Leonurus macranthus Maxim., family Lamiaceae (syn. Labiatae), is a perennial herbaceous plant mainly distributed in northeast Asia. The dried aerial parts of L. macranthus are used as motherwort locally for the treatment of menoxenia, dysmenorrhea, amenorrhea, lochia, edema, oliguresis, and ulcerations [1, 2]. Our previously phytochemical investigations of this plant revealed the presence of diterpenoids, lignans, flavonoids, coumarin, and phenolic acids [2, 3]. As a continued program to search for bioactive diterpenoids from medicinal plants [2, 4, 5], four new diterpenoids including one cis clerodane-type (1) and three highly oxygenated labdane-type diterpenes (2-4) (Fig. 1) were isolated from the acetone extract of the aerial parts of L. macranthus. Herein, the isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.

The acetone extract of the aerial parts of L. macranthus was processed by solvent partitioning with n-hexane and CH₂Cl₂, successively. The CH₂Cl₂-soluble fraction was repeatedly subjected to silica gel, Sephadex LH-20, and Lichroprep RP-C18 gel column chromatography (CC) and semipreparative RP-C18 HPLC to afford one new cis clerodane-type (1) and three new labdane-type diterpenes (2-4). Detailed experimental procedures and full spectroscopic data for compounds 1-4 can be found in Supporting information.

Macranthin H (1) was obtained as a colorless gum, [α]_D²¹ -30. Its ¹³C NMR and negative-ion HRESIMS data, exhibiting a deprotonated molecular ion peak at m/z 321.2425, corresponded to a molecular formula of C₂₀H₃₄O₃ (calcd. for C₂₀H₃₃O₃: 321.2435), with four indices of hydrogen deficiency. The IR absorption band at 3419 cm^-1 was indicative of the presence of hydroxy group. The ¹H NMR data (Table 1) displayed characteristic resonances of five methyls [δ_H 1.07, 1.28, 1.67, and 1.68 (s, each 3H); δ_H 0.99 (d, 3H, J = 6.5 Hz)], one oxygenated methylene [δ_H 4.07 (2H, m)], two oxygenated methines [δ_H 3.57 (dt, 1H, J = 11.0, 4.0 Hz); δ_H 4.07 (m, 1H)], and two olefinic protons [δ_H 5.34 (m, 1H); δ_H 5.35 (m, 1H)]. The ¹³C NMR data (Table 1) showed 20 carbon resonances comprising five methyls, five sp³ methylenes, four sp³ methines, two sp³ quaternary carbons, and four sp² carbons. These functionalities accounted for two out of the four indices of hydrogen deficiency, requiring the presence of two rings in 1. The aforementioned data suggested that 1 is a clerodane diterpenoid [8]. The ¹H NMR and ¹³C NMR spectroscopic data (Table 1) of 1 were comparable to those of kolavenol [8], which was previously isolated from the root bark of Entada abyssinica. The significantly deshielded C-2 (δ_C 69.6) and C-7 (δ_C 70.5) resonances as well as the HMBC correlations from H₂-1/H-10 to C-2, from H-2 to C-3/C-4, from H₂-6/H-8/H₃-17 to C-7, and from H-7 to C-17 (Fig. 2) indicated that C-2 and C-7 are oxygenated. The planar structure of 1 was further supported by HMBC correlations from H-3 to C-5/C-18, from H₂-12 to C-11/C-14, from H₂-15 to C-13/C-14, from H₃-16 to C-12/C-13/C-14, from H₃-18 to C-3/C-5, H₃-19 to C-4/C-6/C-10, and from H₃-20 to C-8/C-10/C-11, as well as ¹H-¹H COSY correlations of H-10/H₂-1, H₂-1/H-2, H-2/H-3, H₂-6/H-7, H-7/H-8, and H-8/H₃-17 (Fig. 2). In the NOESY spectrum (Fig. 3), the NOE correlation between H₃-19 and H-10 supported the cis-fusion of A/B rings in 1. The NOE correlations of H-7/H₂-11, H-7/H₃-17, H-7/H₃-19, H-10/H₂-11, H-10/H₃-19, H₂-11/H₃-19, and H₃-19/6α indicated that these hydrogens are cofacial and α-oriented, while NOEs of H-8/H₃-20, H-8/H-6β, and H-1β/H-6β suggested that these protons are β-cofacially oriented. The presence of NOE correlation of H-2/H-10 supported 2-OH to be β-oriented. NOEs of H₂-15/H₃-16, H₂-12/H-14 indicated E configuration of the double bond between C-13 and C-14. Therefore, the structure of 1 (macranthin H) was characterized as 2β, 7β-dihydroxy-(5α, 8α, 9β, 10α)-cleroda-3, 13E-dien-15-ol.

It is worth to note that the cis-fusion of rings A/B is rarely encountered in clerodane-type diterpenoids [9]. Compound 1 represents the first example of cis clerodane-type diterpene in the plants of Leonurus genus.

Macranthin I (2) gave a molecular formula of C₂₀H₃₂O₄ based on the ¹³C NMR data and a deprotonated ion peak at m/z 335.2235 [M-H]^- (calcd. for C₂₀H₃₁O₄: 335.2228) in the negative-ion HRESIMS. The IR spectrum indicated the presence of hydroxy (3432 cm^-1) and carboxyl carbonyl (1693 cm^-1) functionalities. The ¹H NMR data (Table 1) displayed resonances of three methyls [δ_H 0.88, 1.21, 1.67 (s, each 3H)], two oxygenated methylenes [δ_H 3.95 (d, 1H, J = 12.0 Hz), 3.91 (d, 1H, J = 12.0 Hz); δ_H 4.07 (d, 2H, J = 6.5 Hz)], and two olefinic protons [δ_H 5.36 (t, 1H, J = 6.5 Hz); δ_H 5.57 (br s, 1H)]. The ¹³C NMR data (Table 1) of 2 showed 20 carbon resonances due to three methyls, eight methylenes (two oxygenated), four methines (two olefinic), and five quaternary carbons (one carbonyl, two olefinic). Detailed analysis of the ¹H NMR and ¹³C NMR spectroscopic data (Table 1) of 2showed a close structural resemblance to leojaponin B [10], which was previously isolated from Leonurus japonicus. The major difference was the replacement of the C-17 methyl group in leojaponin B by a hydroxymethyl group in 2. This deduction was supported by the significantly deshielded H₂-17 [δ_H 3.95 (d, 1H, J = 12.0 Hz), 3.91 (d, 1H, J = 12.0 Hz); Δδ_H +2.31, 2.27] and C-17 (δ_C 66.3; Δδ_C+43.0) resonances as well as the HMBC correlations from H₂-17 to C-7/C-8/C-9 (Fig. 2). The 2D structure of 2 was also supported by ¹H-¹H COSY correlations of H₂-1/H₂-2, H₂-2/H₂-3, H-5/H₂-6, H₂-6/H-7, H-9/H₂-11, H₂-11/H₂-12, and H-14/H₂-15 (Fig. 2). The relative configuration of 2 was established on the basis of NOESYdata (Fig. 3). The NOE correlations of H₃-20/H-6β and H-5/H-6α supported the trans A/B junction in 2. NOEs of H-5/H-6α, H-5/H₂-11, and H-5/H₃-18 indicated that these protons are on the same face and α-oriented, while the correlations of H₃-20/H-6β and H₃-20/H-9 suggested that these protons are β-oriented. The NOE correlations of H₂-15/H₃-16, H₂-12/H-14 indicated that the double bond between C-13 and C-14 is E configuration. Thus, the structure of 2 (macranthin I) was elucidated as 15, 17-dihydroxylabd-7, 13E-dien-19-oic acid.

Compound 3 was obtained as colorless gum, [α]_D²¹-30. Its molecular formula, C₂₄H₃₈O₈, was deduced from the HRESIMS (m/z 455.2645 [M + H]⁺, calcd. for C₂₄H₃₉O₈: 455.2639) and ¹³C NMR spectroscopic data, indicating six indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3545 cm^-1) and carbonyl (1731 cm^-1) functionalities. The ¹H NMR data (Table 1) displayed characteristic resonances for three tertiary methyl [δ_H 1.05, 1.26, and 1.45 (s, each 3H)], one secondary methyl [δ_H 1.06 (d, 3H, J = 6.5 Hz)], two oxygenated methylene [δ_H 3.62 (m, 1H), 3.67 (m, 1H); δ_H 4.42 (d, 1H, J = 11.5 Hz), 4.46 (d, 1H, J = 11.5 Hz)], two oxygenated methine [δ_H 3.80 (br s, 1H); δ_H 5.15 (d, 1H, J = 2.5 Hz)], and two O-acetyl [δ_H 2.04, 2.12 (s, each 3H)] groups. The ¹³C NMR data (Table 1) of 3 showed 24 carbon resonances due to six methyl (δ_C 10.0, 20.7, 21.1, 21.2, 22.4, 27.5), seven sp³ methylene (δ_C 26.2, 28.5, 30.4, 40.1, 46.2, 60.0, 68.7), four sp³ methine (δ_C 46.4, 47.9, 70.7, 78.7), four quaternary sp³ (δ_C 44.4, 44.7, 85.6, 98.4), two ester carbonyl (δ_C 171.3 and 172.8), and one keto carbonyl (δ_C 206.9) carbons. These functionalities accounted for three out of the six indices of hydrogen deficiency, requiring the presence of a tricyclic system in 3. The aforementioned data suggested that 3 is a highly oxygenated tricyclic spirolabdane diterpenoid [2, 11]. The ¹H NMR and ¹³C NMR spectroscopic data (Table 1) of 3 were comparable to those of macranthin A [2], except for the absence of resonances of an acetoxy group in 3. The deshielded resonances of H-6 (δ_H 5.15) and H₂-19 (δ_H 4.42, 4.46) indicated that two O-acetyl groups are located at C-6 and C-19, respectively. This was confirmed by the HMBC correlations from H-6 and H₂-19 to the acetoxy carbonyl carbons at δ_C 171.3 and 172.8, respectively (Fig. 2). The planar structure of 3 was further supported by ¹H-¹H COSY correlations of H₂-1/H₂-2, H₂-2/H-3, H-5/H₂-6, H-8/H₃-17, H₂-11/H₂-12, and H₂-14/H₂-15 (Fig. 2). The relative configurations at C-3, C-4, C-5, C-6, C-8, C-9, C-10, and C-13 were the same as those of macranthin A on the basis of NOESY data (Fig. 3). Thus, the structure of 3 was defined as 6β, 19-diacetoxy-9, 13-epoxy-3α, 15-dihydroxylabdan-7-one, and this compound was named 15-O-deacetylmacranthin A.

Compound 4 was isolated as a colorless gum, [α]_D²¹ -22, and its ¹³C NMR and positive-ion HRESIMS data showing a protonated ion peak at m/z 455.2641 [M + H]⁺ established a molecular formula of C₂₄H₃₈O₈, indicating six indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3421 cm^-1), ester carbonyl (1737 cm^-1), and α, β-unsaturated carbonyl (1655 cm^-1) functionalities. The ¹H NMR data (Table 1) exhibited resonances for four tertiary methyl [δ_H 1.10, 1.25, 1.47, and 1.83 (s, each 3H)], two oxygenated methylene [δ_H 3.73 (m, 1H), δ_H 3.76 (m, 1H); δ_H 4.69 (d, 1H, J = 11.5 Hz), 4.82 (d, 1H, J = 11.5 Hz)], two oxygenated methine [δ_H 4.19 (d, 1H, J = 3.5 Hz); 5.08 (br s, 1H)], and two O-acetyl (δ_H 2.05 and 2.07, s, each 3H) groups. The ¹³C NMR spectrum showed 24 carbon resonances including six methyls, seven methylenes (two oxygenated), three methines (two oxygenated), and eight quaternary carbons (one ketocarbonyl, two ester carbonyl, two olefinic, and one oxygenated). The aforementioned data suggested that compound 4 is a highly oxygenated bicyclic labdane diterpenoid similar to macranthin F [2]. The ¹H NMR and ¹³C NMR spectroscopic data (Table 1) were highly similar to those of macranthin F [2], with the exception of the deshielded H-3 resonance (δ_H 5.08; Δδ_H +1.28) and the presence of an additional acetyl group (δ_H 2.05; δ_C 172.3, 20.7), indicating 4 to be the 3-O-acetyl derivative of macranthin F. This deduction was confirmed by the HMBC correlation from H-3 to the carbonyl carbon (δ_C172.3) of the acetyl group (Fig. 2). The 2D structure of 4 was further supported by ¹H-¹H COSY correlations of H₂-1/H₂-2, H₂-2/H-3, H-5/H₂-6, H-8/H₃-17, H₂-11/H₂-12, and H₂-14/H₂-15 (Fig. 2). The relative configuration of 4 was established on the basis of the NOESY data (Fig. 3). The NOE correlations from H₃-20 to H₂-19 and from H-5 to H₃-18 indicated that the A/B rings are trans-fused. NOE correlations of H-6/H-5, H-5/H-1α, and H-6/H₃-18 indicated these protons are cofacial and α-oriented, while NOEs of H-3/H₂-19, H₂-19/H₃-20 were consistent with these protons being β-cofacially oriented (Fig. 3). However, the configuration at C-13 could not be assigned. Accordingly, the structure of 4 was defined as 3α, 19-diacetoxy-6β, 13, 15-trihydroxylabd-8(9)-en-7-one, named 3-O-acetylmacranthin F.

Compounds 1-4 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using Griess assay [2, 6, 7]. Compounds 1 and 4 showed weak inhibition of NO production with IC₅₀ values of 35.8 ± 3.6 μmol/L and 48.6 ± 4.8 μmol/L, respectively. Indomethacin (IC₅₀ = 30.6 ± 2.2 μmol/L) was used as a positive control. Compounds 2 and 3 were inactive (< 50% inhibition at 100 μmol/L, the highest concentration tested). In order to investigate whether the inhibitory activities of compounds 1 and 4 were due to cytotoxicity, their effects on cell proliferation/viability using the MTT method were measured. These two compounds (up to 100 μmol/L) did not show any cytotoxicity with LPS treatment for 24 h.

In summary, one cis clerodane-type (1) and three highly oxygenated labdane-type diterpenes (2-4) were isolated from the aerial parts of Leonurus macranthus. The cis-fusion of rings A/B is rarely encountered in clerodane-type diterpenoids. Compound 1 represents the first example of cis clerodane-type diterpene in the plants of Leonurus genus. Compounds 1 and 4 exhibited weak inhibition of NO in LPS-stimulated BV-2 microglial cells with IC₅₀ values of 35.8 ± 3.6 μmol/L and 48.6 ± 4.8 μmol/L, respectively.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 81573572, 81530097) and New Century Excellent Talents in University (No. NCET-13-0693).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2017.11.033.