The acetone–water (4:1) extract (116 g) of the whole plants of Nemophila menziesii Hook. et Arn. was subjected to a Diaion HP-20 column to yield approximately separated fractions Frs. 1A-1G using a methanol–water mobile phase solvent system. Compounds 1–9 were isolated from Frs. 1C [MeOH: water (2:3)] and 1D [MeOH: water (3:2)] using reversed-phase HPLC. Compounds 1–9 comprised a hydroquinone, monoterpenoids, and either one or two glucosyl moieties (Fig. 1).

The HPLC sugar analyses after acid hydrolysis of 1–9 showed that the glucosyl moieties were D-glucose [5], and the coupling constant value J = 7.0–7.5 Hz indicated their β-orientations. That is, 1–9 were meroterpenoids characterized as di- or mono-glycosides according to the number of β-D-glucosyl moieties.

The molecular formula of 1 (C₂₈H₃₆O₁₃) was confirmed by (+)-HRFABMS ([M + Na]⁺ ion at m/z 603.2042, calculated for C₂₈H₃₆O₁₃Na: 603.2053). The ¹H and ¹³C NMR data for 1 are listed in Table 1. In the ¹H NMR spectrum of 1, three methyl [δ_H 2.24 (3H, s, H-10′), 1.56 (3H, s, H-9′), and 1.50 (3H, s, H-8′)], olefinic [δ_H 7.95 (1H, s, H-2′), 7.06 (1H, d, J = 9.0 Hz, H-6), 6.93 (1H, d, J = 9.0 Hz, H-5), and 6.77 (1H, s, H-5′) and glycosyl [δ_H 5.07–3.35] proton resonances were observed. In the aliphatic region of the ¹³C NMR spectrum of 1, three methyl (δ_C 25.8, C-9′; 25.6, C-8′; 16.6, C-10′), one quaternary (δ_C 49.0, overlapping, C-7′), and two sets of glucosyl (δ_C 102.3, 75.3, 78.8, 71.5, 78.2, 62.6 and δ_C 102.1, 75.3, 78.7, 71.4, 78.1, 62.6) carbon resonances were observed. In its olefinic region, there were 12 carbon resonances indicating the presence of two phenyl moieties (δ_C 148.9, C-1; 131.5, C-2; 142.0, C-3; 151.1, C-4; 113.5, C-5; 115.9, C-6; 130.6, C-1′; 127.8, C-2′; 123.9, C-3′; 156.2, C-4′; 109.0, C-5′; 154.9, C-6′). In the HMBC spectrum, two methyl (H-8′ and H-9′) and H-5′ protons were long-range coupled with the quaternary C-7′; the methyl protons and H-5 were long-range coupled with C-3, which suggested the two phenyl moieties were connected to the quaternary carbon. The HMBC correlations from the other methyl and olefinic protons (Fig. 2) suggested the presence of the 3′,7′,7′-tri-methyl-1,4,4′-trioxy-fluorene skeleton. The NOESY correlation between H-2′ and H-10′ and that between H-5′ and H-8′ supported this conclusion. The anomeric protons at δ_H 5.07 (1H, d, J = 7.5 Hz) and 4.99 (1H, d, J = 7.5 Hz) were HMBC long-range coupled with C-1 and C-4, respectively, which showed the 1,4-diglucosyl of fluorene. From these data, the chemical structure of 1 was established as illustrated in Fig. 1.

Compound 2 had a molecular formula of C₂₈H₃₆O₁₄ (HRFABMS negative m/z 595.2042; [M–H]^– ion at m/z, calcd for C₂₈H₃₅O₁₄: 595.2027), with one more oxygen atom than that in 1. There is no H-5′ resonance in the ¹H NMR spectrum, and an oxygenated olefinic carbon (δ_C 143.0, C-5′) was observed in the ¹³C NMR spectrum of 2, instead of δ_C 109.0 in that of 1. Therefore, 2 was confirmed to be a hydroxy derivative of 1 as shown in Fig. 1.

The molecular formula of 3 (C₂₈H₄₂O₁₃) was confirmed using (+)-HRFABMS, which showed an [M + Na]⁺ ion at m/z 609.2515 (calcd. for C₂₈H₄₂O₁₃Na: 609.2523). The ¹H NMR spectrum of 3 showed three methyl [δ_H 1.72 (3H, s, H-10′), 1.00 (3H, s, H-9′), and 0.69 (3H, s, H-8′)], o- and m- coupling system olefinic [δ_H 6.99 (1H, d, J = 3.0 Hz, H-3), 6.94 (1H, dd, J = 9.0, 3.0 Hz, H-5), 7.04 (1H, d, J = 9.0 Hz, H-6)] proton resonances were observed. In the ¹³C NMR spectrum, 12 glycosidic, 10 terpenoid, and six phenyl carbon resonances were observed. These data, except for that of the sugar moieties were similar to those of conitriol which was one of the meroterpenoids isolated from the Ascidian Aplidium conicum. [6] The HMBC correlations (Fig. 2), including those from H-10′ to C-2′ (δ_C 127.5), C-3′ (δ_C 134.1), C-4′ (δ_C 32.4); from H-8′ and H-9′ to C-6′ (δ_C 52.0); from H-3 to C-1 (δ_C 151.6), C-5 (δ_C 116.4), C-1′ (δ_C 34.3), supported that the aglycone of 3 was conitriol.

The 1,4-diglucosyl moiety was confirmed by the HMBC correlations between the anomeric protons at δ_H 4.85 (1H, d, J = 7.0 Hz) and 4.72 (1H, d, J = 7.5 Hz) and C-1 and C-4 (δ_C 154.4), respectively. The relative configuration of C-1′ and C-6′ was confirmed as the Z-configuration by the coupling constant J_1′-6′ = 4.5 Hz and the NOESY correlation, [6–8]. Based on the determined structures, the most stable conformations were calculated for all the possible absolute configurations at C-1′ and C-6′ on the cyclohexene ring (Figure S2). Thereafter, the dihedral diagonals were obtained and applied to the Karplus equation (1′S6′R: J_1′-6′ = 4.37 Hz; 1′S6′S: J_1′-6′ = 7.61 Hz; 1′R6′R: J_1′-6′ = 1.56 Hz; 1′R6′S: J_1′-6′ = 3.57 Hz), which supports its Z-configuration. Furthermore, their expected ECD spectra were calculated, and the C-1′ absolute configuration of 3 was confirmed to be S by the strong negative Cotton effect at approximately 205 nm in the ECD spectrum by comparison with the calculated data and experimental data of 3 (Fig. 3A). The chemical structure of compound 3 was confirmed to be as shown in Fig. 1.

The molecular formula of 4 (C₂₈H₄₂O₁₃) was confirmed using (+)-HRFABMS ([M + Na]⁺ ion at m/z 609.2515, calcd. for C₂₈H₄₂O₁₃Na: 609.2523), which was identical to that of 3. The ¹H and ¹³C NMR spectra of 4 are similar to those of 3 (Table 1), suggesting that the aglycone of 4 is an enantiomer of the aglycone of 3. In the ECD curve of 4, a positive Cotton effect was observed at approximately 205 nm, indicating the 1′R-configuration (Fig. 3A). Therefore, the structure of 4 was elucidated as shown in Fig. 1.

The molecular formula of 5 (C₂₂H₃₀O₈) was determined using (–)-HRFABMS, which showed an [M–H]^– ion at m/z 421.1867 (calcd. for C₂₂H₂₉O₈: 421.1863). The ¹H and ¹³C NMR spectra of 5 (Table 1) were similar to those of 3 and 4. Conversely, 5 had additional olefinic protons and carbons (δ_H 4.50, 1H, s, H-8′; 4.28, 1H, s, H-8′; δ_C 149.5, C-7′; 110.5, C-8′) and an oxygenated carbon and corresponding proton resonances (δ_H 4.12, 1H, br d, J = 3.0 Hz, H-4′; δ_C 69.2, C-4′) and only one set of the glucosyl moiety. In the HMBC spectrum, H-2′ (δ_H 5.61, 1H, dd, J = 5.0, 1.0 Hz), H-6′ (δ_H 2.80, 1H, ddd, J = 13.5, 5.0, 2.5), and H-10′ (δ_H 1.87, 3H, s) protons were long-range coupled with C-4′, suggesting a hydroxy group bonded to C-4′. The HMBC correlations from H-8′ to C-6′ (δ_C 40.3), C-7′, and C-9′ (δ_C 23.6) indicated the C-7′–C-8′ double bond. The anomeric proton resonance at δ_H 4.65 (1H, d, J = 7.5 Hz) was long-range coupled with C-1, suggesting that the glycosyl group was bonded to C-1. The relative and absolute configurations of C-1′S and C-6′R of 5 were confirmed by the same procedure used to confirm those of 3 and 4 (Fig. 3A and Figure S2). Although it was weak, a NOESY correlation was observed between H-4′ and H-6′, suggesting C-4′ was in the R-configuration. From these data, the structure of 5 was elucidated, as shown in Fig. 1.

The molecular formula of 6 (C₂₈H₄₀O₁₄) was confirmed by (–)-HRFABMS, which showed a [M–H]^– ion at m/z 599.2323 (calcd. for C₂₈H₃₉O₁₄: 599.2340). The IR absorption at 1683 cm⁻¹ suggested the presence of the α,β-unsaturated carboxylic acid moiety. In the ¹H NMR spectrum of 6, the proton resonances of H-3 (δ_H 6.89, 1H, d, J = 3.0 Hz), H-5 (δ_H 6.87, 1H, br s), and H-6 (δ_H 7.08, 1H, d, J = 9.0 Hz) indicated the presence of the 1,2,4-trisubstituted benzene. The two 3H singlet proton resonances (δ_H 1.80 and 1.74) suggested the presence of two methyl groups. In the ¹³C NMR spectrum of 6, a carbonyl (δ_C 171.7, C-8′), 10 olefinic (δ_C 152.2, C-1; 133.6, C-2; 119.1, C-3; 154.4, C-4; 115.7, C-5; 117.7, C-6; 124.6, C-2′; 136.4, C-3′; 143.7, C-6′; 128.9, C-7′), and 5 aliphatic (δ_C 29.2, C-1′; 39.4, C-4′; 28.2, C-5′; 16.2, C-9′; 12.5, C-10′) carbon resonances were observed. The HMBC correlations from H-3 to C-1, C-5, and C-1′, from H-6 to C-1, C-2, C-4, and C-5, from the two anomeric protons (δ_H 4.79, overlapping) to C-1 and C-4 indicated the 1,4-diglucosyl-2-C-substituted benzene moiety. Furthermore, the HMBC correlation from the two methyl groups (H-9′ to C-2′, C-3′, C-4′) and (H-10′ to C-6′, C-7′, C-8′) and from the two olefinic protons (H-2′ to C-2, C-1′, C-4′, C-9′) and (H-6′ to C-4′, C-5′, C-7′, C-8′, C-10′) established the structure of the side chain. The NOESY correlation between H-1′ and H-9′ and between H-5′ and H-10′ indicated a E-configurations of the C-2′–C-3′ and the C-6′–C-7′ double bonds. The data show the chemical structure of 6 (Fig. 1).

In the UV spectra of compounds 7–9, common absorption peaks at 250–251 nm and 303–304 nm were observed, suggesting that they had similar skeletons. Compound 7 was a prenylated phenyl glycoside, similar to 6. Its negative-mode HRFABMS (m/z 585.2535 [M–H]^–, calcd for C₂₈H₄₁O₁₃: 585.2547) established its molecular formula as C₂₈H₄₂O₁₃. In the ¹H NMR spectrum of 7, one set of E-configured olefinic proton (δ_H 7.02, 1H, d, J = 16.5 Hz, H-1′; 6.27, 1H, d, J = 16.5 Hz, H-2′) and three singlet methyl proton (δ_H 1.66, H-8′; 1.60, H-10′; 1.35, H-9′) resonances were observed instead of those of H-1′ methylene, H-2′ olefinic, and two methyl protons (H-9′, H-10′) in 6. The ¹³C NMR spectrum of 7 suggested that C-8′ was a methyl instead of a carbonyl as in 6. Although it was difficult to determine the absolute configuration of C-3′ owing to its chain structure and low yield, we attempted to use ECD. The experimental spectrum was compared with the calculated values of the stable conformations of each 3′R-7 and 3′S-7. The negative and positive Cotton effect curves of the calculated data in the 220–240 nm and 240–300 nm ranges, respectively, were in good agreement with the experimental curves (Fig. 3B). However, the 200–220 curves conflict with each other. Therefore, ECD of the simpler modeled structures of 7 (7a and 7b) were calculated (Fig. 3B) and compared with the experimental ECD spectrum of 7; the 3′R calculated curves were in better agreement with the experimental curve, suggesting a 3′R configuration of 7. Therefore, 250–280 nm may be a key region. (Fig. 3B and Figure S3 and S4).

The molecular formula C₂₈H₄₂O₁₃ of compound 8 was identical to that of 7 as obtained from HRFABMS (negative) m/z 585.2540 [M–H]^– (calcd. for C₂₈H₄₁O₁₃: 585.2547). In the ¹H NMR spectrum of 8, methylene (δ_H 3.54, 2H, t, J = 6.5 Hz, H-9′) and methine (δ_H 2.36, 1H, m, H-3′) proton resonances were observed instead of the H-9′ methyl in 7. Similar to 7, the stable conformations of 3′R-8 and 3′S-8 and their ECD data were calculated and compared with the experimental ECD spectrum of 8 (Fig. 3C). The 3′R calculated curves were in better agreement with the experimental curve, suggesting a 3′R configuration of 8. Although the calculated data of the simpler modeled structures of 8 (8a and 8b) were not fully conclusive, 240–270 nm and/or 190–210 nm appeared to be the key regions (Fig. 3C).

Compound 9 showed a molecular formula of C₂₈H₄₂O₁₄ [HRFABMS (negative) m/z 601.2493 [M–H]^– (calcd. for C₂₈H₄₁O₁₄: 601.2496), with one more oxygen atom than that of compound 8. In the ¹H NMR spectrum of 9, an oxygenated methylene (δ_H 4.02, 2H, m, H-10′) proton resonance was observed instead of that of the H-10′ methyl as observed for 8. The NOESY correlation between H-5′ and H-10′ and between H-6′ and H-8′ indicated a Z-configuration of the C-6′–C-7′ double bond. The experimental ECD spectrum of 9 was similar to that of 8, indicating its 3′R-configuration (Fig. 3C). From the above results, the chemical structures of 7–9 were those shown in Fig. 1.

The meroterpenoids obtained in this study (1–9) are chemical structures characterized by being a hybrid of the hydroquinone moiety and the typical monoterpene moieties, including carvacrol (1 and 2), α-terpineol (3 and 4), carveol (5), and linalool (7). Compounds 6–9 have prenyl chain structures and are derivatives of geranylhydroquinone isolated from Aplidium; [6, 7] 6–9 are similar to the starting material for shikonins, which are the main constituents of the medicinal drug Lithospermum Root (Lithospermum erythrorhizon, Boraginaceae) [9]. Furthermore, compounds 3–5 may be applied in the order of the biosynthetic pathways of cannabinoids identified from Cannabis sativa, including tetrahydrocannabinol (THC) and cannabidiol (CBD) (Scheme 1). [10] Compounds 1 and 2 have a fluorene skeleton. Given their biosynthetic pathways, their structures are very similar to those of cannabinol (CBN). CBN has an ether bond with an olivetol moiety, and it appeared that in compounds 1 and 2, the bond between C-3 and C-7′ forms via a similar pathway (Scheme 1). Because CBN is easily produced via oxidation and dehydration from THC [10], it is possible that non-oxidized compounds (3a–6a) may exist in this plant. Although the specific mechanisms and enzyme involvement have not been established, compounds 1 and 2 are presumably derived from 3 and 4, while compounds 6–9 are likely derived from 6a, through a series of oxidation steps. The most important feature of Nemophila meroterpenoids is their glycosylated form, as such compounds are relatively rare. Second, many of them are oxidized compared with the general terpenoid moieties. Because glucosidation occurred only at the two hydroxyl groups of hydroquinone, it was assumed to be caused by hydroquinonization, and the absence of glycosylated hydroxy groups in all the obtained derivatives, except for the hydroquinone moieties, suggested that glucosidation occurred at the 2-geranylhydroquinone stage (Scheme 1).

The above (Scheme 1) provides hints for considering the biosynthetic pathways of plants that contain meroterpenoids and should be investigated in future studies.

To investigate the biological activities of the isolated meroterpenoids, with reference to the anti-inflammatory and/or sedative effects of shikonins [11] and cannabinoids [12] and the inhibitory activities against cholinesterase of many meroterpenoids [4], the anti-degranulation activity and toxicity to mouse RAW264.7 macrophage cells, fatty acid amide hydrolase (FAAH) inhibitory activity, and acetylcholinesterase (AChE) inhibitory activities were evaluated. Compound 6 significantly regulated nitric oxide (NO) production from the LPS-stimulated RAW264.7 cells at 100 µg/mL with no toxicity, showing inhibition of degranulation (Fig. 4).

The cytotoxicity of compound 2 may be attributed to the presence of catechol; however, the details are unknown. For the FAAH inhibitory test, the IC₅₀ values were estimated as 1, 2, and 5, and the compounds showed very low activity relative to that of the positive control JZL 195 hydrochloride (Table 2). No active compounds inhibited AChE in this study. Currently, among the Nemophila meroterpenoids, no significant biological activity has been revealed. One of the reasons why no strong activity was found in this study may be partly because 1–9 were all glycosides. The activities of aglycones alone or their function as glycosides in plants are issues to be addressed in future studies.