1. Introduction
Leonurus japonicus Houtt. (Lamiaceae) is an annual or biennial herbaceous plant widely distributed and cultivated in China. The dried herb is used in Traditional Chinese Medicine for the treatment of various diseases, especially menstrual disturbances, dysmenorrhea, and amenorrhea [
1]. Our previous investigation on the plant resulted in the isolation of 19 secondary metabolites, including sesquiterpenoids, coumarins, lignans and phenylpropanoids [
2,
3]. In continuation of our research on bioactive natural products from
L. japonicus, we have now carried out another study on the same EtOH extract. The present study has led to the isolation of diterpenoids and ergosterols, including a new norditerpenoid, (−)-3
α-acetoxy-6
β-hydroxy-15,16-dinorlabd-8(9)-ene-13-yne-7-one (
1), and four known compounds, leoheteronin F (
2) [
4], (22
E,24
R)-5
α,8
α-epidioxyergosta-6,9(11),22-trien-3
β-ol (
3) [
5], (22
E,24
R)-5
α,8
α-epidioxyergosta-6,22-dien-3
β-ol (
4) [
5], and (22
E)-ergosta-6,9,22-triene-3
β,5
α,8
α-triol (
5) [
6] (
Figure 1). Compound
1, an unusual bicyclic diterpenoid possessing a new 15,16-dinorlabd carbon skeleton, showed coagulant activity by shortening the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and increasing the fibrinogen (FIB) levels. This paper reports the isolation, structure elucidation, and bioassays of these compounds.
Figure 1.
Structures of compounds 1–5.
Figure 1.
Structures of compounds 1–5.
2. Results and Discussion
Compound
1 was obtained as a colorless oil, and its IR spectrum showed the presence of OH (3,456 cm
−1), acetylenic bond (3,291 and 2,120 cm
−1), carbonyl (1,728 and 1,659 cm
−1) and double bond (1,607 cm
−1) groups. The molecular formula C
20H
28O
4 of
1, with seven degrees of unsaturation, was indicated by the HRESIMS peak at
m/
z 355.1883 [M+Na]
+ (calcd. for C
20H
28O
4Na, 355.1885). The
1H-NMR spectrum of
1 displayed resonances attributable to five tertiary methyl groups [
δH 1.00 (H
3-17), 1.42 (H
3-18), 1.48 (H
3-19), 1.81 (H
3-20), and 1.99 (H
3-2ʹ)], a monosubstituted acetylenic bond [
δH 2.47 (t,
J = 2.4 Hz, H-14)], two oxymethines [
δH 4.60 (t,
J = 3.0 Hz, H-3), 4.19 (dd,
J = 4.2, 3.0 Hz, H-6)], an aliphatic methine [
δH 1.97 (d,
J = 3.0 Hz, H-5)], and four aliphatic methylenes between
δH 1.70 and 2.60 (
Table 1). It also displayed an exchangeable resonance assignable to a secondary hydroxy proton at
δH 4.53 (d,
J = 4.2 Hz, OH-6). The
13C-NMR and DEPT spectra of
1 showed a total of 20 carbon resonances corresponding to the above protonated units and six quaternary carbons, including a conjugated carbonyl (
δC 198.1) and a conjugated tetrasubstituted double bond (
δC 166.5 and 129.6) which implied an
α,
β-unsaturated ketone system for compound
1. The presence of an acetoxyl group in
1 was indicated by the signals of the ester carbonyl (
δC 170.4) in the
13C-NMR and the methyl (
δH 1.99) in the
1H-NMR. In addition, the signals of the sp quaternary carbon (
δC 83.9) and sp methine (
δC 70.6,
δH 2.47) were characteristic of a terminal alkynyl group. These spectroscopic data suggested that
1 was an unusual bicyclic dinorditerpenoid possessing an
α,
β-unsaturated ketone system, a terminal alkynyl unit, and substitutions of a hydroxyl and an acetoxyl group.
The above conjecture was confirmed by 2D NMR data analysis. The proton and corresponding carbon resonances in the 2D NMR spectra of
1 were assigned by the gHSQC experiment. The H
2-1/H
2-2/H-3, H-5/H-6, and H
2-11/H
2-12 coupling correlations in the gCOSY spectrum revealed three vicinal coupled systems (
Figure 2). In the HMBC spectrum, two- and three-bond correlations of H-3/C-1, C-2, C-4, C-5, C-17, C-18, and C-1ʹ; H-6/C-4, C-5, C-7, C-8, and C-10; OH-6/C-5 and C-6; H
2-11/C-8, C-9, C-10, C-12, and C-13; H-14/C-12; H
3-17 and H
3-18/C-3, C-4, and C-5; H
3-19/C-1, C-5, C-9, and C-10; H
3-20/C-7, C-8, and C-9 (
Figure 2), in combination with the vicinal coupled signals, indicated that the planar structure of
1 was 3-acetoxy-6-hydroxy-15,16-dinorlabd-8(9)-ene-13-yne-7-one.
Table 1.
1H- (600 MHz) and 13C-NMR (150 MHz) data of 1 (in Me2CO-d6, δ in ppm, J in Hz).
Table 1.
1H- (600 MHz) and 13C-NMR (150 MHz) data of 1 (in Me2CO-d6, δ in ppm, J in Hz).
No. | δH | δC | No. | δH | δC |
---|
1 | 1.73 m | 31.7 | 12 | 2.37 m | 18.5 |
2 | 2.16 m, 1.72 m | 23.5 | 13 | – | 83.9 |
3 | 4.60 t (3.0) | 78.5 | 14 | 2.47 t (2.4) | 70.6 |
4 | – | 38.0 | 17 | 1.00 s | 27.5 |
5 | 1.97 d (3.0) | 48.5 | 18 | 1.42 s | 24.4 |
6 | 4.19 dd (4.2, 3.0) | 71.4 | 19 | 1.48 s | 22.1 |
7 | – | 198.1 | 20 | 1.81 s | 11.9 |
8 | – | 129.6 | 1ʹ | – | 170.4 |
9 | – | 166.5 | 2ʹ | 1.99 s | 21.0 |
10 | – | 41.5 | OH-6 | 4.53 d (4.2) | – |
11 | 2.58 t (7.8) | 29.5 | | | |
The configuration of
1 was elucidated on the basis of the NOESY interactions and interproton coupling patterns [
7,
8,
9]. A relatively small coupling constant of H-3 (
J = 3.0 Hz) and the distinct NOE between H-3 and H
3-18 (
Figure 2) confirmed the assignment of H-3 as
β-equatorial orientation [
8]. The H-5 and H-6 coupling constant (
J = 3.0 Hz) placed the two protons
cis to each other in an
α-axial,
α-equatorial orientation, leaving the OH-6 as
β-axial, which was confirmed by the NOE correlations of H
3-17 with H-5 and H-6. These data conclusively proved the new structure of
1 as (−)-3
α-acetoxy-6
β-hydroxy-15,16-dinorlabd-8(9)-ene-13-yne-7-one.
Figure 2.
(a) Key 1H, 1H-COSY and HMBC correlations of 1; (b) Key NOESY correlations of 1.
Figure 2.
(a) Key 1H, 1H-COSY and HMBC correlations of 1; (b) Key NOESY correlations of 1.
The coagulant activity of the compounds was examined by monitoring activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) levels. A summary of the results for compound
1 is shown in
Table 2. At concentrations of 1 mg/mL and 0.1 mg/mL, compound
1 shortened the APTT, PT, and TT and increased the content of FIB. Compared to the normal saline control group, they were statistically significant (
p < 0.05 or
p < 0.01), except for the PT at 0.1 mg/mL. Although there was no statistical difference in the APTT, PT, and TT between the group of 0.01 mg/mL and the normal saline group, treatment with compound
1 at this concentration resulted in obviously increased FIB (
p < 0.05). The above results showed that compound
1 had coagulant activity via multiple pathways. Other isolated compounds were inactive in the blood coagulation assay at the concentrations of 1 mg/mL, 0.1 mg/mL, and 0.01 mg/mL.
Table 2.
Coagulant activity of compound 1 a.
Table 2.
Coagulant activity of compound 1 a.
Sample | Dose | APTT (s) | PT (s) | TT (s) | FIB (g/L) |
---|
Control | Normal saline | 31.22 ± 6.20 | 9.98 ± 0.54 | 53.24 ± 5.91 | 1.59 ± 0.08 |
Compound 1 | 1 mg/mL | 28.88 ± 5.57 ** | 9.87 ± 0.47* | 49.94 ± 3.46 * | 1.64 ± 0.03 * |
| 0.1 mg/mL | 29.66 ± 4.90 * | 9.94 ± 0.44 | 50.77 ± 4.43 * | 1.66 ± 0.03 * |
| 0.01 mg/mL | 31.38 ± 4.87 | 10.00 ± 0.49 | 53.28 ± 3.98 | 1.65 ± 0.06 * |
3. Experimental
3.1. General
NMR spectra were recorded on a INOVA-500 or Bruker-AVIIIHD-600 spectrometers. HRESIMS were measured with a Waters Synapt G2 HDMS. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). Optical rotations were measured with a Perkin-Elmer 341 plus. Column chromatography was performed with silica gel (200–300 mesh, Yantai Institute of Chemical Technology, Yantai, China), MCI gel CHP 20P (75–150 μm, Mitsubishi Chemical, Co., Tokyo, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on an instrument consisting of a Cometro 6000LDS pump and a Cometro 6000PVW UV/VIS detector with an Ultimate (250 × 10 mm) preparative column packed with C18 (5 μm). TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China).
3.2. Plant Material
The L. japonicus herb was collected in May of 2012 from a field in Wenjiang District, Chengdu City, Sichuan Province, China. Plant identity was verified by Prof. Min Li (Chengdu University of TCM, Sichuan, China). A voucher specimen (SYMC-0522) was deposited at the School of Pharmacy, Chengdu University of TCM, China.
3.3. Extraction and Isolation
The air-dried herb of L. japonicus (20 kg) was extracted with 95% EtOH (3 × 160 L) at room temperature for 3 × 72 h. The ethanolic extract was evaporated under reduced pressure to yield a dark brown residue (1.2 kg). The residue was suspended in H2O and then successively partitioned into EtOAc (400 g) and n-BuOH (160 g) fractions. The EtOAc extract was subjected to silica gel CC using a gradient elution of petroleum ether–acetone (100:1–0:1) to afford nineteen fractions (F1−F19) based on TLC analysis. F6 was further separated by MCI with a gradient of increasing MeOH (50%–100%) in water to yield six subfractions F6-1−F6-6. The successive separation of F6-4 with Sephadex LH-20 (petroleum ether−CHCl3−MeOH, 5:5:1) and with PTLC (petroleum ether−EtOAc 5:1) yielded 1 (6 mg) and 2 (18 mg). F6-6 was purified via silica gel CC over petroleum ether–EtOAc (40:1–5:1) to get F6-6-1–F6-6-4. Separation of F6-6-2 by repeated PTLC (petroleum ether−EtOAc 5:1) and reversed-phase semipreparative HPLC (80% MeCN in H2O) yielded 3 (7 mg), 4 (21 mg), and 5 (26 mg).
(−)-3α-Acetoxy-6β-hydroxy-15,16-dinorlabd-8(9)-ene-13-yne-7-one (
1): Colorless oil,

= −17.3 (
c = 0.10, MeOH); IR νmax: 3456, 3291, 2941, 2874, 2120, 1728, 1659, 1607, 1461, 1375, 1248, 1128, 1047, 930 cm
−1; ESI-MS
m/z 355.2 [M+Na]
+; HRESI-MS:
m/z 355.1883 [M+Na]
+ (calcd. for C
20H
28O
4Na, 355.1885);
1H- and
13C-NMR data see
Table 1.
3.4. Blood Coagulation Assay [10]
SD rats were lightly anesthetized with ether. Blood was immediately taken from the femoral artery and anticoagulated with 3.8% trisodium citrate (9:1, v/v). Plasma was obtained by centrifugation of the whole blood at 3,500 g for 10 min. Then, the plasma (200 μL) was mixed with 20 μL of tested compounds or normal saline and incubated for 5 min at 37 °C. PT, APTT, TT, and FIB were determined by an automatic coagulation analyzer (Sysmex CA-500, Kobe, Japan) according to the manufacturer's instructions. Data were expressed as mean ± SEM (standard error of the mean) of at least three independent experiments. Statistical significance between two groups was determined using the Student’s t-test and a p-value of <0.05 was considered as significant.