could be formed by ring-opening between C-5 and C-6 of the iridoid skeleton of hastatoside (5). Therefore, the stereochemistry at C-8 is most likely the same as that of 5. However, this needs to be confirmed by other data. From these data, thus, the structure of 1 was established as 3-(5-(methoxycarbonyl)-2-oxo-2H-pyran-3-yl)butanoic acid, named verbeofflin I.

Table 1. The NMR spectroscopic data of compounds 1 and 2 (MeOH-d₄, ¹H-NMR 500 MHz, ¹³C-NMR 125 MHz).

Position	1		2
	1H	13C	1H	13C
1	-	176.2	5.94 (1H, d, J = 1.5 Hz)	94.1
3	8.68 (1H, d, J = 0.5 Hz)	163.5	7.37 (s)	154.8
4	-	120.9	-	109.7
5	8.00 (1H, d. J = 0.5 Hz)	154.7	-	71.7
6	-	175.9	-	198.4
7	2.71 (1H, dd, J = 6.5, 15.5 Hz)2.48 (1H, dd, J = 8.0, 15.5 Hz)	40.0	-	149.3
8	3.26 (1H, m)	29.6	-	137.2
9	-	135.8	3.20 (1H, like t, J = 1.5 Hz)	53.7
10	1.25 (3H, d, J = 7.0 Hz)	19.2	1.91 (3H, d, J = 1.5 Hz)	11.9
11	-	165.0	-	167.8
1ꞌ			4.45 (1H, d, J = 8.0 Hz)	100.2
2ꞌ			3.00–3.28 (1H, m)	71.9
3ꞌ			3.00–3.28 (1H, m)	77.8
4ꞌ			3.00–3.28 (1H, m)	74.7
5ꞌ			3.00–3.28 (1H, m)	78.9
6ꞌ			3.00–3.28 (1H, m)3.56 (1H, dd, J = 6.5, 12 Hz)	63.1
OCH3	3.83 (3H, s)	52.9	3.61 (3H, s)	52.1

Table 1. The NMR spectroscopic data of compounds 1 and 2 (MeOH-d₄, ¹H-NMR 500 MHz, ¹³C-NMR 125 MHz).

Position	1		2
	1H	13C	1H	13C
1	-	176.2	5.94 (1H, d, J = 1.5 Hz)	94.1
3	8.68 (1H, d, J = 0.5 Hz)	163.5	7.37 (s)	154.8
4	-	120.9	-	109.7
5	8.00 (1H, d. J = 0.5 Hz)	154.7	-	71.7
6	-	175.9	-	198.4
7	2.71 (1H, dd, J = 6.5, 15.5 Hz)2.48 (1H, dd, J = 8.0, 15.5 Hz)	40.0	-	149.3
8	3.26 (1H, m)	29.6	-	137.2
9	-	135.8	3.20 (1H, like t, J = 1.5 Hz)	53.7
10	1.25 (3H, d, J = 7.0 Hz)	19.2	1.91 (3H, d, J = 1.5 Hz)	11.9
11	-	165.0	-	167.8
1ꞌ			4.45 (1H, d, J = 8.0 Hz)	100.2
2ꞌ			3.00–3.28 (1H, m)	71.9
3ꞌ			3.00–3.28 (1H, m)	77.8
4ꞌ			3.00–3.28 (1H, m)	74.7
5ꞌ			3.00–3.28 (1H, m)	78.9
6ꞌ			3.00–3.28 (1H, m)3.56 (1H, dd, J = 6.5, 12 Hz)	63.1
OCH3	3.83 (3H, s)	52.9	3.61 (3H, s)	52.1

Figure 2. The key HMBC (H→C) correlations of compounds 1 and 2.

Figure 2. The key HMBC (H→C) correlations of compounds 1 and 2.

Compound 2 was obtained as a white amorphous powder, = −94.5° (c = 0.012, MeOH), with a quasi-molecular ion peak at m/z 441.1001 [M+Na]⁺, indicating that the molecular formula of 2 was putatively C₁₇H₂₂O₁₂, which was further confirmed by its ¹³C-NMR spectrum (Table 1). The UV absorption maximum at 238 nm suggested that 2 had a conjugated double bond and an enol ether, which are characteristic of the -OCO-C=CH-O- chromophore found in iridoids [15]. The ¹H-NMR and ¹³C-NMR spectra of compound 2 showed similar features to those of hastatoside [12]. The differences between the ¹H-NMR data of 2 and that of hastatoside were signals at δ_H 1.91 (3H, d, J = 1.5 Hz, H-10), 3.20 (1H, like t, J = 1.5 Hz, H-9), and the differences between the ¹³C-NMR data of 2 and that of hastatoside were signals at δ_C 137.2 (C-8), 149.3 (C-7), those suggested that 2 possesses a second conjugated double bond, and it can be assigned at C-7-C-8, it was confirmed by the change of chemical shifts of C-6 of compound 2 showed the remarkable upfield shifts by 18ppm, compared to that of hastatoside (δ_C 215), and the HMBC spectrum analysis (Figure 2) displayed the correlation peaks between H-10 and C-7. Furthermore, in HMBC spectrum, the olefinic proton at δ_H 7.37 (H-3) showed cross-peaks with C-11, C-5, and C-1, which suggested that the attachment hydroxyl group was assigned to C-5; A methine proton at δ_H 3.20 (1H, t, J = 1.5 Hz) correlated with C-4, 6, 7 and 10, which implied that the proton should be assigned at C-9; the anomeric proton at δ_H 4.45 (1H, d, J = 8.0 Hz) showed correlations with C-1, which suggested that the sugar moiety was located at C-1. The attachment of the hydroxyl group was assigned to C-7 due to the analysis of the molecular formula. In the meantime, it was proved by the ¹H-NMR spectrum. In the ¹H-NMR, there wasn’t another olefinic proton except for H-3. Confirmation of the stereochemistry of its stereogenic centers was achieved by analysis of J values and comparison with ¹³C-NMR literature data, especially those for chiral centers at C-1, C-5, and C-9, which indicated the β configuration of the hydroxyl group at C-5, consistent with the configuration of this substituent in hastatoside, The cis junction between the two rings and the O-glycosyl residue at C-1 is biosynthetically supported with a β-configuration [16]. From the above information, compound 2 was thus identified as 7-hydroxydehydrohastatoside.

3. Experimental

3.1. Plant Materials

The aerial part of V. officinalis was collected in Chongqing, China, and was identified by Prof. Guoyue Zhong of The Institute of Chinese Materia Medica of Chongqing. A voucher specimen (No. 20070507) was deposited in Shanghai R&D Center for Standardization of Chinese Medicines.

3.2. General Procedure and Regents

¹H (500 MHz), ¹³C (125 MHz), and 2D NMR spectra were obtained on Bruker AV-500 (Bruker Co., Fällanden, Switzerland) instrument with TMS as internal reference, using methanol-d₄ as solvent. Electrospray ionisation (ESI) mass spectra were acquired in the positive and negative ion mode on a LCQ DECAXP instrument (Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyzer. HR-ESI-MS were obtained in the positive ion mode on Waters UPLC Premir Q-TOF (Waters Co., Milford, CT, USA). IR spectra (expressed in in cm⁻¹) were obtained on a Nicolet^TM-380 spectrometer from Thermo Electron. Melting points were measured on a Büchi-Meting-Point-B-540 apparatus (Büchi Co., Flawil, Switzerland), and are uncorrected. Optical rotations were acquired on a Krüss-P800-T polarimeter (Krüss, Co., Hamburg, Germany). TLC plates were HSGF254 SiO₂ from Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China. Column chromatography (CC) silica gel (SiO₂; 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden), ODS (SepaxGPC18, 40−60 m, Sepax Technologies Inc., Newark, NJ, USA) were employed as packing materials, semi-preparative HPLC (Waters 996 detector, Waters 717 autosamper, Waters 600 controller, Milford, MA, USA). All other chemicals were of analytical reagent grade.

3.3. Extraction and Isolation

The aerial part of V. officialis (7.5 kg) was percolated with 90% (v/v) aqueous methanol (200L) for 15 days at room temperature. The 90% MeOH extract was concentrated under reduced pressure to give a residue (995 g), which was stirred with diatomite and silica gel, then extracted separately with petroleum ether, ethyl acetate and methanol. The ethyl acetate extract was concentrated to yield a residue (316 g). The dried ethyl acetate extract (120 g) was subjected to silica gel column chromatography with gradient mixtures of CH₂Cl₂/MeOH (from 100:1 to 1:1), to yield six major Fractions (Fr.1–6). Fr. 2 (13 g) was further subjected to repeated CC (silica gel, CH₂Cl₂/MeOH 18:1, and Sephadex LH-20 MeOH) to afford 1 (51.2 mg) and 3 (890.7 mg). Fr. 3 (21 g) was further subjected to repeated CC (silica gel, CH₂Cl₂/MeOH 15:1, and Sephadex LH-20 MeOH) to afford 5 (912.5 mg). Fr. 5 (32 g) was further subjected to CC (ODS 35% MeOH, and Sephadex LH-20 MeOH), followed by semi-prep. HPLC (MeOH/H₂O, 15:85, 3.0 mL/min) to yield compounds 2 (8.2 mg, t_R 8.2 min), 4 (35.8 mg, t_R 21.6 min).

3.4. Spectra Data

3-(5-(Methoxycarbonyl)-2-oxo-2H-pyran-3-yl)butanoic acid (1): Brown amorphous powder (MeOH), mp 175–176 °C (MeOH). = −57.2° (c = 0.062, MeOH). IR (KBr, cm⁻¹): 3328, 1736, 1720, 1685, 1648, 1624, 1562, 1540, 1458, 1440, 1293, 1133. ESI-MS m/z 241.27[M+H]⁺, 279.22[M+Na]⁺, 502.93[2M+Na]+. HRESI-MS m/z 263.0530 [M+Na]⁺ (C₁₁H₁₂O₆Na). ¹H-NMR and ¹³C-NMR data see Table 1.

7-Hydroxydehydrohastatoside (2): White amorphous powder (MeOH), mp 162–164 °C (MeOH). = −94.5° (c = 0.012, MeOH). IR (KBr, cm⁻¹): 3318, 1733, 1722, 1652, 1628, 1560, 1535, 1448, 1288, 1074. ESI-MS m/z 417 [M−H]⁻, HRESI-MS m/z 441.1001 [M+Na]⁺ (C₁₇H₂₂O₁₂Na). ¹H-NMR and ¹³C-NMR data see Table 1.

4. Conclusions

Two new iridoids were isolated along with three known iridoids from the aerial part of Verbena officinalis L. Verbeofflin I (1) is the new class of secoiridoid in the family Verbenaceae. Biological evaluation of these compounds is under way.