Clinopodium chinense (Benth.) O. Kuntze belongs to the family Labiatae and is a perennial herb distributed in most parts of China. Its aerial parts were used as a folk medicine in China for treatment of influenza, heliosis, allergic dermatitis, dysentery, hematuria, trauma, etc. [1]. Modern pharmacological studies have verified that its aqueous or ethanol extracts exhibited hemostatic, anti-inflammatory, antioxidant, antibiotic activities. In addition, anti-hyperglycemic activity and protective effects in the cardiovascular system have also been reported [2]. Previous phytochemical investigations of this plant indicated the presence of flavonoids, triterpenoid saponins, and volatile oil [3,4]. In our research for bioactive constituents from this medicinal plant, a new prenylated naphthoquinoid, named (3R,4aR,10bR)-3,10-dihydroxy-2,2-dimethyl-3,4,4a,10b-tetrahydro-2H-naphtho[1,2-b]-pyran-5H-6-one (1), as well as six known flavonoids (compounds 2–7) were obtained. Herein, we present the isolation and structure elucidation of compounds 1–7, including the determination of absolute configuration of compound 1by the circular dichroism method, which will provide a standard for absolute configuration determination of structures belonging to this class.

Compound 1 (Figure 1) was obtained in form of colorless crystals with m.p. 151–153 °C. Its molecular formula was determined as C₁₅H₁₈O₄ based on the result of HR-ESI-MS with a quasi molecular ion peak of [M+Na]⁺ at m/z 285.1098 (calcd. 285.1103). The IR spectrum of 1 showed absorptions of hydroxyl (3446 cm⁻¹) and conjugated carbonyl (1680 cm⁻¹) moieties [5]. The ¹H-NMR spectrum of 1 revealed the presence of three ortho-coupled aromatic protons at δ_H 7.41 (1H, d, J = 7.8 Hz, H-7), 7.29 (1H, ps t, J = 7.8 Hz, H-8), 7.11 (1H, d, J = 7.8 Hz, H-9) and aliphatic protons at δ_H 5.14 (1H, d, J = 5.4 Hz, H-10b), 3.99 (1H, t, J = 7.8 Hz, H-3), 2.94 (1H, m, H-4a), 2.86 (1H, dd, J = 15.6, 9.6 Hz, H-5β), 2.59 (1H, dd, J = 15.6, 5.4 Hz, H-5α), 2.24 (1H, dt, J = 12.6, 7.8 Hz, H-4β), 1.63 (1H, ddd, J = 12.6, 7.8, 5.4 Hz, H-4α), 1.17 (3H, s, 11-CH₃), 1.09 (3H, s, 12-CH₃). The ¹³C-NMR spectrum of 1 exhibited 15 carbon signals, which were resolved through a APT experiment into two methyl, two methylene, six methine, and five quaternary carbons, including three oxygen-bearing sp³ carbons at δ_C 88.2 (C-3), 74.3 (C-10b) and 72.1(C-2). ¹H-¹H COSY correlations were observed between H-8 and H-7, H-9; H-4a and H-10b, H-4, H-5; H-4 and H-3, respectively. These correlations established the existence of two isolated proton spin-systems (Figure 2). HMBC correlations from 11-Me and 12-Me to C-2, C-3 indicated that 11-Me, 12-Me and C-3 were all connected with C-2, whereas correlations from H-10b to C-10, C-10a, C-6a and C-2 suggested that C-10b was connected with C-10a, and C-2 was linked to C-10b through a oxygen atom. The cross-peaks of H-5 and H-7 with C-6, H-5 with C-6a demonstrated that C-5 was connected with C-6, and C-6 with C-6a. The correlations from H-9 and H-10b to C-10 verified that the hydroxyl group was linked to C-10 (Figure 2). Based on the above analysis, compound 1 was assigned as a prenylated naphthoquinoid skeleton and was similar to that of 2,2-dimethyl-3-hydroxy-3,4,4a,10b-tetrahydro-5H-naphtho[1,2-b]-pyran-6-one [6]. The only difference was an additional hydroxyl group at position 10 in compound 1.

The relative stereochemistry of 1 was deduced from its NOESY spectrum. The NOESY correlations between H-3 and H-10b, H-10b and H-4a (Figure 3) indicated that the junction of B/C ring adopted a cis configuration, suggesting H-3, H-10b, H-4a were located on the same side of the ring C.

In association with the observed NOESY correlation between H-3 and H-10b as well as the fact that this NOESY correlation could not be observed if the ring C accepted a chair form, suggesting a boat form for the ring C. The CD spectrum of 1displayed a positive Cotton effect at 257 nm and a relatively strong negative Cotton effect at 200 nm, as well as a very weak positive Cotton effect at 343 nm, which was similar to that of cis-isoshinanolone, indicating the 10bR, 4aR configuration [7]. Therefore, compound 1, a rarely seen natural prenylated naphthoquinoid, was elucidated as (3R,4aR,10bR)-3,10-dihydroxy-2,2-dimethyl-3,4,4a,10b-tetrahydro-2H-naphtho[1,2-b]-pyran-5H-6-one. Moreover, the established CD method regarding determination of its absolute configuration should prove useful for the structural elucidation of its analogues.

Besides the new compound, six known flavonoids, namely apigenin (2) [8], luteolin (3) [9], neoeriocitrin (4) [10], naringenin (5) [11], narirutin (6) [12], and didymin (7) [13], were isolated from the plant and their structures determinated by comparing their spectroscopic data with those reported in literature. To the best of our knowledge, neoeriocitrin was isolated for the first time from the species C. chinense.

A new prenylated naphthoquinoid, named (3R,4aR,10bR)-3,10-dihydroxy-2,2-dimethyl-3,4,4a,10b-tetrahydro-2H-naphtho[1,2-b]-pyran-5H-6-one (1), was isolated from the aerial parts of Clinopodium chinense (Benth.) O. Kuntze. To the best of our knowledge, there were only five natural sources of this skeleton in Lippia sidoides [5], and Oroxylum indicum Vent [6], respectively. The rarely seen prenylated naphthoquinoid framework represents a new addition to the molecular diversity of C. chinense. Additionally, the deduced absolute configuration of 1 by the circular dichroism method will provide an approach applicable to determining the stereochemistry of other prenylated naphthoquinoid derivatives.