Monoterpenoids from the whole herb of Veronicastrum axillare

Abstract

Context: Veronicastrum axillare (Sieb. et Zucc.) Yamazaki (Scrophulariaceae) embraces varieties of bioactivities such as anti-inflammatory, anti-pyresis and detoxification activity, while little is known of the phytochemical components of this medicinal plant.

Objective: To isolate and identify bioactive constituents from the whole herb of V. axillare.

Materials and methods: Ethanol extract of the whole herb of V. axillare was subjected to successive column chromatography. Chemical structures of the compounds were elucidated by detailed spectroscopic analyses on the basis of NMR, IR and HR-MS data.

Results: A new monoterpenoid, axillacetal A (1) and a known analogue, tarumal (2), were isolated from the whole herb of V. axillare. The structure of tarumal (2) was also revised according to our NMR data.

Discussion and conclusion: This is the first report on the isolation and authentication of novel chemical constituents from V. axillare.

• Axillacetal A
• monoterpenoids
• tarumal
• Veronicastrum axillare

Introduction

Plants of Veronicastrum Heist. ex Farbic. (Scrophulariaceae) are often used as folk medicine for the treatment of ascites in southern China. Modern pharmacological studies have shown that Veronicastrum species have versatile beneficial pharmacological properties, such as anti-inflammatory, analgesic, antibiosis and immunosuppressive activities (Gao et al., 2004; Huang et al., 2009; Zhou & Meng, 1992a). Several kinds of chemical constituents have been isolated from these plants, including flavonoids, phenols, phenylpropanoids and terpenoids (Inouye & Aoki, 1968; Inouye et al., 1969; Lin et al., 1995; Liu et al., 1999; Nohara et al., 2010; Shang et al., 1997; Shimada et al., 1971; Zhao & Shi, 2007; Zhou & Meng, 1992b). Recently, several studies revealed that Veronicastrum axillare exhibited potential antiulcer effects on gastric ulcer in rats induced by ethanol (Du et al., 2013; Shen et al., 2012). In the course of our searching for novel bioactive components from Veronicastrum species, a new monoterpenoid, axillacetal A (1) and a known analogue, tarumal (2), were isolated from the whole herb of Veronicastrum axillare, a species in Zhejiang, China. This paper describes the isolation and structural elucidation of the isolated compounds.

Materials and methods

General

Analytical TLC was performed on silica-gel plates (Yan-tai Institute of Chemical Technology, Yantai, China), with dichloromethane (CH₂Cl₂)/methanol (MeOH) 30:1 as an eluent; visualization under UV light, and by spraying with 7% H₂SO₄/C₂H₅OH, followed by heating. Column chromatography (CC) was carried out on silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China). Preparative thin layer chromatography was performed on silica gel (10–40 μm, Yantai Institute of Chemical Technology). Sephadex LH-20 was obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Optical rotation density (ORD) was determined on a JASCO P-1020 spectropolarimeter. UV spectra were determined with a Shimadzu UV-260 spectrophotometer, in MeOH, with λ_max (log ε) in nm. IR spectra were obtained on an Avatar 360-ESP spectrophotometer (Thermo Nicolet, Abbott Park, IL), as KBr pellets, in cm⁻¹. ¹H and ¹³C NMR spectra were recorded with a DRX-300 spectrometer, in CDCl₃ in ppm, J in Hz. HR-ESI-MS spectra were determined with a Bruker APEX 7.0 TESLA FT-MS apparatus.

Plant material

The whole plants of V. axillare were collected from Jiande, Zhejiang Province, PR China, in July 2011. A voucher specimen (#201107), identified by Prof. Lu-Ping Qin, is deposited at the Herbarium of Materia Medica, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, PR China.

Extraction and isolation

The air-dried, whole herb (8.5 kg) of V. axillare was reflux-extracted exhaustively with 80% EtOH/H₂O for three times. The EtOH extract was concentrated in vacuo to yield a semi-solid (1.7 kg), which was suspended in H₂O (1.7 L), and extracted with CH₂Cl₂ (3 × 1.0 L). The combined organic-phase was concentrated to yield a residue (55 g), part of which (45 g) was subjected to CC (850 g Gel; PE/EtOAc gradient) to afford four fractions (fractions 1–4). Fraction 3 was eluted with PE/EtOAc 5:1→1:1 to give four subfractions I–IV). Subfraction II was subjected to a Sephadex LH-20 column (CH₂Cl₂:MeOH, 40:60; 0.5 L, v/v) to give compound 2 (68.4 mg). Subfraction IV was subjected to Sephadex LH-20 (CH₂Cl₂:MeOH, 40:60; 0.5 L, v/v), and then with PTLC (PE/EtOAc 3:1) to afford compound 1 (9.1 mg).

Axillacetal A (1)

(Rel-3R,5R,6S)-3-methoxy-8-methyl-4,5,6,7-tetrahydro-2H-cyclopenta[b]furan-9-carbaldehyde; 1): yellow oil; [α]= −7.5° (CHCl₃, c 0.10); IR (KBr) ν_max: 2925, 2854, 1735, 1667 cm⁻¹; UV (MeOH) λ_max (log ε): 288 nm (1.06); ¹H (CDCl₃, 300 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data (Tables 1 and 2); (+)-HRESIMS m/z [M + H]⁺ 183.1014 (calcd for C₁₀H₁₅O₃ 183.0943).

Table 1. ¹H NMR data of compounds 1, tarumal and 2 (compounds 1 and 2 at 300 MHz, tarumal at 400 MHz, in CDCl₃ at 27 °C, δ in ppm, J in Hz).

Position	1	Tarumal	2
1	9.96 (s, 1H)	9.98 (s, 1H)	9.94 (s, 1H)
3	4.99 (dd, J 3.0 and 2.7, 1H)	5.00 (d, J 5.1, 1H)	4.96 (d, J 4.8, 1H)
4a	2.11 (m, 2H)	2.32 (dd, J 13.7 and 9.8, 1H)	2.28 (m, 1H)
4b		1.88 (ddd, J 13.7, 5.2 and 5.1, 1H)	1.84 (m, 1H)
5	3.54 (m, 1H)	3.60 (m, 1H)	3.56 (m, 1H)
6	4.79 (dt, J 7.2 and 1.8, 1H)	4.65 (dd, J 6.4 and 6.1, 1H)	4.61 (t, J 6.3, 1H)
7a	2.90 (dd, J 20.1 and 7.2, 1H)	2.88 (ddd, J 19.8, 6.4 and 1.2, 1H)	2.82 (ddt, J 19.8, 6.3 and 0.9, 1H)
7b	2.69 (d, J 20.1, 1H)	2.68 (d, J 19.8, 1H)	2.64 (dd, J 19.8 and 0.9, 1H)
10	2.12 (s, 3H)	2.17 (s, 3H)	2.13 (s, 3H)
OMe	3.24 (s, 3H)	3.33 (s, 3H)	3.30 (s, 3H)

Table 2. ¹³C NMR data of compounds 1, tarumal and 2 (compounds 1 and 2 at 125 MHz, tarumal at 100 MHz, in CDCl₃ at 27 °C, δ in ppm).

Position	1	Tarumal	2
1	187.9 (d)	188.0 (d)	187.9 (d)
3	106.4 (d)	105.8 (d)	105.7 (d)
4	37.1 (t)	38.0 (t)	37.9 (t)
5	47.3 (d)	47.3 (d)	47.2 (d)
6	80.4 (d)	78.4 (d)	78.3 (d)
7	49.2 (t)	46.0 (t)	46.0 (t)
8	159.8 (s)	159.6 (s)	159.5 (s)
9	139.0 (s)	139.6 (s)	139.5 (s)
10	14.4 (q)	14.3 (q)	14.2 (q)
OMe	54.9 (q)	54.4 (q)	54.3 (q)

Tarumal (2)

(Rel-3S,5R,6S)-3-methoxy-8-methyl-4,5,6,7-tetrahydro-2H-cyclopenta[b]furan-9-carbaldehyde; 2): yellow oil; [α]= +2° (CHCl₃, c 0.10). IR (KBr) ν_max: 2909, 2853, 1704, 1639 cm⁻¹; UV (MeOH) λ_max log ε): 290 nm (1.12); ¹H (CDCl₃, 300 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data (Tables 1 and 2); (+)-HRESIMS m/z [M + H]⁺ 183.1018 (calcd for C₁₀H₁₅O₃ 183.0943).

Results and discussion

Repeated CC of the CH₂Cl₂ extract from the whole herb of V. axillare yielded compounds 1 and 2 (Figure 1).

Figure 1. Two monoterpenoids from V. axillare.

Axillacetal A (1), obtained as a yellow oil, was assigned the molecular formula C₁₀H₁₄O₃ on the basis of HR-ESI-MS data at m/z 183.1014 ([M + H]⁺), which indicated four degrees of unsaturation. Its IR spectrum revealed the absorption bands for an α,β-unsaturated aldehyde group (1667 and 2854 cm⁻¹). The ¹H NMR spectrum (Table 1) exhibited signals corresponding to an aldehyde proton [δ(H) 9.96, s, 1H], two oxygenated methine protons [δ(H) 4.99, dd, J 3.0 and 2.7, 1H; δ(H) 4.79, dt, J 7.2 and 1.8, 1H], a tertiary methyl group [δ(H) 2.12, s, 3H] and a methoxyl group [δ(H) 3.24, s, 3H]. The ¹³C NMR spectrum (Table 2) of compound 1 showed signals for 10 carbons, characterized by the presence of one aldehyde carbon [δ(C) 187.9], two olefinic carbons [δ(C) 139.0 and 159.8], one acetal carbon [δ(C) 106.4], one oxygenated methine carbon [δ(C) 80.4] and one methoxyl carbon [δ(C) 54.9].

The NMR spectra data of 1 were extremely analogous to those of tarumal. These two compounds possess the same molecular formula, C₁₀H₁₄O₃, indicating that they are structural isomers of each other.

Tarumal was an iridoid isolated from Vitex cymosa Bertero ex Spreng (Verbenaceae) (dos Santos et al., 2001). The UV, IR and NMR (Tables 1 and 2) spectra of compound 2 were almost the same as those of tarumal. Combined with the plus optical rotation values of 2 and tarumal ([α] = +2.0°, c 0.10, 2 in MeOH; [α] = +14.3°, c 1%, tarumal in CHCl₃), we believed that compound 2 and tarumal were of the same chemical substance and should share the same planar structure and stereochemistry. However, observed NOESY correlations among H-5/H-6, H-3/H-5 and H-3/H-6 (Figure 2) indicated the spatial arrangement of H-3 was of β-orientation and on the same side of H-5 and H-6, with the configuration of C-3 in 2 deduced to be S, which was different from that of tarumal. Since the R configuration of C-3 in tarumal was only confirmed by molecular modelling using the program Serena Software (dos Santos et al., 2001), without direct 2D NMR evidences such as NOESY data, we suggest that the reported structure of tarumal should be revised as that of compound 2, (rel-3S,5R,6S)-3-methoxy-8-methyl-4,5,6,7-tetrahydro-2H-cyclopenta[b]furan-9-carbaldehyde.

Figure 2. Key NOESY correlations in compounds 1 and 2.

The major NMR data difference between 1 and 2/tarumal were the splitting pattern and the chemical shift of an acetal proton H-3 (δ(H) 4.99, dd, J 3.0 and 2.7 in 1; δ(H) 5.00, d, J 5.1 in tarumal; δ(H) 4.96, d, J 4.8 in 2) and the chemical shift corresponding to the acetal carbon C-3 (δ(C) 106.4 in 1; δ(C) 105.8 in tarumal; δ(C) 105.7 in 2). All of these data led to the assumption that compound 1 was a 3-epimer of tarumal and 2. The configuration of H-3 in compound 1 was recognized as α-orientation, different from that of 2/tarumal, evidenced by the absent correlations between H-3 [δ(H) 4.99] with H-5 [δ(H) 3.54] or H-6 [δ(H) 4.79] in the NOESY spectrum. Finally, the structure of compound 1 was deduced to be (rel-3R,5R,6S)-3-methoxy-8-methyl-4,5,6,7-tetrahydro-2H-cyclopenta[b]furan-9-carbaldehyde, and it was named axillacetal A.

In conclusion, considering the versatile pharmacological activities of Veronicastrum species, our study was conducted to search for novel bioactive chemical components from V. axillare and led to the isolation of a new monoterpenoid, axillacetal A (1) and a known analogue, tarumal (2), which contributes to the expansion of the chemical knowledge of those Veronicastrum species.

Declaration of interest

The authors report no declaration of interest. The authors are grateful for financial support from the National Natural Science Fund of China (Nos. 81102773 and U1203202).